LC AND LC/MS

Maximize system performance and produce quality results time after time

Agilent HPLC columns and supplies are designed, tested, and manufactured with the same attention to detail you expect from Agilent instruments. That means your LC or LC/MS system will deliver superior qualitative and quantitative results, consistent reproducibility and reliability, and ultra high-sensitivity detection.

LC and LC/MS supplies

• LC capillaries ensure tight, leak-free connections to protect the integrity of your LC flow path from raw sample to analytical results

• LC pumps include isocratic, binary, quaternary, capillary and preparative; All key components can be accessed by simply removing the front cover

• From small- and large-volume injection, to multi-phase sampling, Agilent autosamplers help you process samples more quickly and generate better data

• Fraction collectors allow you to collect only the peaks you want, and are ideal for applications such as prep HPLC and protein/peptide purification

The ZORBAX LC column family

From research... to leading-edge method development... to routine quality assurance... Agilent ZORBAX and Poroshell HPLC columns are optimized for high throughput analysis, and feature the sensitivity, accuracy, and reliability that demanding applications require. Other advantages include:

• A wide selection of chemistries, including Eclipse Plus, Eclipse XDB, StableBond, Extend, Bonus-RP, HILIC and more

• Superior flexibility and scalability with a wide range of particle sizes (1.8, 3.5, 5, and 7 μm) and column dimensions

• Individual column performance reports that document column-to-column and lot-to-lot reproducibility

• Superior particle strength, even with demanding high-pressure applications

Agilent Solutions

PUT MORE THAN 40 YEARS OF RELENTLESS INNOVATION
BEHIND YOUR EVERY RESULT

By continually raising the standards for technologies that support your routine analyses, Agilent’s R&D efforts have led to breakthroughs such as:

• New GC columns that help you achieve higher levels of inertness and column-to-column reproducibility

• LC column choices that deliver the sensitivity and reliability you need for demanding applications

• Cutting-edge sample preparation products that promote reliable extraction and concentration

• Fresh atomic and molecular spectroscopy ideas for identifying and confirming targets and unknowns

Longtime Agilent customers have experienced our commitment firsthand. And now, we look forward to demonstrating how Agilent’s approach to relentless innovation can work to your advantage, too.

CHEMICAL ANALYSIS SOLUTIONS

Food

From high-volume screening of vegetables for large numbers of pesticides to rapid identification of pathogens, Agilent understands the analytical needs of food producers, shippers, and regulators. When a new toxin appears, we deploy substantial resources to quickly help customers develop robust and reliable methods. Agilent’s leading separations, mass spectrometry, and spectroscopy solutions are emerging as valuable food testing techniques.

Environmental

Agilent offers more than 40 years of environmental testing and regulatory expertise. We help government and private labs with the full range of assays, from routine testing of soils for heavy metals to detection of pharmaceuticals in groundwater, in concentrations down to parts per trillion.

Energy and Fuels

Agilent collaborates closely with process industry customers to offer analytical systems that meet their needs for separation, detection, throughput, and support. We’ll even preconfigure custom or standard analyzers so they arrive at the lab ready-to-go. Agilent’s expertise in both chemical analysis and life science is a powerful combination for researching and producing biofuels, including a wide range of analytical techniques for fatty acid methyl esters (FAMEs). Our newly-expanded portfolio also offers powerful tools for developing and producing photovoltaic films and solar panels.

Forensics

Because the careers of world class athletes and many other individuals hinge on drug testing, it’s critical that those doing the testing have the highest level of confidence in the results. Forensics analysts worldwide have grown to depend on Agilent tools for accuracy, reliability, and speed in this high stakes, high-throughput field. Our best selling GC, GC/MS and popular LC and LC/MS are workhorses in forensics labs.

Traditional Lab Informatics

The ways labs generate and store data profoundly affect their efficiency. Agilent offers a rich, integrated suite of software products built on a set of customer-driven architectural values with the Agilent OpenLAB Laboratory Software Suite. OpenLAB delivers superior performance, open systems integration and investment protection. Our commitment is to deliver more value across each step in the life cycle of scientific data – from data collection and analysis to interpretation and management.

Materials Science

Agilent offers a newly expanded portfolio of instruments used for the research, manufacturing and testing of advanced materials, from precision optics to pulp, paper and polymers. Tools for chromatography, atomic absorption spectroscopy, molecular spectroscopy, X-ray crystallography, and nuclear magnetic resonance all support continuous progress in materials science.

LIFE SCIENCE SOLUTIONS

Biopharmaceutical

As “multi-omics” studies gain momentum in the search for new therapeutics, Agilent is uniquely positioned to provide the instruments, reagents, and powerful software needed for performing experiments in multiple disciplines and combining the massive amounts of data into biological insight.

Pharmaceutical

Drug manufacturing requires the accuracy, sensitivity and high throughput of other analytical applications, along with the demands of regulatory record-keeping and validation requirements. Agilent provides a potent combination of rugged, high-throughput tools and unmatched compliance services. Agilent now offers the market-leading family of dissolution apparatus and sampling systems that pair perfectly with our HPLC and UV systems.

Proteomics

Research into how large sets of proteins affect the health of an organism requires special sets of analytical tools. Agilent has built a formidable arsenal of liquid chromatograph/mass spectrometers, bioinformatics systems, multiple affinity protein removal columns, and OFFGEL electrophoresis for protein identification and protein biomarker discovery. Accurate-Mass mass spectrometry and the microfluidic HPLC-Chip/MS are two Agilent innovations speeding the work of proteomics researchers around the globe.

Metabolomics

Collections of small molecules are increasingly being seen as rich sources of biomarkers, but studying metabolites presents many challenges. The need for speed, accuracy, and powerful interpretation capabilities in looking at chemical profile snapshots is underscored because molecules are constantly entering, leaving or changing within the metabolome. Agilent’s GC, LC, NMR and MS portfolios, along with our excellent bioinformatics offerings, user-customizable METLIN metabolite database for LC/MS, and the industry’s first commercial GC/MS retention time locked metabolite library align well with needs of metabolomics researchers.

Genomics

Agilent is a global leader in microarrays, scanners, and reagents used in a wide variety of genomic-based disease research experiments. Our SureSelect Target Enrichment System dominates the category, streamlining next generation sequencing studies worldwide. Agilent offers a wide range of catalog microarrays and a highly-developed capability to produce custom arrays featuring ink jet-based SurePrint fabrication and the eArray on-line design tool. All Agilent microarrays feature highly sensitive, selective 60-mer probes. With as many as eight arrays printed on a standard 1 x 3 in slide, the cost per experiment becomes very affordable.

Life Science Informatics

Mirroring its extensive instrument portfolio, Agilent offers the industry’s most extensive suite of bioinformatics software, helping users derive knowledge from complex genomic, proteomic, metabolomic and other biological data. This includes DNA Analytics for analyzing CGH, ChIP and methylation microarray data. The GeneSpring suite includes informatics software for microarray-based gene expression data, genotyping data, and GeneSpring MS, which are useful for analyzing mass spec data from proteomics and metabolomics experiments and comparing complex datasets to explore biological questions from multiple perspectives.

Lab Automation

To meet the skyrocketing demand for more throughput and automation, Agilent has substantially expanded its lab automation offerings. The Agilent line of liquid handlers and microplate processors are designed to streamline high-volume life science workflows. Agilent is also continually upgrading its advanced autosamplers for LC, GC, LC/MS and GC/MS, adding functionality and speed to reflect the performance of its advanced instruments.

Vacuum Technology

Agilent works with customers to solve vacuum challenges from experiments in high-energy physics to developing systems for producing flat panel displays. Agilent manufactures vacuum systems used in its own mass spectrometry instruments as well as those of other manufacturers. Agilent’s vacuum technology has been proven by the most powerful physics experiment ever built, CERN’s Large Hadron Collider machine, which was used in the discovery of the Higgs boson particle.

AGILENT SERVICE AND SUPPORT

Agilent Service and Support for Instrument Systems

Focus on what you do best

For over 40 years, Agilent has been building and maintaining the instruments you count on to stay competitive and successful. Trust us to protect your investment with a broad portfolio of services, backed by a global network of experienced service professionals dedicated to the productivity of your lab.

Get the Agilent Service Guarantee

Should your instrument require service while covered by an Agilent service agreement, we guarantee repair or we will replace your instrument for free.

No other company offers this level of commitment to keep your lab up and running at peak efficiency.

Laboratory decision makers and users ranked Agilent as their first choice for general laboratory compliance services.

Agilent Advantage Service Plans

The best service available for your Agilent instruments

Agilent offers a flexible range of service plans so that you can choose the level of coverage that is best for your lab.

• Agilent Advantage Gold – Priority-one coverage for ultimate uptime and productivity

• Agilent Advantage Silver – Comprehensive coverage for dependable laboratory operations

• Agilent Advantage Bronze – Total repair coverage at a fixed annual price

• Agilent Repair Service – Basic coverage for reliable instrument repair

Agilent Advantage service plans include Agilent Remote Advisor for real-time remote monitoring and diagnostics. Through secure internet connections, you can interact with Agilent service professionals, receive detailed asset reports, and configure text or email alerts to notify you before problems occur – helping you to maximize instrument uptime and optimize laboratory workflows.

Agilent Compliance Services

Equipment qualification that meets the most stringent requirements

Enterprise Edition Compliance was developed to streamline compliance across your entire lab. Used globally in regulated labs, including standards organizations and regulatory agencies, Enterprise Edition enables you to:

• Improve qualification efficiency by automating protocols across platforms to ensure greater efficiency and minimize regulatory risk

• Standardize your entire compliance operation with robust test designs that work with all your instruments

• Add, remove or reconfigure tests based upon your unique user requirements

• Significantly reduce staff review time with consistently formatted, computer generated, tamper-proof reports

Agilent Education and Consulting Services

Our best minds, working for you

Make the most of your instrument with training and consulting from the same experts who designed the instruments, software and processes you use every day.

• Classroom and on-site training in instrument operation, troubleshooting and maintenance

• Customized consulting services to meet your lab’s unique needs

The Agilent Value Promise — 10 Years of Guaranteed Value

In addition to continually evolving products, we offer something else unique to the industry – our 10-year value guarantee. The Agilent Value Promise guarantees you at least 10 years of instrument use from your date of purchase, or we will credit you with the residual value of the system toward an upgraded model. Not only does Agilent ensure a reliable purchase now, but we also ensure that your investment is just as valuable in the future.

For more detailed information,
please go to www.agilent.com/chem/services
or contact your local Agilent Services and Support representative.

Technical Support at work for you

Have a hardware, software, application, instrument repair or troubleshooting question? Agilent’s technical experts are available to answer your questions. With years of laboratory experience, our technical support specialists can provide in-depth knowledge and experience.

For questions pertaining to supplies found in this catalog, contact your local Agilent office or Authorized Agilent Distributor or visit www.agilent.com/chem/techsupport

Need more information?

Visit www.agilent.com/chem/contactus to:

• Locate your nearest Agilent office or distributor for expert technical support.

• Get fast sales and product assistance by phone. Simply use the scroll-down menu to select your country.

• Receive email assistance using our convenient online forms.

FEATURED PRODUCTS

A Portfolio of Analytical LC Solutions to Match Any Application or Budget

Agilent offers a comprehensive portfolio of solutions for analytical LC that give you uncompromised chromatographic performance while remaining within the confines of your budget. Whatever your application requires – now or in the future – common technology across the portfolio helps you increase laboratory productivity and decrease operational costs. And because it’s from Agilent, you get everything you expect from a chromatography leader with over 40 years of innovative contributions to LC and LC/MS technology.

Agilent 1290 Infinity LC – Infinitely more powerful

With binary and quaternary pump options, The Agilent 1290 Infinity LC is not only the most powerful but also the most adaptive UHPLC system available. No matter what your laboratory requires, the 1290 Infinity LC can handle the widest range of applications. And, with Agilent’s Intelligent System Emulation Technology (ISET) built in, you can execute any legacy HPLC or latest UHPLC method for faster method development or instrument-to-instrument method transfer.

Agilent 1260 Infinity LC – Infinitely more confident

The Agilent 1260 Infinity LC raises the standard in HPLC – without raising the price. It offers new levels of productivity, data quality and robustness to give you highest confidence in your investment. With 600 bar standard pump pressure, 80 Hz standard detector speed and up to 10 times higher UV detection sensitivity, the 1260 Infinity LC prepares you for today’s and tomorrow’s challenges.

Agilent 1220 Infinity LC – Infinitely more affordable

The Agilent 1220 Infinity LC is a high quality, integrated system for routine HPLC and advanced UHPLC analysis, for maximum return on investment. Take advantage of the 600 bar power range up to 5 mL/min and 80 Hz detector speed and prepare your lab to take advantage of latest advances in LC column technology.

Low-flow LC Solutions
with Unmatched Performance and Stability

Low-flow LC separation with high sensitivity detection and low sample consumption is a well-established and proven technology. The low-flow technique is used frequently for applications where limited amounts of sample are available, for analysis of trace level components in complex mixtures, or when an optimized LC/MS combination is required. The Agilent 1260 Infinity Low-flow LC systems offer unsurpassed low-flow performance and stability.

Agilent 1260 Infinity Capillary LC System

Using a unique technology, the Agilent 1260 Infinity Capillary LC System is optimized for capillary LC. A wide range of flow rates expands laboratory flexibility, offering unparalleled sensitivity and reproducibility. The system is ideally suited for both UV and MS applications.

Agilent 1260 Infinity Nanoflow LC System for MS

Nanoflow LC is widely used for high resolution separations and high sensitivity MS detection. The Agilent 1260 Infinity Nanoflow LC System incorporates Agilent’s unique Electronic Flow Control (EFC) with active feedback and real time flow adjustment for constant flow delivery to the column independent of system backpressure. After its first introduction with Agilent capillary and nanoflow pumps, EFC with real time flow control is the gold standard for reliable and robust nanoflow performance.

Agilent 1260 Infinity HPLC-Chip/MS System

The Agilent 1260 Infinity HPLC-Chip/MS System is a microfluidic chip-based technology for nanospray LC/MS. Combined with the high performance Agilent 6000 MS systems, the HPLC-Chip II for LC/MS offers even more overall robustness, reliability and ease-of-use. Applications include proteomics and small molecule analysis. A custom chip program delivers customized success.

Agilent 6000 Series LC/MS Solutions

A comprehensive portfolio of LC/MS instrumentation with exceptional MS performance, spectral quality, and productive data analysis tools to address a full range of qualitative and quantitative applications and match your budget.

Agilent 6100 Series Single Quadrupole LC/MS Systems

Unprecedented performance and reliability, day-after-day

The Agilent 6100 Series Single Quadruple systems deliver superior MS data quality in an easy-to-use, space-saving package. With configurations to fit all budgets, the industry’s most popular single-quad LC/MS complements your existing LC detectors and integrates seamlessly with Agilent’s ChemStation LC control. The 6100 Series Single Quadrupole LC/MS provides complementary information to ultraviolet diode array detection (UV-DAD).

Agilent 6120 – Budget friendly and very easy to use – with Agilent’s 1220 Infinity LC, a perfect workhorse addition for labs just getting into LC/MS.

Agilent 6130 – Flexible, high performance solution ideal for any quantitation application with 3,000 amu mass range and 1 pg sensitivity – now compatible with Agilent Jet Stream Technology.

Agilent 6150 – Unsurpassed data quality for UHPLC and high-throughput screening and qualitative applications, with faster scan speed (10K amu/s) and the power of Agilent Jet Stream Technology.

Agilent 6200 Series Accurate-Mass TOF LC/MS Systems

Unmatched confidence of Ultra High Definition Time-of-Flight (TOF) technology

The Agilent 6200 Series TOF systems deliver unmatched speed, mass accuracy and resolution performance in a compact benchtop design. Superior data quality offers certainty for screening, identification and quantitative studies.

Agilent 6224 – Affordable and easy-to-use instrument with <2 mg/L mass accuracy and 20,000 resolution for routine analysis of a wide variety of molecules such as pesticides, pharmaceuticals, peptides and intact proteins.

Agilent 6230 with Agilent Jet Stream Technology – Offers 5-10x more sensitivity for confident screening of compounds at low concentration.

Agilent 6400 Series Triple Quadrupole LC/MS Systems

Clearly better sensitivity gives better results

The Agilent 6400 Series Triple Quadrupole systems offer unmatched sensitivity and reliability for trace analysis in food safety, environmental samples, biomarker validation, and ADME/DMPK studies for drug candidates. Powered by Agilent MassHunter software, with Optimizer to automatically fine tune parameters for ion transitions, the 6400 Series Triple Quadruple systems offer simplified method development to achieve outstanding results.

Agilent 6420 – Economical, easy to use, with Agilent 1260 Infinity LC, a perfect workhorse instrument for labs with basic needs for quantitative capabilities.

Agilent 6430 – Offers added sensitivity to achieve outstanding performance for most assays.

Agilent 6460 with Agilent Jet Stream Technology – Provides dramatically improved sensitivity for your most demanding quantitative applications.

Agilent 6490 – Incorporates the revolutionary iFunnel technology to give ultimate sensitivity for the most challenging quantitative analyses in pharmaceutical, clinical, food safety, and environmental applications.

Agilent 6500 Series Accurate-Mass Q-TOF LC/MS Systems

Ultra high definition MS/MS, uncompromised performance

The Agilent 6500 Series Q-TOF systems deliver an unmatched combination of mass accuracy, mass resolution, sensitivity, dynamic range and speed. With best-in-class MS and MS/MS mass accuracy, they provide the capabilities you need to profile, identify, characterize, and even quantify samples as diverse as drug impurities, endogenous metabolites, and protein biomarkers.

Agilent 6520 – Easy-to-use instrument for routine analysis of a wide range of compounds day in and day out.

Agilent 6530 with Agilent Jet Stream Technology – Provides 5-10x more sensitivity for screening of low level compounds.

Agilent 6538 – Provides enhanced resolution in a bench top instrument for confident characterization of complex samples.

Agilent 6540 with Agilent Jet Stream Technology – Combines the highest levels of sensitivity with enhanced resolution to address the most challenging applications.

Agilent ZORBAX 300 StableBond Columns

ZORBAX 300 StableBond columns are an ideal choice for the reproducible separations of proteins and peptides. 300 StableBond columns are unmatched in their durability at low pH, such as with the TFA containing mobile phases typically used for protein and peptide separations. For LC/MS separations at low pH, 300 StableBond columns can also be used with formic acid and acetic acid mobile phase modifiers.

View Here.

Agilent Poroshell Columns

As new biopharmaceutical research and manufacturing practices focus more and more on peptides and proteins, and as time to market becomes increasingly critical, the rapid HPLC separation of biomolecules becomes increasingly valuable. The speed and resolution benefit shown here by Agilent Poroshell columns improve chances that the desired separation will be achieved while facilitating separation analysis times.

For Poroshell 120, View Here.
For Poroshell 300, View Here.

Agilent LC Capillaries

Agilent LC capillary starter kits contain the most often used capillaries and fittings. Our genuine flexible stainless steel capillaries make the best connection in your LC system, no matter the brand. The kits are for use with 3-4 or 1-2 mm id columns, as well as for Micro LC columns. The free Cybertool, which contains over 30 tools, is useful in every laboratory.

View Here

Agilent ZORBAX RRHD HILIC Plus

HILIC can have distinct advantages over traditional RPLC in terms of LC/MS sensitivity, due to the use of highly organic mobile phases. These mobile phases have higher volatility than traditional RPLC mobile phases, making HILIC well suited for applications with mass spectrometers. Agilent ZORBAX RRHD HILIC Plus is the column to choose.

View Here

AGILENT PARTS AND SUPPLIES

General LC Supplies

Agilent offers a wide range of supplies for operation and maintenance of LC systems. These products have been carefully designed or selected by Agilent to work with your Agilent instruments for maximum performance and uptime.

LC Tools

Your Agilent LC system arrives with a full complement of tools needed to perform general maintenance and operation procedures. Should you need additional or replacement tools, Agilent offers a selection of high-precision, high-quality, stainless steel tools, to avoid any deformation of the screws or nuts.

Hex keys, Rheotool 5064-8211

Mounting tool, 0100-1710

HPLC system tool kit, G4203-68708

Compact tool kit, G4296-68715

Torque wrench, 2-25 Nm, G4220-20012

Plastic tubing cutter, 8710-1930

Tubing clip, 5042-9967

LC Tools
Description | Part No.
Tool kit hex keys, Rheotool Includes 3 hex keys, 4 mm, 1.5 mm, and 9/64 in, with straight or T-handle plus Rheotool | 5064-8211
Torque wrench adapter Used with nanoliter flow cell for Diode Array Detector to mount capillaries of cell (P/N G1315-68714) | G1315-45003
Insert tool (seal wash option) | 01018-23702
Mounting tool for flangeless nut | 0100-1710
Mounting clamp | 5021-1866
Velocity regulator | 5062-2486
USB memory stick | G4208-68700
Compact flash card | 01100-68700
HPLC system tool kit | G4203-68708
Compact tool kit | G4296-68715
Torque wrench, 2-25 Nm | G4220-20012
Bit kit for torque wrench | 5023-0282
PEEK Luer lok needle assembly | 5190-0924
Plastic tubing cutter | 8710-1930
Blades for plastic cutter, 5/pk | 8710-1931
Tubing clip | 5042-9967
Tubing clip | 5042-9954
Open end wrench, 14 mm | 8710-1924

HPLC In-Line Filters

Column inlet frit contamination can increase column backpressure and reduce efficiency. Microbore column blockages are a particular problem, due to the small diameter of the inlet frit. To prevent blockages, always use the appropriate filters in your LC system. Agilent offers two types of high pressure in-line filter kits for use with any HPLC system.

RRLC in-line filter, 5067-1551

Low dispersion in-line filter, 01090-68702

Semi-prep filter, 5064-8273

High pressure semi-prep filter, 5022-2165

1290 Infinity LC in-line filter, 5067-4638

Solvent Filters/Degassers

An added benefit of filtering solvents is that degassing occurs at the same time. This is particularly beneficial if you do not have an on-line degasser in your system. The benefits of solvent filtration:

• Degasses eluents as particulates are removed

• Prevents the formation of spurious peaks within the detector due to solvent outgassing at the low-pressure end of the chromatograph

• Increases solvent inlet lifetime

• Eliminates pump downtime caused by air locks and particulates in check valves

• Decreases piston wear, while increasing column life

Glass solvent filter degasser, 3150-0577

Solvent Filters/Degassers
Description | Part No.
HPLC solvent filter/degasser assembly | 3150-0577
Replacement Parts for 3150-0577 |
Glass funnel, 250 mL | 5188-2743
PTFE coated sieve | 5188-2744
PTFE seal | 5188-2745
Funnel base, glass | 5188-2746
Filter Membranes |
Regenerated cellulose filter membranes Diameter 47 mm, pore size 0.45 μm, 100/pk | 3150-0576
Nylon filter membranes Diameter 47 mm, pore size 0.45 μm, 100/pk | 9301-0895
PTFE filter membranes Diameter 47 mm, pore size 0.45 μm, 10/pk | 3150-0509

Agilent Rack for LC Systems

Reclaim critical bench space with the stable, robust rack for LC systems. The sturdy and open design offers complete protection for your sensitive LC system and easy maintenance for fast, safe access to instruments and cables. It is designed for all Agilent LC modules stacks and features:

• Easy assembly saves time and expense (see diagram)

• Adjustable shelves allow full customization for all Agilent LC modules

• Open design ensures proper airflow management and distribution of equipment and cabling

Description | Part No.
Agilent rack for LC systems | 5001-3726

Agilent rack for LC systems, 5001-3726

TIPS & TOOLS

The Agilent rack can be used to optimize your 1290 Infinity LC for ultra-low dispersion, which can enhance performance of high-efficiency columns. Further information can be found in application note 5990-9502EN at www.agilent.com/chem/library

LC Standards

LC Standards
Description | Part No.
Caffeine standards kit for LC OQ/PV Includes one 10 mL ampoule: 125.0 μg/mL; four 5 mL ampoules: 5.0, 25.0, 250.0 and 500.0 μg/mL caffeine in water | 8500-6762
Caffeine standards kit for capillary OQ/PV Includes 5 ampoules, 5 mL: 2.0, 4.0, 20.0, 100.0, 200.0 μg/mL caffeine in water | 5065-4420
Caffeine OQ/PV sample for dissolution test, 150 mg/L caffeine in water, 500 mL | 5042-6476
Caffeine standard, 250 μg/mL | G4218-85000
Enterprise Edition caffeine standard kit | 5190-0488
Fluorescence detector calibration sample, 1 g glycogen | 5063-6597
RI detector OQ/PV test sample Includes 5 ampoules, 5 mL: 5, 10, 15, 25, and 50 mg/mL glycerin in water | 5064-8220
Isocratic and gradient standards Contains 0.15% diethylphthalate, 0.01% biphenyl, and 0.03% terphenyl in MeOH (w/w). Gradient standard includes 0.32% dioctyl phthalate as well. Two 0.5 mL ampoules of each. | 01080-68702
Isocratic standard, 0.5 mL ampoule | 01080-68704
RRLC Check out sample, 1 mL ampoule | 5188-6529
Chip cube high mass reference (HP-1221), 0.5 mL | G1982-85001
Chip cube high mass solvent (FC-70), 25 mL Fluorinert | G1982-85002
Chip cube low mass reference sample, 1 g Methyl stearate | G1982-85003
ESI+APCI LC demo sample Contains 5 x 1 mL ampoules with 33 ng/μL crystal violet, 77 ng/μL carbazole, 300 ng/μL 9-phenanthrol, 1 ng/μL 1-hexanesulfonic acid sodium salt in water/methanol 60:40 | G1978-85000
ES-TOF biopolymer reference standard kit Contains 7 x 2 mL ampoules with 5 mM purine, 1 M ammonium formate, 0.5 mM HP-0285, 0.1 mM HP-0321,0.2 mM HP-1221, 0.2 mM HP-1821, 0.5 mM HP-2421 | G1969-85003
HSA peptide standard mix kit 2 vials with 6 lyophilized peptides | G2455-85001

Caffeine OQ/PV sample for dissolution test, 5042-6476

LC Capillaries

Your LC system’s components are only as reliable as the connections between them

Think of your LC system as a chain from analyte... to pump... to column... to detector... to waste. Every link must operate at maximum efficiency, or the whole chain risks failure – compromising your results.

Agilent’s unique laser welding process ensures that the capillary ends are absolutely flat, eliminating any chance of capillary-induced dead volume.

Agilent LC capillaries:
Your link to analytical success

At Agilent, we invest heavily in the quality of our capillary connections. All are engineered and manufactured to the same quality standards as our columns and instruments, so you can protect the integrity of your results at every step of your LC flow path.

Using our flexible stainless steel and polymer capillaries and fittings can provide:

• Tight, leak-free connections

• Zero dead volume connections

• An inert surface (when using polymer or PEEK/stainless steel bio-inert capillaries)

• High flexibility without sacrificing durability

• Easy cutting to the exact length you need (PEEK tubing)

• Predefined lengths for specific flow path locations (capillaries)

In addition, all Agilent capillaries are precision cut with square ends, are burr-free, have no inner-diameter distortion, and come in a variety of materials to suit your needs.

Engineering of Agilent Capillaries

The same professional engineers in our LC manufacturing facility in Germany who design our industry-leading LC instruments, also play a critical role in developing capillaries and fittings for your instrument. Their attention to detail helps you to get the best performance possible for your applications.

Our LC manufacturing tools – like high-end, special laser-cutting machines – result in completely tight, smooth and perfect-cut capillaries. Our broad selection of capillaries is made only from the highest-quality materials and will meet any of your application needs.

Avoid chromatographic issues – like peak broadening and system leaks – by choosing Agilent’s selection of premium capillaries. Agilent is committed to Fast LC and high-productivity performance, together with accurate quantitation.

Advantages for Bio Capillaries

• Laser-welded capillary tip for precise capillary cut

• Metal-free connection for all HPLC applications

• PEEK and stainless steel design allows you to exceed typical pressure limit reached with conventional polymer

Recommended HPLC Stack Configuration

Agilent 1290 Infinity LC: default stacking and capillary tubing configuration

Agilent 1290 Infinity LC: with LC system rack and ultra-low dispersion optimizations

Syntax for capillary description

The tables below will be your guide to identifying the proper specifications for your capillary. On all capillaries, dimensions are noted in id (mm), length (mm) and where applicable, volume (μL). When you receive your capillary, these abbreviations are printed on the packaging.

Using the guide: This fitting is coded as “SPF”, for Swagelok, PEEK, Finger-tight.

Type
Key | Description
Capillary | Connection capillaries
Loop | Loop capillaries
Seat | Autosampler needle seats
Tube | Tubing
Heat exchanger | Heat exchanger

Material
Key | Description
SS | Stainless steel
Ti | Titanium
PK | PEEK
FS/PK | PEEK-coated fused silica*
PK/SS | Stainless steel-coated PEEK**
PTFE | PTFE
FS | Fused silica

*Fused silica in contact with solvent

**PEEK in contact with solvent

Fitting Left/Fitting Right
Key | Description
W | Swagelok + 0.8 mm Port id
S | Swagelok + 1.6 mm Port id
M | Metric M4 + 0.8 mm Port id
E | Metric M3 + 1.6 mm Port id
U | Swagelok union
L | Long
X | Extra long
H | Long head
G | Small head SW 4 mm
N | Small head SW 5 mm
F | Finger-tight
V | 1200 bar
B | Bio
P | PEEK

The type gives some indication on the primary function, like a loop or a connection capillary.
The material indicates which raw material is used.
The fitting left/right indicate which fitting is used on both ends of the capillary.

At-a-glance color-coding keys

The color of your capillary will help you quickly identify the capillary id – see the chart to the right for reference.

Color-coding key for Agilent capillary tubing
Internal Diameter in mm | Color code
0.015 | Orange
0.025 | Yellow
0.05 | Beige
0.075 | Black
0.1 | Purple
0.12 | Red
0.17 | Green
0.20/0.25 | Blue
0.3 | Grey
0.50 | Bone White

Tip: As you move to smaller-volume, high efficiency columns, you’ll want to use narrow id tubing, as opposed to the wider id tubing used for conventional HPLC instruments.

Agilent capillary supplies are made from a variety of top-quality materials to suit your lab’s every need

Stainless Steel: good resistance to pitting corrosion

Stainless steel is ideal for most standard applications – except where bio-inertness is required, in which case we recommend PEEK-lined or Bio-inert titanium capillaries. Agilent’s 0.6 mm od flexible grade 316L stainless steel capillaries (chrome/nickel/molybdenum bearing grade) are also much easier to handle than conventional, rigid 1.6 mm od capillaries.

Titanium: high inertness for biological applications

Analyzing metal-sensitive proteins and biotherapeutics presents challenging solvent conditions for LC instruments. In addition, bio-molecules tend to bind non-specifically to surfaces. For these reasons, bio-inert titanium is the best choice for these applications. Titanium is biocompatible, making Bio-inert Titan capillaries perfect for applications where bio-inertness is paramount.

Stainless Steel-coated PEEK: high-pressure bio-inertness and robustness

In bio-chromatography, capillaries and connectors should be inert to ensure the lowest interaction with protein samples. They must also be highly robust to withstand harsh cleaning procedures.

Unfortunately, metal-free PEEK capillaries can only withstand pressures of up to 200 bar in a thermostatically controlled cabinet with acetonitrile; even then, flexibility is compromised. To meet the growing need for bio-inertness, robustness, and higher operating pressures, Agilent has engineered a bio-inert PEEK liner clad with high-strength stainless steel to withstand pressures of at least 600 bar. This same technology is used in Agilent capillary fittings – giving you a strong, metal-free, capillary/connector flow path for bio-inert applications.

PEEK-coated fused silica: rugged and pliable

Since their introduction in the early 1980s, fused silica capillaries have become the industry standard for many GC and LC applications – as well as capillary electrophoresis. Agilent fused-silica capillaries are made from high-purity silicon dioxide, and coated with PEEK for strength, durability, and pliability.

PEEK: durable and abrasion-resistant

Agilent PEEK capillaries are best for standard and bio-inert applications. PEEK (polyetheretherketone) is a thermoplastic polymer that resists mechanical and solvent damage, even at high temperatures. Because it is less vulnerable to corrosion than stainless steel, PEEK can be used in place of stainless steel when the capillary’s external diameter is 1/16 in or less. It also resists abrasion, making it an excellent coating for fused silica capillaries. Tip: Use our color-coded PEEK fittings to track inlets and outlets of valves, columns, and detectors.

To learn more about Agilent LC capillary supplies, or to order now, visit www.agilent.com/chem/LCcapillaries

Agilent capillaries for routine applications

*At ambient temperature with water

**With acetonitrile at non-ambient temperature

Fittings for a strong, capillary flow path

Agilent offers more than 20 fitting varieties for Swagelok-type or metric M4/M3-type connections. Depending on your application, different materials must be used:

• Stainless steel or PEEK delivers permanent high-pressure sealing performance for connections such as valves, heaters, and columns

• Stainless steel ensures permanent high-pressure sealing and optimal performance throughout your LC system up to 1200 bar

• Finger-tight fittings (polymeric for 400 bar and polyketone for 600 bar) are a convenient option; They allow easy end fitting adjustment, so you can seat the capillary into the column properly, preventing extra-column voids and leaks

• High-pressure fittings, which can be used with pressures up to 1200 bar, can be removed and replaced

• It’s a good idea to use stainless steel nuts and ferrules for instrument connections, and PEEK nuts and ferrules for column and guard column connections, since these are changed most frequently

Agilent fittings for leak-free connections

Fitting type | Advantages/Tips

Swagelok-type fittings

• Suitable for most connections

• Available in a variety of combinations:

• One piece or multiple pieces with nut + front and back ferrules

• Stainless steel, PEEK, polyketone, or a stainless steel/PEEK combination

Metric M4/Metric M3 | • For micro valve connections
Stainless steel | • At least 1200 bar • Most popular material for permanent, high-pressure sealing • Use our slitted socket wrench (Part No. 8710-2391 or 5023-0240) for optimal tightness

1200 bar removable fittings for 1290 Infinity LC

• 1200 bar

• Available in standard, long, and extra-long sizes for compatibility with columns that have different sized nuts

• Removable and replaceable

• Use our slotted socket wrench (Part No. 8710-2391 or 5023-0240) for optimal tightness

PEEK

• Up to 400 bar (at ambient temperature with water)

• Easy, finger-tight column connections

• Ideal for frequently changed connections, such as column connections

• Pressure is less critical

Polyketone | • <600 bar (600 bar pressure rating) • Easy, finger-tight column connections • Fits stainless steel tubing

*Capillary is intended to be cut to the right length for your need.

**Calibration capillary assembly

*Capillary is intended to be cut to the right length for your need.

Stainless steel fittings (S), 5062-2418

Finger-tight PEEK fitting (SPF), 0100-1516

Stainless steel back ferrule, 5180-4114

Images shown are capillary fittings. For complete information on fittings, View Here.

Material
Key | Description
SS | Stainless steel
S | Swagelok 1.6 mm port id
SH | Swagelok 1.6 mm port id, long head
SL | Swagelok 1.6 mm port id, long
SLV | Swagelok 1.6 mm port id, long, 1200 bar
SX | Swagelok 1.6 mm port id, extra-long
M | Metric M4 0.8 mm port id

Stainless steel fittings (S), 5062-2418

Stainless steel extra long fitting (SX), 5065-9967

1200 bar removable long fitting (SLV), 5067-4738

1200 bar removable fitting (SV), 5067-4733

PEEK fittings, plugs (MP), 5065-4410

Images shown are capillary fittings. For complete information on fittings, View Here.

*1.6 mm = 1/16 in

Material
Key | Description
SS | Stainless steel
S | Swagelok 1.6 mm port id
U | Swagelok union
SL | Swagelok 1.6 mm port id, long
SLV | Swagelok 1.6 mm port id, long, 1200 bar
SX | Swagelok 1.6 mm port id, extra-long
M | Metric M4 0.8 mm port id

Stainless steel fittings (S), 5062-2418

Images shown are capillary fittings. For complete information on fittings, View Here.

*1.6 mm = 1/16 in

Stainless steel extra long fitting (SX), 5065-9967

Stainless steel ferrule (M), 5067-1557

Images shown are capillary fittings. For complete information on fittings, View Here.

*1.6 mm = 1/16 in

Material
Key | Description
SS | Stainless steel
PK | PEEK
PK/SS | PEEK and Stainless steel
Ti | Titanium
S | Swagelok 1.6 mm port id
U | Swagelok union
SL | Swagelok 1.6 mm port id, long
SLB | Swagelok 1.6 mm port id, long bio
SV | Swagelok 1.6 mm port id, 1200 bar
SLV | Swagelok 1.6 mm port id, long, 1200 bar
SX | Swagelok 1.6 mm port id, extra-long

Stainless steel fittings (S), 5062-2418

Stainless steel extra long fitting (SX), 5065-9967

Stainless steel ferrule (M), 5067-1557

Images shown are capillary fittings. For complete information on fittings, View Here.

1200 bar removable fitting (SV), 5067-4733

1200 bar removable long fitting (SLV), 5067-4738

Titanium fitting (SLB), G5611-60502

Bio-inert Low Dispersion Heat Exchanger, G5616-60050

Images shown are capillary fittings. For complete information on fittings, View Here.

Stainless steel fittings, male (G), 5063-6593

Ferrule and stainless steel lock ring (W), 5065-4423

PEEK fittings, plugs (MP), 5065-4410

Double winged PEEK nut & ferrule (WPF), 5065-4422

Images shown are capillary fittings. For complete information on fittings, View Here.

Loop Capillaries
Volume (μL) | Agilent Autosampler | Part No.
8 | G1389A | G1375-87303
| G1377A | G1375-87315
20 | G1367E, G4226A | G4226-60310
40 | G1367D | G1377-87310
| G1367E, G4226A | 5067-4703
| G1377A | G1377-87300
| G1389A | G1329-87302
100 | G1313A, G1329A/B, 1120, 1220 Infinity LC | 01078-87302
| G1367A/B/C | G1367-87300
| G1367E, G4226A | 5067-4710
| G5667A | G5667-60310
900 | G1329A/B, G2260A | G1313-87303
5000 | G2260A | G2260-68711

Material
Key | Description
FS/PK | Fused silica/PEEK
W | Swagelok 0.8 mm port id
WG | Swagelok 0.8 mm port id, small head SW 4 mm
MP | Metric M4 0.8 mm port id, PEEK
WPF | Swagelok 0.8 mm port id, PEEK, finger-tight

Material
Key | Description
SS | Stainless steel
S | Swagelok 1.6 mm port id
PK | PEEK
PTFE | PTFE

Stainless steel fittings (S), 5062-2418

Finger-tight PEEK fitting (SPF), 0100-1516

Images shown are capillary fittings. For complete information on fittings, View Here.

Tubing

PEEK Tubing

• Flexible and easy to cut to desired lengths

• Color coded for easy tracking

• Accepts both stainless steel and PEEK fittings

• 1/16 in od

Accessories
Description | Part No.
Plastic tubing cutter | 8710-1930
Blades for plastic cutter, 5/pk | 8710-1931
Fitting screws, stainless steel, 10-32, 4 mm, 5/pk | 5065-9948
PEEK ferrule and stainless steel ring for 2 mm tube, 5/pk | 5065-9950
Union, PEEK for 1/8 in od tubing | 0100-2410
Waste adapter, 1200 Series autosamplers, gray | G1313-43216

Plastic tubing cutter, 8710-1930

Fitting screws, 5065-9948

PEEK ferrules and SS rings, 5065-9950

Rigid Capillary Tubing

• Squarely cut, pre-cleaned and ready to use

• Use with stainless steel fittings and ferrules (P/N 5062-2418) or PEEK fittings (P/N 0100-1516)

Fittings and Unions

Your best value: Agilent multi-use capillary and fitting kits with FREE cybertool

Agilent starter kits contain the most widely used capillary tubing, Swagelok connectors, and fittings in a variety of sizes, so you can find just the right length to minimize your connections and tubing volume. We’ve also included our flexible stainless steel capillaries to help you make the best LC connections, regardless of equipment brand. Plus, as a special bonus, all multi-use kits (the first three listed here) feature a FREE cybertool that puts more than 30 lab essentials at your fingertips.

For high-efficiency columns, it’s best to use narrow-diameter red tubing (0.12 mm id), instead of conventional green (0.17 mm id) tubing.

Capillary and fittings kits

Description | Contents | Part No.

Capillary/fitting starter kit for 1100 Capillary LC System Multi-use kit, a collection of various capillaries and tools for use in the lab.

Kit includes:

Qty 2 – Fused silica/PEEK capillary, 50 μm, 55 cm

Qty 1 – Fused silica/PEEK capillary, 50 μm, 20 cm

Qty 1 – Fused silica/PEEK capillary, 100 μm, 110 cm

Qty 2 – Fused silica/PEEK capillary, 50 μm, 50 cm

Qty 2 – Fused silica/PEEK capillary, 50 μm, 40 cm

Qty 4 – 4 mm stainless steel fitting, male 10-32

Qty 4 – 1/32 in PEEK ferrule and stainless steel lock ring

Qty 4 – PEEK fittings for μ-valves

Qty 4 – Double winged PEEK nuts and 1/32 in ferrules

Cybertool

5065-9938

Capillary/fitting starter kit, 0.12 mm id Multi-use kit, a collection of various capillaries and tools for use in the lab.

Kit includes:

Qty 1 – PEEK capillary, 0.13 mm id, 1.5 m

Qty 4 – Stainless steel capillary, 0.12 x 105 mm

Qty 4 – Stainless steel capillary, 0.12 x 150 mm

Qty 2 – Stainless steel capillary, 0.12 x 170 mm

Qty 2 – Stainless steel capillary, 0.12 x 200 mm

Qty 2 – Stainless steel capillary, 0.12 x 220 mm

Qty 2 – Stainless steel capillary, 0.12 x 280 mm

Qty 1 – Stainless steel capillary, 0.12 x 400 mm

Qty 3 – Stainless steel ZDV union

Tubing cutter for PEEK capillaries

1/16 in Stainless steel fittings, 10/pk

1/16 in PEEK fittings, color, 10/pk

1/16 in PEEK fittings, 10/pk

Rheotool

Cybertool

5065-9937

Capillary/fitting starter kit, 0.17 mm id Multi-use kit, a collection of various capillaries and tools for use in the lab.

Kit includes:

Qty 1 – PEEK capillary, 0.18 mm id, 1.5 m

Qty 4 – Stainless steel capillary, 0.17 x 105 mm

Qty 4 – Stainless steel capillary, 0.17 x 150 mm

Qty 2 – Stainless steel capillary, 0.17 x 200 mm

Qty 2 – Stainless steel capillary, 0.17 x 280 mm

Qty 1 – Stainless steel capillary, 0.17 x 400 mm

Qty 3 – Stainless steel ZDV union

Tubing cutter for PEEK capillaries

1/16 in Stainless steel fittings, 10/pk

1/16 in PEEK fittings, color, 10/pk

1/16 in PEEK fittings, 10/pk

Rheotool

Cybertool

5065-9939

Capillary starter kit, 0.17 mm BIO

Kit includes:

Qty 1 – 1.5 m PEEK tubing

PEEK finger-tight fitting, 10/pk

Colored finger-tight PEEK fittings, 10/pk

Qty 3 – Bio-inert union, 600 bar

Qty 1 – Plastic tubing cutter

Qty 1 – Rheotool socket wrench, 1/4 in

Qty 1 – Ti capillary, 0.17 x 400 mm

Qty 2 – PK/SS capillary, 0.17 x 105 mm

Qty 2 – PK/SS capillary, 0.17 x 150 mm

Qty 1 – PK/SS capillary, 0.17 x 300 mm

Qty 2 – PK/SS capillary, 0.17 x 200 mm

Multifunction tool

G5611-68710

Rapid Resolution High Throughput capillary kit
Used for converting an Agilent 1200 instrument to the RRLC configuration, to enable use of high efficiency columns (to 600 bar). Can also be used for Agilent 1100 instruments.

Kit includes:

Qty 1 – PEEK fitting long for 1/32 in od capillaries

Qty 1 – Stainless steel capillary, 0.12 x 280 mm

Qty 1 – Stainless steel capillary, 0.12 x 150 mm

Qty 1 – Stainless steel capillary, 0.12 x 70 mm

Qty 1 – Needle seat capillary, 12 μL x 0.12 mm

Qty 1 – PEEK capillary, 0.125 x 550 mm

5065-9947

Low dispersion capillary kit for G1316C

Kit includes:

Qty 1 – Flexible tubing, 280 mm, 0.12 mm id

Qty 1 – Heater Long Down 0.12 id (1.6 μL internal)

Qty 1 – Carrier for heat exchanger TCC SL Plus

5067-4633

1200 Infinity Series capillary kit 0.12 mm id, G1316C
for installing valves G4231A (2 position/6 ports – 600 bar)
and G4231B (2 position/6 ports – 1200 bar)

Kit includes:

Qty 1 – Column clip set, eight colors

Qty 1 – Stainless steel capillary 0.12 x 340 mm

Qty 1 – Stainless steel capillary 0.17 x 700 mm

Qty 2 – Stainless steel capillary 0.12 x 90 mm

Qty 2 – Stainless steel capillary 0.12 x 150 mm

Qty 2 – Stainless steel capillary 0.12 x 280 mm

Qty 1 – Stainless steel capillary 0.12 x 120 mm

Qty 1 – Stainless steel capillary 0.12 x 200 mm

Qty 1 – Heater Long Up 0.12 id (1.6 μL internal)

Qty 1 – Heater Long Down 0.12 id (1.6 μL internal)

Qty 2 – Carrier for heat exchanger TCC SL Plus

5067-4646

1200 Infinity Series capillary kit 0.17 mm id G1316C
for installing a 2 position/10 port valve G4232A (600 bar)

Kit includes:

Qty 2 – PEEK tubing, 1/32 in od, 0.4 mm id, 450 mm

Qty 1 – Column clip set, eight colors

Qty 3 – Stainless steel capillary 0.17 x 150 mm

Qty 1 – Stainless steel capillary 0.17 x 340 mm

Qty 4 – Stainless steel capillary 0,17 x 90 mm

Qty 2 – Stainless steel capillary 0.17 x 280 mm

Qty 1 – Stainless steel capillary 0.17 x 250 mm

Qty 1 – Stainless steel capillary 0.17 x 700 mm

5067-5103

1200 Infinity Series capillary kit 0.12 mm id G1316C
for installing a 2 position/10 port valve G4232B (1200 bar)

Kit includes:

Qty 1 – Stainless steel capillary 0.12 x 120 mm

Qty 2 – Stainless steel capillary 0.12 x 150 mm

Qty 1 – Stainless steel capillary 0.12 x 200 mm

Qty 2 – Stainless steel capillary 0.12 x 280 mm

Qty 1 – Stainless steel capillary 0.12 x 340 mm

Qty 1 – Stainless steel capillary 0.17 x 700 mm

Qty 2 – Stainless steel capillary 0.12 x 90 mm

Qty 1 – Column clip set, eight colors

Qty 1 – Heater Long Up 0.12 id (1.6 μL internal)

Qty 1 – Heater Long Down 0.12 id (1.6 μL internal)

Qty 2 – Carrier for heat exchanger TCC SL Plus

5067-4682

1200 capillary kit for 0.12 mm id

Kit includes:

Qty 1 – Stainless steel capillary, 0.12 x 130 mm

Qty 2 – Stainless steel capillary, 0.12 x 170 mm

Qty 1 – Stainless steel capillary, 0.12 x 210 mm

Qty 1 – Stainless steel capillary, 0.12 x 300 mm

Qty 3 – Stainless steel capillary, 0.12 x 500 mm

Qty 1 – Stainless steel capillary, 0.12 x 700 mm

Qty 1 – Stainless steel capillary, 0.12 x 340 mm

Qty 1 – Low carry over seat

Qty 1 – DAD heat exchanger capillary, 0.12 x 310 mm

G1316-68716

Stainless steel flexible capillary tubing kit

Kit includes:

Qty 10 – 1.6 mm (1/16 in) Stainless steel back ferrules

Qty 10 – 1.6 mm (1/16 in) Stainless steel front ferrules

Qty 10 – Stainless steel fittings

Qty 3 – Stainless steel Swagelok nut, 0.12 x 105 mm

Qty 1 – Stainless steel capillary, 0.12 x 150 mm

Qty 1 – Stainless steel capillary, 0.12 x 280 mm

5061-3304

Stainless steel flexible capillary tubing kit

Kit includes:

Qty 2 – Stainless steel capillary, 0.12 x 35 mm

Qty 3 – Stainless steel capillary, 0.12 x 105 mm

Qty 1 – Stainless steel capillary, 0.12 x 280 mm

5061-3315

1200 Infinity Series low dispersion capillary kit for installing a 6 position/14 port valve G4234A (600 bar) and G4234B (1200 bar)

Kit includes:

Qty 1 – Stainless steel capillary, 0.12 x 250 mm, with removable fitting

Qty 1 – Stainless steel capillary, 0.12 x 340 mm, with removable fitting

Qty 1 – Stainless steel capillary, 0.12 x 500 mm, with removable fitting

Qty 8 – Stainless steel capillary, 0.12 x 130 mm, with removable fitting

Qty 1 – Stainless steel capillary, 0.17 x 150 mm, with 2 long pre-swaged fittings

Qty 4 – Stainless steel capillary, 0.12 x 170 mm

Qty 2 – PEEK tubing, 1/32 in od, 0.4 mm id, 450 mm

Qty 2 – PEEK fitting, special for Chip-LC

Qty 1 – Column clip set, eight colors

Qty 2 – Heater Long Up 0.12 mm id (1.6 μL internal)

Qty 2 – Heater Long Down 0.12 mm id (1.6 μL internal)

Qty 2 – Carrier for heat exchanger TCC

Qty 2 – Fitting holder assembly

5067-4729

Ultra-low dispersion capillary kit for the 1290 Infinity LC

Kit includes:

Qty 1 – Stainless steel capillary, 0.075 x 220 mm SV/SLV

Qty 1 – Stainless steel capillary, 0.075 x 340 mm SV/SLV

Qty 1 – Low dispersion needle seat for 1290 Infinity LC

Qty 1 – Heater Long Up 0.075 mm (nominal 1.0 μL)

Qty 1 – 1290 Infinity LC Low Dispersion Kit Note

5067-5189

Ultra-low dispersion capillary kit, 5067-5189

Stainless steel fittings (S), 5062-2418

PEEK fittings (SPF), 0100-1516/5063-6591

Stainless steel long fittings (SL), 5065-4454

Finger-tight PEEK fitting (SPF), 0100-1516

Stainless steel extra long fitting (SX), 5065-9967

PEEK long fittings (SPFL), 5062-8541

Stainless steel nut, 5061-3303

Finger-tight PEEK fittings (SPF), 5065-4426

Stainless steel front ferrules, 5180-4108

Double winged fitting (SPF), 5042-6500

Stainless steel back ferrule, 5180-4114

PEEK RheFlex fittings (SPF), 0100-1631

1200 bar removable fitting (SV), 5067-4733

PEEK RheFlex fittings (SPF), 0100-2175

1200 bar removable long fitting (SLV), 5067-4738

Stainless steel blanking nut, 01080-83202

1200 bar removable extra long fitting (SXV), 5067-4739

Material
Key | Description
S | Swagelok 1.6 mm port id
SL | Swagelok 1.6 mm port id, long
SX | Swagelok 1.6 mm port id, extra-long
SV | Swagelok 1.6 mm port id, 1200 bar
SLV | Swagelok 1.6 mm port id, long, 1200 bar
SLB | Swagelok 1.6 mm port id, long bio
SXV | Swagelok 1.6 mm port id, extra-long, 1200 bar
SPF | Swagelok 1.6 mm port id, PEEK, finger-tight
SPLF | Swagelok 1.6 mm port id, PEEK, long, finger-tight

Stainless steel nut and PEEK ferrule, 5067-1540

Ferrule and stainless steel lock ring (W), 5065-4423

PEEK ferrule, 5067-1547

PEEK fittings, plugs (MP), 5065-4410

Finger-tight polyketone fitting (SPF), 5042-8957

Double winged PEEK nut & ferrule (WPF), 5065-4422

M4 stainless steel screw, 5067-1558

PEEk fitting, long (WPFL), 5022-6536

Stainless steel ferrule (M), 5067-1557

Fitting screws, 5065-9948

Plastic blank nut, 0100-1259

PEEK ferrules and SS rings, 5065-9950

Stainless steel screw, 5063-6593

Material
Key | Description
S | Swagelok 1.6 mm port id
SP | Swagelok 1.6 mm port id, PEEK
SPF | Swagelok 1.6 mm port id, PEEK, finger-tight
W | Swagelok 0.8 mm port id
G | Small head SW 4 mm
WG | Swagelok 0.8 mm port id, small head SW 4 mm
MP | Metric M4 0.8 mm port id, PEEK
WPF | Swagelok 0.8 mm port id, PEEK, finger-tight

ZDV union, 5022-2145

Adapter, PEEK, 0100-2298

ZDV universal union, 5022-2184

Barbed Y-connector PP, 5065-9971

ZDV union with fittings, 0100-0900

Union, female to female, 5042-8517

ZDV union, PEEK with fittings, 0100-2441

High flow union, 5022-2133

Adapter, male Luer to female, 5042-8518

PEEK adapter, 0100-1847

Adapter, female to male, 5023-1803

Bio-inert union, 600 bar, 5067-4741

Micro T-connector, PEEK, 5042-8519

Unions
Description | Use With | Part No.
ZDV union, no fittings | Nano LC | 5022-2145
ZDV universal union, stainless steel, no fittings | Standard LC | 5022-2184
ZDV union, with fittings | Standard LC | 0100-0900
ZDV union, PEEK with fittings | Bio-applications | 0100-2441
High flow union, no fittings | Prep LC | 5022-2133
PEEK adapter 1/4-28 to 10-32 |  | 0100-1847
Adapter, PEEK int. 1/4-28 to ext. 10-32 |  | 0100-2298
Barbed Y-connector PP for 3/16 in id tube, 10/pk |  | 5065-9971
Adapter, union PEEK 1/4-28 |  | 5042-8517
Adapter, male Luer to female 1/4-28 |  | 5042-8518
SS adapter Swagelok to 1/4-28 |  | 5023-1803
T-connector, PEEK, swept volume 0.57 μL | For 1/16 in od tubing | 5022-2144
Micro T-connector, PEEK, swept volume 29 mL, with 1/32 in id fittings |  | 5042-8519
Bio-inert union, stainless steel with PEEK insert 600 bar | Bio-applications | 5067-4741

Tips and tools for creating the best possible connections

How do I tighten fittings correctly?

The chart below describes the steps you’ll need to follow.

Fitting type | First Connection | Further connection

Stainless steel

1.   Slide the screw, along with the back and front ferrules, onto the capillary.

2.   Insert capillary into the port until it is completely seated in the end fitting.

3.   Finger-tighten the nut until the capillary does not rotate.

4.   Tighten the nut ½ to ¾ turn with a slitted socket wrench or Rheotool (P/N 8710-2391). If you are using a torque wrench, tightening torque should be between 1.5 and 3.0 Nm)

Finger-tighten, then tighten an extra ¼ to ½ turn with a slitted socket wrench or Rheotool (P/N 8710-2391). (If using a torque wrench, tightening torque should be between 1.5 and 3.0 Nm).

Polymeric finger-tight: PEEK and polyketone

1.   Slide the screw and ferrule onto the capillary.

2.   Insert capillary into the port until it is completely seated in the end fitting.

3.   Finger-tighten the nut until the capillary does not rotate.

4.   Make sure the capillary cannot be easily pulled out.

Additional tightening if necessary

See “good connections” step by step

1200 bar removable fitting

1.   Slide the screw, along with the back and front ferrules, onto the capillary.

2.   Insert capillary into the port until it is completely seated in the end fitting.

3.   Finger-tighten the nut until the capillary does not rotate.

4.   Tighten the nut about ¾ turn with a socket wrench.

5.   For stainless steel capillaries, if using a torque wrench, tightening torque should be between 1.0 and 1.2 Nm.

6.   For stainless steel coated PEEK capillaries, do not exceed 0.8 Nm.

Finger-tighten, then tighten an extra ¼ to ½ turn with a socket wrench.

For stainless steel capillaries, if using a torque wrench, tightening torque should be between 1.0 and 1.2 Nm.

For stainless steel coated PEEK capillaries, if using a torque wrench, do not exceed 0.8 Nm.

PEEK/stainless steel

1.   Slide the screw, along with the back and front ferrules, onto the capillary.

2.   Insert capillary into the port until it is completely seated in the end fitting.

3.   Finger-tighten the nut until the capillary does not rotate.

4.   Tighten the nut about ½ turn with a socket wrench.

Finger-tighten, then tighten an extra ¼ to ½ turn with a socket wrench.

How do I prepare the perfect fitting connection?

Problems with stainless steel tubing connections are commonly mistaken for column issues – and are the source of many calls to Agilent’s technical support line.

Connection problems often arise because different manufacturers supply different types of fittings, as you can see in the following diagram:

Common HPLC column connectors by manufacturer

Ideally, you should use the fittings recommended by your column manufacturer. Most analytical reversed phase columns are compatible with Swagelok or Parker-type fittings when correctly seated in the column.

Stainless steel fittings are the best choice for permanent, high-pressure sealing. Agilent recommends Swagelok-type fittings with front and back ferrules, because they deliver the best performance for Agilent LC systems – and can be used on most instrument connections, including valves, heaters, and column connections.

For lower-pressure operation, finger-tight polymeric fittings allow you to easily adjust the end-fitting to seat the capillary into the column properly – helping avoid extra-column voids and leaks. These connectors can be tightened without wrenches. High-pressure fittings, which are designed to be removed and resealed, are also available for pressures up to 1200 bar.

How do I align the connection properly?

The importance of correct tubing length (relative to the distance from the end of the tubing to the bottom of the ferrule) cannot be overstated. If the tubing is too long, the ferrule will not seat properly and leaks will occur. Likewise, if the tubing is not pushed in far enough, a void occurs, creating extra-column volume that acts as a “mixing chamber”, which can cause peak tailing, and/or poor peak shape.

Always make sure you use the correct fittings, and that all fittings are properly seated in the column end fitting – especially if you use columns from different manufacturers.

Examples of incorrect fittings

A good connection, step by step

This connection uses a Swagelok-type fitting which is good for connections to the instrument. For column connections, it is ideal to use polymeric finger-tight fittings, which are removable, or the 1200 bar removable fitting.

1.   Select a nut that is long enough for the fitting you’ll be using.

2.   Slide the nut over the end of the tubing.

3.   Carefully slide the ferrule components on after the nut, then finger-tighten the assembly while making sure the tubing is completely seated in the bottom of the end fitting.

4.   Use a wrench to gently tighten the fitting; this will force the ferrule to seat onto the tubing, ½ to ¾ turn with a wrench. Do not over-tighten! That will shorten the useful life of the fitting.

5.   Once you are sure your fitting is complete, loosen the nut and inspect the ferrule for correct position on the tubing.

Note:  Avoid re-using a capillary on a different location. The position of the fitting is done the first time it is screwed on a port and all the ports are not strictly identical.

What are the benefits of a small internal diameter?

The roughness of the internal capillary surface is a function of the capillary’s outer diameter. A smaller outer diameter provides better smoothness for the internal capillary surface.

Agilent’s unique stainless steel LC capillary connections for liquid chromatography are designed for small inner diameters, reducing backpressure and blocking. To make these connections compatible with standard 1/16 in capillaries, we weld a tight, flat sleeve at both ends. Agilent’s unique laser welding process ensures that the capillary ends are absolutely flat, eliminating any chance of additional dead volume.

Which capillary connection size is right for me?

Choose the shortest practical length and narrowest diameter that your application and system allows. For 4.6 mm id columns, 0.17 mm capillaries are usually sufficient, but for narrower id columns like 2.1 mm, 0.12 mm id capillaries are used to keep sample dispersion as low as possible. There are also new ultra-low dispersion capillaries (0.075 mm id) for use with the 1290 Infinity LC.

How can I get rid of extra-column volume effects?

Extra-column volume effects can be caused by capillaries that are too long, so try a shorter- length capillary. For low-volume, high efficiency columns (e.g., Agilent ZORBAX Eclipse Plus C18, 2.1 x 50 mm, 1.8 μm), replace 0.17 mm id (green) capillaries with 0.12 mm id (red).

For additional information on extra-column volume and its effect, see our application note “Reduce Tubing Volume to Optimize Column Performance” at www.agilent.com/chem/library/applications/5990-4964EN.pdf

What should I do about high backpressure?

High backpressure issues are usually not caused by capillaries. However, you should check to make sure the capillary isn’t blocked, and replace if necessary, as part of your troubleshooting.

How can I reduce peak broadening, related to my capillary?

In addition to optimizing the length and diameter of the capillary, proper positioning in the fitting is important. The distance between the end of the capillary and the bottom of the ferrule may be too long or too short (creating a void), resulting in a poor connection. This can cause leaks or peak shape issues, such as broadening when the sample mixes in the void. A re-usable fitting can be adjusted, but with stainless steel a new fitting will need to be made.

How do I eliminate detector spikes and bubbles?

Check for air leaks at the capillary connections, and tighten as needed.

Pump Supplies

Regular pump maintenance helps lower operating costs and generate precise results that make you feel confident.

You can count on Agilent isocratic, binary, quaternary, capillary, and preparative pumps for superior flow and composition stability. And by following a regular maintenance routine, you can also count on maximum uptime and a steady, accurate solvent flow for the life of the pump.

Pump Routine Maintenance Procedures

• Replace the seals and pistons

• Replace the PTFE frit

• Replace the cartridge in the Active Inlet Valve

• Clean the outlet ball valve

• Clean or replace the solvent inlet frits

Routine pump maintenance should be done on a regular basis to keep your Agilent LC system performing at its optimum. You can perform all maintenance procedures at once or as needed. Some parts may need to be replaced more than others depending upon your application and solvent preparation procedures.

Sapphire piston and seals

     Pump Routine Maintenance Procedures
Symptom | Cause | Solution
Solvent dripping out of waste outlet when valve closed | Leak on pump head | Exchange the purge valve frit or the purge valve
Pressure ripple unstable | Dirty active inlet valve cartridge | Run leak test for verification and exchange the active inlet valve cartridge
| Leak on pump head | Run leak test for verification and exchange the outlet ball valve sieve or the complete valve
Gradient performance problems, intermittent pressure fluctuations | Solvent filter is blocked | Change the solvent filter
A pressure drop of >10 bar across the frit (5 mL/min H₂O with purge valve open) indicates blockage | Dirty frit | Exchange the purge valve frit or the purge valve
Leaks at lower pump head side | High seal wear | Run leak test for verification and exchange the pump seals
Unstable retention time |  |
Pressure ripple unstable |  |
Seal lifetime shorter than normally expected | Scratch on plunger | Check plungers while changing the seals
Loss of wash solvent | Leaky wash seals | Exchange the wash seals

Purge valve assembly, G1311-60009

Purge valve with PTFE frit, G4280-60061

PTFE frits, 01018-22707

Purge Valves
Description | Comments | Part No.
Purge valve with PTFE frit, 400 bar | For G1310A, G1311A, G1312A, G1376A, G2226A | G1311-60009
Purge valve with PTFE frit | For 1120 | G4280-60031
Purge valve with PTFE frit | For G1311C, G1312C, 1220 | G4280-60061
Purge valve with PTFE frit | For G1310B, G1311B, G1312B, G4302A, | G1312-60061
PTFE frit | For G1310A, G1311A, G1312A, G1376A, G2226A, 1120, 1220, 1220, G1310B, G1311B, G1312B, G1311C, G1312C, G4202A | 01018-22707
Seal cap | Not suitable for outlet ball valves with integrated seal | 5067-4728
Seal for purge valve | For 1120 | 0905-1192
Purge valve actuator | For 1120 | G4280-60033
Bio-inert purge valve | For G5611A | G5611-60061
5 position / 7 port rotor seal | For G4220A, G4220B | 5068-0005
5 position / 7 port stator | For G4220A, G4220B | 5068-0004
Multi purpose valve head | For G4204A | 5067-4174
Stator for 5067-4174 multi purpose valve head | For G4204A | 5068-0122
Rotor for 5067-4174 multi purpose valve head | G4204A | 5068-0123
Stainless steel filter assembly with PEEK ring, 2 μm pore size | For G1361A | 5022-2192
Open end wrench, 14 mm | To remove purge valve | 8710-1924

[picture] | TIPS & TOOLS Information on maintenance procedures can be found at www.agilent.com/chem/LCmaintenancenotes

Inlet and Outlet Valves

Inlet Valves
Description | Comments | Part No.
Active inlet valve without cartridge | For G1310A, G1311A, G1312A, G1376A, G2226A, G1312B, G1312C | G1312-60025
Cartridge for active inlet valve 400 bar | For G1310A, G1311A, G1312A, G1376A, G2226A, G1312C | 5062-8562
Cartridge for active inlet valve 600 bar | For G1312B | G1312-60020
Bio-inert active inlet valve | For G5611A | G5611-60025
Bio-inert cartridge for active inlet valve, 600 bar | For G5611A | G5611-60020
Inlet valve for 1290 quaternary pump | For G4204A | G4204-60022*
Valve assemblies (inlet/outlet) for prep pumps | For G1361A | G1361-60012
Passive inlet valve | For G1310B, G1311B, G1311C, 1120, 1220, G4302A | G1312-60066*
Passive inlet valve | For G4220A, G4220B | G4220-60022*
Seal cap | Not suitable for outlet ball valves with integrated seal | 5067-4728

*Inlet valve with integrated seal.

Outlet Valves
Description | Comments | Part No.
Outlet ball valve | For G1310A, G1311A, G1312A, G1376A, G2226A, 1120, 1220, G1310B, G1311B, G1311C, G1312B, G1312C, G4302A | G1312-60067*
Outlet ball valve | For G4220A, G4220B, G4204A | G4220-60028*
Bio-inert outlet ball valve | For G5611A | G5611-60067*
Valve assemblies (inlet/outlet) for prep pumps | For G1361A | G1361-60012
Seal cap | Not suitable for outlet ball valves with integrated seal | 5067-4728

*Outlet ball valve with integrated seal.

Active inlet valve without cartridge, G1312-60025

Cartridge, 400 bar, 5062-8562

Passive inlet valve, G1312-60066

Seal cap, 5067-4728

Outlet ball valve, G1312-60067

Outlet ball valve, G4220-60028

TIPS & TOOLS Information on maintenance procedures can be found at www.agilent.com/chem/LCmaintenancenotes | [picture]

Pistons and Seals

Agilent pistons are made from a high purity, monocrystalline sapphire for maximum durability. Although ceramic pistons can be manufactured at a lower cost, ceramic is a sintered, polycrystalline material, which can cause undesirable variations during the production process. Agilent sapphire pistons are:

• Meticulously cut at just the right angle, making them durable – and long lasting

• Precisely aligned in their stainless steel holder to minimize wear on the piston and seal

Agilent seals are designed to fit snugly around our pistons, and are capable of adapting to a wide range of flow rates and pressures. Agilent piston seals are:

• Spring-loaded and engineered to deliver optimal performance over highly dynamic flow and pressure ranges

• Manufactured from a proprietary polymer blend, and feature a spring made from the same high-quality stainless steel that is used in our pump’s flow path

The combination of our piston and seal has undergone extensive testing under temperature stress, with all common HPLC solvents, and in many instruments. More importantly, they yield consistent, reproducible results.

Sapphire piston and seals

Pistons
Description | Comments | Part No.
Sapphire plunger | For G5611A, 4302A | 5067-4695
Zirconium based ceramic plunger | For G4220A, G4220B, G4204A | 5067-4678
Sapphire plunger | For G1310A, G1311A, G1312A, G1376A, G2226A, 1120, 1220, G1310B, G1311B, G1312B, G1311C, G1312C | 5063-6586
Sapphire plunger | For G1361A | G1361-22402

Seals
Description | Comments | Part No.
Bio-inert piston seal | For G5611A | G5611-21503
Piston seal polyethylene | For G4220A, G4220B, G4204A | 0905-1719
Piston seal graphite filled PTFE (reversed-phase) (2/pk) | For G1310A, G1311A, G1312A, G1376A, G2226A, 1120, 1220, G1310B, G1311B, G1312B, G1311C, G1312C, G4302A | 5063-6589
Piston seal Polyethylene (normal phase) | For G1310A, G1311A, G1312A, G1376A, G2226A, 1120, 1220, G1310B, G1311B, G1312B, G1311C, G1312C, G4302A | 0905-1420
Piston seal | For G1361A | 5022-2188

Piston seals, 5063-6589

Specific parts for 1290 Infinity LC pumps
Description | Comments | Part No.
Jet Weaver 35 μL/100 μL | For G4220A, G4220B, G4204A | G4220-60006
Jet Weaver 380 μL | For G4220A, G4220B, G4204A | G4220-60012
Clamp for in-line filter | For G4204A | G4204-40000
Outlet filter 1290 quaternary pump | For G4204A | G4204-60004
Inlet valve for flush pump |  | 5067-4717
Outlet valve for flush pump |  | 5067-4716
Tubing kit 270 mm, 2/pk |  | 5067-4661
Tubing kit 140 mm, 2/pk |  | G4220-60035
Shut-off valve | For G4220A, G4220B, G4204A | 5067-4124
Inline pressure relief valve kit | For G4220A, G4220B | G4212-68001
Solvent selection valve bridge tube |  | 5067-4697
Pressure relief valve | For G4220A, G4220B | G4212-60022
PEEK seal for inlet weaver assembly | For G4204A | G4204-40005
Support ring | For G4220A, G4220B | G4220-60015
Seal keeper | For G4220A, G4220B | G4220-60016

TIPS & TOOLS Information on maintenance procedures can be found at www.agilent.com/chem/LCmaintenancenotes | [picture]

Seal Wash

The routine use of highly concentrated buffer solutions (100 mM) will reduce the life of seals and pistons in your pump. Counteract the problem with one of Agilent’s seal wash kits, which flush the back of the seal with a wash solvent. Note: Water/isopropanol (90/10) is recommended as the wash solvent.

Seal Wash
Description | Comments | Part No.
Peristaltic pump cassette with silicone tubing | For 1100/1200/1200 RRLC and 1260 Infinity LC pumps | 5042-8507
Silicone tubing, 1 mm id, 3 mm od, 5 m |  | 5065-9978
Peristalitic pump with ChemSure tubing | For 1100/1200/1200 RRLC and 1260 Infinity LC pumps | 5065-9952
ChemSure tubing for peristaltic pump |  | 5042-8954
Wash Seal and Wash Keeper
Wash seal | For 1100/1200/1200 RRLC, 1120 and 1260/1220 Infinity LC pumps | 0905-1175
Seal keeper | For 1100/1200/1200 RRLC and 1120 pumps | 5001-3743
Bio-inert seal keeper | For G5611A LC pump | G5611-26210
Wash seal gasket, 6/pk | For 1100/1200/1200 RRLC, 1120 and 1260/1220 Infinity LC pumps | 5062-2484
Wash seal PE | For 1290/1260/1220 Infinity LC pump | 0905-1718
Support ring | For 1290/1260/1220 Infinity LC pump | G4220-63010
Support ring | For 1290 Infinity pump without seal wash | G4220-63015
Backup ring for seal holder | For 1290/1260/1220 Infinity LC pump | G4220-24013
Seal keeper | For 1290/1260/1220 Infinity LC pump | G4220-26210

Peristaltic pump, 5042-8507

Peristalitic pump with ChemSure tubing, 5065-9952

[picture] | TIPS & TOOLS Information on maintenance procedures can be found at www.agilent.com/chem/LCmaintenancenotes

Solvent Reservoir and Supplies

Solvent Reservoir and Supplies
Description | Part No.
Solvent Reservoir
Solvent reservoir, 1 L | 9301-1420
Solvent reservoir, 1 L, with cap | 9301-1421
Solvent reservoir, 1 L, F29/32 | 9301-0656
Solvent reservoir, amber, 1 L | 9301-1450
Solvent bottle, clear, 2 L, 2 inlets | 5065-4421
Solvent bottle, amber, 2 L | 9301-6341
Solvent bottle, clear, 2 L | 9301-6342
Bottle Head Assembly
Bottle head assembly for screw bottle | G1311-60003
Bottle head assembly for F29/32 tapered solvent bottle | G1312-68716
Bottle head assembly with tubing and filter For capillary and nano systems (with stainless steel solvent filter) | G1376-60003
Bottle head assembly for prep system | G1361-60022
Bottle head assembly | G4220-60007
Bottle cap with 3-hole insert | 5063-6531

Solvent reservoirs

Prep bottle, 5065-4421

Solvent bottle, amber, 9301-6341

Cleaning the Solvent Filter

If the filter is in good condition, the solvent will freely drip out of the solvent tube (hydrostatic pressure). If the solvent filter is partially blocked, only very little solvent will drip out of the solvent tube.

Caution: Small particles can permanently block the capillaries and valves of the module.

• Always filter solvents

• Never use the module without solvent inlet filter

Glass filter, 5041-2168

Frits and Adapters
Description | Part No.
Solvent mixer, 1100 Series | G1312-87330
Solvent mixer, short, 200 μL | 5067-1565
Frit adapter, PTFE, for 4.7 mm od tubing | G1361-23205
Frit adapter, PTFE, for 3.2 mm od tubing For G2258A Dual Loop Autosampler | G2258-23201
O-ring, Viton, 30 mm | 0905-1516
Stainless steel filter assembly with PEEK ring, 2 μm pore size | 5022-2192

Filter frit adapters, 5062-8517

Solvent inlet filter, 01018-60025

Safety Caps

Open or partially covered solvent bottles can lead to the evaporation of solvents and harmful solvent vapors. Prevent solvent evaporation and possible chemical spills with solvent safety caps from Agilent. These safety caps have been designed for optimal sealing with an integrated exhaust valve providing pressurization during solvent extraction and allowing proper solvent flow to your HPLC system. The exhaust valve contains a PTFE membrane to prevent contamination of your solvents from dirt and dust particles.

• Designed to fit all solvent bottles

• Constructed of PTFE and PFA for high chemical resistance

• Caps rotate freely, preventing tube twisting during bottle exchange

• Available in GL40, GL45, S60 and NS29/32 thread sizes

Installation Details for Solvent Safety Caps

Installation Guide for Standard Safety Caps – 4 Simple Steps to Install a Safety Cap

1. Guide the solvent tube through the fitting on the safety cap

2. Re-connect the solvent inlet filter to the solvent tube (open end)

3. Screw the safety cap on top of your solvent bottle

4. Adjust the length of the solvent tube in the solvent bottle and fix the tube by tightening the fitting

Installation Instructions for Safety Caps with Stopcocks

These caps have a shut-off valve which can be closed. This keeps the solvent tube in the bottle during maintenance activities with no risk of having solvent dropping out of the flow path.

4 Simple Steps to Install a Safety Cap with Stopcocks

1. Cut the solvent tube in 2 parts

2. Connect the upper part to the fitting (red or blue) on the safety cap (Top)

3. Cut a suitable length and connect the lower part to the fitting on the safety cap (Bottom)

4. Screw the safety cap on top of your bottle

10 L waste can assembly, S60, 5043-0243

Safety Cap I, 5043-0223

Safety Cap II, 5043-0224

Safety Caps and Accessories
Description | Kit Contents | Part No.
Safety Cap II with 2 ports – NS29/32 | Includes 1 safety cap, 2 fittings 3.2 mm PFA, 1 venting valve with 1 μm PTFE membrane | 5043-0221
Safety Cap I with 1 port – GL 45 | Includes 1 safety cap, 1 fitting 3.2 mm PFA, 1 venting valve with 1 μm PTFE membrane | 5043-0223
Safety Cap II with 2 ports – GL 45 | Includes 1 safety cap, 2 fittings 3.2 mm PFA, 1 venting valve with 1 μm PTFE membrane | 5043-0222
Safety Cap I with 1 port for prep – GL45 | Includes 1 safety cap, 1 fitting 4.7 mm PFA, 1 venting valve with 1 μm PTFE membrane | 5043-0300
Safety Cap I with 1 stopcock – GL45 | Includes 1 safety cap, 1 fitting 2.3 mm PFA, 1 fitting 2.3 mm PTFE, 1 venting valve with 1 μm PTFE membrane | 5043-0225
Safety Cap II with 2 stopcocks – GL 45 | Includes 1 safety cap, 2 fittings 2.3 mm PFA, 2 fittings 2.3 mm PTFE, 1 venting valve | 5043-0224
Safety Cap IV with 4 ports – 1 leak port – GL45 | Includes 1 safety cap, 4 fittings 2.3 mm PFA, 4 fittings 1.6 mm PFA, 4 fittings 2.3 mm PFA, 1 leak hose. Must be used with charcoal filter, P/N 5043-0230. | 5043-0226
Safety Cap IV with 4 ports – 1 leak port – S60 | Includes 1 safety cap, 4 fittings 2.3 mm PFA, 4 fittings 1.6 mm PFA, 1 leak hose. Must be used with charcoal filter, P/N 5043-0230. | 5043-0227
5 L waste can GL45 with 4 ports and 1 leak port | Includes 5 L waste can, 1 safety cap (5043-0226), 2 ports collector PTFE. Must be used with charcoal filter, P/N 5043-0230. | 5043-0242
10 L waste can S60 with 4 ports and 1 leak port | Includes 10 L waste can, 1 safety cap (5043-0227), 2 ports collector PTFE. Must be used with charcoal filter, P/N 5043-0230. | 5043-0243
Safety Waste Set S50 | Includes 5 L space saving waste can, 1 safety cap, 4 fittings 2.3 mm PFA, 1 x 6.4 mm tubing connector, Charcoal filter (48 g) | 5043-0831
Venting valve with 1 μm PTFE membrane* |  | 5043-0232
Thread Adapter GL45-GL40 (PTFE) |  | 5043-0234
Thread Adapter PTFE, GL45-GL38 |  | 5043-0272
Thread adapter GL45-GPI38-23, (PTFE) |  | 5043-0832
Safety funnel + cover S60, PEHD, conductive |  | 5043-0828
Safety funnel + cover S60, PEHD |  | 5043-0829
Tool for fitting |  | 5043-0830

* Valve change is recommended every six months

Safety Cap IV for GL45 bottles, 5043-0226

Safety Cap IV for S60 threaded waste bottles, 5043-0227

5 L waste can assembly, GL45, 5043-0242

10 L waste can assembly, S60, 5043-0243

Replacement Parts for Safety Caps
Description | Unit | Part No.
Fitting for 3.2 mm tube, PFA | 6/pk | 5043-0255
Fitting for 2.3 mm tube, PFA | 5/pk | 5043-0228
Fitting for 1.6 mm tube, PFA | 5/pk | 5043-0229
Charcoal filter, 48 g |  | 5043-0230
Screw plug 1/4 in, PTFE |  | 5043-0231
Venting valve with 1 μm PTFE membrane* |  | 5043-0232
Screw plug 1/8 in, PTFE | 5/pk | 5043-0233
2 ports collector, PTFE |  | 5043-0235
5 L waste can, GL45 |  | 5043-0236
10 L waste can, S60 |  | 5043-0237
3 port collector |  | 5043-0238
Adapter for two 3.2 mm tubes |  | 5043-0239

* Valve change is recommended every 6 months

2.3 mm fittings to connect waste tubes from the purge valve, 5043-0228

1.6 mm fitting to connect wash tubes going to the peristaltic pump of the High Performance ALS/Wellplate Sampler, 5043-0229

Charcoal filter, 48 g, 5043-0230

Screw plug, 1/4 in, PTFE, 5043-0231

Replacement PTFE filter (replace every six months), 5043-0232

Screw plug, 1/8 in, PTFE, 5043-0233

2 ports collector (PTFE), 5043-0235

Vacuum Degassers

A vacuum degasser is recommended for:

• Maximum sensitivity in the low UV wavelength range

• High injection precision

• High retention time reproducibility

• Flow rates below 0.5 mL/min

Vacuum Degasser Care

• To generally clean the vacuum degasser tubing, flush the system with isopropanol

• Flush the degasser with water after using buffers

• Speed solvent changes by drawing solvent through the degasser and tubing with syringe adapter kit

Vacuum Degassers
Description | Comments | Part No.
Tubing kit, degasser to pump, 4/pk, 30 cm pieces of tubing with screws and bushings | For G1322A, G1379A/B | G1322-67300
Inlet tubing | For Agilent 1260 Infinity Quaternary Pump VL | 5067-5378
Mounting tool for flangeless nut | For G1322A, G1379A/B | 0100-1710
Plastic tubing cutter | For G1322A, G1379A/B | 8710-1930
Tefzel ferrules and SS lock rings, 1/8 in, 10/pk | For G1322A, G1379A/B | 5063-6598
PPS nuts, 1/8 in, 1/4-28 thread, 10/pk | For G1322A, G1379A/B | 5063-6599
Union, 1/4-28 thread, polypropylene | For G1322A, G1379A/B | 5022-2155
PTFE solvent tubing, 5 m, 1.5 mm id, 3 mm od | For G1322A, G1379A/B | 5062-2483
Disposable syringes, 20 mL, 10/pk | For G1322A | 5062-8534
Syringe adapter, 1/16 in od, 2 in long | For G1322A | 9301-1337
Micro vacuum degasser tubing kit | For G1379A | G1379-67310
Micro vacuum degasser tubing kit | For G1379B | 5042-8922
Online degasser accessory kit Includes 8 screws, 8 bushings, 4 markers, tubing, syringe and syringe adapter | For G1322A, G1379A/B | G1322-68705
Vaccum chamber (2 channel) | For G1379A, G1379B | 5067-4798
Tubing kit for 5067-4798 vaccum chamber | For G1379A, G1379B | 5067-5380
Vaccum chamber replacement kit Includes two 5067-4798 and one 5067-5380 | For G1379A, G1379B | 5067-5383

1200 Series Vacuum Degasser

Mounting tool, 0100-1710

Plastic tubing cutter, 8710-1930

Ferrules and rings, 5063-6598

PPS nuts, 5063-6599

Pump Kits

Pump Kits
Description | Kit Contents | Part No.
Start-up Kits
Pump start-up kit For 1100/1200/1220 RRLC pumps | Includes 1 outlet cap, 5 PTFE frits, 4 piston seals, 1 outlet gold seal, 2 glass solvent inlet filters, 20 μm, and 1 cartridge for active inlet valve | G1311-68710
Nanoflow LC start-up kit | Includes PEEK coated fused silica capillaries, column and fittings to start up a Nanoflow LC System | G2228-68700
Seal Wash Kits
Active seal wash kit | Includes 2 wash seal gaskets, 2 pump seals, peristaltic pump (includes pump cassette and motor), 2 seal keepers, 2 support ring assemblies, seal insert tool and silicone tubing | G1311-68711
Active seal wash kit | Includes 4 wash seal gaskets, 4 pump seals, 2 peristaltic pumps (includes pump cassette and motor), 4 seal keepers, 4 support ring assemblies, seal insert tool and silicone tubing | G1312-68711
Continuous seal wash kit For 1100/1200/1200 RRLC pumps | Includes 2 wash seal gaskets, 4 m flex tubing, 2 pump seals, 1 flow regulator, 2 seal keepers, 2 support ring assemblies, 20 mL luer lock syringe, seal insert tool and abrasive paper | 01018-68722
Seal wash kit for 1260 Capillary/Nano pump | Includes 3 adaptor luer/barbs, 2 film washers, 1 insert tool, 2 seal pumps, 1 clamp hose, 2 seal wash, 1 syringe, 2 support ring 1290 Infinity LC w/seal wash, 2 seal keepers for 1290 Infinity LC, and 2 backup rings for seal keeper 1290 Infinity | G1376-60005
Seal wash PM kit For 1260 Infinity LC pumps | Includes 2 PTFE wash seals (P/N 0905-1175), 2 gasket wash seals (P/N 01018-07102) | G1310-68742
PM kit for seal wash option | Includes 2 wash seals and 1 pack of 6 wash seal gaskets | G1310-68731
Preventive Maintenance Kits
For 1260 Infinity LC isocratic or quaternary and 1220 pumps | Includes 1 PTFE pump seal (P/N 0905-1503), PTFE frits, 5/pk (P/N 01018-22707), 1 seal cap (P/N 5067-4728) | G1310-68741
For 1260 Infinity LC binary pump | Includes 1 PTFE pump seals (P/N 0905-1503), PTFE frits, 5/pk (P/N 01018-22707), 1 seal cap (P/N 5067-4728), sieves for outlet valve, 10/pk (P/N 5063-6505) | G1312-68741
For 1100/1200 isocratic or quaternary pumps | Includes piston seal, PTFE frits, and 2 seal caps | G1310-68730
For 1290 Infinity pump | Includes pump seal exchange tool, torque wrench, and hex bit | 5067-4699
For Bio-inert quaternary pump | Includes bio-inert piston seal, 5/pk PTFE frits, seal cap assembly, film washer, peristaltic pump, silicone tubing, Bio-inert wash seal | G5611-68741

Pump start-up kit, G1311-68710

Pump Kits
Description | Kit Contents | Part No.
Preventive Maintenance Kits
For 1100/1200 binary pump | Includes 4 piston seals, PTFE frits, 2 sieves, and 3 seal caps | G1312-68730
For G1376A capillary pump | Includes 4 pump seals, 1 stainless steel frit, and 4 seal cap assemblies | G1376-68710
For 1100/1200 and 1260 Infinity LC prep pump | Includes 1 filter cup (P/N 3150-0942), 4 seal prep flange (P/N 5022-2188), 1 filter assembly (P/N 5022-2192), 1 peristaltic pump | G1361-68710
For 1120 manual injector systems | Includes 1 piston seal, 5 PTFE frits, 1 PEEK rotor seal, and 2 seal cap assemblies | G4280-68710
For 1220 manual injector systems | Includes piston seals, PTFE frits, rotor seal, and 2 seal cap assemblies | G4280-68750
For 1220 automated injector systems | Includes piston seals, PTFE frits, rotor seal, 2 seal cap assemblies, needle and needle seat | G4280-68770
For 1120 automated injector systems | Includes 1 piston seal, 5 PTFE frits, 1 Vespel rotor seal, 1 needle, 1 needle seat, and 2 seal cap assemblies | G4280-68730
Extended PM kit For 1100/1050/1200 pumps | Includes 2 piston seals (P/N 5063-6589), PTFE frits, 5/pk (P/N 01018-22707), cartridge active inlet valve (P/N 5062-8562), outlet ball valve (P/N G1312-60067) and 2 pistons (P/N 5063-6586) | 5065-4499
Accessory Kits
1260 Infinity LC binary pump accessory kit | Includes 1 tubing assembly (P/N 5063-6527), 1 CAN cable (P/N 5181-1519), 1 RRLC system configurator, 1 stainless steel capillary, 400 x 0.17 mm (P/N G1312-87303), 1 stainless steel capillary, 700 x 0.17 mm (P/N G1312-87304) | G1312-68755
1100/1200 pump accessory kit | Includes 3 wrenches, 5 PTFE frits, tubing, capillary and wrist strap | G1311-68705
Pump configuration kit for G1312B with G1158B 2 position/6 port valve | Includes side cover with fixed rail, top and right cover for pump housing, and 6 connecting capillaries. Allows automatic switching between different delay volumes to optimize the system for 2.1 mm id or 4.6 mm id columns | G1312-68726
Capillary pump accessory kit | Includes purge valve and holder, hex keys 2.5 and 3 mm, 2 wrenches 1/2 in x 1/16 in, wrenches 1/4 in x 5/16 in and 14 mm, wrist strap, torque adaptor and stainless steel frit, 0.5 μm | G1376-68705
Accessory kit prep pump/gradient G1361A | Includes stainless steel connecting capillaries, solvent mixer, 2 L solvent bottle, bottle head assembly, filter, glass stop valve, stainless steel union, tubing and other parts | G1361-68707
Online degasser accessory kit | Includes 8 screws, 8 bushings, 4 markers, tubing, syringe and syringe adapter | G1322-68705
Extended flow range kit, 100 μL/min | Includes all parts to go from 20 μL/min to 100 μL/min flow rate in a capillary LC system | G1376-68707
Manual prep injection valve kit, stainless steel | Includes position sensing, 10 mL loop, 25 mL syringe, ring mounting bracket, start cable and SS connecting capillaries, 0.5 mm id, 40 cm and 60 cm | 5065-9922

Outlet ball valve, G1312-60067

Autosampler Supplies

Your Agilent autosampler is designed to deliver accurate measurements, precise injection volumes, and high-quality data. And by following a regular schedule of preventive maintenance, you can ensure a lifetime of defensible results.

     Autosampler Maintenance Schedule
Procedure | When to Perform | Time Required
Exchanging the needle assembly | When needle shows indication of damage or blockage | 15 minutes
Exchanging the seat assembly | When the seat shows indication of damage or blockage | 10 minutes
Exchanging the metering seal | When autosampler reproducibility indicates seal wear | 30 minutes

*Includes seal and stator face

Injection Valve Assembly, 0101-0921

Stator for 2 position/6 port injection valve, 600 bar, 0101-1417

Stator for 2 position/6 port ultra high pressure valve, 1200 bar, 5068-0006

Needles and Needle Seats

The needle should be replaced when it becomes bent, burred or blunt, or when it is leaking or plugged. You should suspect a leak if you notice a trail of buffer crystals on the needle seat. The needle seat can become blocked if the sample contains particulates, as this is the first restriction that the sample experiences. If this occurs, try backflushing the needle seat capillary.

Seat Assembly 0.17 mm id standard autosampler, G1329-87017

Seat Assembly 0.12 mm id standard autosampler, G1329-87012

Accessories
Description | Use With | Part No.
Seat adapter | G1313A, G1329A, G1389A, G2260A, 1120 and 1220 Infinity LC | G1313-43204
Finger caps, for Autosampler Gripper, 15/pk | G1313A, G1329A, G1389A, G2260A, 1120 and 1220 Infinity LC | 5063-6506
Tool for micro seat capillary mounting | G1377A | G1377-44900

Needle seat, PEEK 600 bar, with seat capillary, G1367-87012

Seat assembly for 1290/1260 Infinity LC, G4226-87012

Needle Assembly for 1290/1260 Infinity LC, G4226-87201

Metering Device Supplies

Infrequently, the metering device seal and piston may need replacement if you see loss in injection volume precision or metering device leaking.

Piston seals, 5063-6589

Sample loop, 01078-87302

Loop capillary, 20 μL, G4226-60310

Loop Capillaries
Description | Agilent Autosampler | Part No.
Stainless steel loop capillary, 100 μL | G5667A | G5667-60320
| G1313A | 01078-87302
| G1329A/B |
| 1120 |
| 1220 Infinity LC |
| G1367A/B/C | G1367-87300
| G4226A | 5067-4710
| G1367E |
Stainless steel loop capillary, 900 μL | G1329A/B | G1313-87303
| G2260A |
Loop capillary, 40 μL | G1367D | G1377-87310
| G4226A | 5067-4703
| G1367E |
| G1377A | G1377-87300
| G1389A | G1329-87302
Loop capillary, 20 μL | G4226A | G4226-60310
| G1367E |
Loop capillary, 8 μL | G1389A | G1375-87303
| G1377A | G1375-87315
Loop capillary, 5 mL | G2260A | G2260-68711

Autosampler Trays

Autosampler Trays
Description | Part No.
For G1313A, G1329A/B, 1120, 1220 Infinity LC samplers
100 position tray for 2 mL vials | G1313-44510
100 position tray for 2 mL vials, thermostattable | G1329-60011
40 position tray for 2 mL vials | G1313-44512
15 position tray for 6 mL vials | G1313-44513
External vial tray for 17 vials (disposal position) | G1313-60004
Disposal tube for external vial tray | G1313-27302
For G1367A/B/C/D/E, G2258A, G4226A
Well plate tray, 2 well plates, 10 vials (supports 50 mm plates) | G2258-60011
Vial plate for 54 x 2 mL vials, 6/pk | G2255-68700
Vial plate for 15 x 6 mL vials | 5022-6539
For G1367A/B/C/D/E
100 position tray for micro vials Also for G4226A | G4226-60021
Plate for 27 Eppendorf Safe-lock tubes, 0.5/1.5/2 mL | 5022-6538
For G2257A
8.5 in well plate rack, 2/pk For 16 shallow well plates, 4 deep well plates (max 48 mm height) or 6 vial racks | G2255-68709
10 in well plate rack, 2/pk For 20 shallow well plates (max height 16 mm), not compatible with deep well plates | G2255-68710
8.5 in well plate rack extension Includes 3 racks for 3 x 16 shallow well plates, 2 x 4 deep well plates (max 48 mm height) or 3 x 6 vial racks | G2255-68720
10 in well plate rack extension Includes 3 racks for 3 x 20 shallow well plates (max height 16 mm), not compatible with deep well plates | G2255-68730
For G2250A
205H rack, two 96-deep well plates | G2250-04504
200 rack, 13 x 100 mm tubes (9 mL), 96 | G2250-04503
207 rack, 16 x 100 mm tubes (12 mL), 75 | G2250-04502
209 rack, 12 x 32 mm tubes (12 mL), 96 | G2250-04501
94A special holding 1100 tray | G2250-04500

Vial plate, G2255-68700

Vial plate, 5022-6539

Plate for 27 Eppendorf Safe-lock tubes, 5022-6538

Autosampler Kits

Autosampler Kits
Description | Kit Contents | Part No.
Preventive Maintenance Kits
For G1329B autosamplers | Includes 1 PEEK rotor seal, 1 needle seat, 1 needle | G1313-68719
For G1313A, G1329A autosamplers | Includes 1 Vespel rotor seal, 1 needle seat, 1 needle, 2 metering seals and 15 finger caps | G1313-68709
For G1313A, G1329A samplers | Includes 1 Vespel rotor seal, 1 needle seat, 1 needle | G1313-68730
For G1367A/B autosamplers | Includes 1 Vespel rotor seal, 1 needle seat, 1 needle, 1 peristaltic pump cartridge, 1 seal tight nut | G1367-68730
For G1367C/D autosamplers | Includes 1 needle assembly, 1 low carry over seat, 1 peristaltic pump, 1 rotor seal | G1367-68734
For G1367E autosampler | Includes 1 PEEK rotor seal, 1 needle seat, 1 needle, 1 peristaltic pump cartridge, 1 metering seal | G1367-68741
For 1220 manual injector systems | Includes piston seals, PTFE frits, gold seals and rotor seal | G4280-68750
For 1220 automated injector systems | Includes piston seals, PTFE frits, gold seals, rotor seal, needle and needle seat | G4280-68770
For 1120 manual injector systems | Includes 1 piston seal, 5 PTFE frits, 2 gold seals, 4 outlet caps and 1 PEEK rotor seal | G4280-68710
For 1120 automated injector systems | Includes 1 piston seal, 5 PTFE frits, 2 gold seals, 4 outlet caps, 1 Vespel rotor seal, 1 needle and 1 needle seat | G4280-68730
For G4226A HiP autosampler | Includes 1 needle seat, 1 needle, 1 rotor 2 position/6 ports, 1 peristaltic pump, 1 metering seal | G4226-68735
Extended PM kit For G1313A, G1329A autosamplers | Includes 1 Vespel rotor seal, 1 needle seat, 1 needle, 1 metering seal, 1 stator face | 5065-4498

Maintenance kit, G1313-68709

PM kit for 1220 manual injector systems, G4280-68750

Autosampler Kits
Description | Kit Contents | Part No.
Door Replacement Kits
Cabinet upgrade kit for 1260 Infinity LC sampler | Includes side panel, top cover and front door | G1329-68736
Cabinet kit for G1367E and G4226A | Includes side panel, base plate and top cover | 5067-4662
Door replacement kit for 1260 Infinity LC sampler | Includes front and side doors | G1329-68737
Door repair kit for G1367E and G4226A | Includes front door | G4226-67001
Light protection kit for G1329A | Includes opaque front and side doors and front cover | G1329-68718
Door replacement kit for G1329A | Includes transparent front and side doors | G1329-68727
Upgrade Kits
Multidraw upgrade kit for G1313A/G1327A/G1329A autosamplers | Includes 500 μL capillary, 1500 μL capillary and ZDV union | G1313-68711
Large volume injection kit for the Agilent 1290 Infinity autosampler G4226A | 80 μL seat capillary | G4216-68711

Cabinet upgrade kit, G1329-68736

Door replacement kit, G1329-68737

Fraction Collector Supplies

Agilent fraction collectors are designed to process data in real-time for instantaneous and precise fraction collection, while increasing throughput on your purification system. So you can be certain that you are getting the highest degree of recovery and purity for your fractions – even with low flow rates.

     Fraction Collector Maintenance Schedule
Procedure | When to Perform
Analytical and Preparative Fraction Collector Maintenance
Replace the inlet/waste tubing | Once per year – or when you notice signs of damage or wear
Replace the valve-to-needle tubing | Once per year – or when you notice signs of damage or wear
Exchange the preparative needle assembly | When the needle shows signs of damage or blockage
Exchange the analytical needle assembly | When the needle shows signs of damage or blockage, or when using the short needle assembly with tall test tubes (>45 mm)
Exchange the diverter valve | When the valve is leaking or not switching properly
Exchange the internal tray | When the flow delay sensor no longer works
Repair or exchange a funnel within the internal tray or funnel tray | When defective, leaky, blocked or contaminated
Micro Fraction Collector/Spotter Maintenance
Replace fraction collector capillary | At least every six months or when worn, blocked or damaged
Exchange the capillary guiding assembly | When bent or damaged
Exchange the internal tray | When the flow delay sensor no longer works properly
Exchange the flap septum and waste tubing | At least every six months or when defective or contaminated

Micro fraction collector

Funnel tray for G1364C fraction collector, G1364-84532

Collection plate, showing 96-position closing mat, 5042-1389

Vial plate, G2255-68700

Plate for 27 Eppendorf Safe-lock tubes, 5022-6538

Vial plate, 5022-6539

[picture] | TIPS & TOOLS Information on maintenance procedures can be found at www.agilent.com/chem/LCmaintenancenotes

G1364D Micro Fraction Collector Supplies
Description | Part No.
MALDI spotting adapter for G1364D | G1364-83205
Well plate adapter assembly for G1364C/D | G1364-60021
Flap septum, PEEK, for internal tray | G1364-27107
Fused silica/PEEK capillary, 25 μm, 50 cm | G1364-87304
Fused silica/PEEK capillary, 50 μm, 50 cm | G1364-87305
Fused silica/PEEK capillary, 100 μm, 50 cm | G1364-87306
Waste tube, PTFE, 20 cm, 1.4 mm id, 2.0 mm od | G1364-86711
MALDI plate carrier Bruker | 5022-6541
MALDI plate carrier Bruker PAC | 5022-6546
MALDI plate carrier ABI | 5022-6542
MALDI plate carrier ABI Opti-TOF | 5023-0238
MALDI plate carrier Agilent | 5022-6543
MALDI plate carrier Micromass | 5022-6544
Target plate for AP-MALDI LC/MS | G1972-60025
Calibration plate Bruker | 5023-0208
Calibration plate ABI 192 | 5023-0209
Calibration plate ABI 10x10 & 20x20 | 5023-0213
Calibration plate Agilent | 5023-0214
Calibration plate Micromass | 5023-0215
On-line matrix kit for MALDI spotting Includes BCD board/cable, syringe, needles, adapters, connector and capillary | G1364-68706
Adapter, female to female 1/4-28 | 5042-8517
Adapter, male Luer to female 1/4-28 | 5042-8518
Syringe, glass, 1 mL, 1/4-28 connector | 5181-1541
Micro T-connector, PEEK, swept volume 29 nL, with 1/32 in id fittings | 5042-8519
MALDI spotting tips, PTFE, 10/pk | G1364-81701

MALDI spotting adapter, G1364-83205

Well plate adapter assembly, G1364-60021

MALDI plate carrier Bruker, 5022-6541

Calibration plate Bruker, 5023-0208

Union, female to female, 5042-8517

Adapter, male Luer to female, 5042-8518

Micro T-connector, PEEK, 5042-8519

Valve Supplies

Agilent’s industry-leading Manual Injection Valves are designed to ensure trouble-free operation with your HPLC System.

Our valves also feature patented “Make-Before-Break” architecture that allows you to switch between LOAD and INJECT positions without interrupting the flow. So you can analyze more samples in less time.

Injection Valve Assembly, 0101-0921

Valve Maintenance Notes

• Vespel is a polyimide with low wear and high chemical resistance. Vespel tolerates a pH range of 0 to 10. More basic solutions dissolve Vespel, which damages the rotor seal

• PEEK offers a high chemical resistance and versatility, and will tolerate the entire pH range from 0 to 14

• Tefzel is recommended for use in applications where PEEK cannot be used, such as methylene chloride or DMSO in higher concentrations

     pH Range | 0-7 | 7-10 | 10-14
Vespel |  |  |
PEEK |  |  |
Tefzel |  |  |

Injection Valves

*Includes seal and stator face

Stator for 2 position/6 port ultra high pressure valve, 1200 bar, 5068-0006

Switching Valve Supplies

A set of valve types specially designed for Agilent HPLC systems allows you to extend your HPLC applications. New valve offerings give you:

• More flexibility in solvent selection and column selection

• New automation capabilities in sample preparation

• Increased sample throughput through alternating column regeneration

• Increased separation performance with multidimensional chromatography

Switching valve

Valve head, 2 position/6 port, 600 bar, 5067-4137

2 position/6 port ultra high pressure valve, 5067-4117

Valve head, 2 position/10 ports for ultra high pressure, 1200 bar, 5067-4118

Stator for 2 position/6 port ultra high pressure valve, 600 bar, 0101-1417

Rotar seal, 2 position/6 port, 600 bar for C1316B, 0101-1409

Manual Injection Valves

Agilent provides the latest developments in LC injection technology from Rheodyne.

• Continuous flow path with “Make-Before-Break” design

• Sample capacity

• Choice of stainless or PEEK flow path

• Easy access to fittings due to wide 30° port angles

Series 7725i and 9725i Analytical Injection Valves

Stainless steel (SS) 7725i and PEEK 9725i valves are the most popular injection valves for analytical HPLC. Features include:

• A 20 μL loop (installed). Loops are also available in stainless steel or PEEK from 5 μL to 5 mL (10 mL for PEEK)

• Make-Before-Break (MBB) technology allows switching without flow interruption

• Wide 30° port angles offer easier access to fittings

• Built-in position sensing switch provides the chromatograph with a reproducible start signal

Series 3725i-038 and 3725i Preparative Injection Valves

The series 3725i-038 (stainless steel) and 3725i (PEEK) are the most suitable manual valves for large sample volumes, high flow rates, and preparative columns sized 1.0-10 cm in diameter.

• Versatile ports accommodate 1/8 in (3.2 mm) and 1/16 in (1.6 mm) od tubing.
Note: 1/16 in od tubing requires an adapter, P/N 5067-1503

• 1.0 mm diameter passages allow flow rates up to 800 mL/min with virtually no pressure drop

• Make-Before-Break technology allows switching without flow interruption

• High reproducibility for both partial-filling and complete-filling methods

• Sample range is 100 μL to 20 mL (10 mL loop is installed)

• Flow range is 10 to 800 mL/min

• Built-in position sensing switch gives the chromatograph a reproducible start signal

7725i manual injection valve, 5063-6502

Manual Injection Valve Replacement Parts

• Rotor seals wear with use and need routine replacement

• Stators only need replacement if the ports are damaged

• PEEK rotor seals are incompatible with concentrated nitric and sulfuric acids

Manual Injection Valve Sample Loops

The right mix of injection valve sample loops are available for your application needs. Agilent offers factory-cut and finished loops of the highest quality.

• Stainless steel loops are square cut and free of burrs for a flush connection

• Flexible PEEK loops have a clean, straight cut for low dead volume connections

Stainless steel sample loops

Stainless Steel Sample Loops

• Sample loops for Rheodyne 7725 Series and 7125 Series valves are not interchangeable due to the change in port angle

• Actual volumes can differ due to tolerance of metal tubing bore

• Accuracy of large metal loops is ±5%, intermediate loops ±10%, small loops ±30%

PEEK Sample Loops

• Inert to most organic solvents

• Wall thickness, temperature, exposure time and concentration of organic solvents affect the durability of PEEK tubing

• Concentrated nitric acid and sulfuric acid weaken PEEK tubing

• THF, methylene chloride and DMSO cause PEEK to swell

• Actual volumes can differ because of tolerance of tubing bore

• Accuracy of large PEEK loops is ±14%, intermediate loops ±21%, small loops ±65%

PEEK sample loops

Syringes for Manual Injection

Agilent syringes for manual injection valves have a blunt-tip point style needle to prevent damaging the valve’s internal parts. They can be used with any type/brand of manual injection valve.

Syringe, 100 μL FN LC tip, 5190-1508

TIPS & TOOLS

Agilent has made vial, cap and septum selection easy with its new Interactive Vial Selection Tool, available online in both desktop and mobile versions. The tool identifies the right vial and closures for your particular application, and provides the rationale for the choices offered. Visit www.agilent.com/chem/SelectVials

Thermostatted Column Compartment Supplies

Thermostatted Column Compartment Supplies
Description | Part No.
Capillary system for 0.12 mm id use | G1316-68744
Rapid Resolution High Throughput capillary kit Used for converting an Agilent 1200 instrument to the RRLC configuration, to enable use of high efficiency columns (to 600 bar). Can also be used for Agilent 1100 instruments. | 5065-9947
1200 capillary kit for 0.12 mm id | G1316-68716
High temperature heat exchanger, 1.6 μL, 0.12 mm id, “R” | G1316-80002
High temperature heat exchanger, 1.6 μL, 0.12 mm id, “L” | G1316-80003
Heat exchanger/cooler, 1.5 μL, 0.12 mm id | G1316-80004
Carrier for heat exchanger 1290 Infinity TCC and 1200 Series TCC SL | G1316-83200
Column Identification Module (CIM), 3/pk | 5062-8588
Column clamp, 6/pk | 5063-6526
Column holder for micro LC columns | 5001-3702
Column connecting capillary with fittings, 7 cm, 0.12 mm id, 1/16 in male/male | G1316-87303
Column connecting capillary with fittings, 9 cm, 0.17 mm id, 1/16 in male/male | G1316-87300
Column connecting capillary with fittings, 18 cm, 0.12 mm id, 1/16 in male/male | G1313-87304
Column connecting capillary with fittings, 18 cm, 0.17 mm id, 1/16 in male/male | G1313-87305
PEEK tubing, 1/32 in od, 0.4 mm id, 450 mm, Micro valve to waste | 5022-6503
Thermal column insulation enclosure kit | G1316-60001

High temperature heat exchanger, G1316-80002

High temperature heat exchanger, G1316-80003

Heat exchanger/cooler, G1316-80004

Column Identification Module, 5062-8588

Capillary Tubing Kits

Capillary kits are available for easy ordering and setup of the switching valves. They include all capillaries and fittings for specific applications, as well as bulk PEEK capillaries and a capillary cutter to add maximum flexibility.

Capillary Tubing Kits
Application | Valve Kit | Part No.
Column regeneration Capillaries: 0.17 mm id | G1157A | G1156-68711
Column regeneration Capillaries: 0.25 mm id | G1157A | G1156-68713
Capillary kit column regeneration intern Capillaries: 0.17 mm id | 2 position/10 port 400 bar valve, for G1316A/B | G1316-68711
Column selection Capillaries: 0.17 mm id | G1159A | G1156-68712
Sample enrichment Capillaries: 0.17 mm id | G1316A #055 | G1316-68710
Sample enrichment Capillaries: 0.17 mm id | G1158A | G1156-68714
Solvent selection Flow rate up to 10 mL/min | G1160A | G1160-68706 5067-4601*

*Use for method development applications. Kit contains longer tubing.

Capillary Kits for Internal Switching Valves
Description | Use With | Part No.
Capillary kit column switching valve | 2 position/6 port 400 bar valve, for G1316A/B | G1316-68708
Capillary kit column regeneration intern | 2 position/10 port 400 bar valve, for G1316A/B | G1316-68711
Column regeneration kit for μ-LC columns | 2 position/10 port CSV 600 bar, for G1316B | G1316-68721

Capillary Kit Contents
Part No. | Description | Quantity
5067-1595 | Heater long-up 0.12 id, 1.6 μL internal | 4
| Heater long-down 0.12 id, 1.6 μL internal | 4
| Carrier for heat exchanger, TCC SL Plus | 4
| Flexible tubing, 280 mm, 0.12 mm id | 2
| SS capillary, 340 x 0.12 mm, m/m, n-s/n-s | 1
| SS capillary, 280 x 0.12 mm, ps/ps, 1 long nut, 1 short nut | 8
| SS capillary, 280 x 0.12 mm, ps-ns, 2 long nuts, 1 short nut | 8
| SS capillary, 280 x 0.17 mm, ps/ps, 1 long nut | 1
| Flexible capillary, 0.12 x 500 mm, no fittings | 1
| Fitting holder assembly | 4
| Column clip set, eight colors | 2
| Long fittings and ferrules, SS, 10/pk | 1

[picture] | TIPS & TOOLS For internal switching valves for your TCC, see here

Capillary Kit Contents
Part No. | Description | Quantity
5067-1596 | Capillary, 0.17 x 90 mm, 1/16 in male/male | 6
| Flexible tubing, 400 mm, 0.17 mm id | 1
| Flexible capillary, 0.17 x 280 mm, no fitting | 2
| Plastic fitting | 8
| Long fittings and ferrules, SS, 10/pk | 2
| SS capillary, 280 x 0.17 mm, ps/ps, 2 long nut | 1
| SS capillary, 280 x 0.17 mm, ps/ps, 1 long nut, 1 short nut | 6
| SS capillary, 500 x 0.17 mm, ps-ns, 2 long nuts, 1 short nut | 6
| Flexible capillary, 0.17 mm id x 600 mm | 1
| Column clip set, eight colors | 2
| VHP-fitting standard length (10 each = 1/pk) | 6
5067-1597 | Heater long-up 0.12 id, 1.6 μL internal | 3
| Heater long-down 0.12 id, 1.6 μL internal | 3
| Carrier for heat exchanger, TCC SL Plus | 3
| Flexible tubing, 280 mm, 0.12 mm id | 2
| SS capillary, 340 x 0.12 mm, m/m, n-s/n-s | 1
| SS capillary, 280 x 0.12 mm, ps/ps, 1 long nut, 1 short nut | 6
| SS capillary, 400 x 0.12 mm, ps-ns, 2 long nuts, 1 short nut | 6
| SS capillary, 280 x 0.17 mm, ps/ps, 2 long nut | 1
| Flexible capillary, 0.12 x 500 mm, no fittings | 1
| Column clip set, eight colors | 2
| Fitting holder assembly | 3
| Long fittings and ferrules, SS, 10/pk | 1
5067-4601 | Bottle head assembly | 4
| Tefzel ferrules/SS rings, 1/8 in, 10/pk | 1
| Flexible tubing, 1 ea / 1 meter | 5
| PPS nuts, 1/8 in, 1/4-28 thread, 10/pk | 1
| Long fittings and ferrules, SS, 10/pk | 5
5067-4646 | SS capillary, 0.12 mm id, 340 mm pre-swaged | 1
| SS capillary, 0.17 mm id, 700 mm pre-swaged | 1
| SS capillary, 0.12 mm id, 90 mm pre-swaged | 2
| SS capillary, 0.12 mm id, 150 mm pre-swaged | 2
| SS capillary, 0.12 mm id, 280 mm pre-swaged | 2
| SS capillary, 0.12 mm id, 120 mm pre-swaged | 1
| SS capillary, 0.12 mm id, 200 mm pre-swaged | 1
| PTFE flexible tubing | 1
| Heat exchanger, long-up, 1.6 μL | 1
| Heat exchanger, long-down, 1.6 μL | 1
| Carrier for heat exchanger, TCC SL Plus | 2
| Fitting holder assembly | 1
| Column clip set, eight colors | 1
5067-4682 | SS capillary, 120 x 0.12 mm, ns ns, 1 large, 1 large | 1
| SS capillary, 150 x 0.12 mm, ns ns, 1 large, 1 large | 2
| SS capillary, 200 x 0.12 mm, ns ps, 1 short, 1 large | 1
| SS capillary, 280 x 0.12 mm, ns ns, 1 large, 1 large | 2
| SS capillary, 340 x 0.12 mm, ps ns, 1 short, 1 large | 1
| SS capillary, 0.17 mm id, 700 mm pre-swaged | 1
| SS capillary, 90 x 0.12 mm, ns ns, 1 short, 1 large | 2
| Column clip set, eight colors | 1
| Fitting holder assembly | 2
| Heater long-up 0.12 id, 1.6 μL internal | 1
| Heater long-down 0.12 id, 1.6 μL internal | 1
| Carrier for heat exchanger, TCC SL Plus | 2
5067-4729 | SS capillary, 0.8 x 0.11 mm, 340 mm, RF/M4 | 1
| SS capillary, 0.8 x 0.11 mm, 5000 mm, RF/M4 | 1
| SS capillary, 0.8 x 0.11 mm, 130 mm, Ig RF/M4 | 8
| SS capillary, 0.8 x 0.17 mm, 150 mm, Ig M4/M4 | 1
| SS capillary, 0.8 x 0.11 mm, 250 mm, Ig ps/M4 | 1
| Flexible PEEK tubing | 2
| Heater long-up 0.12 id, 1.6 μL internal | 2
| Heat exchanger, long-down, 1.6 μL | 2
| Carrier for heat exchanger, TCC SL Plus | 2
| Fitting holder assembly | 2
| Column clip set, eight colors | 1
5067-4730 | SS capillary, 340 x 0.17 mm, ps-ns, 1 short, 1 long | 1
| SS capillary, 0.17 mm id, 700 mm pre-swaged | 1
| SS capillary, 90 x 0.17 mm, ns-ns, 1 short, 1 long | 4
| SS capillary, 150 x 0.17 mm, ns-ns, 1 large, 1 large | 2
| SS capillary, 280 x 0.17 mm, ns-ns, 1 large, 1 large | 2
| SS capillary, 120 x 0.17 mm, ns-ns, 1 large, 1 large | 1
| SS capillary, 200 x 0.17 mm, ns-ps, 1 short, 1 large | 1
| PTFE flexible tubing | 1
| Column clip set, eight colors | 1
5067-4800 | SS capillary, 340 x 0.11 mm, SW-1/16/M4 | 1
| SS capillary, 0.17 mm id, 700 mm, SW-1/16/M4 | 1
| SS capillary, 90 x 0.11 mm, SW-1/16/M4 | 2
| SS capillary, 0.8 x 0.11 mm, 150 mm, large RF/M4 | 2
| SS capillary, 0.8 x 0.11 mm, 280 mm, large RF/M4 | 1
| SS capillary, 0.8 x 0.17 mm, 150 mm, large M4/M4 | 1
| SS capillary, 0.8 x 0.12 mm, 250 mm, large ps/M4 | 1
| PEEK tubing, 1/32 in od, 0.4 mm id, 450 mm | 2
| Heat exchanger, long-down, 1.6 μL | 1
| Heater long-up 0.12 id, 1.6 μL internal | 1
| Carrier for heat exchanger, TCC SL Plus | 2
| Fitting holder assembly | 2
| Column clip set, eight colors | 1
5067-4767 | Capillary, 400 x 0.17 mm Bio | 1
| Capillary, 300 x 0.17 mm Bio | 2
| Fitting holder assembly | 2
| Column clip set, eight colors | 1
| Finger-tight fittings, long, 1/16 in, 10/pk | 1
5067-4769 | Capillary, 400 x 0.17 mm Bio | 1
| Capillary, 300 x 0.17 mm Bio | 4
| Fitting holder assembly | 2
| Column clip set, eight colors | 1
| Finger-tight fittings, long, 1/16 in, 10/pk | 1
5067-5103 | SS capillary, 340 x 0.17 mm, ps-ns SW-M4 | 1
| SS capillary, 0.17 mm id, 700 mm, SW-1/16/M4 | 1
| SS capillary, 90 x 0.17 mm, SW-1/16/M4 | 2
| SS capillary, 90 x 0.17 mm, RF/M4 | 2
| SS capillary, 0.8 x 0.17 mm, 150 mm, Ig RF/M4 | 2
| SS capillary, 0.8 x 0.17 mm, 280 mm, Ig RF/M4 | 2
| SS capillary, 0.8 x 0.17 mm, 150 mm, large M4/M4 | 1
| SS capillary, 0.8 x 0.12 mm, 250 mm, large ps/M4 | 1
| PEEK tubing, 1/32 in od, 0.4 mm id, 450 mm | 1
| Column clip set, eight colors | 1

Detector Supplies

Agilent wavelength detectors combine exceptional flexibility with superior instrument control, data communication, and analytical capabilities. This section shows you how to maintain your detector’s high level of selectivity and sensitivity.

Detector Maintenance Tips
Symptom | What To Do | Additional Information
Lamp does not ignite | Exchange the lamp | Perform a wavelength calibration test and an intensity test after lamp replacement
Noise exceeds application limit | Exchange the flow cell | Perform a wavelength calibration test after flow cell replacement
Drift exceeds application limit | Exchange the lamp | Perform a wavelength calibration test and a pressure tightness test after flow cell replacement
Leaky flow cell (For G4212 only) | Exchange the flow cell | Perform a wavelength calibration test after flow cell replacement
Leaky flow cell (For all G1314/G1315/G1365 detectors) | Clean or exchange the flow cell | Perform a wavelength calibration test and a pressure tightness test after flow cell replacement
Lower intensity (For G4212 only) | Exchange the flow cell | Perform a wavelength calibration test after flow cell replacement
Lower intensity (For all G1314/G1315/G1365 detectors) | Clean or exchange the flow cell | Perform a wavelength calibration test and a pressure tightness test after flow cell replacement

Certified Lamps

• All lamps are tested for noise and drift specifications, correct operating voltage, light intensity and proper alignment

• Improved coating process increases Agilent lamp lifetimes up to 50%

• Agilent deuterium lamps are designed with a much narrower aperture providing increased light intensity and decreased noise – translating into an appreciably higher signal-to-noise ratio

• By providing higher sensitivity, Agilent lamps can extend detection capabilities and improve qualification at trace levels – for more than 2,000 hours of use

Agilent’s lamps are manufactured in an ISO 9001 certified environment and are fully traceable throughout every step of the production process. Each lamp is then tested to ensure it meets Agilent’s performance specifications. Test equipment is regularly calibrated using optical standards certified by NIST (National Institute of Standards and Technology) or PTB (Physikalisch-Technische Bundesanstalt).

Detector Lamps
Description | Comments | Part No.
Variable Wavelength Detector (VWD)
Long life Deuterium lamp with RFID tag | For G1314D/E/F | G1314-60101
Long life Deuterium lamp | For G1314A/B/C, 1120 and 1220 Infinity LC | G1314-60100
Diode Array Detector (DAD)/Multiple Wavelength Detector (MWD)
Long life HiS Deuterium lamp (8-pin) with RFID tag | For G4212A/B | 5190-0917
Long life Deuterium lamp with RFID tag | For G1315C/D and G1365C/D | 2140-0820
Long life Deuterium lamp | For G1315A/B and G1365A/B | 2140-0813
Long life Deuterium lamp | For G1315A/B and G1365A/B | 5182-1530
Deuterium lamp | For G1315A/B and G1365A/B | 2140-0590*
Tungsten lamp | For G1315A/B/C/D and G1365A/B/C/D | G1103-60001

*Standard lamp for 1000 hours of use only

Deuterium lamp, G1314-60100

Long life HiS Deuterium lamp, 5190-0917

Deuterium longlife lamp, 2140-0813

Long life Deuterium lamp, 5182-1530

Deuterium lamp, 2140-0590

Tungsten lamp assembly, G1103-60001

Variable Wavelength Detector (VWD)

*For more information about what is included in each kit, see here.

Variable wavelength detector

Diode Array Detector (DAD)/Multiple Wavelength Detector (MWD)

Cleaning or Replacing DAD/MWD Flow Cells

• A decrease in detector performance or unusual noise levels may mean you have dirty flow cell windows

• Clean and reassemble one side of the flow cell before beginning the other side to prevent mixing the front and rear gaskets, which have different hole diameters

• While cleaning or replacing flow cell windows, if the washers fall out of the window assembly, they must be inserted in the correct order with a PTFE ring to prevent any leaks from the flow cell window

• Clean the cell body with water or isopropanol

• After opening the cell you should always use a new gasket

Diode array detector (DAD)/Multiple wavelength detector (MWD)

*For more information about what is included in this kit, see here.

Max-Light cartridge cell, G4212-60008

Ultra-low dispersion flow cell, G4212-60038

Max-Light cartridge cell, interior view, 60 mm path for high concentrations, G4212-60007

Max-Light HDR flow cell, interior view, to show short 3.7 mm path length for high concentrations, G4212-60032

[picture] | TIPS & TOOLS Learn more about how different flow cells impact your chromatography, and the High Dynamic Range (HDR) Flow Cell. See application note 5991-0115EN at www.agilent.com/chem/library

80 nL and 500 nL Flow Cell Supplies
Description | Unit | Part No.
Fitting screw | 10/pk | 5063-6593
Double winged nuts and 1/32 in ferrules | 10/pk | 5065-4422
1/32 in ferrule and stainless steel lock ring, lite touch | 10/pk | 5063-6592
Union adjustment tool | 2/pk | 5022-2146
Universal ZDV union, stainless steel, no fittings | 2/pk | 5022-2184
Torque wrench adapter |  | G1315-45003
Open end wrench, 4 mm |  | 8710-1534

Stainless steel fittings, male (G), 5063-6593

Double winged PEEK nut & ferrule (WPF), 5065-4422

ZDV universal union, 5022-2184

Wrench, open end, for use with PEEK-coated fused silica capillaries, 8710-1534

500 nL Flow Cell and Replacement Parts
Description | Comments | Part No.
500 nL flow cell | Contains quartz flow cell with 10 mm path length and 500 nL volume and connecting capillaries, max 50 bar pressure | G1315-68724
Sealing kit | Includes torque adapter, 2 cell seal assemblies, 5 LiteTouch front and back ferrules | G1315-68715
Quartz cell body, 10 mm |  | G1315-80001
Cell seal assembly, 500 nL |  | G1315-87101
Fused silica/PEEK capillary, 100 μm id, 30 cm long | Inlet | G1315-87333
Fused silica/PEEK capillary, 50 μm id, 40 cm long | Inlet | G1315-87323
Fused silica/PEEK capillary, 100 μm id, 12 cm long | Outlet | G1315-87338
Fused silica/PEEK capillary, 50 μm id, 12 cm long | Outlet | G1315-87328

80 nL Flow Cell and Replacement Parts
Description | Comments | Part No.
80 nL flow cell | Contains quartz flow cell with 6 mm path length and 80 nL volume and connecting capillaries, max 50 bar pressure | G1315-68716
Sealing kit for 80 nL flow cell | Includes torque adapter, 2 cell seal assemblies, 5 LiteTouch front and back ferrules and 5 sleeves for 360 μm od capillaries | G1315-68725
Quartz cell body, 80 nL, 6 mm path length |  | G1315-80002
Fused silica/PEEK capillary, 50 μm id, 40 cm long | Inlet | G1315-87323
Fused silica/PEEK capillary, 50 μm id, 12 cm long | Outlet | G1315-87328
Fused silica/PEEK capillary, 25 μm id, 20 cm long | Inlet | G1315-87313
Fused silica/PEEK capillary, 25 μm id, 60 cm long | Outlet | G1315-87318

Preparative Flow Cells and Replacement Parts
Description | Part No.
Preparative flow cell, 0.3 mm, 20 bar, quartz | G1315-60017
Preparative flow cell, 0.06 mm, 20 bar, quartz | G1315-60018
PTFE tubing, 0.8 mm id, 2 m | G1315-67301
PTFE tubing, 0.5 mm id, 0.8 m | G1315-67302
Cell housing | G1315-27705
1/16 in finger-tight PEEK fitting, 2/pk | 0100-1516
Quartz body, 0.3 mm | G1315-80004
Quartz body, 0.06 mm | G1315-80003
Prep flow cell, stainless steel, 3 mm, 120 bar | G1315-60016
Stainless steel connecting capillary, 0.5 mm, 250 mm | G1315-87305

Finger-tight PEEK fitting (SPF), 0100-1516

Detector Maintenance Kits

Detector Maintenance Kits
Description | Kit Contents | Part No.
Variable Wavelength Detector (VWD)
Standard “D” type flow cell kit | Includes 2 windows, 2 gaskets #1, 2 gaskets #2 | G1314-65061
Semi-micro flow cell kit | Includes 2 windows, 4 gaskets: 2 standard #1, 1 semi-micro #1, 1 semi-micro #2 | G1314-65056
Micro flow cell kit | Includes 2 windows, 2 gaskets #1, 2 gaskets #2 | G1314-65052
Cell repair kit, semi-micro | Includes window screw kit, 4 mm hexagonal wrench and seal kits | G1315-68713
High-pressure flow cell kit | Includes 2 windows, 2 Kapton gaskets and 2 PEEK rings | G1314-65054
Diode Array Detector (DAD)/Multiple Wavelength Detector (MWD)
Cell repair kit | Includes window screw kit, 4 mm hexagonal wrench and seal kit | G1315-68712
Cell repair kit, semi-micro | Includes window screw kit, 4 mm hexagonal wrench and seal kits | G1315-68713
Sealing kit for 500 nL flow cell | Includes torque adapter, 2 cell seal assemblies, 5 lite touch front and back ferrules | G1315-68715
Sealing kit for 80 nL flow cell | Includes torque adapter, 2 cell seal assemblies, 5 lite touch front and back ferrules and 5 sleeves for 360 μm od capillaries | G1315-68725

Other Detectors

G4218A 1200 Series Evaporative Light Scattering Detector Supplies
Description | Part No.
Standard flow nebulizer | G4218-20000
Semi-micro flow nebulizer | G4218-20001
Large flow nebulizer | G4218-20002
Micro flow nebulizer | G4218-20003
RRLC nebulizer | G4218-20004
Nebulization chamber, glass | G4218-40000
Black plastic nut, 13 mm diameter, glassware | G4218-40010
Black plastic nut, 22 mm diameter, glassware | G4218-40011
Black exhaust tube, 2.5 m | G4218-40110
Bulkhead | G4218-40130
Cartridge, 0.01 μm for gas regulator | G4218-40150
Pneumatic tube with stainless steel fitting | G4218-40220
Drain tube with stainless steel fitting | G4218-40100
Gas regulator with 0.01 μm filter and manometer | G4218-60100
Seal kit for nebulization chamber | G4218-68010
Caffeine standard, 250 μg/mL | G4218-85000

G1362A 1100/1200 Series Refractive Index Detector (RID) Supplies
Description | Part No.
Tubing kit Includes 300 mm recycle valve to recycle port, 200 mm recycle valve to waste port, 120 mm purge valve to recycle valve, 270 mm purge valve to sample cell, 170 mm purge valve to reference cell | G1362-68709
Interface tubing kit Includes 1/8 in ferrule, 1/3 in nut, PTFE tubing | G1362-68706
Interface capillary, 400 mm, 0.17 mm id | G1362-87300
Restriction capillary, 0.17 mm id | G1362-87301

1200 Series Evaporative Light Scattering Detector

Standard flow nebulizer, G4218-20000

Cartridge for gas regulator, G4218-40150

G1321A/B 1100/1200 Series Fluorescence Detector (FLD) Supplies
Description | Part No.
Detector lamp | 2140-0600
Flow cell, 8 μL, 20 bar | G1321-60005
Flow cell, 4 μL | G1321-60015
Cuvette kit, 8 μL, 20 bar Includes tubing, stainless steel fitting, front and back ferrule, PEEK fitting, syringe needle and syringe | G1321-60007
Cut-off filter kit: |
389, 408, 450, 500, 550 nm | 5061-3327
380, 399, 418, 470, 520 nm | 5061-3328
280, 295, 305, 335, 345 nm | 5061-3329
Corrugated tubing, polypropylene, 6.5 mm id, 5 m | 5062-2463
PTFE tubing, FEP, 0.7 mm id, 5 m | 5062-2462
1/16 in finger-tight PEEK fitting, 2/pk | 0100-1516
Column connecting capillary with fittings, 380 x 0.17 mm | G1315-87311
1/16 in stainless steel front ferrule, 10/pk | 5180-4108
1/16 in stainless steel back ferrule, 10/pk | 5180-4114
1/16 in stainless steel fitting, 10/pk | 5061-3303
Fluorescence detector calibration sample, 1 g glycogen | 5063-6597
Open end wrench, 1/4 and 5/16 in | 8710-0510
Glass syringe | 9301-1446
Syringe needle | 9301-0407

Flow cell for G1321A fluorescence detector, G1321-60005

Finger-tight PEEK fitting (SPF), 0100-1516

Stainless steel front ferrules, 5180-4108

Back ferrules 1/16 in, 5180-4114

1100/1200 Series Chip LC Supplies

1100/1200 Series Chip LC Supplies
Description | Part No.
Rotor, inner valve, 3 grooves, chip LC | G4240-23705
Rotor, outer valve, 5 grooves, chip LC | G4240-25206
PEEK fitting, special for chip LC | G4240-43200
Fused silica/PEEK capillary, 15 μm, 90 cm Nano pump to chip cube | G4240-87300
Fused silica/PEEK capillary, 25 μm, 105 cm Micro well plate sampler to chip cube | G4240-87301
Fused silica/PEEK capillary, 100 μm, 100 cm Chip cube to waste | G4240-87302
Fused silica/PEEK capillary, 75 μm, 100 cm Syringe pump to chip cube | G4240-87303
Fused Silica/PEEK capillary, 50 μm, 50 cm | G4240-87304
Inline micro filter kit, 0.5 μm, PEEK Use with chip cube LC system | 5067-1582
Fitting with 0.5 μm PEEK frit, 10/pk | 5067-1584
PEEK fitting for use with 1/32 in od, 10/pk | 5067-1585
PEEK sample transfer capillary, 25 μm, 100 cm Micro inline filter to chip cube (Phospho-Chip application) | G4240-87309
PEEK capillary, 25 μm, 10 cm Micro well plate samper to micro inline filter (Phospho-Chip application) | G4240-87310

Bio-inert Supplies

Agilent 1260 Infinity Bio-inert Quaternary LC Supplies

For your challenging bio-molecule analyses, the new 1260 Infinity Bio-inert Quaternary LC System sets new standards in performance, reliability, and robustness. Analysis of proteins and biotherapeutics usually presents the most challenging solvent conditions for any LC instrument. In addition, bio-molecules tend to bind unspecifically to surfaces, requiring tedious procedures. To address these needs, Agilent designed this application-specific LC instrument for bio-molecular analysis – without any compromise in performance – built on the proven Agilent 1200 Infinity platform technology.

The Agilent 1260 Infinity Bio-inert Quaternary LC features bio-inertness for all components without exception. The sample flow path through autosampler, capillaries and a variety of detectors are completely metal-free, with only PEEK and ceramic components coming into contact with your bio-molecule. Thus, the uncertainty of secondary interaction for proteins and peptides with surfaces which can result in peak tailing, low recovery and decreased column lifetime is minimized – and your confidence maximized.

1260 Bio-inert Quaternary Pump Parts
Description | Part No.
Bio-inert purge valve | G5611-60061
Bio-inert active inlet valve | G5611-60025
Bio-inert cartridge for active inlet valve, 600 bar | G5611-60020
Bio-inert outlet ball valve | G5611-60067
Sapphire plunger | 5067-4695
Bio-inert piston seal | G5611-21503
Bio-inert wash seal | 0905-1731
Bio-inert seal keeper | G5611-26210
Bio-inert support ring | G5611-63010
Preventative maintenance kit for Bio-inert quaternary pump | G5611-68741

TIPS & TOOLS For information on the family of complementary Biocolumns, View Here. | [picture]

Stator face, ceramic, 0100-1851

1260 Bio-inert High Performance Autosampler Parts
Description | Part No.
2 position/6 port valve head, 600 bar | 5067-4131
Rotor seal, 3 grooves, max 600 bar | 0101-1416
Bio-inert stator | 5068-0060
Stator face, ceramic | 0100-1851
Bio-inert needle, 600 bar | G5667-87200
Tool for needle adjustment | G5667-40500
Bio-inert needle seat assembly, 600 bar | G5667-87017
Sapphire plunger | 5067-4695
Bio-inert piston seal | G5611-21503
Sample loop, 100 μL, Bio-inert | G5667-60320

1260 Bio-inert Valve Parts
Description | Use With | Part No.
Rotor seal, 3 grooves, max 600 bar | Bio-inert 2 position/6 port switching valve | 0101-1409
Bio-inert stator | Bio-inert 2 position/6 port switching valve | 5068-0060
Stator face, ceramic | Bio-inert 2 position/6 port switching valve | 0100-1851
Bio-inert rotor, 2-position/10-port, 600 bar |  | 5068-0041
Bio-inert stator, 2-position/10-port, 600 bar |  | 5068-0040
Bio-inert rotor seal, 4 column PEEK | Bio-inert 4 column selection valve | 5068-0045
Bio-inert PEEK tube from valve to needle |  | G5664-86703
Bio-inert PEEK tube valve to detector |  | G5664-86706
Bio inert 12 position/13 port, solvent selection valve | G4235A | 5067-4159

Rotor seal, 2 position/6 port, 600 bar for G1316B, 0101-1409

Bio-inert union, 600 bar, 5067-4741

1260 Bio-inert Detector Parts
Description | Use With | Part No.
Bio-inert standard flow cell, with RFID tag | G1315C/D and G1365C/D | G5615-60022
Bio-inert max light cartridge cell | G4212A/B | G5615-60017
Bio-inert max light cartridge cell | G4212A/B | G5615-60018
Bio-inert FLD flow cell | G1321B | G5615-60005
PEEK tubing |  | 0890-1763

1260 Bio-inert Fittings
Description | Part No.
Bio-inert union, stainless steel with PEEK insert 600 bar | 5067-4741

1260 Bio-inert Column Compartment
Description | Part No.
Bio-inert low dispersion heat exchanger | G5616-60050

Bio-inert low dispersion heat exchanger, G5616-60050

LC/MS Supplies

Combined with Agilent’s industry-leading LC systems, our single quadrupole, ion trap, triple quadrupole, TOF and Q-TOF LC/MS solutions combine world-class performance with legendary reliability and ease-of-use.

This section contains all of the mass spectrometry supplies you need to keep your LC mass spectrometer running at peak performance.

   LC/MS Maintenance Schedule

Procedure | When to Perform
Flush the nebulizer | Daily or at the end of each shift to flush traces of samples and buffers out of the tubing, valves, and nebulizer.
Clean the electrospray spray chamber | Daily or anytime you suspect carryover contamination from one sample or analysis to another.
Replace the electrospray nebulizer needle | When the needle is plugged. Common symptoms of a plugged needle are increased LC backpressure, off-axis spraying, or dripping from the nebulizer.
Clean the APCI spray chamber | Daily or anytime you suspect carryover contamination from one sample or analysis to another.
Replace the APCI nebulizer needle | When the needle is plugged. Common symptoms of a plugged needle are increased LC backpressure or off-axis spray from the nebulizer.
Clean the multimode source | Daily or anytime you suspect carryover contamination from one sample or analysis to another, or when you must access the end cap and capillary cap for cleaning and inspection.
Check calibrant levels | Monthly or weekly if you tune the LC/MS frequently.

LC/MS Preventive Maintenance Kit

For your convenience, the LC/MS Preventive Maintenance Kit has the recommended common supplies needed for most Agilent LC/MS systems. Unique source parts should be ordered separately.

Agilent 6100 Series Single Quadrupole LC/MS, G6140A

LC/MS Preventive Maintenance kit
Description | Part No.
LC/MS Preventive Maintenance kit | 5190-1443
Foreline pump (rotary pump) oil, Inland 45, 1 L, for E1M18/E2M28 | 6040-0834
Oil mist filter element for E2M18 | 1535-4970
Filter element, 5 μm, 5/pk | 0100-2051
Spring, canted coil, 4/pk | 1460-2571
Big hydrocarbon trap, 1/4 in fittings | BHT-4
Rotor seal, Vespel, pH 0 to 10 | 0100-1855

Foreline pump oil, 6040-0834

Oil mist filter element for E2M18, 1535-4970

[picture] | TIPS & TOOLS Save ordering time and money with the LC/MS PM Kit! It contains the common supplies specified in Agilent service engineer preventive maintenance procedure for LC/MS platforms.

Corona needle APCI, G1947-20029

TIPS & TOOLS

ES nebulizer (original) is compatible with the following ion sources:

• ESI G1948A with Serial Number < US91801994

• ESI G1948B with Serial Number < US91201787

• Multimode G1978A with Serial Number < US90800804

• Multimode G1978B with Serial Number < US90700787

• Dual ESI G3251A with Serial Number < US91200355

• Dual ESI G3251B with Serial Number < US91200355

Electron multiplier replacement horn, 05971-80103

LC/MS Foreline Pump Supplies
Description | Part No.
Oil mist filter kit for E1M18/E2M28 | 3162-1056
Oil mist cartridge filter for MS40+ | G1960-80039
Oil return kit | 3162-1057
Foreline pump (rotary pump) oil, Inland 45, 1 L, for E1M18/E2M28 | 6040-0834
Foreline pump (rotary pump) oil, 4 L, for E1M18/E2M28 | 6040-0798
Foreline exhaust adapter | 59980-20134
Hose clamp | 1400-0563
Oil mist filter element for E2M18 | 1535-4970
KF25 clamp, stainless steel | 0100-0549
KF25 coseal (inside clamp) | 0100-1597
Exhaust tubing | 0890-1727
Pump oil drip pan | G1946-00034

Adding foreline pump (rotary pump) oil, 4 L

LC/MS Common Supplies*
Description | Part No.
Common Parts |
Filter element, 5 μm, 5/pk | 0100-2051
Rotor seal, Tefzel, pH 0 to 14 | 0100-1854
Rotor seal, Vespel, pH 0 to 10 | 0100-1855
Inlet filter assembly | G1946-60180
SSV long drain tubing assembly | G1969-60086
Spring, canted coil | 1460-2571
Cleaning Supplies |
Abrasive mesh, 4000 grit | 8660-0827
Capillary cleaning wire for dip tube | G1946-80054
Cleaning powder, dielectric capillary, Alconox | 5190-1401
Cloths, lint-free | 05980-60051
Cotton swabs, 100/pk | 5080-5400
Gas Purifiers |
Big hydrocarbon trap, 1/4 in fittings | BHT-4
Big moisture trap, 1/4 in fittings | BMT-4
Big universal trap, 1/4 in fittings | RMSN-4
Big universal trap, 1/8 in fittings, Nitrogen | RMSN-2
Tools |
LC/MS tool kit | G1946-60157
Nebulizer adjustment fixture | G1946-20215
Nebulizer 25X magnifier | G1946-80049
Plastic tubing cutter | 8710-1930
Screwdriver, Torx T15 | 8710-1622
Screwdriver, Torx T20 | 8710-1615
Open end wrench, 1/4 and 5/16 in | 8710-0510
Wrench, 1/2 and 7/16 in | 8710-0806
Needle nose pliers, pointed serrated jaws | 8710-0004
3 mm wrench for nebulizer needle adjustment | 8710-2699

*These parts are common to all LC/MS systems

Quiet Cover

Agilent has a solution to the frequent maintenance of LC/MS and ICP-MS rough pumps (visual check of oil levels, oil changes, oil additions, clean-up of oil leaks, etc.), as well as the inherent noise produced by the pumps. Quiet Covers are designed for easy movement, maintenance, and better living with rough pumps used with Agilent and other LC/MS systems.

• Locking castors to move heavy pump for maintenance

• No tools necessary to remove sectioned cover for easy access to pump

• Built in lift-and-tilt lever raises end of pump to drain oil

• Removable drip pan with well and hand holds to collect and transport oil

• Sound absorbing cabinet with resistant foam insulation to reduce pump noise

• Pump mounted to minimize vibration

• 2 Integrated fans maintain temperature inside cover

• LEDs and audible alarm if temperature exceeds 35 °C limit

• Maximum ambient temperature of 35 °C when airflow is neither restricted nor recycled

• Standard one-year warranty; installation and familiarization included with new LC/MS or ICP-MS orders

Please check www.agilent.com/chem/quietcover for the most up-to-date instrument compatibility guide.

Please confirm rough pump used in your Agilent system to ensure compatibility.

Quiet Cover MS, G6011A

Quiet Cover
Description | Compatible Pump Models | Part No.
Quiet Cover | BOC Edwards Pumps, E2M28, E2M18, or E1M18 | G3199B
Quiet Cover MS | Agilent MS40+ | G6011A
Quiet Cover DS | Agilent DS202, DS302, DS402, and DS602 | G6012A
Quiet Cover TS | Agilent TS300, TS300INV, TS600, TS600INV, and TS800 | G6013A
Quiet Cover GC/MS* | Agilent DS42, Pfeiffer Duo 2.5 | G6014A

*This model does not contain all features.

Quiet Cover DS, G6012A

Quiet Cover MS, G6011A

LC/MS Standards Kits

LC/MS Standards Kits
Description | Part No.
Caffeine standards kit for LC/MS OQ/PV | 8500-6917
Caffeine standards kit for LC/MS-Trap OQ/PV | 5065-9908
Sulfa drug standards kit for LC/MS OQ/PV | 5188-6523

LC/MS Application Kit Standards
Description | Part No.
LC/MS pesticide checkout mixture | 5190-0469
LC/MS pesticide comprehensive mixture | 5190-0551
LC/MS toxicology checkout mixture | 5190-0556
LC/MS toxicology comprehensive mixture | 5190-0555
LC/MS vet drug checkout mixture | 5190-0443
LC/MS vet drug comprehensive mixture | 5190-0554

LC/MS Calibrant Mixes
Description | Part No.
Calibrant, ES | G2421-60001
APCI/APPI calibrant solution, 100 mL | G2432A
ESI tuning mix for ion trap, 100 mL | G2431A
ES-TOF tuning mix, 100 mL | G1969-85000
APCI-L low concentration tuning mix, 100 mL | G1969-85010
MMI-L low concentration tuning mix, 100 mL | G1969-85020

CROSSLAB PARTS AND SUPPLIES

Agilent CrossLab offers a growing portfolio of HPLC supplies manufactured for seamless performance with a variety of non-Agilent analytical instruments in your lab. Look inside this selection guide to find a wide range of products for your applications.

We currently support:

• Waters

• Shimadzu

• Dionex*

• CTC Analytics

• And more to come

Our growing portfolio includes the following products:

• Autosampler syringes

• Capillaries, tubing, and fittings

• Detector lamps

• Performance Maintenance kits

• Pump supplies

• Sample loops

• Valve supplies

• Vials and closures

• Well plates and sealing mats

Agilent CrossLab is more than supplies:

• Over 40 years of chromatography expertise

• The right supplies for both routine and challenging applications

• Hassle-free operations and reproducible results

• High-quality products manufactured to Agilent standards

• Technical and application support

• Dependable worldwide availability and delivery

• Convenience of consolidating purchasing

• 90-day risk free money back guarantee

*Dionex is now a part of Thermo Scientific

CrossLab Detector Lamps

Our detector lamps are designed for precise alignment and thermal stability

Agilent CrossLab lamps are designed and built to be compatible with a wide variety of detectors, including Variable Wavelength Detectors (VWD), Multiple Wavelength Detectors (MWD), and Diode Array Detectors (DAD) or Photodiode Array Detectors (PDA). All the lamps adhere to the tightest specifications for consistent quality and reproducible performance over the entire lifetime of the lamps. Test equipment is regularly calibrated using optical standards certified by NIST (National Institute of Standards and Technology) or PTB (Physikalisch-Technische Bundesanstalt).

Long-life deuterium lamp, 8005-0705

• Manufactured in an ISO 9001 certified environment

• Quartz glass bulbs for extended lifetime

• Individually tested for light intensity, noise and drift, correct operating voltage, and proper alignment for low lamp-to-lamp variability

• Tight QA/QC with traceability for each lamp throughout every step of the product process

• Both deuterium lamps and tungsten lamps available

Deuterium lamps

• High output stability and intensity for extended detection capabilities and improved qualification at trace level

• Guaranteed lifetime of 2,000 hours

Tungsten lamps

• Offer coverage in the visible wavelength range for high sensitivity detection

• Average lifetime range from 1,200 to 5,000 hours depending on operation conditions such as operation cycles, soft start, and operation voltage

TIPS & TOOLS Each time after a replacement of a detector lamp, it is recommended to perform a wavelength calibration test and an intensity test after warm up. | [picture]

TIPS & TOOLS An Agilent CrossLab Supplies Selection Tool is available to help you choose the correct supplies for your non-Agilent instrument. For more information, visit www.agilent.com/chem/SelectCrossLab | [picture]

CrossLab Vials and Closures

Agilent CrossLab vials and closures are thoroughly tested to ensure the highest level of quality. Additionally, CrossLab vials are designed for use in a wide range of non-Agilent GCs and LCs, including those from Bruker (formerly Varian), Dionex (now Thermo Scientific), PerkinElmer, Shimadzu, and Thermo Scientific. They are:

• Manufactured in an ISO 9001 certified facility

• Made from First Hydrolytic Type 1 Class A or Class B borosilicate glass, which conforms to US FDA, USP, and EU Pharmacopeia standards

• Protected by proprietary packaging with a crush barrier to reduce vial breakage

• Packaged in material that has been tested and selected for cleanliness

• Subjected to rigorous end-of-line sampling and quality control procedures to ensure all vials remain within specifications

• Compatible with a wide variety of autosamplers regardless of make and model

Snap top vials with write-on spot, 8010-0025

For Agilent CrossLab Vials and Closures ordering information please see the General Chromatography catalog (publication # 5991-1059EN) www.agilent.com/chem/library

[picture] | TIPS & TOOLS Easy, Reliable pH Testing, Designed for Chromatographers Agilent now offers a full line of pH meters and electrodes. Designed for chromatographers, these pH meters offer intuitive user design and exceptional ruggedness for your lab. Learn more at www.agilent.com/chem/phmeters

CrossLab Well Plates and Sealing Mats

Choose the cleanest well plates and sealing mats for your autosamplers

96-well plate, 8010-0534

Advantages of Agilent CrossLab well plates:

• Made from polypropylene

• Free from RNase, DNase, Endotoxins, and human DNA

• Chemically resistant to common solvents

• Autoclavable (121 °C/20 min)

• Alpha-numeric grid

• Compatible with pipetting workstations and multi-channel pipettes

• Raised rims are available with 0.5 mL and 1.0 mL deep well plates for efficient sealing and a reduced risk of cross-contamination during rigorous vortexing

• Available in 96- or 384-well plate format

• 96-well plates range from 0.2 mL to 2 mL

• 384-well plates available in 0.1 mL and 0.25 mL

• Stackable

Advantages of Agilent CrossLab sealing mats:

• Protect well contents during storage

• Free from RNase, DNase, Endotoxins, and human DNA

• Highly flexible for a tight seal and exact fit

• Insets in every well eliminate the need for glue during connection

• Pierceable with pipette tips (0.5 mL and 1.0 mL sealing mats)

• Prevent solvent evaporation

• Made from thermoplastic elastomer (TPE) or ethylene-vinyl acetate (EVA)

• Compatible with common solvents

For Agilent CrossLab Well Plates and Sealing Mats ordering information please see the General Chromatography catalog (publication # 5991-1059EN) www.agilent.com/chem/library

Cleanliness of well plates and sealing mats: Agilent CrossLab vs. the competition

The chemical extraction experiments demonstrate the superior cleanliness of the Agilent CrossLab well plates and sealing mats compared to the competition, making them ideal for LC/MS applications.

LC/MS ESI positive chromatogram comparison. Reagent blank: 1:1 Acetonitrile/H₂O with 0.1% formic acid

CrossLab Autosampler Syringes and Manual Syringes

Agilent’s CrossLab HPLC syringe portfolio contains a broad selection of syringe styles and volumes to provide what you need for accurate and effective sampling. From autosampler syringes in both large or small volume, to syringes for pump priming, CrossLab syringes meet all form, fit, and function criteria to support your HPLC systems.

• Accuracy to within ±1% of nominal volume with a precision of 1% at 80% of the total volume

• A chemically inert fluid path of stainless steel, borosilicate Type I glass, or PTFE

• Robust design for long lifetime

Agilent CrossLab Syringe Features

Syringe terminations at the end of the syringe barrel function as the interface between the syringe and its mating connection such as the needle. Terminations are offered in a number of different needle and connection configurations to accommodate a broad range of applications.

Fixed Needle

• Economical option for injections

• Preferred for applications requiring trace level samples

• Needle is cemented into the glass syringe barrel at a point corresponding to the zero graduation mark

• Dead volume is limited to the needle’s internal volume

• Not autoclavable

• Recommended for use where probability of needle bending is minimal

Removable Needle

• Versatile option for injections

• Needle seats precisely at the zero graduation mark

• Needle can be replaced if damaged or clogged

• Allows for a removable needle without increasing dead volume

• Ideal for when there is a risk of needle clogging

• Autoclavable when disassembled (repeated autoclaving shortens syringe life)

PTFE Luer Lock

• PTFE, male Luer taper with nickel-plated brass locking hub for use with Kel-F needles or metal hub needles, and universal connectors

• Autoclavable when disassembled, except on 25 mL or greater syringes (repeated autoclaving shortens syringe life)

Chem

• ¼-28 UNF male fitting thread connection

• Used for low-volume applications where system dead volume must be minimized

• Can be screwed directly into injection valves

Note: Most manual HPLC injection valves are designed to be used with a 22-gauge, or a 22s-gauge blunt needle point style 3.

[picture] | TIPS & TOOLS Replace syringe if dirt is noticeable, syringes cannot be cleaned, or plunger does not slide easily.

[picture] | TIPS & TOOLS Follow manufacturer’s recommendation for syringe cleaning and regularly inspect syringe barrel for sample build-up and needle tip for wear.

Autosampler Routine Maintenance

Routine autosampler maintenance should be performed on a regular basis to keep your HPLC system performing at its optimum level. You can perform all maintenance procedures at once or as needed. Some parts may need to be replaced more often than others depending upon your application and solvent preparation procedures.

Regular autosampler maintenance helps lower operating costs and generate precise results with the utmost confidence. By following a regular maintenance routine, you can count on maximum uptime during the life of your autosampler.

Waters Autosampler Routine Maintenance Procedures

• Replace the metering syringe

• Rebuild the injector seal pack and replace the needle

• Rebuild the high-pressure motorized valves

• Replace the in-line filter insert

• Adjust the seal pack seal valves

Waters Autosampler Routine Maintenance and Troubleshooting
Symptom | Cause | Solution
Poor injection reproducibility | Low sample level in vials | Confirm sample level in the vials is at minimum a quarter full
| Worn metering syringe | Replace the metering syringe assembly
| Worn high-pressure motorized valve seals | Rebuild the high-pressure motorized valves
| Leaking waste valve | Replace the waste valve
| Worn injector seals | Rebuild the injector seal pack, replace the needle, and adjust the seal pack seal valves
Leaking syringe | Worn metering syringe | Replace the metering syringe assembly
Sample carryover | Needlewash solvent exhausted | Refill the needlewash solvent reservoir
| Dirty needlewash seals | Rebuild the seal pack and adjust seal pack seal valves
| Faulty needlewash valve | Replace the needlewash valve
Sample vials filling during injection cycle | Worn high-pressure motorized valve seal | Rebuild the high-pressure motorized valves
Low peak response | Low sample level in vials | Confirm sample level in the vials is at minimum a quarter full
| Worn metering syringe | Replace the metering syringe assembly
| Worn high-pressure motorized valve seals | Rebuild the high-pressure motorized valves
| Leaking waste valve | Replace the waste valve
| Worn injector seals | Rebuild the injector seal pack, replace the needle, and adjust the seal pack seal valves

Shimadzu Autosampler Routine Maintenance Procedures

• Replace the sample metering drive plunger seal

• Replace the sample needle

• Replace the needle seat seal

• If equipped, replace the rinse port septum

• If equipped, clean the high-pressure valve and replace the high-pressure valve rotor seal

• If equipped, clean the low-pressure valve and replace the low-pressure valve rotor seal

• If equipped, clean the injection valve and replace the injection valve rotor seal

• Adjust the needle if necessary

Shimadzu Autosampler Routine Maintenance and Troubleshooting
Symptom | Cause | Solution
Poor injection reproducibility | Low sample level in vials | Confirm sample level in the vials is at minimum a quarter full
| Worn metering seal | Replace the metering plunger seal
| Damaged metering plunger | Replace the metering plunger
| Worn high-pressure valve seals | Rebuild the high-pressure valve
| Worn low-pressure valve seals | Rebuild the low-pressure valve
| Leaking waste valve | Replace the waste valve
| Damaged sample needle | Replace the sample needle and needle seat seal
Sample carryover | Needlewash solvent exhausted | Refill the needlewash solvent reservoir
| Dirty needlewash seals | Rebuild the seal pack and adjust seal pack seal values
| Contaminated rinse port | Clean and flush the rinse port
Low peak response | Low sample level in vials | Confirm sample level in the vials is at minimum a quarter full
| Worn metering plunger seal | Replace the metering plunger seal
| Damaged metering plunger | Replace the metering plunger
| Worn high-pressure valve seals | Rebuild the high-pressure valve
| Leaking waste valve | Replace the waste valve
| Damaged sample needle | Replace the sample needle and needle seat seal

Dionex* Autosampler Routine Maintenance Procedures

• Replace the sample metering syringe

• Replace the sample needle

• Replace the needle seat seal

• If equipped, clean the injection valve and replace the injection valve rotor seal

• Replace the buffer tubing

• Replace the capillary tubing

• Fill the syringe reservoir

• Adjust the needle if necessary

*Dionex is now a part of Thermo Scientific

Dionex Autosampler Routine Maintenance and Troubleshooting
Symptom | Cause | Solution
Poor injection reproducibility | Low sample level in vials | Confirm sample level in the vials is at minimum a quarter full
| Worn metering syringe seal | Replace the metering syringe
| Worn syringe valve | Replace the syringe valve
| Worn injection valve seal | Rebuild the injection valve
| Leaking waste valve | Replace the waste valve
| Leaking fittings | Check all the fittings for leaks
| Damaged sample needle | Replace the sample needle and needle seat seal
Sample carryover | Needlewash solvent exhausted | Refill the needlewash solvent reservoir
| Dirty needlewash seals | Rebuild the seal pack and adjust seal pack seal valves
| Contaminated rinse port | Clean and flush the rinse port
Leaking syringe | Damaged syringe | Replace the syringe
| Worn syringe valve | Check for leaks and replace if necessary
Low peak response | Low sample level in vials | Confirm sample level in the vials is at minimum a quarter full
| Worn syringe valve | Replace the metering syringe
| Damaged syringe valve | Check the syringe valve for leaks and replace if necessary
| Worn injector valve seal | Rebuild the injection valve
| Leaking waste valve | Replace the waste valve
| Leaking fittings | Check all the fittings for leaks
| Damaged sample needle | Replace the sample needle and needle seat seal

CrossLab Pump Supplies

Proper pump maintenance helps ensure precise, consistent results and lower operating costs

Sapphire plunger assembly, 8005-0538

Regular pump maintenance helps lower operating costs and ensure precise and consistent results. By following a regular maintenance routine, you can count on maximum uptime, steady and accurate solvent flow, pressure stability throughout the life of a pump, and keep your HPLC system in its optimum condition.

Plunger seal replacement kit, 8005-0541

CrossLab Plungers and Seals

Plungers (or pistons) and seals are two of the most important components of a pump. All Agilent CrossLab plungers combined with seals undergo extensive testing under temperature stress with common HPLC solvents for reproducible results.

Face seals replacement kit, 8005-0536

Plungers:

• Available in sapphire or ceramic

• Sapphire plungers are made from high purity monocrystalline sapphire for optimal concentricity and resistance to wear

• Are meticulously cut and polished for great durability and long life

Seals:

• Manufactured from ultra-high-molecular-weight polyethylene (UHMWPE) or graphite-filled polytetrafluoroethylene (GFP)

• Designed to provide precise sealing around the plungers

• Engineered to deliver top performance over highly dynamic flow and pressure ranges

[picture] | TIPS & TOOLS Plungers (or pistons) should be exchanged on a regular basis in combination with seals to maintain the best sealing surface and durability for optimal instrument performance.

[picture] | TIPS & TOOLS Depending on your applications and solvent preparation protocols, some parts may need to be replaced more often than the others.

CrossLab Check Valves

Check valves are key components for a pump, and their lifetime is often related to the solvents used. A defective valve can lead to pressure fluctuation, inconsistent flow, and a noisy baseline.

Agilent CrossLab check valves and assembly components:

• Made to operate at different pressures (up to 16,000 psi, or 1,100 bar) per specifications from the Original Equipment Manufacturers (OEMs)

• Made from a variety of materials, such as stainless steel, titanium, ceramic, and PEEK, depending on OEM specifications

• Carefully assembled to ensure reliability and consistent performance

• Each ruby or ceramic ball and sapphire or ceramic seat is manufactured under extremely tight tolerances to ensure proper sealing within the operating pressure range

TIPS & TOOLS Don’t forget to check your check valve regularly and replace check valve cartridges to ensure proper pump operations. | [picture]

HPLC Pump Routine Maintenance

Routine pump maintenance should be performed on a regular basis to keep your HPLC system performing at its optimum level. You can perform all maintenance procedures at once or as needed. Some parts may need to be replaced more often than others depending upon your application and solvent preparation procedures.

Regular pump maintenance helps lower operating costs and generate precise results with the utmost confidence. By following a regular maintenance routine, you can count on maximum uptime and a steady, accurate solvent flow for the life of the pump.

[picture] | TIPS & TOOLS Pump seals should be replaced when there are leaks on the bottom of the pump head, when retention times are inconsistent, or when the pressure ripple is unstable.

Sapphire plunger assembly, 8005-0523

Waters Pump Routine Maintenance Procedures

• Replace the seals and plungers (or pistons)

• Replace the in-line filter insert

• Replace the check valve cartridges

• Replace the seal wash seals and tube seals

• Replace the solvent inlet frits

Waters Pump Routine Maintenance Procedures
Symptom | Cause | Solution
Pressure ripple unstable | Dirty check valve cartridge(s) | Run static leak test to verify and exchange the check valve cartridge(s)
| Leak on pump head | Run static leak test to verify and exchange the in-line filter insert and plunger (or piston) seals
Gradient performance problems Intermittent pressure fluctuations | Blocked solvent filter(s) | Change the solvent filter(s)
A pressure drop of greater than 10 bar (150 psi) across the in-line filter (5 mL/min H₂O during wet priming) | Dirty in-line filter | Exchange the in-line filter insert
Leaks at lower pump head side Unstable retention time Pressure ripple unstable | High seal wear | Run leak test to verify and exchange the pump seals and in-line filter insert
Seal lifetime shorter than normally expected | Scratch on plunger | Check plungers while changing the seals
|  | Exchange the plungers if damaged or scratched
Loss of wash solvent | Leaky wash seals | Exchange the wash seals

Shimadzu Pump Routine Maintenance Procedures

• Replace the seals and plungers (or pistons)

• Replace the in-line filter cartridge

• Replace the check valve cartridges

• Replace the seal wash seals

• Replace the solvent inlet frits

Plunger seal, 8001-0502

Shimadzu Pump Routine Maintenance and Troubleshooting
Symptom | Cause | Solution
Pressure ripple unstable | Dirty check valve cartridge(s) | Run Pump Pressure test to verify and exchange the check valve cartridge(s)
| Leak on pump head | Run Pump Pressure test to verify and exchange the in-line filter insert and plunger (or piston) seal
|  | Check inlet and outlet fittings for leaks
|  | Tighten all fittings and re-run the Pump Pressure test
Gradient performance problems Intermittent pressure fluctuations | Solvent filter(s) is blocked | Change the solvent filter(s)
A pressure drop of greater than 10 bar (150 psi) across the in-line filter (5 mL/min H₂O during wet priming) | Dirty in-line filter | Exchange the in-line filter insert
Leaks at lower pump head side Unstable retention time Pressure ripple unstable Broad peaks or peak tailing | High seal wear | Run Pump Pressure test to verify and exchange the pump seals and in-line filter cartridge
Seal lifetime shorter than normally expected | Scratch on plunger | Check plungers while changing the seals
|  | Exchange the plungers if damaged or scratched
Loss of wash solvent | Leaky wash seals | Exchange the wash seals

Dionex* Pump Routine Maintenance Procedures

• Replace the purge valve cap seal

• Replace the seals, seal rings, and plungers (or pistons)

• Replace the in-line filter cartridge

• Replace the check valve cartridges

• Replace the seal wash seals and seal wash tubing

• Replace the mixing chamber gasket

• Replace the solvent inlet filter frits

*Dionex is now a part of Thermo Scientific

Vespel rotor seal, 8002-0602

Dionex Pump Routine Maintenance and Troubleshooting
Symptom | Cause | Solution
Pressure ripple unstable | Dirty check valve cartridge(s) | Run Pump Pressure test to verify and exchange the check valve cartridge(s)
| Leak on pump head | Run Pump Pressure test to verify and exchange the in-line filter insert and plunger (or piston) seal
|  | Check inlet and outlet fittings for leaks
|  | Tighten all fittings and re-run the Pump Pressure test
Gradient performance problems, Intermittent pressure fluctuations | Solvent filter(s) is blocked | Change the solvent filter(s)
A pressure drop of greater than 10 bar (150 psi) across the in-line filter (5 mL/min H₂O during wet priming) | Dirty in-line filter | Exchange the in-line filter insert
Leaks at lower pump head side Unstable retention time Pressure ripple unstable Broad peaks or peak tailing | High seal wear | Run Pump Pressure test to verify and exchange the pump seals and in-line filter cartridge
Seal lifetime shorter than normally expected | Scratch on plunger | Check plungers while changing the seals
|  | Exchange the plungers if damaged or scratched
Loss of wash solvent | Leaky wash seals | Exchange the wash seals and seal wash tubing

CrossLab Valve Supplies

Agilent CrossLab valve supplies work seamlessly with your HPLC systems for reliable, accurate, and reproducible results.

Rotor seals:

A variety of materials for a wide range of applications

A rotor seal is a polymeric disk that creates a high-pressure seal against the stator or stator face seal. A variety of materials are available for different applications. It is important to replace rotor seals on a routine basis to prevent excessive wear. The recommended replacement interval for most rotor seals is once per year for preventive maintenance or as needed depending on stresses imposed by specific applications.

• Vespel: Vespel is a polyimide with low wear and high chemical resistance. Recommended for use with solutions under pH 10; because more basic solutions dissolve Vespel over time and damage the rotor seal

• PEEK (Polyetheretherketone): PEEK offers high resistance, versatility, and applicability for the entire pH range between 0 and 14

• Tefzel: Recommended for use in applications where PEEK cannot be used, such as higher concentrations of methylene chloride or dimethyl sulfoxide (DMSO)

Rotor, 6-port valve, 8001-0601

TIPS & TOOLS For any solution above pH 10, a PEEK rotor seal is recommended. | [picture]

TIPS & TOOLS PEEK rotor seals are incompatible with concentrated nitric and sulfuric acids. | [picture]

Stators

Stator, 6-port valve, 8001-0604

• Available in 316 stainless steel, PEEK, and ceramic depending on the specifications of OEMs

• Typically require replacement only if the ports or sealing surfaces become damaged, which can be prevented by using proper injection needles.

Sample Loops

Stainless Steel Sample Loops:

• Have burr-free and square-cut ends to ensure a flush connection to valve ports

• Actual volumes might differ due to tolerance of metal tubing bore

• Accuracy of large metal loops (1.0 mm, 0.040 in bore) is ± 14%, intermediate loops (0.5 mm, 0.020 in bore) is ± 21%, and small loops (0.2 mm, 0.007 in bore) is ± 65%

PEEK Sample Loops

• Alternatives to stainless steel sample loops

• Clean, straight cuts for easy valve installation and low dead volume connections

• Inert to almost all organic solvents and biocompatible

• Actual volumes might differ due to tolerance of metal tubing bore

• Accuracy of large PEEK loops (0.8 mm, 0.030 in bore) is ± 5%, intermediate loops (0.5 mm, 0.020 in bore) is ± 10%, and small loops (0.2 mm, 0.007 in bore) is ± 30%

• Wall thickness, temperature, concentration of organic solvent, and solvent exposure time affect the durability of PEEK tubing

[picture] | TIPS & TOOLS Concentrated nitric acid and sulfuric acid weaken PEEK tubing while tetrahydrofuran (THF), methylene chloride, and DMSO cause swelling.

TIPS & TOOLS

Since both standards and unknowns are usually analyzed using the same sample loops, knowledge of the actual, accurate volume is rarely needed. If the sample loop volume must be known, it is best to calibrate the loop in place on the valve so the flow passages in the valve are taken into account.

CrossLab Performance Maintenance (PM) Kits

PM kits make it easier to keep your instruments running at peak performance

Many instrument failures are caused not by functional breakdowns, but by a lack of performance maintenance. As suggested by a recent study, a regular performance maintenance program can reduce instrument failure rates up to 25%, and is critical to ensure optimal overall system operations.

515 pump PM kit, 8005-0913

2960/2965 PM kit, 8005-0915

The Effect of Performance Maintenance

Agilent CrossLab PM kits contain the normal wear parts, tools for disassembly/reassembly, and instructions necessary to keep your systems operating at peak performance. The kits provide a convenient, cost-effective way to make sure your instruments are properly maintained. Each kit contains all the parts you need to:

• Boost system efficiency and reliability

• Reduce unplanned downtime and repair costs

• Increase accuracy by enhancing precision and sensitivity

• Comply with regulatory requirements, or meet quality accreditation standards

• Extend instrument useful life

• Eliminate individual part ordering

• PM kits available for pumps, valves, autosamplers, and HPLC systems

General HPLC Supplies

CrossLab LC Capillaries and Tubing

Your link to analytical success

Agilent CrossLab offers a range of capillaries and tubing made from stainless steel and PEEK. Used in combination with the right fittings, they are ideal for LC system plumbing, providing an inert surface, tight, leak-free connections, and zero dead volumes.

Stainless steel capillaries

• Made of ultra-clean stainless steel tubing

• Ideal for most standard applications, and the best choice for reliable high-pressure sealing

• Smooth inner surface for the lowest backpressure

• Precision cutting for burr-free, square-cut ends without inner-diameter distortion

• Both ends accept standard fittings

• Predefined lengths for specific path locations to avoid peak broadening

• Individually tested to ensure reproducibility

• Available in pre-swaged and non pre-swaged

[picture] | TIPS & TOOLS Always use the smallest capillary internal diameter and the shortest length to keep sample dispersion as low as possible.

PEEK tubing

• Flexible and easy to cut to desired lengths

• Ideal for frequently changed connections

• Resists mechanical and solvent damage, even at high temperatures

• Biocompatible and inert to almost all organic solvents

• A durable alternative to stainless steel connections

• Accepts both stainless steel and PEEK fittings

CrossLab Fittings

The right LC fittings make the best LC connections

To ensure leak-free connections, and to prevent the loss of peak shape and resolution, always use the recommended fitting style for columns, valves and unions.

With Agilent CrossLab fittings, you get the confidence and choice that come with:

• Robust design for long life and smooth, leak-free operation

• Stainless steel nuts and ferrules for high-pressure systems

• Polymer fittings for biocompatible applications

CrossLab HPLC In-Line Filters

Help stop time-wasting blockages

Column inlet frit contamination can increase backpressure and reduce efficiency. Microbore column blockages are of particular concern, due to the frit’s small diameter.

To help prevent time-wasting contamination and blockages, the Agilent CrossLab portfolio offers two types of LC filters made from clean, high-quality stainless steel parts:

• In-line solvent filters, installed between the LC pump and injector, prevent blockages by removing particles from solvents before they reach the injector

• Column inlet filters, positioned immediately before an LC column, remove particles from both the injection system and sample

Troubleshooting Guide for HPLC Systems

The at-a-glance tables can help you pinpoint and solve the most common problems for your HPLC systems and ensure maximum instrument uptime and productivity.

HPLC Troubleshooting
Symptom Type | Possible Cause | Solution
Baseline disturbance at void time | Positive/negative – Difference in refractive index of injection solvent | Use mobile phase for sample solvent
Detector leaks | Plugged inlet frit | Replace seals/gaskets
Drifting baseline | Positive direction – Contaminant buildup/elution | Flush column, clean up sample, use pure solvents
| Positive/negative – Difference in refractive index of injection solvent | Use mobile phase for sample solvent
| Negative direction (gradient) – Absorbance of “A” mobile phase solvent | Use non-absorbing or HPLC-grade or better solvent
| Positive direction (gradient) – Absorbance of “B” mobile phase solvent | Use non-absorbing or HPLC-grade or better solvent
| Random – Temperature changes | Insulate column and tubing
| Random – Temperature changes | Thermostat column and tubing
| Wavy or undulating – Temperature changes in room | Monitor room temperature and control
Ghost peaks | Peaks from previous injection | Flush column to remove contaminants
| Contamination | Sample cleanup or pre-fractionation
| Unknown interferences in samples | Sample cleanup or pre-fractionation
| Ion-pair – Upset equilibrium | Prepare sample in actual mobile phase to minimize disturbance
| Peptide mapping – Oxidation of TFA | Prepare fresh daily; use anti-oxidant
| Reversed-phase – Contaminated water | Check suitability of water by running different amount through reversed-phase column and measure peak height with elution; use HPLC grade solvents
| Spikes – Bubbles in solvent | De-gas solvents
High column backpressure | Column blockage, adsorbed sample | Better sample cleanup; use guard column
| Mobile phase viscosity too high | Use lower viscosity solvents or higher temperature
| Particle size too small | Use larger dp packing
| Plugged inlet frit | Replace column
| Plugged inlet frit | Reverse solvent flow
Leaks | Subtle – White powder at fitting/loose fitting | Tighten fittings, cut tubing, or replace ferrules
Leaks, injection valve | Catastrophic – Worn valve rotor | Replace rotor in valve
Leaks, column or other fittings | Catastrophic – Loose fittings | Tighten or replace fittings
Leak, pump | Catastrophic – Pump seal failure | Replace pump seal
Negative peaks | RI detector – solute refractive index less than solvent | No problem; reverse polarity to make positive
| UV detector – solute absorbance less than mobile phase | Use mobile phase with lower UV absorbance; do not recycle solvent too long
Noisy baseline | Random – Contaminant buildup | Flush column; clean up sample; use HPLC-grade solvent
| Continuous – Detector lamp problem | Replace detector lamp
| Occasional – External electrical interference | Use voltage stabilizer for LC system
Peak doubling | Sample volume too large | Reduce the volume e.g. by half and re-inject
| Injection solvent too strong | Use weaker injection solvent or mobile phase
| Blocked frit | Replace and use 0.5 μm porosity in-line filter
| Column void or channeling | Replace column; for some columns, fill in void with packing
| Unswept injector flowpath | Replace injector rotor
| Void at head of column | Replace column, top off column with packing
| Column overloaded with sample | Use higher capacity stationary phase Increase column diameter Decrease sample size
| Single peak – interfering components | Sample cleanup; pre-fractionation
Peak tailing | Beginning of peak doubling | See “peak doubling”
| Unswept dead volumes | Minimize number of connections Ensure injector seal is tight Ensure fittings are properly seated
| Basic compounds – Silanol interactions | Choose endcapped bonded phase Switch to polymeric phase
| Basic substances – Silanol interactions | Use stronger mobile phase or add competing base (e.g. TMA)
| Silica-based – Column degradation | Use specialty column; polymeric column or sterically protected
Peaks are broad | Injection volume too large | Decrease solvent strength of injection solvent to focus solute
| Peak dispersion in injector valve | Introduce air bubble in front/back of sample to decrease dispersion
| Sampling rate of data system too slow | Increase frequency of sampling
| Slow detector time constant | Adjust time constant to match peak width
| Mobile phase viscosity too high | Increase column temperature
| Detector cell volume too large | Use smallest possible cell volume with no heat exchanger in system
| Injector volume too large | Decrease injection volume
| Long retention times | Use gradient elution or stronger mobile phase
Pressure fluctuation | Leaky check valve | Replace check valve
| Pump seal leaks | Replace pump seals
| Buildup of particulates | Filter sample; in-line filter; filter mobile phase
Pressure increasing | Buildup of particulates | Filter sample; in-line filter; filter mobile phase
| Water/organic systems – buffer precipitation | Test buffer-organic mixtures; ensure compatibility
Retention beyond total permeation volume | Size exclusion – Specific interactions | Add mobile phase modifiers or change solvent
Retention times changing | Column temperature varying | Thermostat column; insulate column; ensure lab temperature constant
| Equilibration time insufficient with gradient run or changes in isocratic mobile phase | Make sure at least 10 column volumes pass through column after solvent change or gradient conclusion
| Selective evaporation of mobile phase component | Less vigorous helium sparging; keep solvent reservoirs covered; prepare fresh mobile phase
| Buffer capacity insufficient | Use >20 mM concentration of buffer
| Inconsistent on-line mobile phase mixing | Ensure gradient system delivering constant composition; check vs. manual prep of mobile phase
| Contamination buildup | Occasionally flush column with strong solvent to remove contaminants
| First few injections – Adsorption on active sites | Condition column by initial injection of concentrated sample
Retention times decreasing | Flow rate increasing | Check pump to make sure correct; if not, reset
| Column overloaded with sample | Decrease sample size
| Loss of bonded stationary phase | Keep mobile phase pH between 2 and 8.5
Retention times increasing | Flow rate is slowing | Fix leaks in liquid lines, replace pump seals, check for pump cavitation or air bubbles
| Active sites on silica packing | Use mobile phase modifier
| Loss of bonded stationary phase | Keep mobile phase pH between 2 and 8.5
| Mobile phase composition changing | Make sure mobile phase container is covered
| Active sites on silica packing | Add competing base to mobile phase
| Active sites on silica packing | Use higher coverage packing for stationary phase
Sensitivity problem | Peaks are outside of linear range of detector | Dilute/concentrate to bring into linear region
| First few sample injections – Absorption of sample in loop or column | Condition loop/column with concentrated sample
| Autosampler flow lines blocked | Check flow and make sure there are no blockages
| Injector sample loop underfilled | Make sure that loop is overfilled with sample
| Sample-related losses during preparation | Use internal standard during sample prep; optimize sample prep method
Slow column equilibration times (ion-pairing) | Equilibration time slow for long-chain ion-pairing reagents | Use shorter alkyl chain ion-pair reagent

CrossLab Supplies for Waters HPLC Systems

The cross references to the original equipment manufacturer (OEM) part numbers listed here serve as a recommendation that the Agilent CrossLab products are viable alternatives to OEM products. CrossLab products are compatible with the corresponding OEM instruments, although in some cases, the CrossLab products may have slightly different designs as compared to the OEM counterparts. All CrossLab supplies are backed by Agilent’s 90-day money-back warranty.

Long-life deuterium lamp, 8005-0705

Long-life deuterium lamp, 8005-0704

Long-life deuterium lamp, 8005-0702

Autosampler syringe, gas tight, 8005-0417

The cross references to the original equipment manufacturer (OEM) part numbers listed here serve as a recommendation that the Agilent CrossLab products are viable alternatives to OEM products. CrossLab products are compatible with the corresponding OEM instruments, although in some cases, the CrossLab products may have slightly different designs as compared to the OEM counterparts. All CrossLab supplies are backed by Agilent’s 90-day money-back warranty.

Plunger seal replacement kit, 8005-0535

Plunger seal replacement kit, 8005-0541

Face seals replacement kit, 8005-0536

Seal wash face seal kit, 8005-0539

The cross references to the original equipment manufacturer (OEM) part numbers listed here serve as a recommendation that the Agilent CrossLab products are viable alternatives to OEM products. CrossLab products are compatible with the corresponding OEM instruments, although in some cases, the CrossLab products may have slightly different designs as compared to the OEM counterparts. All CrossLab supplies are backed by Agilent’s 90-day money-back warranty.

The cross references to the original equipment manufacturer (OEM) part numbers listed here serve as a recommendation that the Agilent CrossLab products are viable alternatives to OEM products. CrossLab products are compatible with the corresponding OEM instruments, although in some cases, the CrossLab products may have slightly different designs as compared to the OEM counterparts. All CrossLab supplies are backed by Agilent’s 90-day money-back warranty.

The cross references to the original equipment manufacturer (OEM) part numbers listed here serve as a recommendation that the Agilent CrossLab products are viable alternatives to OEM products. CrossLab products are compatible with the corresponding OEM instruments, although in some cases, the CrossLab products may have slightly different designs as compared to the OEM counterparts. All CrossLab supplies are backed by Agilent’s 90-day money-back warranty.

2960/2965 PM kit, 8005-0915

515 pump PM kit, 8005-0913

Rheodyne 7725(i) PM kit, 8005-0904

Rheodyne 9125 PM kit, 8005-0906

The cross references to the original equipment manufacturer (OEM) part numbers listed here serve as a recommendation that the Agilent CrossLab products are viable alternatives to OEM products. CrossLab products are compatible with the corresponding OEM instruments, although in some cases, the CrossLab products may have slightly different designs as compared to the OEM counterparts. All CrossLab supplies are backed by Agilent’s 90-day money-back warranty.

The cross references to the original equipment manufacturer (OEM) part numbers listed here serve as a recommendation that the Agilent CrossLab products are viable alternatives to OEM products. CrossLab products are compatible with the corresponding OEM instruments, although in some cases, the CrossLab products may have slightly different designs as compared to the OEM counterparts. All CrossLab supplies are backed by Agilent’s 90-day money-back warranty.

Please visit www.agilent.com/chem/CrossLabHPLC for up-to-date info for the growing portfolio of CrossLab LC Supplies for your non-Agilent HPLC systems.

For Agilent CrossLab Vials and Closures ordering information please see the General Chromatography catalog (publication # 5991-1059EN) www.agilent.com/chem/library

For Agilent CrossLab Well Plates and Sealing Mats ordering information please see the General Chromatography catalog (publication # 5991-1059EN) www.agilent.com/chem/library

CrossLab Supplies for Shimadzu HPLC Systems

Long-life deuterium lamp, 8001-0701

Long-life deuterium lamp, 8001-0702

Long-life deuterium lamp, 8001-0704

The cross references to the original equipment manufacturer (OEM) part numbers listed here serve as a recommendation that the Agilent CrossLab products are viable alternatives to OEM products. CrossLab products are compatible with the corresponding OEM instruments, although in some cases, the CrossLab products may have slightly different designs as compared to the OEM counterparts. All CrossLab supplies are backed by Agilent’s 90-day money-back warranty.

Autosampler syringe, 8001-0401

Replacement syringe plunger, 500 μL, 8001-0403

The cross references to the original equipment manufacturer (OEM) part numbers listed here serve as a recommendation that the Agilent CrossLab products are viable alternatives to OEM products. CrossLab products are compatible with the corresponding OEM instruments, although in some cases, the CrossLab products may have slightly different designs as compared to the OEM counterparts. All CrossLab supplies are backed by Agilent’s 90-day money-back warranty.

Plunger seal, 8001-0502

Looking for Performance Maintenance Kits for Rheodyne Valves? Please see here.

Rotor, 6-port valve, 8001-0601

Stator, 6-port valve, 8001-0604

PEEK rotor, low pressure valve (LPV), 8001-0608

The cross references to the original equipment manufacturer (OEM) part numbers listed here serve as a recommendation that the Agilent CrossLab products are viable alternatives to OEM products. CrossLab products are compatible with the corresponding OEM instruments, although in some cases, the CrossLab products may have slightly different designs as compared to the OEM counterparts. All CrossLab supplies are backed by Agilent’s 90-day money-back warranty.

The cross references to the original equipment manufacturer (OEM) part numbers listed here serve as a recommendation that the Agilent CrossLab products are viable alternatives to OEM products. CrossLab products are compatible with the corresponding OEM instruments, although in some cases, the CrossLab products may have slightly different designs as compared to the OEM counterparts. All CrossLab supplies are backed by Agilent’s 90-day money-back warranty.

The cross references to the original equipment manufacturer (OEM) part numbers listed here serve as a recommendation that the Agilent CrossLab products are viable alternatives to OEM products. CrossLab products are compatible with the corresponding OEM instruments, although in some cases, the CrossLab products may have slightly different designs as compared to the OEM counterparts. All CrossLab supplies are backed by Agilent’s 90-day money-back warranty.

The cross references to the original equipment manufacturer (OEM) part numbers listed here serve as a recommendation that the Agilent CrossLab products are viable alternatives to OEM products. CrossLab products are compatible with the corresponding OEM instruments, although in some cases, the CrossLab products may have slightly different designs as compared to the OEM counterparts. All CrossLab supplies are backed by Agilent’s 90-day money-back warranty.

Please visit www.agilent.com/chem/CrossLabHPLC for up-to-date info for the growing portfolio of CrossLab LC Supplies for your non-Agilent HPLC systems.

For Agilent CrossLab Vials and Closures ordering information please see the General Chromatography catalog (publication # 5991-1059EN) www.agilent.com/chem/library

For Agilent CrossLab Well Plates and Sealing Mats ordering information please see the General Chromatography catalog (publication # 5991-1059EN) www.agilent.com/chem/library

CrossLab Supplies for Dionex HPLC Systems

Long-life deuterium lamp, 8002-0701

The cross references to the original equipment manufacturer (OEM) part numbers listed here serve as a recommendation that the Agilent CrossLab products are viable alternatives to OEM products. CrossLab products are compatible with the corresponding OEM instruments, although in some cases, the CrossLab products may have slightly different designs as compared to the OEM counterparts. All CrossLab supplies are backed by Agilent’s 90-day money-back warranty.

Chem (1/4-28 UNF screw threads), gas tight, 8002-0405

Removable needle, gas tight, 8002-0401

The cross references to the original equipment manufacturer (OEM) part numbers listed here serve as a recommendation that the Agilent CrossLab products are viable alternatives to OEM products. CrossLab products are compatible with the corresponding OEM instruments, although in some cases, the CrossLab products may have slightly different designs as compared to the OEM counterparts. All CrossLab supplies are backed by Agilent’s 90-day money-back warranty.

The cross references to the original equipment manufacturer (OEM) part numbers listed here serve as a recommendation that the Agilent CrossLab products are viable alternatives to OEM products. CrossLab products are compatible with the corresponding OEM instruments, although in some cases, the CrossLab products may have slightly different designs as compared to the OEM counterparts. All CrossLab supplies are backed by Agilent’s 90-day money-back warranty.

Vespel rotor seal, 8002-0602

Stainless steel stator, 8002-0603

The cross references to the original equipment manufacturer (OEM) part numbers listed here serve as a recommendation that the Agilent CrossLab products are viable alternatives to OEM products. CrossLab products are compatible with the corresponding OEM instruments, although in some cases, the CrossLab products may have slightly different designs as compared to the OEM counterparts. All CrossLab supplies are backed by Agilent’s 90-day money-back warranty.

Looking for Performance Maintenance Kits for Rheodyne Valves? Please see here.

The cross references to the original equipment manufacturer (OEM) part numbers listed here serve as a recommendation that the Agilent CrossLab products are viable alternatives to OEM products. CrossLab products are compatible with the corresponding OEM instruments, although in some cases, the CrossLab products may have slightly different designs as compared to the OEM counterparts. All CrossLab supplies are backed by Agilent’s 90-day money-back warranty.

The cross references to the original equipment manufacturer (OEM) part numbers listed here serve as a recommendation that the Agilent CrossLab products are viable alternatives to OEM products. CrossLab products are compatible with the corresponding OEM instruments, although in some cases, the CrossLab products may have slightly different designs as compared to the OEM counterparts. All CrossLab supplies are backed by Agilent’s 90-day money-back warranty.

The cross references to the original equipment manufacturer (OEM) part numbers listed here serve as a recommendation that the Agilent CrossLab products are viable alternatives to OEM products. CrossLab products are compatible with the corresponding OEM instruments, although in some cases, the CrossLab products may have slightly different designs as compared to the OEM counterparts. All CrossLab supplies are backed by Agilent’s 90-day money-back warranty.

Please visit www.agilent.com/chem/CrossLabHPLC for up-to-date info for the growing portfolio of CrossLab LC Supplies for your non-Agilent HPLC systems.

For Agilent CrossLab Vials and Closures ordering information please see the General Chromatography catalog (publication # 5991-1059EN) www.agilent.com/chem/library

For Agilent CrossLab Well Plates and Sealing Mats ordering information please see the General Chromatography catalog (publication # 5991-1059EN) www.agilent.com/chem/library

CrossLab Supplies for CTC Analytics HPLC Autosamplers

Agilent’s CrossLab portfolio includes a variety of syringes compatible with CTC Analytics HPLC HTS and HTC PAL systems.

The cross references to the original equipment manufacturer (OEM) part numbers listed here serve as a recommendation that the Agilent CrossLab products are viable alternatives to OEM products. CrossLab products are compatible with the corresponding OEM instruments, although in some cases, the CrossLab products may have slightly different designs as compared to the OEM counterparts. All CrossLab supplies are backed by Agilent’s 90-day money-back warranty.

CE and CE/MS

CE Solutions Kits

Agilent continues to introduce new CE solutions kits designed to simplify many of your applications:

• Inorganic anions

• Cations

• Organic acids

• Forensic anions

• μPage

These kits include all you need to begin your CE analyses, including buffers, capillaries, conditioning solutions, test samples, methods and detailed descriptions. Each kit is designed to take advantage of the automation of the Agilent CE system to make your time in the laboratory more efficient. All kits are prepared using the same quality procedures as our buffers and are thoroughly tested and supported.

While the kits have been optimized for use with the Agilent CE system, they may be used with virtually any commercial or home-built CE system.

Inorganic Anion Solutions Kit

The Inorganic Anion Solutions Kit contains all components needed for the analysis of common inorganic anions such as chloride, bromide, iodide, fluoride, sulfate, and phosphate. Applications include the analysis of inorganic ions in:

• Ultra pure water

• Waste water

• High purity chemicals

• Drug formulations

• Pulp and paper solutions

• Semiconductor solutions

Inorganic anion solutions kit, 5063-6511

Using an indirect UV detection system optimized for small anions, analyses are sensitive and rapid, and provide an alternative to traditional ion chromatography. The kit contains buffer, capillaries, test mixture, and instructions.

Inorganic Anion Solutions Kit
Description | Unit | Part No.
Inorganic Anion Solutions Kit |  | 5063-6511
Inorganic anion buffer | 250 mL | 8500-6797
Ultra pure CE water | 500 mL | 5062-8578
0.1 N sodium hydroxide | 250 mL | 5062-8575
1.0 N sodium hydroxide | 250 mL | 5062-8576
Bare fused-silica capillary, 50 μm id, 72 cm long | 2/pk | G1600-62211
Inorganic anion test mixture Includes 1000 ppm each of fluoride, chloride, bromide, nitrite, sulfate and 2000 ppm phosphate | 10 mL | 5062-8524

Note: The following part should be ordered separately for use with the Agilent CE System: Alignment interface for standard 50 μm id capillary (P/N G1600-60210) for 1600 HP³D CE Alignment interface for standard 50 μm id capillary (P/N G7100-60210) for 7100 CE

Inorganic anion solutions kit, 5063-6511

Ultra pure CE water, 5062-8578

1. Thiosulfate

2. Bromide

3. Iodide

4. Chloride

5. Sulfate

6. Nitrite

7. Nitrate

8. Molybdate (VI)

9. Azide

10. Thiocyanate

11. Chlorate

12. Fluoride

Separation of common anions

0.1 N sodium hydroxide, 5062-8575

Inorganic anion test mixture, 5062-8524

Cation Solutions Kit

The Cation Solutions Kit provides everything you need for the analysis of inorganic and low-molecular-mass organic cations. It is specially designed for the separation of alkali metal ions, alkaline-earth metal ions and alkyl amines in a wide range of matrices.

Each kit contains a cation buffer, bare fused silica capillaries, cation standard, CE grade water and a detailed description of the analysis method and most common applications, including detection limits and reproducibility data. The Cation Solutions Kit and the separation methods were developed to fit perfectly with the Agilent CE system and to support its high automation capabilities. The methods are very easy to perform and provide accurate and quantitative analyses.

Cation Solutions Kit
Column Description | Unit | Part No.
Cation Solutions Kit |  | 5064-8206
Cation buffer | 250 mL | 5064-8203
Ultra pure CE water | 500 mL | 5062-8578
Bare fused silica capillary, extended light path bubble factor (3), 50 μm id, 56 cm long | 2/pk | G1600-61232
Cation test mixture | 25 mL | 5064-8205

Note: The following part should be ordered separately for use with the Agilent CE System: Alignment interface for 50 μm id extended light path capillary (P/N G1600-60230) for 1600 HP³D CE Alignment interface for 50 μm id extended light path capillary (P/N G7100-60230) for 7100 CE

1. Ammonium

2. Potassium

3. Sodium

4. Calcium

5. Magnesium

Cations in coffee and energy drinks

Organic Acids Solutions Kit

The Organic Acids Solutions Kit is ideal for the analysis of short alkyl chain carboxylic acids. Employing an indirect UV detection agent optimized for organic acids, the methodology is simple, sensitive, and provides accurate quantitative analysis. Suited for the analysis of organic acids in a wide range of matrices, it is especially useful for determination of organic acids in beverages and food.

Organic Acids Solutions Kit
Description | Unit | Part No.
Organic Acids Solutions Kit |  | 5063-6510
Organic acids buffer | 250 mL | 8500-6785
Ultra pure CE water | 500 mL | 5062-8578
1.0 N sodium hydroxide | 250 mL | 5062-8576
Bare fused-silica capillary, 75 μm id, 72 cm long | 2/pk | G1600-62311
Organic acids test mixture Includes 1000 ppm each of malate, succinate, and lactate | 20 mL | 8500-6900

Note: The following part should be ordered separately for use with the Agilent CE System: Alignment interface for 75 μm id capillary (P/N G1600-60310) for 1600 HP³D CE Alignment interface for 75 μm id capillary (P/N G7100-60310) for 7100 CE

Anions in beer

1. Chloride

2. Sulfate

3. Oxalate

4. Formate

5. Malate

6. Citrate

7. Succinate

8. Pyruvate

9. Acetate

10. Lactate

11. Phosphate

12. Pyroglutamate

Anions in red wine

1. Chloride

2. Sulfate

3. Tartarate

4. Malate

5. Citrate

6. Succinate

7. Pyruvate

8. Acetate

9. Lactate

Organic acids in beer and red wine

Forensic Anions Solutions Kit

This highly focused kit was developed specifically for the analysis of poisonous compounds, such as cyanide, azide, selenate, arsenate, and arsenite. In cases of poisoning, analytical tools are needed to determine the identity of toxins quickly and accurately. A rapid determination of anionic toxins in adulterated foods and beverages is possible using CE with indirect UV detection. Forensic and other anions can be detected within 15 minutes with minimal sample preparation.

Forensic Anions Solutions Kit
Description | Unit | Part No.
Forensic Anions Solutions Kit | 5 x 50 mL | 5064-8208
Basic anion buffer | 50 mL | 5064-8209
Ultra pure CE water | 500 mL | 5062-8578
Bare fused-silica capillary, 50 μm id, 104 cm long | 2/pk | G1600-64211
Inorganic anion test mixture Includes 1000 ppm each of fluoride, chloride, bromide, nitrite, sulfate and 2000 ppm phosphate | 10 mL | 5062-8524

Note: The following part should be ordered separately for use with the Agilent CE System: Alignment interface for standard 50 μm id capillary (P/N G1600-60210) for 1600 HP³D CE Alignment interface for standard 50 μm id capillary (P/N G7100-60210) for 7100 CE

1. Cyanide

2. Chloride

3. Nitrite

4. Nitrate

5. Sulfate

6. Azide

7. Selenate

8. Carbonate

9. Fluoride

10. Arsenate

11. Phosphate

12. Glutamate

13. Acetate

14. Arsenite

15. Lactate

Analysis of an anion standard with the Forensic Anions Solutions Kit

μPAGE Solution Kits

μPAGE poly-acrylamide gel-filled capillaries are the most direct vehicles to transfer all of your applications from slab gel to CE, utlizing the automation, high speed, high resolution, and quantitative advantages of CE. The capillaries are ideal for high resolution separations of oligonucleotides, single-stranded and double-stranded DNA fragments, polymerase chain reaction (PCR) products, sequencing reaction products and oligosaccharides.

μPAGE capillaries are available in three different pore sizes. The size of the molecular sieving pores is controlled by the monomer concentration (%T) and the degree of polymer cross-linking (%C). Gels with higher %T and %C values have smaller pores and are, therefore, more effective at resolving smaller molecules. μPAGE-10 (10%T, 0%C) capillaries provide high resolution capabilities for separation of antisense therapeutic agents, primers and probes, as well as nucleotides.

μPAGE-5 (5%T, 5%C) allows single base resolution of oligonucleotides [pd(A)] ranging from 20 to 150 bases.

For your convenience, μPAGE capillaries and μPAGE buffers can be purchased together or separately. To achieve the highest reproducibility and provide optimal longevity, use μPAGE buffer with μPAGE capillaries.

μPAGE Starter Kits
Includes 3 μPAGE capillaries, 75 cm total length, 50 cm effective length, oligonucleotide standard and μPAGE buffer
Kit as defined by type of μPAGE capillary | ID (μm) | Part No.
μPAGE-10 (10%T, 0%C) μPAGE pd(A)₂₅₋₃₀ oligonucleotide standard for μPAGE-10 kit μPAGE buffer, 2 x 237 mL | 100 | 192-1311
μPAGE-5 (5%T, 5%C) μPAGE pd(A)25-30, 40-60 oligonucleotide standard for μPAGE-3 and μPAGE-5 kits μPAGE buffer, 2 x 237 mL | 75 | 192-5211
μPAGE-3 (3%T, 3%C) μPAGE pd(A)25-30, 40-60 oligonucleotide standard for μPAGE-3 and μPAGE-5 kits μPAGE buffer, 2 x 237 mL | 75 | 192-3211

μPAGE Basic Kits
Includes 3 μPAGE capillaries, 75 cm total length, 50 cm effective length
Kit as defined by type of μPAGE capillary | ID (μm) | Part No.
μPAGE-10 (10%T, 0%C) μPAGE pd(A)₂₅₋₃₀ oligonucleotide standard for μPAGE-10 kit | 100 | 191-1311
μPAGE-5 (5%T, 5%C) μPAGE pd(A)25-30, 40-60 oligonucleotide standard for μPAGE-3 and μPAGE-5 kits | 75 | 191-5211
μPAGE-3 (3%T, 3%C) μPAGE pd(A)25-30, 40-60 oligonucleotide standard for μPAGE-3 and μPAGE-5 kits | 75 | 191-3211

Note: The μPAGE capillaries are not pre-aligned for the G1600A CE and G7100 CE systems. To cut them to the correct length, use the CE column cutter (P/N 5183-4669). To create detection window, use the Window Etching Tool (P/N 590-3003).

μPAGE Buffer Solutions and Oligo Standards
Kit as defined by type of μPAGE capillary | Part No.
μPAGE tris-borate and urea buffer for μPAGE-10, 4 x 237 mL | 590-4005
μPAGE tris-borate and urea buffer for μPAGE-3 and μPAGE-5, 4 x 237 mL | 590-4001
μPAGE pd(A)25-30, 40-60 oligonucleotide standard for μPAGE-3 and μPAGE-5, 3 x 50 μL | 590-4000

Single base pair separation of pd(A) on a 3%T, 3%C μPAGE capillary

Secondary peaks of identical base length as the primary peaks, but retarded in electromigration due to loss of the terminal 5’ phosphate group

Oligonucleotide samples with or without terminal 5 phosphate group

CE and CE/MS Capillaries

Standard Bare Fused-Silica Capillaries

Fused-silica capillaries are the heart of CE. Pre-aligned capillaries from Agilent Technologies are designed and optimized for ease of use and reliability. All capillary ends are cut to a smooth, mirror-like finish. In addition, the polyimide outer coating is removed from the ends. These processes ensure minimal sample adsorption and help maintain sharp peak shapes. All capillaries have a pre-made detection “window” and a built-in alignment stopper that allows rapid and precise insertion in the alignment interface.

CZE of a tryptic digest of recombinant human growth hormone using a standard fused-silica capillary with 75 μm internal diameter

[picture] | TIPS & TOOLS Different inner diameters of capillaries need to use different alignment interfaces to guarantee optimal detection. The color coding of the capillary and the alignment interface allow you to easily match the correct interface with the capillary.

Extended Light Path (Bubble Cell) Bare Fused-Silica Capillaries

Use Agilent Technologies extended light path capillaries (“bubble” cell capillaries) to improve sensitivity 3- to 5-fold over standard capillaries. With extended light path capillaries, the inner diameter is increased only at the detection window, offering the sensitivity of a wide inner diameter capillary and the low current generation of a narrow one.

Resolution is not sacrificed when used with matching optical alignment interfaces from Agilent.

Through a computer-controlled proprietary process, the diameter is increased three to five times, with a manufacturing precision better than 3%. Take advantage of this process to extend the detection pathlength of 25 μm id capillaries to 125 μm, 50 μm to 150 μm, and 75 μm to 200 μm.

Electroosmotic flow maintains the “plug” flow in the bubble. Optical slits matched to the zone geometry maintain resolution.

25 μm with 125 μm bubble

Analysis of cold medicine ingredients in a standard capillary (25 μm id) and an Agilent Extended Light Path Capillary

TIPS & TOOLS Use narrow 25 and 50 μm id “bubble” cell capillaries for highly conductive buffers without sacrificing sensitivity. | [picture]

Universal Bare Fused-Silica Capillaries

Universal Bare Fused-Silica Capillaries have a window, 75 cm effective length and 363 μm od, fitting into any CE instrument. To cut them to the correct length we recommend using the CE column cutter (P/N 5183-4669).

Bulk Fused-Silica Capillaries
ID (μm) | Total Length (m) | Part No.
20 | 5 | 160-2660-5
50 | 5 | 160-2650-5
75 | 5 | 160-2644-5

Polyvinyl Alcohol (PVA) Coated Capillaries

PVA coated capillaries contain a permanently adsorbed layer of polyvinyl alcohol. This coating minimizes hydrophobic and electrostatic solute/wall interactions and eliminates electroosmotic flow (EOF). Using a proprietary deposition process, the PVA coating is stable over a wide pH range, even under basic conditions, from pH 2.5 to 9.5. This stability allows the use of many common CE buffers. Because the silica surface is covered, many proteins and amines can be analyzed without the peak tailing found with uncoated capillaries. In addition, since EOF is eliminated, cumbersome washing procedures are unnecessary and migration time reproducibility may be improved.

Each batch of PVA coated capillaries is rigorously tested by Agilent Technologies and includes a representative electropherogram to assure quality.

The color coding of the capillary (alignment stopper) and the alignment interfaces allow you to easily combine the correct interface with the capillary. Capillaries for non-Agilent CE systems have removable alignment stoppers without color code.

PVA coated capillaries can be used for a variety of applications, including protein analysis at physiological pH, isoelectric focusing, and small anion analysis without the need for flow-reversal agents in the buffer.

PVA coating is available in standard capillaries, or in Agilent Extended Light Path Capillaries (“bubble” cell capillaries) for high sensitivity applications. Both capillary types are available in longer lengths for use in non-Agilent systems.

PVA is also available for use with the High Sensitivity Detection Cell for even further improved HPLC-like sensitivity. In addition, PVA coated capillaries are offered for CE-MS applications. The capillaries are provided with a normally positioned detection window to allow tandem UV-Vis and MS detection for improved sample identification.

1. Cytochrome C

2. Lysozyme

3. Lactoglobulin A

4. Lactoglobulin A

5. Lactoglobulin B

Use of PVA coated capillaries to reduce protein adsorption

1. Benzidine

2. Aniline

3. o-Toluidine

4. p-Cl-Aniline

5. 2-Aminonaphthalene

6. 4,4-Methylene-bis(2-chloroaniline)

7. 3,3-Dichlorobenzidine

8. 2-Amino-5-azotoluene

9. 2-Methyl-5-nitroaniline

CZE analysis of basic amines using PVA coated capillaries (decomposition products of azo dyes)

Analysis of meat proteins by c-IEF using PVA capillaries

Note: PVA coated capillaries for CE/MS have a blue alignment stopper matching the blue color code of the alignment interface for MS-UV detection. The alignment stopper of the 50 μm id PVA capillary for CE/MS has a black dot for easy identification.

Note: When extended pathlength capillaries are used in non-Agilent systems, loss of resolution may be found if the axial slit width is not reduced. In Agilent systems, the alignment interface contains properly matched slits to maintain resolution.

CEP Coated Capillaries

CEP coated capillaries contain a permanently bonded polymer coating. This CEP coating shields the silanol functionality of the capillary surface and helps prevent sample adsorption. Additionally, EOF is nearly eliminated, making the capillary ideal for applications such as DNA separations with sieving polymer buffers.

Elimination of EOF also simplifies analysis of anions and organic acids by direct UV detection. Without EOF reduction, highly mobile ions such as nitrate can migrate in the opposite direction to the slower, longer chain acids.

The CEP coated capillary is stable from pH 2 to 8. It can be used with borate buffers, offering a different surface functionality to help alleviate sample adsorption. Each batch of CEP coated capillaries is rigidly tested by Agilent Technologies and each capillary includes a representative electropherogram to assure quality.

Restriction fragment separation (36–2645 bp)

Cross-linked and Bonded μSIL Capillaries

μSIL-FC and μSIL-DNA Capillaries with Windows

A series of coated capillaries specifically designed for CE, which are prepared by cross-linking and bonding a novel, proprietary fluorocarbon (FC) polymer. μSIL-FC capillaries are chemically inert, hydrophobic, and stable from pH 2.5-10.

These capillaries are a must-have for cIEF, protein, peptide and carbohydrate separations, as well as replaceable gel CE applications such as oligonucleotides, DNA fragments, and PCR product separations.

μSIL-DNA capillaries are also coated with an FC polymer but have a 75 μm id to accommodate the viscosity of entangled polymer solutions. All μSIL capillaries are batch tested to ensure the highest performance and reproducibility.

μSIL-WAX Capillaries with Windows

μSIL-WAX features a modified, polyethylene oxide, hydrophilic coating made through a special cross-linking and bonding process. The coating effectively masks active silanol sites, offering exceptional efficiency, resolution, peak shape and reproducibility. The highly stable coating and near-zero EOF of μSIL-WAX makes the capillary ideal for CE-MS, and protein and peptide separations from pH 2-5.

μSIL-DNA, 199-2602

Bulk μSIL-DB Capillaries

μSIL-DB coated capillaries are available as μSIL-DB-1 and μSIL-DB-17. In combination with a cellulose based buffer system, μSIL-DB coated capillaries have been widely used in cIEF applications, PCR product and DNA fragment separation, and many other CE applications which require reduced EOF.

Analysis of Allelic ladder with μSIL-DNA

Analysis of Myoglobin tryptic digest using μSIL-WAX

Capillary Electrochromatography (CEC) Capillaries

Capillary electrochromatography is a hybrid of CE and LC and can be performed in the Agilent CE system. Using CE capillaries packed with LC stationary phases, CEC offers the loadability and selectivity of LC and the high efficiency of CE.

Using the high pressure capabilities of the Agilent CE system, both ends of the CEC capillary can be pressurized. This process prevents outgassing upon application of high voltage and significantly extends capillary lifetime.

Use CEC to improve resolution of solutes, which are difficult to resolve by HPLC, for hydrophobic solutes which cannot be solubilized in MEKC buffers, or for reduced sample and solvent consumption compared to HPLC.

CEC C8 Capillaries, 2/pk, 5063-6535

1. Chlorothiazide

2. Hydrochlorothiazide

3. Chlorthalidone

4. Hydroflumethiazide

5. Bendroflumethiazide

6. Bumetanide

Buffer: 50 mmol/
Na₂HPO₄, pH 2.5

Capillary Electrochromatography of diuretic test mixture

(courtesy of Dr. Melvin Euerby, Astra Charnwood, UK)

1. Thiourea

2. Methylparaben

3. Ethylparaben

4. Propylparaben

5. Butylparaben

6. Pentylparaben

7. Naphthalene

8. Biphenyl

9. Fluorene

10. Phenanthrene

11. Anthracene

12. Fluoranthene

Capillary Electrochromatography of parabens and aromatics

TIPS & TOOLS CEC capillaries require an Agilent CE system with external gas supply capabilities. | [picture]

Alignment Interfaces and Capillary Cassette

Agilent Technologies alignment interfaces are an integral part of the Agilent diode-array detection (DAD) system. These interfaces contain optical slits which are precisely matched to the capillary inner diameter for optimized sensitivity and linear detection range.

In combination with the capillary cassette, alignment interfaces simplify capillary exchange, protect the fragile detection window and ensure exact alignment of the window in the detector. Quick-change cassette allows capillary exchange in less than one minute.

Note: The color code of the alignment interface must match the color code of the capillary’s built-in alignment stopper.

Alignment interface for standard capillary, G1600-60310

Capillary cassette, G7100-60002

Capillary cassette
Description | G7100 CE Part No. | G1600 CE Part No.
Capillary cassette | G7100-60002 | G1600-60002

Note: Only use G7100-60002 cassette in G7100 and G1600-60002 cassette in G1600. Never mix cassettes.

Optical filter for DAD
Description | G7100 CE Part No. | G1600 CE Part No.
Optical filter for DAD 260 nm, for DNA analysis with polyacrylamide filled capillaries and oligonucleotide analysis | G7100-62700 | G1600-62700

[picture] | TIPS & TOOLS Cassette and interfaces accept all commercially available capillaries (~365 μm od).

Instrument Parts and Supplies

High Sensitivity Detection Cell

The Agilent high sensitivity detection cell – a technological leap which extends sensitivity by an order of magnitude – provides a solution to sensitivity limitations often encountered in CE. This improvement will substantially increase the utility of CE for impurity analysis of chiral drugs, biologicals, and compounds of environmental interest, among others.

The high linear range allows quantification of both <0.1% impurities and the main component in one run. This is useful for all impurity determinations and is especially useful for determining chiral excess.

The high sensitivity detection cell for the Agilent CE system not only improves detection sensitivity more than 10-fold over standard capillaries, but also extends linearity beyond 2000 mAU and provides unsurpassed spectral fidelity. These improvements are a result of a proprietary micromachined design which increases the detection pathlength from 75 μm to 1200 μm while dramatically reducing stray light.

The high sensitivity detection cell has a design comprised of a fused-silica cell body and removable capillaries. The light path through the cell is made from black fused-silica which significantly minimizes stray light and defines the aperture for the diode-array spectrometer. In addition, the reflective interior functions as a “lightpipe,” ensuring almost 100% transmission of light which entered the cell. These properties result in enhanced linearity and unsurpassed spectral fidelity with the diode-array detector.

High Sensitivity Detection Cell

Characteristics of the Agilent High Sensitivity Detection Cell

• Up to 10-fold increase in signal-to-noise

• Detector linearity beyond 2000 mAU for accurate quantitative analysis

• Decoupled design allows replaceable capillaries and reduced cost operation

• Special capillary geometry ensures maintenance of peak symmetry

• Full diode-array spectral capabilities

• Design fits all Agilent CE instruments

High Sensitivity Detection Cell
Description | G7100 CE Part No. | G1600 CE Part No.
High sensitivity cell kit Includes detection cell, 75 μm id inlet capillary (72 cm) and outlet capillary (8.5 cm) pair, capillary cassette, fittings (3 fitting screws with seals, 2 fitting caps), cleaning solution, CE Partner CD-ROM | G7100-68723 | G1600-68723
CE cell fitting kit Includes 3 fitting screws, 2 fitting caps |  | G1600-63200
Replacement detection cell |  | G1600-60027
Cell cleaning fluid, 1 L |  | 5062-8529

Capillary Kits for High Sensitivity Detection Cell
Description | Effective Length (cm) | G1600 CE Part No.
75 μm capillary kit with 8.5 cm outlet | 56 | G1600-68716
| 72 | G1600-68715
| 88 | G1600-68714
PVA coated 75 μm capillary kit with 8.5 cm outlet | 56 | G1600-68319

Agilent high sensitivity detection cell vs. 75 μm standard capillary for the CZE separation of naphthalene sulfonic acids

CE/MS Accessories

The CE/MS Adapter Kit simplifies coupling the Agilent CE system with MS systems equipped with an electrospray ionization (ESI) source. Integral to this kit is the CE/MS cassette, which completely thermostats the capillary until it exits the CE system. The cassette offers multiple capillary paths that vary the capillary length. A method development configuration uses online diode array detection and MS. For rapid or routine MS analysis, the detector can be bypassed to decrease the total capillary length and reduce analysis time. The CE/MS adapter kit can be used with the complete Agilent 6000 Series mass spectrometers, or virtually any electospray-MS platform.

The CE/MS cassette completely thermostats the capillary until it exits the CE system. Methods development configuration uses online diode array detection (DAD) and MS. For rapid or routine MS analysis the DAD can be by-passed to decrease the total capillary length and reduce analysis time.

The CE/MS Sprayer Kit includes the electrospray needle and splitter assembly, which allows the direct connection of the CE instrument with Agilent and other electrospray MS systems. The CE/MS Sprayer Kit needs the CE/MS Adapter Kit to fully support CE/MS coupling.

CE with tandem UV-Vis and MS detection allows the analysis of complex mixtures. Analyte mixtures are separated and the components detected via UV-Vis absorption, allowing preliminary identification based on peak elution time and UV-Vis spectra, or both, when compared to a standard. Online coupling to electrospray-ionization mass spectrometry (ESI-MS) then reveals unambiguous information on the solute’s molecular weight, and possibly structure.

Interfacing the capillary requires an electospray needle which is not included in this kit but in the CE-ESI-MS Nebulizer Kit. For coupling with non-Agilent MS please contact the MS vendor.

CE/MS Adapter Kit
Description | Part No.
CE/MS Adapter Kit For interfacing the Agilent CE system with a mass spectrometer Includes parts below, which can be ordered separately* | G1603A
CE/MS interface cassette, metallic, for G1600 and G7100 CE | G1600-60013
CE/MS alignment interface for 360 μm od capillaries, nonmetallic, for G1600 CE | G1600-60400
CE/MS alignment interface for 360 μm od capillaries, nonmetallic, for G7100 CE | G7100-60400
Bare fused-silica, 50 μm id, 125 cm long, 2/pk | G1600-67311

*Interfacing the capillary requires an electrospray needle which is not included in this kit

1. Leu-Enkephalin, MW 555.6

2. Met-Enkephalin, MW 573.7

3. VYV, MW 379.4

4. Angiotensin II, MW 1046.2

CE/MS of 4-component peptide mixture (210 fmol)

CE/MS Sprayer Kit
Description | Unit | Part No.
CE/MS Sprayer Kit Includes CE/MS test sample (5 g quinine sulfate dihydrate) and the parts listed below |  | G1607A
ES needle assembly |  | G1607-60041
CE-ESI sprayer |  | G1607-60001
Splitter assembly |  | G1607-60000
PEEK ferrule, 360 μm for CE/MS Sprayer |  | 5022-2141
Nut, finger-tight fitting and ferrule | 2/pk | 0100-1543
Flex loc element | 2/pk | 1520-0401
Gasket | 1/pk | G1607-20030
Ion kit (ammonium acetate) | 5 x 5 mL | 8500-4410

CE Standards & Reagents

Premade buffers help eliminate the time-consuming buffer preparation process. All Agilent buffers and reagents are designed to meet the stringent demands of CE. Manufactured under GLP/GMP conditions in ISO 9001 facilities, each is shipped with assay information and verification of purity. Chemicals are all electrophoresis grade, with nearly all ionic and organic impurities removed. Solutions are prepared under Class 10 clean room conditions and prefiltered through 0.2 μm filters to ensure removal of particulates. Superior quality control ensures reproducible results bottle-to-bottle and batch-to-batch.

In addition to a set of kit buffers, which are specially designed for dedicated applications, Agilent offers a series of basic CZE buffers covering a broad pH range. The product portfolio also includes special buffers for protein analysis and for Micellar Electrokinetic Chromatography (MEKC). Cleaning and conditioning solutions complete the offering.

Ultra pure CE water, 5062-8578

Ultra Pure CE Water
Description | Volume (mL) | Part No.
Ultra pure CE water | 500 | 5062-8578

Capillary Conditioning Solutions
Description | Volume (mL) | Part No.
0.1 N sodium hydroxide | 250 | 5062-8575
1.0 N sodium hydroxide | 250 | 5062-8576
0.1 N phosphoric acid | 250 | 5062-8577

0.1 N sodium hydroxide, 5062-8575

CZE Buffers for Charged Analytes
Description | Volume (mL) | Part No.
50 mM sodium phosphate buffer, pH 2.5 | 250 | 5062-8571
50 mM sodium phosphate buffer, pH 7.0 | 250 | 5062-8572
50 mM sodium tetraborate buffer, pH 9.3 | 250 | 5062-8573
20 mM sodium tetraborate buffer, pH 9.3 | 100 | 8500-6782

50 mM sodium phosphate buffer, pH 2.5, 5062-8571

CZE Buffers for Proteins
Description | Volume (mL) | Part No.
50 mM phosphate, 0.05% hydroxyethyl cellulose buffer, pH 2.5 | 250 | 8500-6786
150 mM phosphate, 200 mM ammonium sulfate buffer, pH 7.0 | 250 | 8500-6787

MEKC Buffers for Neutral and Charged Analytes
Description | Volume (mL) | Part No.
50 mM sodium tetraborate, 100 mM sodium dodecyl sulfate buffer, pH 9.3* | 250 | 5062-8574

*Dilute with 50 mM sodium tetraborate, pH 9.3 (P/N 5062-8573) to reduce SDS concentration without affecting the tetraborate composition or pH

Plating Bath Analysis Buffer
Description | Volume (mL) | Part No.
Plating bath analysis buffer | 250 | 5064-8236

μPAGE Buffer Solutions and Oligo Standards
Description | Part No.
μPAGE tris-borate and urea buffer for μPAGE-10, 4 x 237 mL | 590-4005
μPAGE tris-borate and urea buffer for μPAGE-3 and μPAGE-5, 4 x 237 mL | 590-4001
μPAGE pd(A)25-30, 40-60 oligonucleotide standard for μPAGE-3 and μPAGE-5, 3 x 50 μL | 590-4000

The total fluorimetry spectrum of the 50 mM borate buffer pH 9.2 verifies that the solution is free of fluorescence-active impurities (1 and 2 = Rayleigh stray light of zero and first order, 3 = Raman stray light).

CZE analysis of a peptide mixture using pre-made 50 mM sodium phosphate buffer, pH 2.5

CE System Start-up and Test Kits

Chemical test kits and validation packages are available to help comply with regulatory and quality standards. The Installation Qualification (IQ) Chemical Kit and Hardware Start-Up Kits, which are shipped with new instruments, are useful for rapidly verifying system functionality. For rigorous testing, the Operational Qualification (OQ)/Performance Verification (PV) Kit can be used to verify DAD noise, drift, linearity, wavelength accuracy and replenishment functionality. The OQ/PV kit is only part of the validation services available from Agilent Technologies. When implemented by qualified Agilent personnel, our service packages can be used to help validate your Agilent CE system.

CE System Start-up and Test Kits

CE System Start-up and Test Kits
Description | Part No.
CE Installation Qualification (IQ) Kit Includes buffer (20 mM borate, pH 9.3, 100 mL), test sample (4-(hydroxy)-acetophenone, 2 mL), capillary conditioning solution (0.1 N sodium hydroxide, 100 mL) | 5063-6514

CE Operational Qualification Performance Verification (OQ/PV) Chemical Kit

Includes buffer (20 mM borate, pH 9.3, 100 mL), test samples (0.1, 0.5, 1.0, and 5.0 mM 4-(hydroxy)-acetophenone, 2 mL ea.), capillary conditioning solution (0.1 N sodium hydroxide, 100 mL), test capillary (L 48.5 cm, I 40 cm, id 50 μm), diskette with methods, sequence, spectral library. Note: Method is supported for G1600 only.

5063-6515

CE OQ/PV Chemicals Only Kit Includes buffer (20 mM borate, pH 9.3, 100 mL), test samples (0.1, 0.5, 1.0, and 5.0 mM 4-(hydroxy)-acetophenone, 2 mL) | 5063-6520

IQ and OQ/PV test method

Sample: | p-Hydroxyacetophenone
Buffer: | 20 mM Borate pH 9.2
Capillary: | I = 40 cm, L = 48.5 cm, 50 μm id
Injection: | 250 mbar*s
Voltage: | 30 kV
Temperature: | 20 °C
Detector: | 192/4 nm

Vials and Caps for CE
Description | Unit | Part No.
Crimp/snap top vial, 1 mL, Polypropylene, crimp/snap top | 100/pk | 5182-0567
Clear, wide opening crimp/snap top vial, 2 mL | 100/pk | 5182-9697
Clear, wide opening crimp/snap top glass vial, 2 mL | 500/pk | 5183-4623
Amber, wide opening crimp/snap top vial, write-on spot, 2 mL | 100/pk | 5183-4619
Crimp/snap top vial, 250 μL | 1000/pk | 9301-0978
Snap caps PEO (polyethylene olefin for chemical resistance) | 100/pk | 5181-1507
Snap caps PEO (polyethylene olefin for chemical resistance) | 500/pk | 5181-1513
Snap caps PUR (polyurethane for resealing)* | 100/pk | 5181-1512
Snap caps PUR (polyurethane for resealing)* | 500/pk | 5042-6491

*PUR caps are recommended to help prevent sample or buffer evaporation even after multiple injections

Instrument Supplies
Description | Unit | Part No.
Long life HiS Deuterium lamp (8-pin) with RFID tag |  | 5190-0917
Deuterium lamp |  | 2140-0585
Electrode assembly, standard (for G1600 only) |  | G1600-60007
Electrode assembly, short (for G1600 only) |  | G1600-60033
Electrode assembly, standard (for G7100 only) |  | G7100-60007
Electrode assembly, short (for G7100 only) |  | G7100-60033
Electrode O-ring, silicone | 5/pk | 5062-8544
Electrolyte bottle, 500 mL |  | 9300-1748
Electrolyte bottle, 100 mL |  | 5042-6478
Electrolyte bottle cap |  | 9300-1747
Bottle sealing O-ring |  | 0905-1163
Glass filter, solvent inlet, 20 μm |  | 5041-2168
Filter frit adapter, 3 mm | 4/pk | 5062-8517
Bottle cap plug |  | G1600-23223
Air filter, 5 μm |  | 3150-0619
Pre-puncher |  | G1600-67201
Screws for pre-puncher/insulation plate holding | 10/pk | G1600-62402

Screws for pre-puncher/insulation plate holding, G1600-62402

Snap caps, polyurethane, 5181-1512, 5042-6491

Electrode assembly, standard (for G1600 only), G1600-60007

Electrode O-ring, silicone, 5062-8544

Electrolyte bottle, 500 mL, 9300-1748

Filter frit adapters, 5062-8517

Air filter, 5 μm, 3150-0619

Pre-puncher, G1600-67201

Accessories
Description | Part No.
CE accessory kit Includes electrode tool, screwdriver, fuses, air filter, glass frit, vials and caps alignment interfaces (red and green) standard and 50 μm id capillaries: L 64.5 cm, Standard: L 64.5 cm, Extended Light Path: L 48.5 cm | G7100-68705
Rack for 12 mm, 2 mL vials, holds 50 vials per rack, 5/pk | 9301-0722
CE column cutter | 5183-4669
Diamond blade replacement kit for CE column cutter | 5183-4670
Capillary tubing cutter, 4/pk | 5181-8836

Vial rack, 9301-0722

Window Etching Tool

The window etching tool is designed for fast, convenient and reproducible preparation of detection windows on fused-silica capillaries. The polyimide coating is removed without destroying the inner polymeric coating. The tool contains three glass blocks with fine grooves, precisely controlling the size of the windows.

Description | Part No.
Window etching tool, 3/pk | 590-3003

CE column cutter, 5183-4669

Window etching tool, 590-3003

Troubleshooting

Basic Capillary Electrophoresis Troubleshooting
Symptom | Possible Cause | Solution(s)
Unstable Current
Variable or no current | Air bubble formed in capillary | Flush capillary, ramp voltage to limit initial heating, and/or degas buffers.
| Clogged capillary | Flush capillary with absorbing solution (such as NaOH). A “step” on the baseline should be observed when viewing the online signal at 200 nm. If still plugged, flush manually with syringe or high pressure gas.
| Broken capillary | Replace capillary.
| No or incorrect solution in buffer vials | Fill/change buffer vials.
| Large volume injection | Normal situation. Current should stabilize during analysis.
Unstable Baseline
Spikes in baseline | Precipitates in buffer | Filter buffer through 0.2 or 0.45 μm filter.
| Micro air bubbles in buffer | Degas buffer by ultrasonication or vacuum.
| Precipitation of sample | Verify that sample components are sufficiently soluble in buffer.
Noisy baseline | Optical slit in capillary interface is occluded | Clean slit with methanol or water. View under magnifier.
| Aging deuterium lamp | Use DAD test to measure lamp output and time-on. Replace if necessary.
| Data acquisition rate too high | Determine peak width and decrease acquisition rate if appropriate.
| Improper reference wavelength | Acquire UV spectrum during analysis. Use lowest wavelength possible without impinging where sample absorbs. Also use wide bandwidth.
| Buffer absorbs at detection wavelength | Use minimally UV-absorbing buffers such as phosphate and borate, especially below 210 nm.
Drifting baseline | Improper capillary alignment | Re-seat capillary cartridge in detector block.
| Unequilibrated temperature | Allow 10-20 minutes for equilibration after opening top cover.
| Lamp recently ignited | Allow 15-30 minutes for equilibration after igniting lamp.
Poor Peak Efficiency
Broad peaks | Sample overloading | Decrease sample injection or concentration.
| Excessive Joule heating | Reduce voltage, buffer conductivity, or capillary id.
Skewed peaks | Mismatched sample buffer ion mobilities | Match mobilities or increase difference between buffer and sample conductivity.
| Sample overloading | Decrease sample injection or concentration.
Tailing peaks | Adsorption to capillary wall | Use pH extremes, high buffer concentrations, polymer additives, or coated capillary.
Poor Migration Time Reproducibility
Adsorption to capillary walls | Changes in EOF caused by buffer (especially phosphates and detergents) or sample adsorption | Condition capillary and allow sufficient equilibration time. Replace capillary.
Hysteresis of wall charge | Caused by conditioning capillary at high (or low) pH and employing a low (or high) pH runnning buffer | Avoid pH differences. Allow sufficient equilibration time.
Changes in buffer composition | pH changes due to electrolysis | Replenish buffer.
| Buffer evaporation | Tightly cap buffer vials and reduce carousel temperature.
| Conditioning solution waste flushed into outlet reservoir | Use separate vial to collect waste.
| Conditioning solution carried over into buffer vial | First dip capillary in separate buffer or water vial.
Buffer reservoirs not level | Generation of laminar flow | Level liquid in reservoirs. If not replenishing buffer, do not use inlet vial for flushing capillary.
Different silanol content of capillary batches | Different wall charge and variations in EOF | Measure EOF and normalize.
Temperature changes | Changes in viscosity and EOF | Use system with capillary thermostatting.
Poor Peak Area Reproducibility
Sudden application of high voltage | Heating, thermal expansion of buffer, and expulsion of sample | Ramp separation voltage or inject buffer plug after sample.
Sample evaporation | Increasing sample concentration and peak area | Cap vials and/or reduce temperature of sample carousel.
Instrumental limitations | System rise time significant proportion of injection time | Increase injection time.
Sample carry-over | Extraneous injection | Use capillary with flat, smooth injection end. Remove polyimide from end of capillary.
Zero-injection caused by simply dipping the capillary in the sample | Extraneous injection | Cannot be totally eliminated. Increase injection amount to minimize effect.
Sample adsorption to capillary walls | Distorted peak shape (tailing) Non-eluting sample | Change buffer pH. Increase buffer concentration. Use additive such as cellulose or coated capillary.
Low signal-to-noise ratio | Integration errors | Optimize integration parameters. Increase sample concentration. Use peak height.
Temperature changes of capillary environment | Changes in viscosity and injection amount | Use system with capillary thermostatting.

LC AND LC/MS COLUMNS

LC and LC/MS Columns

The largest portfolio of Fast LC columns, and a broad family of phases across all particle sizes for exceptional flexibility and scalability

Whether you are performing conventional or ultra-fast chromatography, separating biomolecules, or analyzing complex basic compounds, you can trust Agilent for the industry’s highest-performing columns that deliver the fast, reproducible results you need – all engineered with Agilent’s unparalleled quality and reliability.

• Poroshell 120 columns – high efficiency and high resolution with up to 50% less pressure than sub-2 μm columns.

• ZORBAX Rapid Resolution High Definition (RRHD) columns – 1.8 μm columns feature improved packing processes to achieve stability up to 1200 bar for use with the Agilent 1290 Infinity LC and other UHPLC instruments and are available in more than 14 phases, plus HILIC.

• ZORBAX Eclipse Plus columns – C18 and C8 columns deliver superior peak shape, while the phenyl-hexyl bonded phase and C18 bonded phase for PAH separations expand selectivity options for more applications. All Eclipse Plus phases are available in Fast LC/UHPLC RRHD and RRHT columns, 1.8 μm. For scalability, the Eclipse Plus C18 phase is very similar to the Poroshell 120 EC-C18 phase.

• In addition to Poroshell 120 and RRHD columns, ZORBAX Rapid Resolution High Throughput (RRHT) columns are a third Fast LC option with over 140 1.8 μm columns choices. RRHT columns are available in 2.1, 3.0 and 4.6 mm ids, all with 600 bar stability.

And remember, when you choose Agilent ZORBAX LC columns, you get more than just a dependable product. You also get over 40 years of expertise – along with unmatched technical support – from the world’s largest chromatography supplier. On the web, by phone or in person, Agilent helps you solve the problems that can slow you down and get in the way of your results.

COLUMNS FOR SMALL MOLECULE SEPARATIONS

Table of Contents

LC and LC/MS Columns for Small Molecule Separations

HPLC Column Selection

Fast Columns for Reversed-Phase HPLC/UHPLC

Poroshell 120

ZORBAX Rapid Resolution High Definition 1.8 μm

ZORBAX Rapid Resolution High Throughput 1.8 μm

Agilent UHPLC Guards

Columns for Reversed-Phase Analytical HPLC

ZORBAX Eclipse Plus

ZORBAX Eclipse PAH

ZORBAX Eclipse XDB

ZORBAX 80Å StableBond

ZORBAX Rx

ZORBAX 80Å Extend-C18

ZORBAX Bonus-RP

ZORBAX Original Reversed-Phase Columns

Kits for Analytical HPLC

Pursuit

Polaris

TC-C18(2) and HC-C18(2)

PLRP-S

Preparative HPLC Columns

Agilent Prep LC Columns

ZORBAX PrepHT

Pursuit and Pursuit XRs Prep

Polaris Prep Columns

Load & Lock

Columns for Other HPLC Techniques

ZORBAX HILIC Plus

Normal-Phase Columns

ZORBAX Ion-Exchange Columns – SAX and SCX

Hi-Plex Columns for Carbohydrate Analysis

Appendices

Quick Guide to USP Designations for HPLC Columns

Oligo Solutions

StratoSpheres DNA Cartridges

TOP, TOP-DNA and TOP-RNA Cartridges

HPLC Column Selection

To use the column selection guide diagram below, simply follow the path for your analyte and mobile phase. At the far right, follow your final column selection to the pages indicated.

Quick Guide to Agilent Reversed-Phase Bonded Phases

ZORBAX RP-HPLC Columns | Recommended Uses and Applications

Poroshell 120

• Superficially porous particles for high efficiency at low pressure

• Sub-2 μm efficiency with a 2.7 μm particle

• Endcapped and non-endcapped C18 and C8 phases, and a variety of other phases, for selectivity optimization

• Compatible with 400 bar and 600 bar LC’s

Eclipse Plus | • Excellent first choice for method development

Available in RRHD (1200 bar) and RRHT (600 bar) configurations, 1.8 μm

• Long life from pH 2-9 for reliable separations of basic, acidic and neutral compounds

• Superior peak shape with basic compounds

• High resolution and efficiency with 1.8, 3.5 and 5 μm columns

• Rigorous QA/QC testing for greater long-term reproducibility

Eclipse XDB | • Four selectivity choices for flexible method development

Available in RRHD (1200 bar) and RRHT (600 bar) configurations, 1.8 μm

• High performance over a wide pH range (2-9)

• Good peak shape for acids, bases and neutrals

• Long lifetime with eXtra Dense Bonding and double endcapping

• Fast, ultra-fast, and high resolution separations using 1.8 and 3.5 μm columns

• Choices from capillary to prep

StableBond (SB) | • Basic, acidic, neutral compounds

Available in RRHD (1200 bar) and RRHT (600 bar) configurations, 1.8 μm

• Exceptional stability at low pH (1-2)

• Use of high temperature (up to 90 °C for C18, 80 °C for C8, C3, Phenyl, CN, and Aq) and low pH as an added selectivity tool

• Widest selection of bonded phases for different selectivity (C18, C8, C3, CN, Phenyl, Aq)

• Uses mobile phases for LC/MS with formic acid, acetic acid, or TFA

• Uses mobile phases with TFA for peptide and protein separation

• Rapid separations using 1.8 and 3.5 μm columns

ZORBAXRx Available in RRHD (1200 bar) and RRHT (600 bar) configurations, 1.8 μm | • General separation of basic, acidic and neutral compounds at low pH with different selectivity than SB columns • Rx-C8 is the same as SB-C8

Bonus-RP

Available in Fast LC/UHPLC RRHD (1200 bar) and RRHT (600 bar) configurations, 1.8 μm

• Separating basic compounds in higher aqueous mobile phases

• General separation of basic, neutral, acidic compounds at mid-range pH or low pH; especially stable at low pH

• Separating peptides for different selectivity

• Rapid separations using 3.5 μm columns

Extend C-18

Available in Fast LC/UHPLC RRHD (1200 bar) and RRHT (600 bar) configurations, 1.8 μm

• Separating basic compounds above their pKa in free base form; separation of basic, acidic, neutral compounds at high pH; up to pH 11.5

• Uses ammonium hydroxide as mobile phase additive with LC/MS with small molecules or peptides

• Separating at high, mid-range and low pH for selectivity changes

• Rapid separations using 3.5 μm columns

Original ZORBAX Columns | Recommended Uses and Applications
ZORBAX | • General separation of basic, acidic, neutral compounds at low pH with different selectivity than SB columns; higher number of active silanols than SB • “Mixed mode” separation at more neutral pH values • Available in ODS, C8, CN and ODS “Classic” (non-endcapped)

Information about biocolumns can be found in the section beginning here

TIPS & TOOLS

The LC Handbook: Guide to LC Columns and Method Development

This handy guide makes it easy to choose the right LC column, and contains plenty of tips and tricks to make your job easier and more productive (publication # 5990-7595EN).

Request a copy or download a mobile copy at www.agilent.com/chem/lchandbook

Quick Guide to Additional Agilent Reversed-Phase Columns

Pursuit Family | Recommended Uses and Applications

Pursuit HPLC

• Full range of phases, including C18 and C8

• Diphenyl utilizes strong dipole-dipole hydrogen bonding and pi-pi mechanisms for different selectivity with aromatic compounds

• PFP provides excellent separation of polar (halogenated) analytes and positional isomers under standard reversed-phase conditions

Pursuit XRs and Pursuit XRs Ultra | • Offer larger surface area and smaller pore size, in complementary phases to Pursuit family • Ultra offers stability to 600 bar, due to special hardware and loading
Polaris Family | Recommended Uses and Applications
C18-A and C8-A Available in 3.0, 5.0, and 10 μm (C18-A only) | • C18-A and C8-A offer alternate selectivities for general polar applications • Designed with hydrogen-bond-accepting endcapping
Amide-C18 | • Subtle alternative selectivity due to the absence of steric protection
Available in 3.0 and 5.0 μm | • Utilize an embedded amide, similar to ZORBAX Bonus-RP
C18-Ether and C8-Ether Available in 3.0 and 5.0 μm | • Endcapped with an ether group to create a more polar surface for selectivity variation
Other Agilent Columns | Recommended Uses and Applications
TC-C18(2) Available in 5 μm | • An excellent choice for mixtures of polar and non-polar compounds, including strong basic compounds
HC-C18(2) Available in 5 μm | • High-value, highly retentive option • Carbon load of 17% • Superior peak shape for basic compounds

ZORBAX Reversed-Phase HPLC Column Selection Flow Chart

For small and large molecules

Most chromatographers use reversed-phase HPLC as one of their key analysis techniques. Reversed-phase HPLC can be used to analyze ionic and nonionic analytes. Therefore this ZORBAX Column Selection Flow Chart will focus on reversed-phase columns. To more easily select a reversed-phase column for method development of small and large molecules, follow the outline on these pages.

This flow chart provides information on choosing an initial column for method development of small molecule and protein and peptide samples, and includes decisions on bonded phase and column configuration.

Information about biocolumns can be found in the section beginning here

Column and Mobile Phase Guidelines: Reversed-Phase

HPLC columns consist of two parts: the column chemistry and hardware. For the proper column chemistry, consult the catalog section for each type of bonded phase. For choosing column hardware and particle sizes, consult the section on column sizes and rapid separations, including Agilent ZORBAX Rapid Resolution HT, Solvent Saver, Capillary and PrepHT columns.

ZORBAX Rapid Resolution High Throughput (RRHT) Columns

Pore Size Selection

Choose a column packing with small pore (60-120Å) if the solute molecular weight is less than about 3000. Otherwise, use column packing with the 300Å pore size.

Particle Size Selection

The typical particle size for HPLC columns is 5 μm with 3.5 μm and smaller, now common in method development. If high-speed analyses or higher resolution analyses are required, packing with 1.8 μm and 2-3 μm particles can be used. Shorter columns with these particles can produce faster high-resolution separations, with the 1.8 μm particle size providing the highest efficiency and 2.7 μm superficially porous providing similar results. With 1.8, 2.7, 3.5 and 5 μm particle sizes to choose from, start with the smallest particle size for your HPLC or UHPLC – 400 bar, 600 bar, or 1200 bar – to achieve the best results.

Column Configuration

Choosing the best column size for method development has changed dramatically in the past few years. Smaller 3.0 mm id or 2.1 mm id columns are now used more than 4.6 mm id to lower solvent use and achieve compatibility with MS detectors. And shorter 50, 75 and 100 mm long columns can be a great starting choice, with longer columns used only when more resolution is needed or when 3.5 and 5 μm particle sizes are used.

TIPS & TOOLS

Need help selecting the right LC column for your method?
Try the Navigator: A selection tool for LC columns and sample prep.
Look for it online and via your mobile device at http://navigator.chem.agilent.com

Silica, Polymers, and Bonded Phase

Base Material

The base material for an LC column is most often high purity silica material with totally porous particles such as that used in most Agilent columns, including ZORBAX, Pursuit, and Polaris. However, more choices are available, including polymer material with high pH stability used in PLRP-S columns and superficially porous silica particles such as those used in Poroshell 120 columns. The high purity Type B silicas, including the ZORBAX Rx-Sil used in ZORBAX Eclipse Plus, and superficially porous Poroshell 120, are an excellent first choice for most methods. Type A silicas, such as ZORBAX SIL, used in Original ZORBAX columns, are still manufactured and used in many methods.

Bonded Phase

A good first choice for bonded phase is C18 or C8, and the recommended starting column choices are Eclipse Plus C18 or Poroshell 120 EC-C18. These two choices provide excellent peak shape and can be used over the pH range 2-9, accommodating most typical LC and LC/MS mobile phases. If the sample solutes of interest are not adequately separated on these columns, CN and Phenyl columns – including Phenyl, Phenyl-Hexyl and Diphenyl – may offer significant differences in selectivity from straight-chain alkyl phases to effect the separation.

In general, larger solutes, such as proteins, are best separated on short-chain reversed-phase columns (C3, CN, C8) and peptides and small molecules are separated on longer-chain columns (C18). However, there are many cases where this conventional wisdom does not apply. For example, peptides can also be effectively separated using short-chain columns, and hydrophobic peptides can show better recovery on longer-chain phases. Therefore, it is best to initially select a phase in the middle of the hydrophobic spectrum (e.g., C8), then change to a more hydrophobic phase or more hydrophilic phase depending on initial results and solubility properties of your sample.

Polymers

When a column is needed that can operate at very low and very high pH, polymeric packings provide an alternative to silica-based materials. Polymeric particles are good for small-scale chromatography, particularly LC/MS, as they are chemically stable and do not leach soluble or particulate species. Reversed-phase spherical polymeric packings used in Agilent PLRP-S columns, for example, are based on a styrene/divinylbenzene copolymer with an inherently hydrophobic surface. No bonded phase is required for reversed-phase chromatography with polymeric particles. These rigid macroporous particles can be coated and derivatized to give a range of functionalities, including weak and strong cation and anion exchangers.

pH and Mobile Phase

The choice of mobile phase for a reversed-phase system starts with selecting the organic modifier. Acetonitrile is the most commonly used organic modifier. However, selectivity differences and sample retention will vary significantly among mobile phases containing acetonitrile, methanol, and tetrahydrofuran (THF). Sample solubility is likely to differ in such solvents and dictate use of a specific solvent or solvents. UV detection at certain wavelengths is not possible with certain modifiers (e.g., methanol at 200 nm).

Both pH and ionic strength of the aqueous portion of mobile phases are important parameters in developing rugged methods that are not sensitive to small variations in conditions. With ionic compounds, retention of typical species shows significant changes with pH. It is very important to control pH in such reversed-phase systems to stabilize retention and resolution. A pH between 2 and 4 generally provides the most stable conditions for retention vs. small changes in pH, and this pH is recommended for starting method development for most samples, including basic compounds and typical weak acids.

Working with LC/MS

When choosing HPLC columns for LC/MS, chromatographers often need to consider several aspects of their method and separation, typically including resolution, flow rate, and stationary phase choice. Often, for relatively simple analytes, shorter high resolution columns are the best choice. These columns allow for high throughput while maintaining high separation efficiency. Narrow bore Rapid Resolution High Definition (RRHD) for separations (> 600 bar) and Poroshell 120 columns (< 600 bar) offer high resolution even in shorter columns dimensions. For more difficult samples, users should seek longer column lengths.

Since many LC/MS analyses are run at lower flow rates (typically from μL/min flow rates up to 1 mL/min), moving to smaller internal diameter columns is the best choice for the user. Agilent’s Solvent Saver (3.0 mm id) and narrow bore (2.1 mm id) will often result in lower solvent usage for the method, and are excellent options for high resolution and higher sensitivity than the larger id columns.

Most often, the best bonded phase choice is an endcapped C18 phase. Eclipse Plus C18 is a high performance endcapped C18 phase available in sub-2 μm RRHD and RRHT column formats. For fast high-throughput separations with LC/MS, Poroshell 120 EC-C18 is an excellent choice. Poroshell has a larger frit, so it’s well suited for dirtier LC/MS samples, such as blood plasma, which may often clog columns with smaller porosity frits.

Both Eclipse Plus C18 and Poroshell 120 EC-C18 phases are stable over a wide pH range and are compatible with the volatile buffers such as acetic and formic acids.

TIPS & TOOLS

LC Flow Rate Calculator App

This FREE Smartphone app lets you quickly adjust your flow rate to accommodate other method changes.

Download at www.agilent.com/chem/lcapp

Transferring your method to a high efficiency column

High efficiency columns for UHPLC/Fast LC will help you increase your analytical speed and resolution. Depending on the instrument configuration you are using, you may need to make a few adjustments to get the most from these columns.

Because of their high efficiency, very narrow peaks elute from higher efficiency columns quickly. While modern HPLC instrumentation and data systems are able to capture the benefits of these particles, attention to instrumental configuration is important to get the best results.

Steps to transfer your method:

Check the specifications that came with your instrument – Your instrument may already be configured appropriately for high efficiency columns. If not, then continue.

Optimize the data collection rate for LC and LC/MS (at least 40 Hz detector with fast response time for UV) – Set the detector to the fastest setting, then to the second fastest setting and evaluate if the resolution is different.

Use a semi-micro or micro-flow cell – Smaller volume flow cells such as the semi-micro (6 mm/5 μL) or micro (3 mm/2 μL) are recommended for best performance. There are newer cartridge flow cells (e.g. the Ultra Low-Dispersion Max-Light Ultra Flow Cell, P/N G4212-60007) designed to optimize UHPLC instrument performance.

TIPS & TOOLS For the Agilent 1290 Infinity LC, in situations requiring extremely low dead volumes, use the ultra-low dispersion kit, which includes an ultra-low dispersion flow cell and 0.08 mm id capillaries. | [picture]

Minimize tubing volume in the instrument – Use Red (0.12 mm id) tubing instead of Green (0.17 mm id) as it has only half of the volume that the sample has to travel through. This cuts down extra column band broadening. Ensure that your connections are as short as possible. The key locations to check are:

• The autosampler needle seat

• The autosampler to the Thermostatted Column Compartment – or ‘TCC’

• The TCC to the column

• The column to the flow cell, including the internal diameter of the integral flow cell inlet capillary

All of these specific capillaries can be ordered individually from Agilent, in the lengths you need, and for your instrument.

View Here

Scale your gradient profile and injection volume – If using gradient elution, scale the gradient profile and injection volume to the new smaller column to quickly transfer the method and avoid overloading. For isocratic and gradient elution, make sure that you scale the injection volume to match the overall column volume.

Minimize injection sample dispersion in the column – Use an injection solvent with solvent strength that is equivalent to or weaker than the mobile phase, especially when using an isocratic method. This is good practice in general for any column, and more important with high efficiency columns.

[picture] | TIPS & TOOLS See a video that takes you through these steps at www.agilent.com/chem/poroshell120video Also, check out the LC Method Translator Tool at www.agilent.com/chem/lcmethodtranslator

Take care to make proper connections – Agilent recommends Swagelok fittings with front and back ferrules, which give best sealing performance throughout our LC system (use this on the instrument connections, i.e. valves, heaters, etc). Polyketone fittings are highly recommended for up to 600 bar. Use this fitting (P/N 5042-8957) on column connections with Poroshell 120. For RRHD columns, use Agilent’s removable 1200 bar fitting (P/N 5067-4733).

Optimize your flow rate – For Poroshell 120, if you’re using a 2.1 mm id, the suggested starting flow rate is 0.42 mL/min; for 3.0 mm id Poroshell 120 columns, we suggest starting at 0.85 mL/min, and for 4.6 mm id, we suggest starting at 1.5 - 2 mL/min.

1200 bar removable fitting (SV), 5067-4733

Fast analysis of cefepime and related impurities

Column: | Poroshell 120 EC-C18 697975-902 4.6 x 75 mm, 2.7 μm
Instrument: | Agilent 1200 Infinity Series Rapid Resolution LC System
Detector: | DAD, 254 nm

1. Impurity E

2. Cefepime

3. Impurity F

4. Impurity A

5. Impurity B

VHP FITTINGS

Agilent’s 1200 bar removable fitting (for 1/16 in od capillaries) consists of a stainless steel screw, an internal stainless steel ferrule and a front ferrule in PEEK. The fitting can be used throughout the flow path, but because it can be re-used without losing tightness, it is especially suitable for the connection between the heat exchanger and the column. This new and improved fitting replaces the standard stainless steel Swagelok fitting which was not removable. The Very High Pressure (VHP) fitting is available in three sizes – short (P/N 5067-4733), long (P/N 5067-4738) and extra long (P/N 5067-4739). The short fitting is the one that is most commonly used, and will be appropriate 90% of the time. In some cases, if using columns with longer nuts, a longer fitting will be needed.

Agilent LC Columns Overview: Small Molecules

Method Development from pH 1-12

Start method development at low pH (pH 2-3)

With so many column choices available, how do you know where to start your method development? The recommended starting point for method development is using a buffered low pH mobile phase – around pH 2-3. Using a low pH mobile phase most often results in the best peak shape for basic compounds on silica-based columns. At low pH, the silanols on the silica are fully protonated so positively charged basic compounds do not interact strongly. The result is good peak shape. Many acidic compounds are non-charged, maximizing their retention at low pH. These observations are key advantages to method development at low pH.

For standard analytical work, start method development with acetonitrile as the mobile phase organic modifier and 20-50 mM phosphate buffer (pH 2-3) as the aqueous component for non-LC/MS applications. These conditions provide good pH control, necessary for the most reproducible analyses of ionizable compounds. For LC/MS applications formic acid or TFA are good mobile phase additives for low pH.

Optimize solvents and bonded phases at low pH

The initial method development steps may lead very quickly to a satisfactory separation. But if more optimization is needed, acetonitrile can be replaced with methanol or tetrahydrofuran and the separation re-optimized. This step may lead to a satisfactory solution, but if still more selectivity optimization is needed, the column bonded phase can be changed.

At low pH there are many bonded phase choices available for optimization. These include the Eclipse Plus phases as well as the Eclipse XDB family with C18, C8, Phenyl and CN. Alternate choices include five different StableBond bonded phases: SB-C18, SB-C8, SB-Phenyl, SB-CN, and SB-C3. For polar analytes, try Bonus-RP, SB-Aq or the Polaris family, including C18-A, C8-A, C18-Ether and Amide-C18 phases.

It may be necessary at low pH to improve the retention of acidic compounds. For these situations, lower the pH even further, down to pH 1-2, and use StableBond columns. These columns provide the greatest stability at very low pH and provide many selectivity options for achieving the highest resolution separations.

TIPS & TOOLS

LC Method Translator

Use this online tool to quickly factor in changes to column length, diameter, flow rate, and more – and to calculate method adjustments. This is particularly useful for gradient methods.

To download, go to www.agilent.com/chem/lcmethodtranslator

Choose Agilent ZORBAX Eclipse Plus or Poroshell 120 for method development at mid pH (pH 4-9)

There are some samples that may not be resolved at low pH or may have better solubility and stability at mid pH. The Eclipse Plus C18 and Poroshell 120 EC-C18 columns can be used at the mid pH range for method development. The Eclipse Plus column is stable to pH 9 so it is equally reliable at mid pH. These double endcapped columns have two key advantages – good peak shape at low and mid pH, as well as sufficient bonded phase density to protect the column from silica degradation from pH 6-9.

At mid pH, basic compounds (e.g., amines) may still have a positive charge and the silanols on the silica surface may have a negative charge. Therefore covering as many silanols as possible leads to the best peak shape at mid pH. This makes the Eclipse Plus C18 the best starting choice for a column at mid pH. Phosphate buffer is usually the first choice for mobile phase modifier at pH 7 because its buffer range is pH 6.1-8.1. A second choice for mid pH is acetate buffer since it buffers from pH 3.8-5.8 and its volatility makes it a good choice for LC/MS compatibility.

Choose Agilent ZORBAX Extend-C18 columns for method development at high pH (pH 9-12)

At low or mid pH, some separations of basic compounds may still not have enough retention or the desired selectivity. For these samples, high pH separations may be appropriate. Until recently, high pH separations on silica-based columns were avoided because of short column lifetimes, due to dissolution of the underlying silica gel. Special bonded phases such as the ZORBAX Extend-C18, can protect the silica from dissolution, so that a reasonable column lifetime can be achieved and the selectivity advantages of high pH can be explored.

The mobile phase buffer choices at high pH with the Extend-C18 column are organic buffers like triethylamine and ammonium hydroxide. These buffers are best used with methanol as the organic modifier to extend the column lifetime at high pH. This is another good option to consider when working with high pH and PLRP-S columns, which are made from a polymeric material.

EASY, RELIABLE PH TESTING

Agilent offers a full line of pH meters and electrodes. Designed for chromatographers, these pH meters offer intuitive user design and exceptional ruggedness for your lab. Learn more at www.agilent.com/chem/AgilentpH

Method Development Guidelines from Low to High pH

Guard Columns

The Value of Guard Columns

Guard columns can help extend the life of your analytical column. Choosing to use guard columns can help reduce operating expenses, by reducing the frequency of analytical column replacement.

The guard column prevents damage caused by particulate matter and strongly adsorbed material. To maintain an adequate capacity for sample impurities, choose a guard column with an internal diameter similar to the column internal diameter. Ideally, the packing of the guard column should be the same as the analytical column so that the chromatography of the analytical column is not altered.

Guard columns contribute to the separation, so you should include a guard column in-line during method development.

Agilent UHPLC guards provide protection for high-efficiency Poroshell 120 and ZORBAX RRHD and RRHT columns, without reducing performance. Part numbers for all guard columns are incorporated into the different product family tables.

Judging when to replace a guard column can be difficult. As a rough guide, if plate number, pressure or resolution change by more than 10%, the guard column probably needs replacing. You will need to make a judgment call on how often to replace your guard columns based on your application type. It is always preferable to change the guard column sooner rather than later.

UHPLC Guard, 1200 bar, 821725-903

Fast Columns for Reversed-Phase HPLC/UHPLC

The past decade has seen a steady increase in the efficiency and speed of chromatography, starting with smaller particle sizes, that enable higher resolution, and continuing with new technological advances in particle design – superficially porous particles – that enable these same resolution enhancements with lower backpressure.

Designed especially for high-productivity analysis (Fast LC), Agilent ZORBAX and Poroshell columns are the best first choice for any analysis, because they give you:

• The productivity you need to stay ahead of your competition: technological advances like sub-2 μm particles and superficially porous Poroshell 120 columns deliver increased speed and resolution.

• Flexibility and method scalability from lab to lab and around the world – for small molecule and biomolecule analyses.

• Unbeatable chromatographic performance: ZORBAX silica – the base silica used for all ZORBAX and Poroshell 120 columns – is ultra-pure, very strong, and highly uniform for ultimate reliability.

• The broadest range of phases and column configurations to suit your specific application needs.

Recommendations for Fast LC Columns
Your Lab Situation | Agilent Recommends | Rationale
You’re using both UHPLC (1000+ bar) and HPLC instruments (e.g. Agilent 1290 Infinity LC and 1260 Infinity LC – 600 bar) | 1. Poroshell 120 2. ZORBAX RRHD 1.8 μm | Poroshell 120 is an easy column to use on both instrument types. ZORBAX RRHD will help you optimize the capabilities of the 1290 Infinity LC for UHPLC.
Only 400-600 bar HPLCs – Agilent 1200s, Agilent 1100s (400 bar) as well as the 1220 Infinity LC or 1260 Infinity LC (600 bar) | 1. Poroshell 120 2. ZORBAX Eclipse Plus 3.5 μm and 5 μm | With Poroshell 120, you can enhance the performance of older 400-bar instruments, and also get even better performance from newer 600 bar UHPLC instruments. For established methods that you can’t transfer, the ZORBAX Eclipse Plus column will provide exceptional peak shape and performance.
A mix of UHPLC instruments (Agilent 1290 Infinity LC, other 1000+ bar instruments) and some HPLC instruments (e.g. 1200 LC) | 1. ZORBAX RRHD 1.8 μm 2. Poroshell 120 | ZORBAX RRHD can deliver optimum performance on all these instruments. Poroshell 120 can be used on the 600 bar instruments to optimize their performance.

TIPS & TOOLS Agilent CrossLab offers a range of PEEK capillaries and tubing. Used in combination with the right fittings, they provide an inert surface for the Fast LC of sensitive biomolecules. View Here. | [picture]

Poroshell 120

• High efficiency and high resolution, with up to 50% less backpressure than sub-2 μm columns

• 2 μm frit, for rugged performance with dirty samples

• Compatible with 400 bar and 600 bar LCs, as well as UHPLC instruments

• An expanding family of bonded phases to align with the ZORBAX Family, for reliable scalability

• Excellent selectivity and peak shapes

• Designed for exceptional reproducibility

Agilent Poroshell 120 columns are a 2.7 μm particle with a 1.7 μm solid core and 0.5 μm porous outer layer. This small particle size provides high efficiency, similar to sub-2 μm columns, but with 40-50% less pressure. These high efficiency, high resolution columns can be used on any type of LC. The porous outer layer and solid core limit diffusion distance and improve separation speed while the narrow particle size distribution improves efficiency and resolution. The columns can support high pressure and multiple columns can be used for the highest resolution and efficiency possible. The same principles are used in Poroshell 300 columns, ideal for fast, high resolution separations of biomolecules.

[picture] | TIPS & TOOLS Watch the Poroshell 120 Method Transfer Video to learn how easy it is to transfer existing methods to Poroshell 120 at www.agilent.com/chem/poroshell120video

Poroshell 120 Columns

Environmental phenols on Poroshell 120

Column A: | Poroshell 120 EC-C18 695975-902 4.6 x 100 mm, 2.7 μm
Column B: | Eclipse Plus C18 959964-902 4.6 x 100 mm, 1.8 μm

Gradient:

A: Water 0.1% formic acid
B: Acetonitrile 0.1% formic acid
2 mL/min
Initial: 8% B
10 min: 30% B

Detector: | 275 nm, 2 mm flow cell
Injection: | 10 μL

Agilent 1200 SL 40 °C

No pulse damper

No mixer 3 μL heater

Poroshell 120 provides sub-2 μm like efficiency at lower pressure.

1. Hydroquinone

2. Resorcinol

3. Catechol

4. 4-Nitrophenol

5. p-cresol

6. o-cresol

7. 2-Nitrophenol

8. 2,3 Dimethyl phenol

9. 2,5 Dimethyl phenol

10. 1-Naphthol

UHPLC efficiency at HPLC pressures

Column A: | Poroshell 120 EC-C18 695975-302 3.0 x 100 mm, 2,7 μm
Column B: | Eclipse Plus C18 959964-302 3.0 x 100 mm, 1.8 μm
Mobile Phase: | 60% Acetonitrile:40% water
Flow Rate: | 0.58 mL/min
Temperature: | 26 °C
Injection Volume: | 4 μL
Detector: | DAD Sig = 254,4 nm Ref = 360,100 nm
Sample: | RRLC checkout sample (P/N 5188-6529) spiked w/50 μL 2 mg/mL thiourea in water/acetonitrile (65:35)

For this sample of neutral alkylphenones, the Poroshell 120 column delivered >90% of the efficiency attained by the 1.8 μm column. Also note that the pressure on the Poroshell 120 column is about 50% of the pressure on the 1.8 μm column.

HPLC separation of 12 phenols performed in just 5 minutes – and under 400 bar – using an Agilent Poroshell 120 EC-C18 column

Column: | Poroshell 120 EC-C18 699975-902 4.6 x 50 mm, 2.7 μm
Mobile Phase: | Solvent A: Water with 0.1% formic acid Solvent B: Acetonitrile

Gradient:

5% B in 0.8 min
60% B in 6.8 min
1200 SL controlled temperature
at 25 °C 2 mm flow cell

Detector: | DAD, 270 nm

Importantly, the flow rate was kept to 2.5 mL/min, reducing the amount of mobile phase consumed per analysis to about 15 mL.

Agilent Poroshell 120 gives high efficiency, high resolution separations quickly at HPLC pressures.

1. Hydroquinone

2. Resourcinol

3. Catechol

4. Phenol

5. 4-Nitrophenol

6. p-cresol

7. o-cresol

8. 2-Nitrophenol

9. 3,4 di methyl phenol

10. 2,3 di methyl phenol

11. 2,5 di methyl phenol

12. 1-napthol

12 phenols analyzed using a longer (4.6 x 100 mm) Agilent Poroshell 120 EC-C18 column

Column: | Poroshell 120 EC-C18 695975-902 4.6 x 100 mm, 2.7 μm
Mobile Phase: | Solvent A: Water with 0.1% formic acid Solvent B: Acetonitrile

Gradient:

5% B in 2 min
60% B in 17 min
1200 RRLC SL
controlled temperature
at 25 °C 2 mm flow cell

Detector: | DAD, 270 nm

By reducing the flow rate to 2.0 mL/min, the pressure was kept to less than 400 bar improving the separation of a late-eluting peak pair (highlighted) with only a minor increase in analysis time. This separation can be achieved using HPLC or, if a higher flow rate is desired, a UHPLC.

1. Hydroquinone

2. Resourcinol

3. Catechol

4. Phenol

5. 4-Nitrophenol

6. p-cresol

7. o-cresol

8. 2-Nitrophenol

9. 3,4 di methyl phenol

10. 2,3 di methyl phenol

11. 2,5 di methyl phenol

12. 1-napthol

Poroshell 120 EC-C18 for fast UHPLC separations

Column: | Poroshell 120 EC-C18 695975-302 3.0 x 100 mm, 2.7 μm
Mobile Phase: | 65% A: 0.2% Formic acid 35% B: Methanol Isocratic
Flow Rate: | Varies
Temperature: | 26 °C
Detector: | Sig = 220, 4 nm, Ref = Off

This example shows a fast separation using a mobile phase that generates higher pressures. In the top chromatogram, a 3.0 mm id column was used, with a flow rate of 0.5 mL/min and a pressure below 400 bar – making this a typical LC separation.

Although the top separation was fast (just under 6 minutes), the middle and bottom chromatograms show that you can reduce run times to under 3 minutes by increasing the flow rate. These faster analyses will take your pressure to 400-560 bar; look to the Agilent 1200 Infinity Series flexible upgrade options to help you take advantage of UHPLC capabilities.

More viscous solvents like methanol can be used at HPLC or UHPLC pressures.

1. Saccharin

2. Caffeine

3. P-hydroxybenzoic acid

4. Aspartame

5. Dehydroacetic acid

6. Benzoic acid

[picture] | TIPS & TOOLS For a full listing of our LC capillary portfolio, View Here.

ZORBAX Rapid Resolution High Definition (RRHD) 1.8 μm

• High pressure (1200 bar) columns for optimum results with the 1290 Infinity LC or other UHPLC instruments

• 1.8 μm particles deliver maximum resolution for the most defined separations

• Available in 12 ZORBAX phases, including Eclipse Plus C18 for superior peak shape, ZORBAX StableBond C18 for low pH stability, Bonus-RP, Eclipse PAH, Eclipse Plus Phenyl-Hexyl and Extend-C18

• Also available in HILIC Plus

• Achieve the same selectivity on 3.5 and 5 μm ZORBAX columns with the same bonded phase for compatibility with any LC

ZORBAX Rapid Resolution High Definition (RRHD) columns are an expansion of the ZORBAX 1.8 μm particle column line. The new RRHD columns use improved packing processes to achieve stability up to 1200 bar for use with the Agilent 1290 Infinity LC or other UHPLC instruments. RRHD 1.8 μm columns are available in 50, 100 and 150 mm lengths for fast or high resolution – truly high definition – separations of your most complex samples.

ZORBAX Rapid Resolution High Definition (RRHD) 1.8 μm Columns

Separation of licorice root on RRHD columns

Column A: | ZORBAX RRHD SB-C18 857700-902 2.1 x 50 mm, 1.8 μm
Column B: | 858700-902 2.1 x 100 mm, 1.8 μm
Column C: | 859700-902 2.1 x 150 mm, 1.8 μm
Mobile Phase: | 10-100% B/30 min A: 0.1% Formic acid (fa) B: Acetonitrile with 0.1% fa
Flow Rate: | F = 0.4 mL/min
Gradient: | 30 minute gradient on each length
Temperature: | Ambient
Detector: | 280 nm UV
Instrument: | 1290 Infinity LC

Sub 1 minute separations with RRHD columns

Column: | ZORBAX RRHD SB-C18 857700-902 2.1 x 50 mm, 1.8 μm
Gradient: | H₂O (0.05% trifluoroacetic acid)/10-40% ACN/1 min
Temperature: | 60 °C
Injection Volume: | 0.5 μL x 100 ppm each
Detector: | UV, 275 nm
Data Rate: | 160 Hz

1. 2-methyl-4-isothiazolin-3-one

2. 5-chloro-2-methyl-4-isothiazolin-3-one

3. Carbendazim

4. Benzisothiiazol-3(2H)-one

5. 2-phenoxyethanol

6. Methylparaben

New levels of sensitivity and resolution

Column A: | ZORBAX RRHD Eclipse Plus C18 959758-302 3.0 x 100 mm, 1.8 μm
Column B: | ZORBAX RRHD Eclipse Plus C18 959757-302 3.0 x 50 mm, 1.8 μm
Ion Source: | 360 °C, 12 L/min. 50 psi, 3500 V.
Mobile Phase: | A: 0.2% Formic acid in water B:ACN
Temperature: | Ambient, no temperature control (approx 24 °C)
Detector: | Agilent 1290 Infinity LC with 6410 MS/MS
Sample: | 20 μL (10 μL for 50 mm column) of 1 μg/mL standard

Compounds (in elution order) with identifying mass:

1. Acetaminophen, m/z 109

2. Caffeine, m/z 194

3. 2-acetamidophenol, m/z 109

4. Acetanilide, m/z 135

5. Acetylsalicylic acid, m/z 120

6. Phenacetin, m/z 179

7. Salicylic acid, m/z 120

8. Sulindac, m/z 356

9. Piroxicam, m/z 332

10. Tolmetin, m/z 257

11. Ketoprofen, m/z 254

12. Diflunisal, m/z 332

13. Diclofenac, m/z 235

14. Celecoxib, m/z 351

15. Ibuprofen, m/z 160

By transferring your method to an Agilent RRHD column, you can enhance resolution for difficult analyses – allowing you to save time by using shorter columns without compromising performance.

The RRHD column saves analytical time without sacrificing performance.

Selectivity comparison: C18 columns

Column A: | ZORBAX RRHD Eclipse Plus C18 959758-902 2.1 x 100 mm, 1.8 μm
Column B: | ZORBAX RRHD Eclipse XDB-C18 981758-902 2.1 x 100 mm, 1.8 μm
Column C: | ZORBAX RRHD Extend-C18 758700-902 2.1 x 100 mm, 1.8 μm
Column D: | ZORBAX RRHD SB-C18 858700-902 2.1 x 100 mm, 1.8 μm
Mobile Phase: | A: 0.1% HCOOH in H₂O (30%) B: 0.1% HCOOH in CH₃CN (70%)
Flow Rate: | 1 mL/min, isocratic
Temperature: | 30 °C
Sample: | 1 μL

MS2 Scan:

290-390, ESI positive mode,
scan time: 500, fragmentor:
135 V; drying gas: 12 L/min, 325 °C;
nebulizer pressure: 35 psig;
capillary voltage: 3000

Selectivity differences are due to subtle, yet important variations, such as bonding type, endcapping, or the amount and type of silanols on the silica. Other factors that influence selectivity include mobile phase composition, temperature, and pH. (Note that these factors are identical in the following example.)

Here we compared the selectivity of four Agilent ZORBAX RRHD C18 columns using an endocannabinoid analysis method.

1. Anadamine (AEA), m/z 348

2. Palmitoylethanolamide (PEA), m/z PEA

3. 2-arachinoylglycerol (2-AG), m/z 379*

4. Oleoylethanolamide (OEA), m/z 326

*A fifth peak with a mass of 379 was also detected. This impurity is believed to be 1,3 arachidonolyglycerol, a rearrangement of 2-AG.

[picture] | TIPS & TOOLS For full details, see Agilent publication 5990-7166EN, www.agilent.com/chem/library

Selectivity comparison: Phenyl and other columns

Column A: | ZORBAX RRHD Eclipse Plus C18 959758-902 2.1 x 100 mm, 1.8 μm
Column B: | ZORBAX RRHD Eclipse Plus Phenyl-Hexyl 959758-912 2.1 x 100 mm, 1.8 μm
Column C: | ZORBAX RRHD SB-Aq 858700-914 2.1 x 100 mm, 1.8 μm
Column D: | ZORBAX RRHD SB-Phenyl 858700-912 2.1 x 100 mm, 1.8 μm
Mobile Phase: | A: 5% HCOOH in H₂O B:CH₃CN
Flow Rate: | 0.65 mL/min
Gradient: | 10-50% B in 15 min
Temperature: | 30 °C
MS2 Scan: | ESI +, 200-1000

Extracted ion chromatograms from LC/MS scan data of blueberry anthocyanins.

TIPS & TOOLS For full details, see Agilent publication 5990-8470EN, www.agilent.com/chem/library | [picture]

ZORBAX Rapid Resolution High Throughput (RRHT) 1.8 μm

• High pressure (600 bar) columns for ultra high speed or maximum resolution analyses with Rapid Resolution HT columns packed with totally porous, 1.8 μm packings

• Carefully engineered particles deliver maximum resolution at 25% less pressure than other sub-2 μm materials

• Reduce analysis time by up to 95%

• Develop HPLC methods more quickly

• Securely transfer conventional methods with over 140 RRHT column choices

• Analyze complex samples on shorter columns faster and maximize peak capacity

• Matching selectivity in 3.5, 5 and 7 μm particle sizes for complete method scalability

• Short (50 mm long and less) column can be used on some conventional LCs

Agilent ZORBAX Rapid Resolution HT (1.8 μm) columns use a totally porous, 1.8 μm particle to provide maximum resolution in fast, ultra-fast and high resolution analyses. You can reduce analysis time by up to 95% in comparison to 250 mm length columns. With more than 140 RRHT column choices, including the high performance ZORBAX Eclipse Plus and many other ZORBAX column choices (Eclipse XDB, StableBond, Extend, Bonus-RP), methods can be developed quickly or securely transferred to a smaller particle size column with no loss in resolution. The small particle size provides double the efficiency of a 3.5 μm column in the same column length, providing the highest efficiency and resolution possible. This permits the analysis of complex samples on shorter columns with the highest resolution and peak capacity. The 1.8 μm Rapid Resolution HT columns take high-speed, high-resolution HPLC to a new level.

The 600 bar columns can be used with the Agilent1260 Infinity LC System up to this high pressure limit. In addition, the shorter columns can be used on many other LC’s, including the Agilent 1200 Rapid Resolution LC System.

ZORBAX Rapid Resolution High Throughput (RRHT) 1.8 μm Columns

Rapid Resolution HT (RRHT) provides double the efficiency of Rapid Resolution columns

Column A: | ZORBAX Rapid Resolution SB-C18 835975-902 4.6 x 50 mm, 3.5 μm
Column B: | ZORBAX RRHT SB-C18 827975-902 4.6 x 50 mm, 1.8 μm
Mobile Phase: | 25% Water, 75% MeOH
Flow Rate: | 1.5 mL/min
Temperature: | Ambient
Detector: | UV, 254 nm

This figure shows that Rapid Resolution HT columns can provide double the efficiency of a 3.5 μm column in the same column length. This high efficiency can be used for very high-resolution, high throughput analyses.

Increase peak capacity with RRHT columns

Column A: | Eclipse RRHT XDB-C8 928700-906 2.1 x 100 mm, 1.8 μm
Column B: | Eclipse XDB-C18 961753-902 2.1 x 100 mm, 3.5 μm
Mobile Phase: | A: H₂O B: ACN
Peak capacity: | A: 461 B: 343
Flow Rate: | 0.5 mL/min
Gradient: | 0.0 min 50% B 20.0 min 100% B
Temperature: | 40 °C
Detector: | UV, 254 nm
Sample: | Alkylphenones

1. Uracil

2. C₃-Alkylphenone

3. C₄-Alkylphenone

4. C₅-Alkylphenone

5. C₆-Alkylphenone

6. C₇-Alkylphenone

7. C₈-Alkylphenone

8. C₉-Alkylphenone

9. C₁₀-Alkylphenone

10. C₁₂-Alkylphenone

11. C₁₄-Alkylphenone

Reduce analysis time dramatically with Rapid Resolution HT columns

Column A: | Eclipse XDB-C18 990967-902 4.6 x 250 mm, 5 μm
Column B: | Eclipse XDB-C18 963967-902 4.6 x 150 mm, 3.5 μm
Column C: | Eclipse XDB-C18 966967-902 4.6 x 75 mm, 3.5 μm
Column D: | ZORBAX Eclipse XDB-C18 935967-902 4.6 x 50 mm, 3.5 μm
Column E: | Eclipse RRHT XDB-C18 925975-902 4.6 x 50 mm, 1.8 μm
Mobile Phase: | 73% MeOH:27% 20 mM Phosphate Buffer, pH 7.0
Flow Rate: | 1 mL/min
Temperature: | Ambient
Detector: | UV, 254 nm

1. Uracil

2. Naproxen

3. Mefanamic acid

4. Butyl paraben

5. Propranolol

6. Naphthalene

7. Dipropyl phthalate

This figure shows the dramatic reduction in analysis time made possible by using Rapid Resolution HT columns. Chromatogram A shows a separation that takes 11.5 minutes on a 25 cm, 5 μm column. Rapid Resolution (3.5 μm) columns, shown in chromatogram B and C, reduce analysis time substantially, but with a slight compromise in resolution. The Rapid Resolution HT column reduces analysis time to 2.2 minutes, an 80% reduction, while still maintaining baseline resolution.

Long lifetime of RRHT columns at elevated temperatures

Column: | ZORBAX RRHT SB-C18 827700-902 2.1 x 50 mm, 1.8 μm
Mobile Phase: | A: 60% H₂O B:40% ACN
Flow Rate: | 1 mL/min
Temperature: | 80 °C
Detector: | UV, 254 nm
Sample: | QC test mix

[picture] | TIPS & TOOLS The LC Rack from Agilent can help you reduce capillary lengths and minimize extra-column volume. It also protects your instrument and enables you to switch out modules as needed.

Agilent rack for LC systems, 5001-3726

Agilent Fast Guards for UHPLC

• High performance guard columns for Fast LC columns

• Two formats – one for Poroshell 120 columns, stable to 600 bar, RRHD columns, 1.8 μm (stable to 1200 bar), and RRHT columns, 1.8 μm (stable to 600 bar)

Agilent UHPLC Guards are high performance guards designed by Agilent for its Fast LC columns families. Agilent UHPLC Guards use easy-to-install hardware that fits directly on the end of the column; no extra hardware is needed. They are sold in packages of three.

Agilent UHPLC Guards extend the lifetime of analytical columns without diminishing performance.

UHPLC Guard, 1200 bar, 821725-903

[picture] | TIPS & TOOLS Learn about Fast Guards for UHPLC – an easy way to extend the life of your analytical Fast LC column without losing performance. www.agilent.com/chem/fastguardsvideo | [picture]

Other Columns for Reversed-Phase Analytical HPLC

Achieve excellent peak shape and resolution every time – leveraging the industry’s broadest selection of reversed-phase columns

Whether you are using Fast LC or working with more conventional HPLC applications, Agilent’s LC family offers you a range of phases and selectivities to help you perfect your separation.

The ZORBAX Family of phases scales readily to Fast LC columns in the Rapid Resolution High Throughput (RRHT) and Rapid Resolution High Definition (RRHD) families and Poroshell 120 columns, see here.

In this section, we’ll provide overviews of other key analytical columns from Agilent:

ZORBAX Rapid Resolution, 3.5 μm, configurations are an ideal choice for initial method development, providing increased sample throughput for any application when compared to 5 μm columns.

ZORBAX Solvent Saver 3.0 mm id column configurations provide 60% mobile phase reduction over 4.6 mm id columns.

ZORBAX Eclipse Plus HPLC columns are designed to reliably produce superior peak shapes for basic compounds, and are available across all ZORBAX column configurations.

More than 13 additional ZORBAX phases including StableBond, Eclipse PAH, Eclipse XDB, ZORBAX Rx, Extend-C18, Bonus-Rx and Original ZORBAX columns – in total, more than 1400 configurations for reliable scalability and method transfer.

ZORBAX Method Development kits contain three columns for the price of two! Each as a different bonded phase for optimizing selectivity.

ZORBAX Method Validation kits – choose as many columns as you need (or as few) to make method validation easier and less expensive.

Pursuit, Pursuit XRs and Pursuit XRs Ultra columns provide alternate selectivities to the ZORBAX family.

Polaris Columns provide polar-modified phases for routine polar applications.

Other Columns for Reversed-Phase Analytical HPLC.

ZORBAX ECLIPSE Plus

• The ideal column for method development – excellent results for a wide range of compounds

• High level of performance – peak shape, efficiency, resolution, and lifetime – with all sample types: acids, bases and neutrals

• Superior reproducibility with more rigorous QA/QC testing

• Improved, patented silica manufacturing with start-to-finish product control

• Available in 1.8, 3.5, and 5 μm particle sizes for all analytical, high resolution, and fast LC analyses

ZORBAX Eclipse Plus Columns

Agilent ZORBAX Eclipse Plus columns provide the ultimate in performance for silica-based columns. Peak shape is excellent for the most challenging basic compounds, improving efficiency and resolution with these sample types. These results are achieved by improvements in the silica manufacturing and bonding technology, which is completely controlled by Agilent.

Because of their high level of performance, Eclipse Plus columns are the ideal first choice for method development of all samples. If you need to achieve fast method development and superior productivity, then choose a column with high-resolution 1.8 μm particles. For standard methods, conventional 5 μm and Rapid Resolution 3.5 μm columns are your best choice. With all particle sizes, easy method transfer is possible.

With more rigorous QA and QC testing, column lot-to-lot reproducibility is also improved, resulting in long-term reliable results for all analyses.

*Column lifetime will be reduced significantly at pH >7 and temperature >40 °C. At pH 6-9, highest column stability for all silica based columns is obtained by operating at temperatures <40 °C and using lower buffer concentrations in range of 0.01-0.02 M, especially with phosphate and carbonate buffers.

[picture] | TIPS & TOOLS The EC-C18, EC-C8 and Phenyl-Hexyl phases on Poroshell 120 are very similar to Eclipse Plus C18, Eclipse Plus C8 and Eclipse Plus Phenyl-Hexyl phases. View Here.

ZORBAX Eclipse Plus: Best peak shape in the industry without tailing

Column: | Eclipse Plus C18 959996-902 4.6 x 100 mm, 5 μm
Mobile Phase: | A: 60% Water B: 40% Acetonitrile
Flow Rate: | 1.0 mL/min
Temperature: | Ambient
Detector: | UV, 254 nm
Sample: | Pyridine, Phenol

1. Pyridine

2. Phenol

Peak shape and efficiency are better with ZORBAX Eclipse Plus

Column A: | XBridge C18, 4.6 x 150 mm, 5 μm
Column B: | Eclipse Plus C18 959993-902 4.6 x 150 mm, 5 μm
Mobile Phase: | A: 0.1% Formic acid B: 0.1% Formic acid in ACN
Flow Rate: | 1.0 mL/min
Gradient: | 0.0 min 10% B 15 min 30% B
Temperature: | 40 °C
Detector: | UV, 254 nm
Sample: | Sulfonamides

1. Sulfanilamide

2. Sulfadiazine

3. Sulfathiazole

4. Sulfamerazine

5. Sulfmethazine

6. Sulfamethoxazole

Eliminate tailing and maximize resolution with Eclipse Plus Columns

Column A: | Eclipse Plus C18, 4.6 x 50 mm
Column B: | Competitive C18, 4.6 x 50 mm
Mobile Phase: | 65% ACN:35% 25 mM phosphate buffer (pH 7.4)

Superior peak shape and better selectivity with Eclipse Plus means more resolution, easier quantitation and better results in your separations.

1. Nortriptyline

2. Dipropylthalate

Eclipse Plus C18 vs. C8

Column A: | Eclipse Plus C18 4.6 x 50 mm, 5 μm
Column B: | Eclipse Plus C8 4.6 x 50 mm, 5 μm
Mobile Phase: | Water: acetonitrile (30:70)
Flow Rate: | 2.0 mL/min
Temperature: | 30 °C
Detector: | UV, 230 nm
Sample: | Lip balm extract in ACN (melted at 100 °C, cooled and 0.45 μm filtered)

Less retention can save significant time – the C8 is a good choice here.

1. Oxybenzone

2. Internal Std.

3. Octylmethoxycinnamate

Rapid analysis of an analgesic tablet, selectivity differences at pH 2 and pH 7

Column: | Eclipse Plus C8 959946-906 4.6 x 50 mm, 5 μm
Gradient: | 10-60% B/3 min pH 2.7: A: 0.1% Formic acid B: 0.1% fa in ACN pH 7.0: A: 20 mM Na phosphate B: ACN
Sample: | generic Excedrin tablet

Both Eclipse Plus C18 and C8 can be used over a wide pH range to optimize selectivity or analysis time.

1. Acetaminophen

2. Caffeine

3. Acetylsalicylic acid

4. Unknown

Eclipse Plus C8 is less retentive than Eclipse Plus C18

Column A: | Eclipse Plus C8 959996-906 4.6 x 100 mm, 5 μm
Column B: | Eclipse Plus C18 959996-902 4.6 x 100 mm, 5 μm
Mobile Phase: | 80% Methanol 8 mM (total) K₂HPO₄ pH 7
Flow Rate: | 1.0 mL/min
Detector: | UV, 215 nm
Sample: | Amitriptyline 0.05 μg/μL (0.5 μL injection)

A C8 column is typically selected because it will retain less than a C18 column, reducing analysis time.

The Eclipse Plus C8 column shows the same behavior with excellent peak shape on difficult basic compounds.

Fast and ultra-fast analysis of basic compounds on Eclipse Plus

Column A: | Eclipse Plus C18 959941-902 4.6 x 50 mm, 1.8 μm
Column B: | Eclipse Plus C18 959993-902 4.6 x 150 mm, 5 μm
Mobile Phase: | A: 50% 8 mM K₂HPO₄, pH 7 B:50% ACN
Flow Rate: | 1.0 mL/min
Temperature: | Ambient
Detector: | UV, 254 nm
Sample: | Berberine, 0.4 mg/mL, 2 μL

1. Berberine

ZORBAX Eclipse Plus Columns

Agilent HILIC Plus uses the same manufacturing processes as the Eclipse Plus family. See information about ZORBAX HILIC Plus Here.

ZORBAX Eclipse PAH

• High resolution separation of 16 PAHs in EPA Method 610

• Extensive range of particle sizes (1.8, 3.5 and 5 μm) and sizes for fast and high resolution separations

• Each batch of material is specifically tested with PAHs for maximum reproducibility under expected operating conditions

• Excellent performance using the high quality, improved silica of Eclipse Plus columns

• Good for applications requiring “shape selectivity” or the separation of geometric isomers

Agilent ZORBAX Eclipse PAH columns are recommended for the separation of polycyclic aromatic hydrocarbons. PAHs are considered priority pollutants and the analysis of these potentially carcinogenic compounds in water, soil and food is of major importance. Eclipse PAH columns separate all 16 PAHS in EPA method 610 quickly and with high resolution.

ZORBAX Eclipse PAH Columns

High resolution and fast analysis on RRHT Eclipse PAH column

Column: | Eclipse PAH 959941-918 4.6 x 50 mm, 1.8 μm |
Mobile Phase: | A: Water; B: Acetonitrile |
Gradient: | Time (Min) | %B
| 0.00 | 40
| 3.5 | 100
| 5.2 | 100
| 5.5 | 40
| 6.5 | 40
Flow Rate: | 2.0 mL/min
Temperature: | 25 °C
Detector: | DAD 220, 4 nm No Ref. DAD Stop Time = 6.0 min Stop Time = 7.0

ZORBAX Eclipse XDB

• Four selectivity choices for method development optimization

• Good peak shape for basic, acidic and neutral compounds

• High performance over a wide pH range – pH 2-9

• Particle sizes from 1.8 to 7 μm

• Long lifetime with eXtra Dense Bonding and double endcapping

Agilent ZORBAX Eclipse XDB columns – C18, C8, Phenyl and CN – provide four bonded phase choices for method development optimization. These columns provide good peak shape over a wide pH range (2-9) for additional method development flexibility with one family of columns. Eclipse XDB columns can be used for method development at low pH (2-3) and the same column can be used for method development in the mid pH (6-8) region. In the mid pH region residual silanols are more active and tailing interactions are more likely. To overcome these interactions, Eclipse XDB columns are eXtra Densely Bonded and double endcapped through a proprietary process to cover as many active silanols as possible. The result is superior peak shape of basic compounds from pH 2-9. Eclipse XDB columns are available in 1.8, 3.5, 5 and 7 μm particle sizes for high speed, high resolution, analytical and prep scale separations.

eXtra Densely Bonded and Double Endcapped Eclipse XDB Bonded Phase

*Eclipse XDB columns are designed for operation over a wide pH range.At pH 6-9, highest columns stability for all silica based columns is achieved by operating at temperatures <40 °C and using low buffer concentrations in the range of 0.01-0.02 M.

[picture] | TIPS & TOOLS Poroshell 120 EC-CN is very similar to ZORBAX XDB-CN. View Here

Good peak shape over a wide pH range with ZORBAX Eclipse XDB

Column: | Eclipse XDB-C8 993967-906 4.6 x 150 mm, 5 μm
Mobile Phase: | A: pH 3.0 75% 25 mM phosphate buffer 25% ACN B: pH 7.0 90% 20 mM phosphate 10% ACN
Flow Rate: | 1.5 mL/min
Temperature: | 40 °C

ZORBAX Eclipse XDB columns provide good peak shape over a wide pH range and are an excellent choice for method development from pH 2-9.

Column stability testing at pH 3 and 60 °C

Column: | ZORBAX SB-C8 883975-906 4.6 x 150 mm, 5 μm
Column: | Eclipse XDB-C8 993967-906 4.6 x 150 mm, 5 μm

Mobile Phase:

Purge Conditions:
70% 50 mM NaAc-HCl, pH 3.0
30% ACN
Retention Test Conditions:
65% Methanol
35% Water

Flow Rate: | 1.0 mL/min
Temperature: | 60 °C
Sample: | Tricyclic antidepressants

Eclipse XDB columns are stable over a wide pH range. At low pH an Eclipse endcapped column is extremely stable and shows equivalent stability to a non-endcapped column, SB-C8, at pH 3. The columns were purged with a pH 3 mobile phase at 60 °C. Then they were tested with a strongly basic compound to determine if the endcapping or bonded phase had been hydrolyzed from the silica surface. The Eclipse XDB column was very stable, as shown by the consistency of the retention of amitriptyline over the 12,000 column volumes of the test. Another endcapped column shows less stability under these same conditions.

Column stability testing at pH 7.0

Column A: | Competitive C8 SIL-type After 1826 column volumes

Column B:

Eclipse XDB-C8
993967-906
4.6 x 150 mm, 5 μm
Sol-type
After 1843 column volumes

Mobile Phase: | 60% ACN 40% 250 mM Phosphate Buffer, pH 7.0
Flow Rate: | 1.5 mL/min
Temperature: | 60 °C
Sample: | Tricyclic antidepressants

1. Uracil

2. Nortriptyline

3. Doxepin

4. Amitriptyline

5. Trimipramine

Double endcapping, dense bonding and the durable Rx-Sil particles (sol-type) combine to provide long lifetime at pH 7 when compared to single endcapped sil-gel columns used here. The conditions used for this test – high temperature (60 °C) and high salt concentration (250 mM), accelerate the dissolution of silica, causing premature failure of the sil-gel type column.

Selectivity changes for basic compounds with Eclipse XDB and StableBond

Column A: | Eclipse XDB-C8 966967-906 4.6 x 75 mm, 3.5 μm
Column B: | ZORBAX Rx/SB-C8 866953-906 4.6 x 75 mm, 3.5 μm
Mobile Phase: | 70% 25 mM NaH₂PO₄, pH 3.0 30% Methanol
Flow Rate: | 1.0 mL/min
Temperature: | 35 °C

1. Barbital

2. Sulfamethoxazole

3. Caffeine

Eclipse XDB and StableBond columns are based on the same silica but have different bonding and endcapping. Therefore, they can have very different selectivity for the same sample under the same conditions, as this example shows.

Optimize separations with Eclipse XDB selectivity options

Column A: | Eclipse XDB-Phenyl 963967-912 4.6 x 150 mm, 3.5 μm
Column B: | Eclipse XDB-C8 963967-906 4.6 x 150 mm, 3.5 μm
Column C: | Eclipse XDB-C18 963967-902 4.6 x 150 mm, 3.5 μm
Mobile Phase: | 15% H₂O:85% MeOH
Flow Rate: | 1.0 mL/min
Temperature: | 35 °C
Sample: | Sunscreens

1. Oxybenzone

2. Padimate-O

3. Ethylhexylsalicylate

This separation of sunscreens on all three Eclipse XDB bonded phases – C18, C8 and Phenyl – shows that different bonded phases can be used to optimize a separation. While all three bonded phases provide an adequate separation, the Eclipse XDB-Phenyl provides a different peak elution order and a much shorter overall analysis time. All three bonded phases also provide excellent peak shape with no mobile phase additives.

Selectivity for urea pesticides

Column A: | Eclipse XDB-C18 993967-902 4.6 x 150 mm, 5 μm
Column B: | Eclipse XDB-CN 993967-905 4.6 x 150 mm, 5 μm
Column C: | Eclipse XDB-C18 993967-902 4.6 x 150 mm, 5 μm
Mobile Phase: | A. 60:40 MeOH:Water B. 60:40 MeOH:Water C. 77:23 MeOH:Water
Flow Rate: | 1.0 mL/min
Temperature: | 25 °C
Sample: | Urea pesticides

1. Fenuron

2. Monuron

3. MonoLinuron

4. Diuron

5. Linuron

6. Pencycuron

The Eclipse XDB-CN column reduces retention time and provides good selectivity for Urea pesticides when compared to an Eclipse XDB-C18 column.

ZORBAX 80Å StableBond

• Longest column lifetime and best reproducibility for low pH separations – down to pH 1

• Patented stable column chemistry allows use at high temperature and low pH without degradation

• Six different bonded phases provide broad selectivity – SB-C18, SB-C8, SB-CN, SB-Phenyl, SB-C3, and SB-Aq

• High purity (Type B) silica for good peak shape

Agilent ZORBAX StableBond columns use patented, unique, nonfunctional silanes with bulky diisobutyl (SB-C18) or diisopropyl (SB-C8, SB-C3, SB-Phenyl, SB-CN, and SB-Aq) side chain groups that sterically protect the key siloxane bond to the silica surface from hydrolytic attack at low pH. StableBond packing materials are not endcapped in order to provide exceptional stability and to maximize lifetime and reproducibility under acidic mobile phase conditions. The high purity, low acidity silica provides excellent peak shape with acidic, basic and neutral compounds making StableBond columns an excellent choice for low pH method development. ZORBAX StableBond columns are compatible with all common mobile phases, including very high aqueous mobile phases.

Sterically Protected StableBond Bonded Phase

*StableBond columns are designed for optimal use at low pH. At pH 6-8, highest column stability for all silica-based columns is obtained by operating at temperatures <40 °C and using lower buffer concentrations in the range of 0.01-0.02 M. At mid-range pH, Eclipse Plus, Eclipse XDB and Bonus-RP are recommended.

[picture] | TIPS & TOOLS ZORBAX StableBond SB-C18, SB-C8 and SB-Aq phases are also available on Poroshell 120. View Here

StableBond SB-C18 shows excellent stability at low pH and high temperature (pH 0.8, 90 °C)

Column: | ZORBAX SB-C18 883975-902 4.6 x 150 mm, 5 μm
Column: | ZORBAX Rx-C18 883967-902 4.6 x 150 mm, 5 μm
Mobile Phase: | 50% Methanol/50% Water with 1.0% TFA Test Solute: Toluene
Temperature: | 90 °C

As an indicator of column breakdown, retention time of toluene was measured after purging the column with mobile phase. Only the StableBond SB-C18 is unchanged after three working months of use under these very low pH (0.8) and high temperature (90 °C) conditions. ZORBAX Rx-C18 also provides a stable matrix, and can be used as an alternative selectivity to StableBond SB-C18.

Shorter chain ZORBAX SB-CN is also stable at low pH (pH 2.0, 50 °C)

Column: | ZORBAX SB-CN 883975-905 4.6 x 150 mm, 5 μm
Mobile Phase: | 0.1% TFA, pH 2:ACN
Flow Rate: | 1 mL/min
Gradient: | 0-100% ACN
Temperature: | 50 °C
Sample: | 1-phenylheptane @ 50% AC/50% water with 0.1% TFA

ZORBAX StableBond SB-CN and other short chain StableBond bonded phases are also exceptionally stable at low pH. Conventional dimethyl CN and similar bonded phases lack this stability.

SB-CN optimizes retention and resolution

Column A: | ZORBAX SB-C18 866953-902 4.6 x 75 mm, 3.5 μm
Column B: | ZORBAX SB-CN 866953-905 4.6 x 75 mm, 3.5 μm
Mobile Phase: | 30% ACN 70% 25mM NaH₂PO₄, pH 2.5
Flow Rate: | 1.0 mL/min
Temperature: | 35 °C

1. Estriol

2. Daidzen

3. Quercetin

4. Genistein

5. Diethylstilbestrol

The SB-CN column is used here to reduce analysis time by 50%. The retention of the most hydrophobic analyte is cut in half. At the same time, retention of the more polar, early eluting peaks increases slightly.

Five different bonded phases provide selectivity options

Column A: | ZORBAX SB-C18 883975-902 4.6 x 150 mm, 5 μm
Column B: | ZORBAX SB-C8 883975-906 4.6 x 150 mm, 5 μm
Column C: | ZORBAX SB-C3 883975-909 4.6 x 150 mm, 5 μm
Column D: | ZORBAX SB-Phenyl 883975-912 4.6 x 150 mm, 5 μm
Column E: | ZORBAX SB-CN 883975-905 4.6 x 150 mm, 5 μm

Mobile Phase:

0-100% B in 18.8 min
A: 50 mM NaH₂PO₄,
pH 2.5 in 95% H₂O/5% ACN
B: 50 mM NaH₂PO₄,
pH 2.5 in 47%H₂O/53% ACN

Flow Rate: | 1.0 mL/min
Temperature: | 26 °C
Detector: | 254 nm

SB-C3 is just one of the five different StableBond selectivity choices. In this example, optimum resolution is obtained with SB-C3. All are based on the same high purity Rx-SIL. Selectivity changes are therefore dependent only on the bonded phases, making method development more reliable.

ZORBAX Rx

• Recommended for alternate selectivity at low pH relative to Eclipse Plus C18, Eclipse XDB-C18 and StableBond SB-C18; for higher temperature applications, StableBond is recommended

• Higher carbon load than SB-C18 columns (12% vs. 10%)

• High stability and good peak shape for low pH applications (up to pH 8)

• Manufactured using dimethyloctadecylsilane and non-endcapped

• ZORBAX Rx-C8 is the same product as SB-C8

*At pH 6-9 highest column stability for all silica based columns is obtained by operating at temperatures <40 °C and using lower buffer concentrations in the range of 0.01-0.02 M.

Analysis of diazepam on Rx-C18

Column: | ZORBAX Rx-C18 880967-302 3.0 x 250 mm, 5 μm
Mobile Phase: | 35% H₂O:65% MeOH
Flow Rate: | 0.5 mL/min

1. Ethylparaben

2. Diazepam

An Rx-C18 column is used for this USP analysis of diazepam and the internal standard ethylparaben. The Solvent Saver 3.0 mm id Rx-C18 column reduces solvent usage by 60% over what would be used if the analysis was done on a 4.6 x 250 mm column.

ZORBAX 80Å Extend-C18

• High efficiency and long life at high pH – up to pH 11.5

• Unique bidentate bonding and double endcapping provides high pH stability

• More efficiency and better peak shape than polymer-based columns

• Improve retention, resolution and peak shape of basic compounds

• High sensitivity for LC/MS separations of peptides

The Agilent ZORBAX Extend-C18 column uses a novel bidentate C18-C18 bonding technology to make it possible to develop high-resolution separations at high pH with a silica-based column. At high pH, non-charged basic compounds will not interact with the underlying silica. The result is high efficiency separations with superior peak shape and improved resolution. High pH separations are also the best choice for compounds that are more stable or more soluble in high pH solutions. Some of the mobile phase buffer options for high pH include triethylamine, pyrrolidine, glycine, borate and ammonium hydroxide. Ammonium hydroxide at pH 10.5 is an excellent mobile phase modifier for the LC/MS of peptides and small molecules with improved sensitivity compared with TFA containing mobile phase at low pH. The Extend-C18 column is stable from pH 2-11.5 with good peak shape for all types of compounds. Extend-C18 columns also provide an additional selectivity choice at low pH.

TIPS & TOOLS

Always use Agilent Certified Lamps for Best LC Performance

Agilent detector lamps are built to the tightest specifications and quality standards. They are designed to increase light intensity and decrease noise, which improves chromatographic results. Agilent rigorously tests its lamps for lowest lamp-to-lamp variability. Trust Agilent lamps for robust, long-lasting performance and lower cost of ownership. To learn more, visit www.agilent.com/chem/lamps

Basic antihistamines on Extend-C18 at high pH

Column: | ZORBAX Extend-C18 773450-902 4.6 x 150 mm, 5 μm

Mobile Phase:

pH 7:
30% 20 mM Na₂HPO₄ 70% MeOH
pH 11:
30% 20 mM TEA 70% MeOH

Flow Rate: | 1.0 mL/min
Temperature: | Ambient
Detector: | 254 nm
Sample: | Antihistamines

1. Maleate

2. Scopolamine

3. Pseudoephedrine

4. Doxylamine

5. Chlorpheniramine

6. Triprolidine

7. Diphenhydramine

Pseudoephedrine and scopolamine are difficult to retain at low and mid pH. Pseudoephedrine is often analyzed by ion exchange methods. The Extend-C18 column retains these compounds in a noncharged form at high pH and improves resolution.

Long life at high pH with Extend-C18

Column: | ZORBAX Extend-C18 773450-902 4.6 x 150 mm, 5 μm
Mobile Phase: | 20% Methanol 80% 0.1 M Carbonate buffer, pH 10.0
Flow Rate: | 1.0 mL/min
Temperature: | Ambient

At high pH, columns will fail due to silica dissolution. The example here shows extended lifetime of ZORBAX Extend-C18 at high pH in comparison to competitor W. This was measured by the amount of dissolved silica.

Extend-C18 provides good peak shape at low pH

Column: | ZORBAX Extend-C18 773450-902 4.6 x 150 mm, 5 μm
Mobile Phase: | 80% 25 mM NaH₂PO₄, pH 3.0 20% Methanol
Flow Rate: | 1.0 mL/min
Temperature: | 35 °C
Detector: | UV, 254 nm
Sample: | Basic compounds

These basic compounds are separated on the Extend-C18 at low pH with excellent peak shape. The Extend-C18 column can be used at high and low pH.

1. Theobromine

2. Dimethylxanthine

3. Theophylline

4. Caffeine

ZORBAX Bonus-RP

• Excellent peak shape for challenging basic compounds at low and mid pH

• Unique reversed-phase selectivity

• Novel bonding technology with embedded polar group and steric protection

• Usable in 100% aqueous mobile phases

The Agilent ZORBAX Bonus-RP column has a polar amide group embedded in a long alkyl chain. This novel bonding reduces interactions between basic compounds and the silica support, improving peak shape for the most difficult basic compounds. Peak shape and column lifetime are further improved by triple endcapping. In addition, diisopropyl side groups provide steric protection against acid hydrolysis for good lifetime at low pH. The Bonus-RP column provides an alternate selectivity to C18 and C8 alkyl bonded phases.

Unique, Polar Alkyl Bonus-RP Bonded Phase

[picture] | TIPS & TOOLS ZORBAX Bonus-RP is also available on Poroshell 120. View here

Improved peak shape of basic compounds using Bonus-RP

Column: | Alkyl-C8 4.6 x 150 mm, 5 μm
Mobile Phase: | 75% 25 mM NH₄OAc, pH5.5 25% ACN
Flow Rate: | 1.5 mL/min
Temperature: | 40 °C
Detector: | 254 nm

Column: | ZORBAX Bonus-RP 883668-901 4.6 x 150 mm, 5 μm
Mobile Phase: | 80% 25 mM NH₄OAc, pH5.5 20% ACN
Flow Rate: | 1.5mL/min
Temperature: | 40 °C
Detector: | 254 nm

1. Doxylamine

2. Chlorpheniramine

3. Triprolidine

1. Doxylamine

2. Chlorpheniramine

3. Triprolidine

Bonus-RP eliminates peak tailing of these basic compounds in comparison to a typical alkyl C8 bonded phase. In the mid-pH region, residual silanols can interact more strongly with basic compounds to cause peak tailing. The polar group in the Bonus-RP bonded phase eliminates peak tailing of these basic compounds by reducing interactions with residual silanols.

ZORBAX Bonus-RP is stable at low and mid pH

Column: | ZORBAX Bonus-RP 883668-901 4.6 x 150 mm, 5 μm
Mobile Phase: | 60% 25 mM Phosphate Buffer, pH 7.0:40% ACN
Flow Rate: | 1.5 mL/min
Temperature: | 23 °C

Triple endcapping of Bonus-RP enhances stability at pH 7. Each 10,000 column volume is equivalent to approximately one working month.

Dimethyl-C18/amide, Bonus-RP

Column: | ZORBAX Bonus-RP883668-901 4.6 x 150 mm, 5 μm

Mobile Phase:

Aging:
50% MeOH
50% 0.1% TFA
Test:
80% MeOH
20% H₂O

Flow Rate: | 1.0 mL/min

Temperature:

Aging:
60 °C
Test:
23 °C

Sterically protecting side groups provide good low pH stability and longer column lifetime than similar polar alkyl bonded phases.

ZORBAX Bonus-RP provides unique selectivity

Column A: | ZORBAX Bonus-RP 883668-901 4.6 x 150 mm, 5 μm
Column B: | Eclipse XDB-C8 993967-906 4.6 x 150 mm, 5 μm
Mobile Phase: | 75% 25 mM Na Citrate, pH 6 25% MeOH
Flow Rate: | 1.0 mL/min
Temperature: | Ambient
Detector: | 254 nm
Sample: | 3 μL Cephalosporins

1. Cephalexin

2. Cephaclor

3. Cephuroxime

4. Cephoxitin

Peak elution order can change dramatically when using Bonus-RP. In this example, the elution order of the first three peaks changes.

ZORBAX Bonus-RP bonding is also available on Poroshell 120 columns. View Here.

[picture] | TIPS & TOOLS Watch LC troubleshooting videos featuring Agilent chromatographic experts at www.agilent.com/chem/lctroubleshooting

ZORBAX Original Reversed-Phase Columns

Agilent Original ZORBAX columns are made with Type A silica and are useful for many applications of acidic or neutral compounds. These columns have a higher activity level and are therefore useful for separating isomers (e.g. cis-trans, geometric) or other compounds where silanol activity enhances selectivity. These columns are used in many established methods.

Kits for Analytical HPLC

ZORBAX Method Development Kits

Agilent offers a series of kits that allow for fast method development at an attractive price. Each kit contains 3 columns. Six new kits have been added and are recommended for use with the new Agilent Automated Method Development LC. Several of these kits contain Rapid Resolution HT (1.8 μm) columns in a variety of bonded phases for easy method optimization and several kits contain Rapid Resolution (3.5 μm) columns in the same variety of bonded phases. These kits contain some of the Eclipse Plus family of columns for excellent peak shape and optimum performance with a wide variety of compounds.

ZORBAX Method Development Kits

ZORBAX Method Development Kits
Recommended for use with the Agilent Automated Method Development LC System

Description | Part No.
Rapid Resolution HT (RRHT) Selectivity Method Development Kit, 2.1 mm id Includes 2.1 x 50 mm, 1.8 μm, 600 bar columns: one each Eclipse Plus C18, Eclipse Plus Phenyl-Hexyl and Bonus-RP | 5190-1431
Rapid Resolution HT (RRHT) pH Method Development Kit, 2.1 mm id Includes 2.1 x 50 mm, 1.8 μm, 600 bar columns: one each Eclipse Plus C18, SB-C18 and Extend-C18 | 5190-1432
Rapid Resolution HT (RRHT) Selectivity Method Development Kit, 4.6 mm id Includes 4.6 x 50 mm, 1.8 μm, 600 bar columns: one each Eclipse Plus C18, Eclipse Plus Phenyl-Hexyl and Bonus-RP | 5190-1433
Rapid Resolution HT (RRHT) pH Method Development Kit, 4.6 mm id Includes 4.6 x 50 mm, 1.8 μm, 600 bar columns: one each Eclipse Plus C18, SB-C18 and Extend-C18 | 5190-1434
Rapid Resolution Selectivity Method Development Kit, 4.6 mm id Includes 4.6 x 100 mm, 3.5 μm columns: one each Eclipse Plus C18, Eclipse Plus Phenyl-Hexyl and Bonus-RP | 5190-1435
Rapid Resolution pH Method Development Kit, 4.6 mm id Includes 4.6 x 100 mm, 3.5 μm columns: one each Eclipse Plus C18, SB-C18 and Extend-C18 | 5190-1436

ZORBAX Method Development Kits
Description | Part No.
StableBond Method Development Kit Includes 4.6 x 150 mm, 5 μm columns; one each: SB-C18, SB-CN and SB-Phenyl phases | 5183-4624
Fast StableBond Method Development Kit Includes 4.6 x 75 mm, 3.5 μm columns; one each: SB-C18, SB-CN and SB-Phenyl phases | 5183-4625
Eclipse XDB Method Development Kit Includes 4.6 x 150 mm, 5 μm columns; one each: XDB-C18, XDB-C8, XDB-Phenyl phases | 5183-4626
Fast Eclipse XDB Method Development Kit Includes 4.6 x 75 mm, 3.5 μm columns; one each: XDB-C18, XDB-C8 and XDB-Phenyl phases | 5183-4627
pH Method Development Kit Includes 4.6 x 150 mm, 5 μm columns; one each: SB-C18, XDB-C18 and Extend-C18 phases | 5185-5807
Fast pH Method Development Kit Includes 4.6 x 75 mm, 3.5 μm columns; one each: SB-C18, XDB-C18 and Extend-C18 phases | 5185-5808
Aqueous Method Development Kit Includes 4.6 x 150 mm, 5 μm columns; one each: SB-Aq, Bonus RP and SB-C18 | 5185-5809
Fast Aqueous Method Development Kit Includes 4.6 x 75 mm, 3.5 μm columns; one each: SB-Aq, Bonus RP and SB-C18 | 5185-5810

ZORBAX Cartridge Column Starter Kits
Hardware | Description | Part No.
[picture] | ZORBAX C18 Kit Includes one 4.6 x 150 mm, 5 μm Eclipse XDB-C18 column; one 4.6 x 150 mm, 5 μm StableBond C18 column; cartridge holder; mounting tool; replacement filter (2/pk); and open-end wrench | 5183-2021
[picture] | ZORBAX C8 Kit Includes one 4.6 x 150 mm, 5 μm Eclipse XDB-C8 column; one 4.6 x 150 mm, 5 μm StableBond C8 column; cartridge holder; mounting tool; replacement filter (2/pk); and open-end wrench | 5183-2022

ZORBAX Method Validation Kits

ZORBAX Method Validation Kits are supplied to customers who need the same HPLC column type (bonded phase, particle size, configuration) but from different manufacturing lots. To request columns from different lots, contact Agilent Technologies or your local Agilent Authorized Distributor using the following procedure:

• Request Validation Kits (columns from different lots) by using Part Number 899999-888

• Indicate the Part Number of the current column you are using

• Indicate the Lot Number of the current column you are using

• Indicate the number of additional columns needed from different lots (example: you have a current column and may need two additional lots)

• Please fax your request to (302) 993-5354 (United States and Canada) or email to cag_sales-na@agilent.com. You will receive a quote from your Customer Service Representative within 1-2 business days. Delivery of your method validation kit is usually 3 weeks or less from the time your order is placed, depending on lot availability.

Custom HPLC Column Ordering

Columns not listed can be easily ordered using the following procedure:

• Request a Special Products Quotation (SPQ) using Part Number 899999-999

• Indicate column dimensions (example: 4.6 x 50 mm); bonded phase type (example: StableBond C3); particle size (example: 5 μm); and pore size (example: 80Å)

• Please fax your request to (302) 993-5354 (United States and Canada) or email to cag_sales-na@agilent.com. You will receive a quote from your Customer Service Representative within 1-2 business days. Delivery of your custom column is usually 3 weeks or less from the time your order is placed, depending on lot availability.

Custom columns are priced with a minimal surcharge over the price of stocked columns.

Pursuit HPLC Columns

Beginning in drug discovery and drug metabolism, Pursuit columns are ideal for analyzing lead compounds and biological samples. The column’s performance is due to the unique combination of advanced bonding chemistry and ultra-high purity silica. These factors combine to provide rapid separations with excellent first time resolution and symmetrical peaks for polar compounds, whether at pH 1.5 or 10. Additionally, the need for ion-pairing agents such as TFA is often eliminated, thus maximizing the performance of single and parallel multi-channel LC/MS systems.

Culminating in QC, Pursuit is ideal for implementing dependable trouble-free analysis of raw materials and approved drugs. Rigorous control and validation of each step in the manufacturing process ensures column reproducibility. With Pursuit, your laboratory can spend its energy on producing results.

Special columns, such as Pursuit PFP (for very polar compounds) and Pursuit PAH (environmental), give you the extra selectivities you need for your most challenging applications.

Pursuit HPLC Columns

Pursuit

For LC/MS and high throughput applications, the Pursuit column is built on the larger 200Å pore size silica. High ligand density delivers up to 40% faster separations without sacrificing resolution. This is accomplished by optimizing mass transfer with the larger pore size.

Pursuit XRs

Pursuit XRs columns are for performance in analytical R&D, QC and preparative applications. Combining high ligand density with a 100Å pore size, high surface area silica, Pursuit XRs columns are designed to increase productivity, as they offer maximum loadability, excellent stability and easy scalability while maintaining superior resolution.

Pursuit XRs Ultra

For the μultimate in speed and good resolution on any instrument, we designed the Pursuit XRs Ultra around an optimized 2.8 μm particle and an advanced packing procedure. Now you can decrease your run time while maintaining resolution. Lower backpressure allows high flow rates to be used, and the 2.8 μm particles of ultra-pure silica delivers 10-15% higher efficiency than 3 μm columns.

[picture] | TIPS & TOOLS Request custom LC columns online at www.agilent.com/chem/customlccol

Tricyclic antidepressants and benzodiazepines

Column: | Pursuit XRs C18 A6000150X046 4.6 x 150 mm, 5 μm
Mobile Phase: | A:Water+0.1% HCOOH B:MeCN+0.1% HCOOH
Gradient: | 30-40% B in 15 min, hold at 40% B for 15 min
Flow Rate: | 1.0 mL/min
Temperature: | Ambient
Detector: | UV, 254 nm

1. 7-Aminoclonazepam

2. 7-Aminoflunitrazepam

3. Nordoxepin

4. Doxepin

5. Desipramine

6. Imipramine

7. Nortriptyline

8. Amitriptyline

9. Trimipramine

10. Clomipramine

11. Nordiazepam

12. Clonazepam

13. Flunitrazepam

14. Diazepam

Mechanical stability of Pursuit XRs

Column: | Pursuit XRs C18 A6000050X020 2.0 x 50 mm, 5 μm
Sample: | DMSO mix
Mobile Phase: | A: MeOH:water, 10:90 + 0.1% HCOOH B: MeOH:water, 90:10 + 0.1% HCOOH
Gradient: | 0-100% B in 3 min, back to 0% B in 0.5 min, hold at 0% B for 3.5 min
Flow Rate: | 0.4 mL/min
Temperature: | Ambient
Detector: | UV, 254 nm

1. 4-Methoxybenzenesulfonamide

2. Methyl 3-aminothiophene-2-carboxylate

3. Trimipramine

Antifungals

Column: | Pursuit XRs Ultra Diphenyl A7521050X020 2.0 x 50 mm, 2.8 μm
Mobile Phase: | Water+0.1% HCOOH:MeCN+0.1% HCOOH, 80:20
Flow Rate: | 0.4 mL/min
Temperature: | Ambient
Detector: | UV, 254 nm

1. 4-Aminobenzoic acid

2. Sorbic acid

3. Benzoic acid

4. Salicylic acid

Liquid chromatography phase test mixture (LPTM) on Pursuit C8

Column: | Pursuit C8 A3031050X020 2.0 x 50 mm, 3 μm
Mobile Phase: | A: 0.05% HCOOH in water B: 0.05% HCOOH in MeCN
Flow Rate: | 0.6 mL/min
Detector: | UV, 220 nm

1.Aspartame

2.Cortisone

3.Reserpine

4.Dioctyl phthalate

Adrenocorticosteroids on Pursuit PFP and C18

Mobile Phase: | MeCN:water, 22.5:77.5
Flow Rate: | 1.5 mL/min
Temperature: | Ambient
Detector: | UV, 240 nm

1. Triamcinolone

2. Prednisolone

3. Cortisone

4. Methylprednisolone

5. Corticosterone

6. Beclomethasone

7. Prednisolone acetate

8. Triamcinolone acetonide

9. Cortisone acetate

10. Fluocinolone acetonide

Polaris HPLC Columns

In areas like drug discovery where target compounds are increasingly polar, it is critical to have a reversed-phase column that performs well under aqueous conditions. Retention is critical, but cannot come with troublesome secondary interactions. Likewise, phase collapse and shifting retention times need to be avoided. The answer is our Polaris line of polar-modified columns.

From the collapse-resistant pore structure of our base silica, to the “wettability” engineered into the bonded phases, Polaris columns have been designed for high aqueous conditions. The combination of high phase density bonding, ultra pure silica, and silanol shielding leads to excellent peak shape among polar-modified columns.

As a family, Polaris offers a variety of polar modifications in both C18 and C8 chemistries.

Polaris HPLC Columns

Polaris C18-A

Polaris C18-A is the best starting place for separations where the benefits of polar-modified columns are desired. The polar modifications of C18-A help it avoid poor peak shape and retention issues in low organic conditions.

Polaris C8-A

Polaris C8-A offers an alternative selectivity to standard C8 phases and has a lower hydrophobicity than Polaris C18-A, making it ideal for polar samples, or faster overall analysis times.

Polaris C18-Ether

Polaris C18-Ether offers an alternative selectivity to Polaris C18-A and standard C18 phases, and typically delivers increased retention of polar compounds away from the void volume.

Polaris C8-Ether

Polaris C8-Ether offers an alternative selectivity to Polaris C8-A with particular utility for hydrogen bonding compounds.

Selectivity test mix for Polaris columns

Mobile Phase: | MeCN:water 70:30
Flow Rate: | 1.0 mL/min
Temperature: | Ambient
Detector: | UV, 254 nm

1. Uracil

2. Caffeine

3. Phenol

4. Toluene

5. Butylbenzene

6. o-Terphenyl

7. Amylbenzene

8. Triphenylene

LC/MS performance test mix for Polaris C8-A

Column: | Polaris C8-A
| A2011030X030
| 3.0 x 30 mm, 3 μm
Mobile Phase: | A: Water+0.05% HCOOH B: MeCN+0.05% HCOOH
Gradient: | 5-90% B in 3 min and hold for 4 min
Flow Rate: | 0.6 mL/min
Temperature: | Ambient
Detector: | UV, 220 nm

1. Aspartame

2. Cortisone

3. Reserpine

4. Dioctyl phthalate

Agilent TC-C18(2) and HC-C18(2)

For cost-conscious chromatographers who need traditional LC columns and don’t need the individual testing of ZORBAX, Pursuit or Polaris columns, the Agilent TC(2)/HC(2) columns provide an alternative.

TC-C18(2)

Agilent TC-C18(2) is the ideal choice for complex natural product extract samples, traditional medicines and environmental samples or any sample where you need to analyze mixtures of polar and non-polar compounds, including strong basic compounds.

• Lower carbon load – 12%

• Ideal for polar compounds and gradient separations that start at low % organic or cover a wide organic range

• Good choice for samples dissolved in water, or mostly water

• Use with most common mobile phases, including formic acid, acetic acid, trifluoroacetic acid (TFA) and phosphate buffers with acetonitrile and methanol as the organic modifiers

• Excellent performance from pH 2-8

HC-C18(2)

Agilent HC-C18(2) is a more retentive C18 with a higher carbon load. An excellent value alternative to other high carbon load columns, it also provides superior peak shape for basic compounds.

• Higher carbon load – 17% – provides greater retention for moderately polar and non-polar compounds

• Ideal for non-polar compounds and separations that start at mid-level % organic (at least greater than 10% organic)

• Good choice for industrial samples or samples dissolved in organic/mostly organic solvents

• Stable over a very wide pH range (2-9) for maximum flexibility

Agilent HC-C18(2) and TC-C18(2)
Description | Size (mm) | Particle Size (μm) | Part No.
Agilent HC-C18(2) | 4.6 x 250 | 5 | 588905-902
Agilent HC-C18(2) | 4.6 x 150 | 5 | 588915-902
Agilent TC-C18(2) | 4.6 x 250 | 5 | 588925-902
Agilent TC-C18(2) | 4.6 x 150 | 5 | 588935-902
Agilent HC-C18(2) guards, 2/pk | 4.6 x 12.5 | 5 | 520518-904
Agilent TC-C18(2) guards, 2/pk | 4.6 x 12.5 | 5 | 520518-905
Guard Hardware Kit |  |  | 820999-901

TIPS & TOOLS Don’t forget, we have special offers throughout the year. To learn more, visit www.agilent.com/chem/specialoffers | [picture]

PLRP-S HPLC Columns

• Contain durable and resilient polymer particles that deliver reproducible results over longer lifetimes

• Thermally and chemically stable

• Comply with USP L21 designation

• Used in bioscience, chemical, clinical research, energy, environmental, food and agriculture, material science and pharmaceutical industries

• Pore sizes (100Å-4000Å) for separations of small molecules to large complexes and polynucleotides

The PLRP-S family of columns consists of a range of pore sizes and particle sizes, all with identical chemistry and fundamental adsorptive characteristics. The particles are inherently hydrophobic, therefore no bonded phase, alkyl ligand is required for reversed-phase separations. This gives a highly reproducible material that is free from silanols and heavy metal ions. Columns within the extensive product range are suitable for micro separations, including both bottom-up and top-down proteomics, analytical separations, and preparative purifications. In addition, process columns can be packed with bulk media.

Column Specifications
pH Range | 1-14
Buffer Content | Unlimited
Organic Modifier | 1-100%
Temperature Limits | 200 °C
Maximum Pressure | 5-8 μm: 3000 psi (210 bar)
| 3 μm: 4000 psi (300 bar)

PLRP-S Applications
Pore Size | Application
100Å | Small molecules/synthetic biomolecules
300Å | Recombinant peptides/proteins
1000Å | Large proteins
4000Å | DNA/high speed

HPLC of 25 bp DNA ladder

Column: | PLRP-S, 2.1 x 150 mm
Mobile Phase: | A: 0.1 M TEAA
| B: 0.1 M TEAA in 50% water:50% ACN
Flow Rate: | 200 μL/min
Gradient: | 12.5-50% B in 150 min

Polyethylene glycols

Column: | PLRP-S 100Å PL1111-3500 4.6 x 150 mm, 5 μm
Mobile Phase: | A: Water
| B: ACN
Gradient: | 10-30% B in 12 min, held at 30% B for 3 min
Flow Rate: | 1.0 mL/min
Injection Volume: | 10 μL
Sample Conc: | 1 mg/mL
Detector: | ELS (neb=50 °C, evap=70 °C, gas=1.6 SLM)

Exploiting chemical stability – NH₄OH concentration

Column: | PLRP-S 100Å PL1512-5500 4.6 x 250 mm, 5 μm
Mobile Phase: | A: NH₄OH (various mM) in water
| B: NH₄OH (various mM) in ACN
Gradient: | Linear 10-100% B in 15 min
Flow Rate: | 1.0 mL/min
Detector: | ELS (neb=80 °C, evap=85 °C, gas=1.0 SLM)

1. Angiotensin II

2. Angiotensin I

3. Angiotensin III

Alberta Peptide Institute test mix

Column: | PLRP-S 100Å PL1512-5500 4.6 x 250 mm, 5 μm
Mobile Phase: | A: 0.1 % TFA in 99% water:1 % ACN
| B: 0.1% TFA in 70% water:30% ACN
Gradient: | 0-100% B in 30 min
Flow Rate: | 1.0 mL/min
Detector: | UV, 220 nm

1. Ala³-Gly⁴ (free amino)

2. Gly³-Gly⁴ (Nα-acetylated)

3. Ala³-Gly⁴ (Nα-acetylated)

4. Val³-Gly⁴ (Nα-acetylated)

5. Val³-Val⁴ (Nα-acetylated)

Large fibrous proteins

Column: | PLRP-S 300Å PL1512-3801 4.6 x 150 mm, 8 μm
Column: | PLRP-S 1000Å PL1512-3802 4.6 x 150 mm, 8 μm
Mobile Phase: | A: 0.25% TFA in water
| B: 0.25% TFA in 5% water:95% ACN
Flow Rate: | 1.0 mL/min
Gradient: | 20-60% B in 15 min
Detector: | UV, 220 nm

1. Collagen (120,000 MW)

2. Fibrinogen (340,000 MW)

Preparative HPLC Columns

Flexible, cost-effective options for scaling and prep

Whether you are scaling up a routine analytical method, or maintaining precise separations throughout every phase of production, Agilent can help you rise to the challenge.

• Agilent Prep LC columns are a cost-effective prep solution designed for high loadability to purify milligram to gram quantities of product

• ZORBAX PrepHT columns are designed for rapid scale-up from the ZORBAX family of phases

• Scalable prep columns are also available for Pursuit and Polaris columns

• Bulk materials are available for all phases and can be ordered through Agilent’s Custom Ordering Process, www.agilent.com/chem/customlc

Agilent Prep LC Columns

• High loadability for maximum sample purification

• Easy scalability from 4.6 up to 50 mm id for rapid method development

• High throughput 21.2 mm id cartridges for fast purification

• Exceptional column stability and loadability up to pH 10

Agilent Prep LC columns are designed for high loadability to purify milligram to gram quantities of products. Preparative sized columns are available in 21.2, 30, and 50 mm internal diameters with lengths ranging from 50-250 mm. Columns are available in 5 and 10 μm particle sizes with very high efficiency in every dimension. These column choices accommodate almost every preparative sample.

Agilent Prep 21.2 mm id columns are available with Agilent’s Preparative Cartridge Hardware. This reliable cartridge hardware makes it simple to use columns with different lengths to increase sample load. Guard columns are easily integrated onto these columns, providing superior protection of the analysis column. Analytical size 4.6 mm id scalar columns are available for method development and optimization prior to scaling up to larger columns. Bulk material is also available.

Agilent Prep columns are available in a C18 bonded phase suitable for purification of a wide variety of non-polar and polar compounds. Unbonded silica columns are also available.

Superior loadability on Agilent Prep C18 with basic compounds

Column: | Agilent Prep C18 443905-902 4.6 x 150 mm, 5 μm
Mobile Phase: | 50% 0.1%TFA:50% ACN
Flow Rate: | 1 mL/min
Sample: | 10 μL
| Doxepin/Amitriptyline
| 0.5-50 mg/mL

Agilent Prep columns show better resolution and loadability than competitor columns.

Steroids: Easy scalability using Agilent Prep columns

Column A: | Agilent Prep C18
| 443905-102
| 21.2 x 150 mm, 5 μm
Column B: | Agilent Prep C18
| 443905-102V
| 21.2 x 150 mm, 5 μm
Column C: | Agilent Prep C18
| 443905-102
| 21.2 x 150 mm, 5 μm
Column D: | Agilent Prep C18
| 413910-502
| 50.0 x 150 mm, 10 μm
Mobile Phase: | 55% Water:45% ACN
Flow Rate: | 0.7 mL/min
| 14.87 mL/min
| 29.77 mL/min
| 85.37 mL/min
Temperature: | Ambient
Detector: | 240 nm
Sample: | 2 μL
| 42.4 μL
| 170 μL
| 488 μL

Agilent Prep C18 shows excellent scalability, making method transfer simple and predictable.

1. Hydrocortisone

2. Testosterone (in MeOH @ 1mg/mL)

ZORBAX PrepHT Columns

ZORBAX PrepHT

• Easy scale-up from analytical to preparative scale with ZORBAX phases

• Fast preparative separations, up to 2000 mg

• 5 to 7 μm particles for high efficiency and high yield

• Easy to install finger-tight connections seal up to 5000 psi/350 bar

• Use to maintain selectivity of the analytical phase in your prep separations

High purity, high recovery and high throughput can be easily achieved with Agilent ZORBAX PrepHT columns. These are available in a variety of bonded phases – Eclipse XDB, StableBond, Bonus-RP, and Extend-C18 – for optimized resolution and loadability under any conditions.

ZORBAX PrepHT columns are packed with 5 and 7 μm particle sizes for very high resolution. The high resolution allows high loadability, high yield, and high purity of compounds. The larger diameter columns and mechanically stronger ZORBAX particles allow for flow rates up to 100 mL/min, thus increasing throughput.

ZORBAX PrepHT columns are designed for rapid scale-up from analytical to preparative scale without losing resolution. For complex separations on larger columns (21.2 mm id, 150 mm length and longer), Agilent has carefully chosen the 7 μm particle size to achieve a balance between high efficiency and high loadability.

High purity and high recovery with ZORBAX PrepHT columns

Sample: | Antianginal drugs

Mass-based fraction collection using ZORBAX SB-C18 column shows high purity and high recovery of each compound (Application Note publication number 5988-7113EN). The separation of the three antianginal drugs was successfully done in a single run with high recovery and >90% purity. Separations up to 2000 mg are possible depending on the complexity of separation.

1. Nifedipine

2. Nifmodipine

3. Nifsoldipine

ZORBAX PrepHT columns are designed for rapid scale-up from analytical to preparative scale without losing resolution. For complex separations for larger columns (21.2 mm id and higher, 150 mm length and higher), Agilent has carefully chosen the 7 μm particle size to achieve a balance between high efficiency and high loadability.

Scale-up to PrepHT

1. Theobromine

2. Theophylline

3. Caffeine

Pursuit and Pursuit XRs Prep

• Prep-scalable columns for Pursuit and Pursuit XRs columns

• Particle sizes to 10 μm and column diameters up to 50 mm

• High surface area silica

Pursuit and Pursuit XRs Prep columns are designed for high loadability with a high surface area.

Natural products – capsaicin and dihydrocapsaicin on Pursuit XRs C18

Column A: | Pursuit XRs C18
| A6001150X046
| 4.6 x 150 mm, 3 μm
Column B: | Pursuit XRs C18
| A6000150X046
| 4.6 x 150 mm, 5 μm
Column C: | Pursuit XRs C18
| A3002150X046
| 4.6 x 150 mm, 10 μm
Mobile Phase: | CH₃CH:H₂O - 70:30
Flow Rate: | 1.0 mL/min
Temperature: | Ambient
Detector: | 220 nm
Sample: | 1. Capsaicin
| 2. Dihydrocapsaicin

Demonstrating an easy, linear scale-up of natural products from Pursuit XRs C18 3 μm and 5 μm analytical columns to a 10 μm preparative column.

Polaris Prep Columns

• Prep-scalable columns for Polaris phases

• 10.0 and 21.2 mm ids available, with particles up to 10 μm

Scale-up from analytical to prep

Column A: | Polaris C18-A
| A2001050X046
| 4.6 x 50 mm, 3 μm
Column B: | Polaris C18-A
| A2000050X046
| 4.6 x 50 mm, 5 μm
Column C: | Custom column
Mobile Phase: | A: H₂O + 0.1 % NH₄OH, pH 10.0 (25 mM)
| B: CH₃CN
| A:B 65:35
Flow Rate: | 1.0 mL/min
Temperature: | 40 °C
Detector: | 215 nm
Sample: | 1. Pindolol
| 2. Metoprolol
| 3. Propranolol

Bulk materials for prep

Agilent has bulk materials available for all phases. Most materials and quantities can be ordered through the Custom Column and Bulk Ordering process, and can be produced in multiple kg. quantities. Quotes are able to be delivered within 48 hours. Contact your Agilent product specialist for support in placing a custom order.

Load & Lock columns

Load & Lock Preparative HPLC Column Packing Systems

Agilent offers a complete range of Load & Lock column systems for laboratory and process preparative LC. They are designed to let you easily and quickly pack your own preparative high efficiency columns. This is the right solution for applications ranging in scale from development (multigrams) to production (multi-kilo) of pharmaceutical compounds, peptides, and natural products. Our Load & Lock columns have a unique fluid/sample distribution system to maximize productivity. The system provides dynamic axial compression (DAC) and static “locked” axial compression (SAC) and is designed for easy operation to deliver greater convenience.

Laboratory Load & Lock Columns

• Mobile packing station supports three different column sizes

• Runs on compressed air with no need for a power supply

• Quick and easy packing and unpacking within minutes

Agilent’s laboratory scale Load & Lock columns combine excellent packed-bed stability with enhanced flow distribution to deliver the highest quality purification possible with maximum speed, flexibility, and ease of operation. Three different column sizes are supported: 1 in, 2 in and 3 in id. Because the station is powered by compressed air, it is the perfect solution for hazardous environments. The quick-release single bolt clamp offers speedy and easy packing and unpacking within minutes.

Load & Lock Preparative HPLC Column Packing Systems
Description | Water Jacket | Size (mm) | Part No.
Load & Lock 4001 Column | No | 25.0 x 500 | PCG93LL500X25
| Yes | 25.0 x 500 | PCG93LL500X25WJ
| Spare parts kit |  | PCG931AAKIT
Load & Lock 4002 Column | No | 50.0 x 500 | PCG93LL500X50
| Yes | 50.0 x 500 | PCG93LL500X50WJ
| Spare parts kit |  | PCG932AAKIT
Load & Lock 4003 Column | No | 75.0 x 500 | PCG93LL500X75
| Yes | 75.0 x 500 | PCG93LL500X75WJ
| Spare parts kit |  | PCG933AAKIT
Mobile packing station (air driven hydraulic) |  |  | PCG93LLSTAND123

Columns for Other HPLC Techniques

Reproducible results for Normal Phase and beyond

Agilent’s extended family of HPLC columns support every technique, providing you with the Agilent quality you depend on for every application.

• ZORBAX HILIC Plus – good retention of small, polar analytes and high sensitivity for LC/MS -in Fast LC 1.8 μm options

• ZORBAX normal phase columns – bonded and non-bonded silica packings

• ZORBAX ion-exchange columns – based on rugged ZORBAX Silica, stable from pH 2-7

• Hi-Plex columns for carbohydrate analysis – ligand-exchange columns

• Ultron ES Chiral columns – with two complimentary protein-based chiral stationary phases – are an excellent choice for enantiomeric separations. Ideal for many pharmacological applications.

ZORBAX HILIC Plus

• HILIC column for good retention of small, polar analytes

• Based on Eclipse Plus silica for excellent peak shape

• High sensitivity for LC/MS applications

• Recommended for EPA Method 1694

Agilent ZORBAX HILIC Plus columns are for use in hydrophilic interaction chromatography (HILIC) applications, which are typically used for the retention and resolution of small polar compounds. HILIC Plus columns are non-bonded silica columns based on the high performance silica used in ZORBAX Eclipse Plus columns. This silica provides excellent peak shape, critical for many polar, basic analytes. These columns ship prepared for use in HILIC mode – containing acetonitrile:water – in order to reduce the extensive equilibration typically required for HILIC separations. HILIC Plus columns are available in a 3.5 μm particle size for high resolution and in 2.1 and 4.6 mm id for compatibility with mass spectrometers or with standard UV detectors.

Column Specifications
Phase | Pore Size | Surface Area | pH Range
Non-bonded silica | 95Å | 160 m²/g | 0-8.0

Specifications represent typical values only.

[picture] | TIPS & TOOLS Poroshell 120 HILIC is very similar to ZORBAX HILIC Plus. View Here

Separation of group 4 analytes in EPA 1694 on ZORBAX HILIC Plus column

Column: | ZORBAX HILIC Plus
| 959793-901
| 2.1 x 100 mm, 3.5 μm
Mobile Phase: | 90% Acetontrile:10% Water
Flow Rate: | 0.25 mL/min
Gradient: | Linear gradient to 55% acetonitrile in 7 min Held at 55%
Temperature: | 25 °C

Duplicate runs for column USCJP0004; 10 min equilibration between two runs

ZORBAX HILIC Plus
Description | Size (mm) | Particle Size (μm) | Part No.
Analytical | 4.6 x 100 | 3.5 | 959961-901
Analytical | 4.6 x 50 | 3.5 | 959943-901
Narrow Bore | 2.1 x 100 | 3.5 | 959793-901
Narrow Bore | 2.1 x 50 | 3.5 | 959743-901

ZORBAX HILIC Plus RRHD, stable to 1200 bar
Size (mm) | Particle Size (μm) | Part No.
3.0 x 100 | 1.8 | 959758-301
3.0 x 50 | 1.8 | 959757-301
2.1 x 150 | 1.8 | 959759-901
2.1 x 100 | 1.8 | 959758-901
2.1 x 50 | 1.8 | 959757-901

Poroshell 120 HILIC Plus
Size (mm) | Particle Size (μm) | Part No.
2.1 x 50 | 2.7 | 699775-901
2.1 x 100 | 2.7 | 695775-901
2.1 x 150 | 2.7 | 693775-901
3 x 50 | 2.7 | 699975-301
3 x 150 | 2.7 | 693975-301
4.6 x 50 | 2.7 | 699975-901
4.6 x 100 | 2.7 | 695975-901
4.6 x 150 | 2.7 | 693975-901

ZORBAX Normal-Phase Columns

For normal-phase chromatography, the Agilent ZORBAX product line offers a choice of bonded and non-bonded silica packings.

ZORBAX Rx-SIL

• Made from highly pure (> 99.995%) porous silica microspheres (pore size is the space between the solid silica microparticles)

• Available in 1.8 and 5 μm particle sizes

• Stronger than other silica types

• Less acidic than ZORBAX SIL, lower metal content

• Low acidity and low metal content make ZORBAX Rx-SIL ideal for normal-phase separation of polar compounds that exhibit poor peak symmetry on more acidic silica

• Useful for very hydrophilic compounds with high organic mobile phases in HILIC mode

ZORBAX Eclipse XDB-CN

• Made from highly pure Rx-SIL

• Excellent choice for normal-phase applications with basic compounds

• Equilibrates more rapidly than ZORBAX Rx-SIL and is used for many of the same normal-phase applications

ZORBAX CN

• Cyanopropyldimethylsilane monolayer bonded to ZORBAX SIL

• Equilibrates more rapidly than ZORBAX SIL, and used for many of the same normal-phase applications

• Less prone to fouling and less water sensitive than silica

Pursuit XRs Si

• 100Å silica for higher surface area and good loadability

• 14.6% carbon load

• Available in 3 μm, 5 μm and 10 μm

Polaris NH2

• 810Å silica for high surface area and loadability

• 5.5% carbon load

• Available in 3 μm, 5 μm, and 10 μm sizes

• Polar-modified with silanol shielding

• Designed for high-aqueous conditions

Polaris Si-A

• 810Å silica with highest surface area and loadability

• Available in 3 μm, 5 μm, and 10 μm

High resolution normal-phase separation of octylphenoxy ethanol surfactant on ZORBAX CN

Column: | ZORBAX CN
| 880952-705
| 4.6 x 250 mm, 5 μm
Mobile Phase: | Primary: Heptane
| Secondary: 2-Methoxyethanol/Isopropanol (50/50)
Flow Rate: | 2 mL/min
Gradient: | 2-20% Secondary in 10 min, Linear Hold at 20%
Temperature: | 50 °C
Detector: | 278 nm
Sample: | Octylphenoxy (polyethylene oxy)
| Ethanol Surfactant (n=10)

Polaris ordering information can be found here

Pursuit ordering information can be found here

ZORBAX NH2

• Amino-propyl silane phase bonded to ZORBAX SIL

• Used for normal-phase and weak anion-exchange, and reversed-phase HPLC of polar compounds

• Vitamins A and D are separated in the normal-phase mode

• Carbohydrates and sugars are separated in the reversed-phase mode

[picture] | TIPS & TOOLS Pursuit XRs Silica is another choice for normal-phase chromatography. For more information, see here.

Pursuit XRs Si, USP L3
Size (mm) | Particle Size (μm) | Part No.
Semi-Prep Scale
10.0 x 250 | 10 | A6004250X100
Analytical Scale
4.6 x 250 | 10 | A6004250X046
4.6 x 100 | 5 | A6006100X046
4.6 x 50 | 5 | A6006050X046
4.6 x 100 | 3 | A6005100X046
4.6 x 50 | 3 | A6005050X046
2.1 x 100 | 5 | A6006100X021
2.0 x 50 | 3 | A6005050X020
Prep Scale
50.0 x 250 | 10 | A6004250X500
30.0 x 250 | 10 | A6004250X300
21.2 x 250 | 10 | A6004250X212

Polaris HPLC Columns
|  | Polaris | Polaris
Size (mm) | Particle Size (μm) | NH2 | Si-A
50.0 x 250 | 10 |  | A2004250X500
21.2 x 250 | 10 |  | A2004250X212
21.2 x 250 | 5 | A2013250X212 | A2003250X212
21.2 x 150 | 5 |  | A2003150X046
21.2 x 50 | 5 |  | A2003050X212
10.0 x 250 | 5 | A2013250X100 |
4.6 x 250 | 10 |  | A2003250X046
4.6 x 250 | 5 | A2013250X046 |
4.6 x 150 | 5 | A2013150X046 | A2003150X046
4.6 x 100 | 5 | A2013100X046 | A2003100X046
4.6 x 50 | 5 | A2013050X046 | A2003050X046
4.6 x 250 | 3 | A2014250X046 | A2005250X046
4.6 x 150 | 3 | A2014150X046 | A2005150X046
4.6 x 100 | 3 | A2014100X046 | A2005100X046
4.6 x 50 | 3 | A2014050X046 | A2005050X046
4.0 x 250 | 5 | A2013250X040 | A2003250X040
4.0 x 150 | 5 | A2013150X040 | A2003150X040
4.0 x 125 | 5 | A2013125X040 | A2003125X040
3.0 x 250 | 5 | A2013250X030 | A2005250X046
3.0 x 150 | 5 | A2013150X030 | A2003150X030
3.0 x 100 | 5 | A2013100X030 | A2003100X030
3.0 x 50 | 5 |  | A2003050X030
3.0 x 250 | 3 | A2014250X030 | A2003250X030
3.0 x 150 | 3 | A2014150X030 | A2005150X030
3.0 x 100 | 3 | A2014100X030 | A2005100X030
3.0 x 50 | 3 | A2014050X030 | A2005050X030
2.0 x 250 | 5 | A2013250X020 | A2003250X020
2.0 x 150 | 5 | A2013150X020 | A2003150X020
2.0 x 100 | 5 | A2013100X020 | A2003100X020
2.0 x 50 | 5 | A2013050X020 | A2003050X020
2.0 x 30 | 5 | A2013030X020 | A2003030X020
2.0 x 20 | 5 | A2013020X020 | A2003020X020
2.0 x 250 | 3 | A2014250X020 | A2005250X020
2.0 x 150 | 3 | A2014150X020 | A2005150X020
2.0 x 100 | 3 | A2014100X020 | A2005100X020
2.0 x 50 | 3 | A2014050X020 | A2005050X020
2.0 x 30 | 3 | A2014030X020 | A2005030X020
2.0 x 20 | 3 | A2014020X020 | A2005020X020

Ion-Exchange Columns

ZORBAX Ion-Exchange Columns – SAX and SCX

• ZORBAX SAX and 300SCX columns are based on rugged ZORBAX silica

• Stable from pH 2-7

• Provide high efficiency, rapid separations

• Compatible with organic mobile phase modifiers

Agilent ZORBAX Strong Ion-Exchange columns are available as both Strong Anion-Exchange (SAX) and Strong Cation-Exchange (300SCX) columns. Each column is packed with bonded, 5 μm, spherical silica particles for optimum efficiency.

ZORBAX SAX packing has a permanently bonded quaternary amine. A trifunctional organo-silane reagent is used in producing this packing to maximize its stability with aqueous mobile phases. This column is ideal for separation of water-soluble compounds such as aromatic and aliphatic carboxylic acids and sulfonic acids.

ZORBAX SCX packing has 300Å pore size silica particles chemically bonded to an aromatic sulfonic acid group. This column is used for separations of basic, water-soluble compounds and bio-molecules.

Cough/cold remedies on ZORBAX 300SCX

Column: | ZORBAX 300SCX
| 880952-704
| 4.6 x 250 mm, 5 μm
Mobile Phase: | 100 mM NaH₂PO₄(pH 6.5)
Flow Rate: | 3 mL/min
Temperature: | 20 °C
Detector: | 210 nm
Sample: | Cold remedies

1. Pyrilamine

2. Theophylline

3. Glyceryl Guaicolate

4. Caffeine

5. Phenylephrine

Hi-Plex Columns for Carbohydrate Analysis

• Agilent’s recommended column for accurate, low-pressure analysis of typical carbohydrates, providing leading-edge features for reliable quantitative and qualitative analysis

• Enable reduced column operating pressures for repeatable performance and longer column life

• Wide range of ligand counter ions and column configurations meet requirements of challenging organic applications

• Simplified HPLC system requirements through isocratic separation capabilities; excellent batch-to-batch reproducibility for ultimate confidence in your results

• Can be used with water or diluted acid as an eluent

• Available in 8 μm and 10 μm particle sizes in a range of choices for USP media types – L17, L19, L34 and L58

The least complicated LC methods for detecting sugars, sugar alcohols and organic acids call for ligand-exhange columns with a simple mobile phase. However, the wide particle size distribution of conventional resins can lead to high backpressures and reduced productivity.

Hi-Plex columns are engineered with monodisperse sulfonated particles, creating a high-performance media uniquely suited to stringent USP methods for analyzing carbohydrates, alcohols and organic acids. Unlike the ZORBAX NH2 column used for carbohydrate analysis with an acetonitrile:water mobile phase, Hi-Plex ligand-exchange columns provide more resolution for mono- and disaccharides due to the interaction of the hydroxyl groups with the metal ion associated with the cation-exchange functionality of the sulfonic acid group.

Column Specifications
| Temperature | Flow Rate |
Bonded Phase | Range | (mL/min) | Eluent
Hi-Plex Ca | 80-90 °C | 0.6 | Water
Hi-Plex Ca USP L19 | 80-90 °C | 0.3 | Water
Hi-Plex Pb | 70-90 °C | 0.6 | Water
Hi-Plex H for carbohydrates | 60-70 °C | 0.6 | Water
Hi-Plex H for organic acids | 40-60 °C | 0.6 | Dilute Acid
Hi-Plex Ca (Duo) | 80-90 °C | 0.6 | Water
Hi-Plex K | 80-90 °C | 0.6 | Water
Hi-Plex Na (Octo) | 80-90 °C | 0.6 | Water, Sodium Hydroxide
Hi-Plex Na | 80-90 °C | 0.3 | Water

Hi-Plex Column Selection

USP methods specify the type of HPLC media and column dimensions which should be used for the analysis. The Hi-Plex product range has four materials that comply with USP definitions.

Media Type L17

Strong cation-exchange resin consisting of sulfonated, cross-linked styrene-divinylbenzene copolymer in the hydrogen form, 7 to 11 μm in diameter – Hi-Plex H.

Media Type L19

Strong cation-exchange resin consisting of sulfonated, cross-linked styrene-divinylbenzene copolymer in the calcium form, 9 μm in diameter – Hi-Plex Ca and Hi-Plex Ca (Duo).

Media Type L34

Strong cation-exchange resin consisting of sulfonated, cross-linked styrene-divinylbenzene copolymer in the lead form, about 9 μm in diameter – Hi-Plex Pb.

Media Type L58

Strong cation-exchange resin consisting of sulfonated, cross-linked styrene-divinylbenzene copolymer in the sodium form, 6 to 30 μm diameter – Hi-Plex Na and Hi-Plex Na (Octo).

In addition to the standard column sizes, the media is also packed in specific column dimensions for different USP methods, including sugar alcohol analysis.

For some application areas there are several column options, and the choice of the most appropriate Hi-Plex media will depend on sample matrix and exact carbohydrate composition.

Hi-Plex Column Selection
Application Area | Recommended Column
USP Methods Specifying L17 Media | Hi-Plex H
USP Methods Specifying L19 Media | Hi-Plex Ca and Hi-Plex Ca (Duo)
USP Methods Specifying L34 Media | Hi-Plex Pb
USP Methods Specifying L58 Media | Hi-Plex Na and Hi-Plex Na (Octo)
Mono- and Disaccharides | Hi-Plex Ca
| Hi-Plex Pb
| Hi-Plex H
| Hi-Plex Na (Octo)
Anomer Separations | Hi-Plex Ca
Organic Acids | Hi-Plex H
Alcohols | Hi-Plex Ca
| Hi-Plex K
| Hi-Plex H
| Hi-Plex Pb
Adulteration of Food and Beverages | Hi-Plex Ca and Hi-Plex Pb
Food Additives | Hi-Plex Ca and Hi-Plex Pb
Dairy Products | Hi-Plex Ca and Hi-Plex H
Sweetened Dairy Products | Hi-Plex Pb
Confectionery | Hi-Plex Ca and Hi-Plex Pb
Fruit Juice | Hi-Plex Ca
Wine | Hi-Plex H
Wood Pulp Hydrolysates (cellulose/hemi-cellulose) | Hi-Plex Pb
Fermentation Monitoring | Hi-Plex H
Oligosaccharides | Hi-Plex Na
Samples with High Salt Content (molasses) | Hi-Plex Na (Octo)
Oligosaccharides <Dp5 with Monosaccharides | Hi-Plex Ca (Duo)
Corn Syrups | Hi-Plex Na

Analysis of fruit juice

Column: | Hi-Plex Ca
| PL1170-6810
| 7.7 x 300 mm, 8 μm
Mobile Phase: | Water
Flow Rate: | 0.6 mL/min
Temperature: | 85 °C
Detector: | RI

1. Sucrose

2. Glucose

3. Fructose

4. Sorbitol

Organic acid analysis

Column: | Hi-Plex H
| PL1170-6830
| 7.7 x 300 mm, 8 μm
Mobile Phase: | Water with acid as specified
Flow Rate: | 0.6 mL/min
Temperature: | 60 °C
Detector: | ELS (neb=80 °C,
| evap=90 °C,
| gas=0.7 SLM), RI

1. Oxalic acid

2. Citric acid

3. Tartaric acid

4. Succinic acid

5. Lactic acid

USP methods for sugar alcohols

Column: | Hi-Plex Ca USP L19
| PL1570-5810
| 4.0 x 250 mm, 8 μm
Mobile Phase: | Water
Flow Rate: | 0.3 mL/min
Temperature: | 60 °C
Detector: | RI

1. Iso-erythritol

2. Adonitol

3. Arabitol

4. Mannitol

5. Xylitol

6. Dulcitol

7. Sorbitol

Corn syrup, Hi-Plex

Column: | Hi-Plex Na
| PL1171-6140
| 7.7 x 300 mm, 10 μm
Mobile Phase: | Water
Pressure: | 11 bar
Flow Rate: | 0.3 mL/min
Temperature: | 80 °C
Detector: | RI

1. Dp1

2. Dp2

3. Dp3

4. Dp4

5. Dp5

6. Dp6

7. Dp7

8. Dp8

9. Dp9

10. Dp10

11. Dp11

12. Dp12

13. Dp13

14. Dp14

Analysis of sweeteners on Hi-Plex Ca columns

Column: | Hi-Plex Ca
| PL1170-6810
| 7.7 x 300 mm, 8 μm
Mobile Phase: | Water
Flow Rate: | 0.6 mL/min
Temperature: | 85 °C
Detector: | ELSD

Hi-Plex Ca columns are ideal for analyzing most sweeteners, including glucose and fructose (monosaccharides), sucrose (disaccharide), and mannitol and sorbitol (sugar alcohols).

1. Stachyose

2. Raffinose

3. Sucrose

4. Glucose

5. Galactose

6. Fructose

7. Mannitol

8. Sorbitol

Analysis of carbohydates on Hi-Plex H columns

Column: | Hi-Plex H
| PL1170-6830
| 7.7 x 300 mm, 8 μm
Mobile Phase: | Water
Flow Rate: | 0.6 mL/min
Temperature: | 70 °C
Detector: | RI

For carbohydrate analysis of samples containing high levels of organic acids, Hi-Plex H columns deliver sharp, reproducible peaks. Note, however, that some sugars (such as raffinose) can undergo acid hydrolysis even when water is used as the eluent.

1. Maltotriose

2. Lactose

3. Glucose

4. Fructose

Analysis of sugars with high sodium matrix

Column: | Hi-Plex Na (Octo)
| PL1170-6840
| 7.7 x 300 mm, 8 μm
Mobile Phase: | 0.015 M NaOH
Flow Rate: | 0.6 mL/min
Temperature: | 85 °C
Detector: | RI

Food products containing high levels of sodium ions are best analyzed with Hi-Plex Na (Octo) columns. This saves time when sodium hydroxide is used as the eluent with PAD, because it eliminates the need for the post-column addition of sodium hydroxide.

1. Sucrose

2. Glucose

3. Arabinose

USP method for sorbitol

Column: | Hi-Plex Pb USP L34
| PL1170-2820
| 7.7 x 100 mm, 8 μm
Mobile Phase: | Water
Flow Rate: | 0.7 mL/min
Temperature: | 50 °C
Detector: | RI

USP method for sorbitol – a sugar alcohol and alternative sweetener – using mannitol as the internal standard. Hi-Plex Pb columns are recommended for alcoholic drinks that also contain glycerol, as well as sweetened dairy-based food products.

1. Mannitol

2. Sorbitol

Quick Guide to USP Designations for HPLC Columns

The US Pharmacopeia (USP) is a standard source for many pharmaceutical methods. The USP specifies columns by packing materials rather than by manufacturer. The USP has updated its L1 definitions, listed below you will see the most recent definitions and columns that apply. Rapid Resolution High Throughput (RRHT) columns are now choices in the L1, L7, and L11 categories.

Oligo Solutions

StratoSpheres DNA Cartridges

• Greater yields of full length products than controlled-pore glass

• Inert support prevents side reactions and improves quality of the end product

• 1000Å pore size permits synthesis of longer oligonucleotide sequences, up to 70-mer

• Certificate of Analysis offered for every batch

StratoSpheres DNA Synthesis Cartridges make it easy to obtain high-quality synthetic DNA oligonucleotides. The high-yielding polystyrene packing delivers more full-length product than conventional controlled-pore glass supports. In addition, the hydrophobic nature of the polystyrene promotes coupling and minimizes non-specific binding to maximize production efficiency. These high-throughput cartridges deliver very economical oligonucleotide synthesis, and provide the high performance expected from macroporous polystyrene supports. StratoSpheres DNA synthesis cartridges deliver maximum flexibility in high-throughput environments.

StratoSpheres DNA Cartridges

StratoSpheres DNA Cartridges
| Size |
Description | (nmol) | Part No.
StratoSpheres DNA DMT bz dA | 40 | PL3554-1602dAbz
| 200 | PL3554-4602dAbz
StratoSpheres DNA DMT bz dC | 40 | PL3554-1602dCbz
| 200 | PL3554-4602dCbz
StratoSpheres DNA DMT ac dC | 40 | PL3554-1602dCac
| 200 | PL3554-4602dCac
StratoSpheres DNA DMT ibu dG | 40 | PL3554-1602dGibu
| 200 | PL3554-4602dGibu
StratoSpheres DNA DMT dmf dG | 40 | PL3554-1602dGdmf
| 200 | PL3554-4602dGdmf
StratoSpheres DNA DMT dT | 40 | PL3554-1602dT
| 200 | PL3554-4602dT

TOP, TOP-DNA and TOP-RNA Cartridges

TOP, TOP-DNA and TOP-RNA Cartridges

• Superior yield and purity come from proprietary polymeric resins and optimized buffers

• Typical yield is more than 85% and typical purity is over 90%, eliminating the need for multiple sample-loading steps

• Agilent TOP cartridges use up to two thirds less reagent than products from other vendors

TOP, TOP-DNA and TOP-RNA cartridges provide a high-throughput, simple, cost-effective solution for DNA and RNA oligonucleotide purification. The TOP product range incorporates a unique 96-well plate with removable tubes, streamlined gravity flow or vacuum procedure, and proprietary polymeric resin. Agilent’s innovative technology delivers superior yield and purity for standard oligos up to 1 μmol synthesis scale and up to 150-mer in length. Flexibility is assured from a choice of simple gravity flow (for walk-away and low initial setup cost) or vacuum procedure (for fast turnaround – less than 15 minutes for the entire purification process). Up to 10 minutes drying time between each step is permissible with no effect on purification results (drying time after the acetonitrile conditioning step should be kept to a minimum).

TOP and TOP-DNA Cartridges

• Fast throughput improves production efficiency

• Pre-HPLC “sample prep” ability maximizes utility

• Gravity (TOP) or vacuum flow (TOP-DNA) ensures flexibility

TOP-DNA is a high-throughput, simple, fast, cost-effective solution that purifies oligos up to 150-mer in length. Its high binding capacity can purify DNA oligos from 200 nmol to 1 μmol synthesis scales. TOP-DNA can also be used for sample preparation before HPLC purification for very high quality oligos in large-scale analysis. The proprietary polymeric resin is compatible with direct loading of AMA deprotected oligo solutions.

[picture] | TIPS & TOOLS For more information on TOP RNA, view this Application Note on-line: High Performance RNA oligonucleotide purification using Agilent TOP-RNA (publication # 5990-8974EN), www.agilent.com/chem/library

TOP-RNA Cartridges

• A complete solution for RNA oligo purification to enhance productivity

• High throughput and automation friendly, freeing up operator time

• Less reagent use reduces operating costs

With TOP-RNA you can purify short and long RNA oligos, siRNA to 21-mer and long RNA to 60-80-mer. The high binding capacity purifies RNA oligos up to 1 μmol. The proprietary polymeric resin and validated protocol allow deprotection of 2'hydroxyl group without removal of the 5'trityl group.

Table of Contents

LC and LC/MS Columns for Biomolecules

Biocolumn Selection Guidelines

Biomolecule Separations

UHPLC/HPLC Techniques

Reversed-Phase HPLC

ZORBAX 300Å StableBond

ZORBAX 300Å Extend-C18

Poroshell 300

Poroshell 120

PLRP-S

ZORBAX Amino Acid Analysis (AAA) Columns and Supplies

Ion-Exchange Chromatography

Agilent Bio MAb HPLC Columns

Agilent Bio IEX HPLC Columns

PL-SAX Strong Anion-Exchange Columns

PL-SCX Strong Cation-Exchange Columns

Agilent Bio-Monolith Ion-Exchange HPLC Columns

Size Exclusion Chromatography

Agilent Bio SEC-3

Agilent Bio SEC-5

ProSEC 300S

ZORBAX GF-250 and GF-450 Gel Filtration Columns

Affinity Chromatography

Agilent Bio-Monolith Protein A HPLC Columns

Agilent Protein Fractionation System and Proteomics Reagents

Multiple Affinity Removal System

Multiple Affinity Removal System Starter Kits

mRP-C18 High-Recovery Protein Columns

Method Development

ZORBAX Column Methods

Reversed-Phase LC/MS Methods

Bio Ion-Exchange Column Methods

SEC Column Methods

High Sensitivity Capillary Column Methods

Capillary and Nano Columns

2-D LC/MS Analyses Using ZORBAX Capillary and Nano LC Columns

ZORBAX Bio-SCX Series II

MicroBore (1.0 mm id) Columns

Purification – Prep HPLC

ZORBAX PrepHT

PLRP-S for Prep to Process

PL-SAX and PL-SCX for Prep to Process

Peptide Purification

VariTide RPC Columns for Synthetic Peptides

VariPure IPE

Appendices

BioHPLC Columns Literature

ZORBAX 300 Citations

Poroshell 300 Citations

PLRP-S Citations

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COLUMNS FOR BIOMOLECULE SEPARATIONS

From sample purification to analysis, Agilent's biomolecule columns and supplies are easy to integrate into your workflow for a complete, reproducible, and high-quality solution.

In this section of the catalog you will also find advice and tips on solvent choice, mobile phase modification, optimization, and example separations to assist you in column selection and method development.

Agilent has complete solutions for your needs. These include the Agilent 1260 Infinity Bio-inert LC system with a metal-free sample path and the Agilent 1290 Infinity LC, designed to provide highest speed, resolution, and ultra-sensitivity for UHPLC applications, including those utilizing Agilent wide-pore 300Å ZORBAX StableBond columns. Biomolecules may be complex in structure, but their analysis is simplified by using Agilent HPLC columns, systems, and supplies.

What is a biomolecule?

Biomolecules are compounds made by living organisms. They can range in size from amino acids and small lipids to large polynucleotides such as DNA or RNA.

In this section, we deal with the separation of:

Proteins – separation based on size with size exclusion chromatography, charge with ion-exchange chromatography, and hydrophobicity with reversed-phase chromatography.

Peptides – biocolumns for the analysis and purification of the full range of peptides, including hydrophobic, hydrophilic, basic and acidic peptides across the full size range. Also, columns for peptide mapping by HPLC and UHPLC.

DNA/RNA oligonucleotides – reversed-phase and ion-exchange options for DNA and RNA oligos, and with particle pore sizes to cover the full range of oligonucleotide sizes, from small synthetic oligos to large plasmids.

Amino acids – the ZORBAX Eclipse Amino Acid Analysis HPLC columns provide a high efficiency solution for rapid analysis of 24 amino acids. Typical analysis times range from 14 minutes, with a 75 mm column, to 24 minutes with a 150 mm column.

Broad-distribution polymers – analysis of lipids, polysaccharides and drug delivery compounds using polymeric columns and standards to determine their molecular weight distribution and composition. These compounds tend to exhibit broad MW distributions, in contrast to other biomolecules that have narrow MW distributions or a defined molecular weight.

What is a biocolumn?

Biochromatography columns, or biocolumns, are liquid chromatography columns used for the separation of biological compounds such as peptides and proteins, oligonucleotides and polynucleotides, and other biomolecules and complexes. Biocolumns are specifically designed for biomolecule analysis with larger pore sizes to accommodate the larger molecule sizes. Media is designed to minimize non-specific binding of analytes for improved recovery. Separation mechanisms are chosen to either retain biological function so bioactivity is not lost during analysis, or to deliberately denature for primary structure characterization.

Typically, HPLC has been used to separate biomolecules. Now, advanced techniques such as UHPLC are becoming a popular choice because multiple separation mechanisms are needed in the characterization of biomolecules. Therefore, Agilent offers advanced chemistries developed specifically for the separation of biomolecules using size exclusion, reversed-phase, ion-exchange, and affinity functionalities, all of which are covered in this section of the catalog.

Protein Separations

Proteins are complex molecules that require multiple techniques to provide full characterization. They exist as three-dimensional structures and it is this structure that confers their biological activity.

The sequence of the amino acid chains defines the primary structure of the protein. Hydrogen bonding between amino acids of the primary structure then confers a secondary structure typically in the form of alpha helices and pleated sheets. A further series of interactions, hydrogen bonding, ionic, hydrophobic and disulphide bridges, between regions of the secondary structure, then provides the tertiary protein structure, or three-dimensional conformation. If the protein is composed of a number of amino acid chains, the interaction between these chains gives the quaternary structure.

When looking at methods for protein characterization, it is therefore clear from Figure 1 that techniques will be required that characterize the protein in its native state, without disrupting the tertiary and quaternary structures. We also need techniques for assessing the primary amino acid sequence, in the fully denatured state with the three-dimensional structure stripped away.

Figure 1. Schematic showing the various levels of protein structure.

The environment of the protein can influence, stabilize, or disrupt the structure of the protein. Factors to consider include pH, temperature, salt concentrations, aqueous or organic solvent content, and for some proteins, the presence of a stabilizing small molecule or metal ion. Protein structure can also be disrupted by the use of sulfhydryl reducing agents to break -S-S- bonds or chaotropic agent, like urea or guanidine HCI. With the complexity of proteins and the intramolecular interactions that determine the three-dimensional structure, you can also expect that there will be intermolecular associations between protein molecules and other molecular entities and the surfaces with which they come into contact. This can result in protein complexes, aggregation (with possible precipitation), and deposition on surfaces, including those of the HPLC column and system. Therefore, you should consider the handling and environment in which the protein is maintained.

Protein Column Selection Guide

Higher resolution of oxidation study

Column: | ZORBAX RRHD 300SB-C18 857750-902 2.1 x 50 mm, 1.8 μm
Mobile Phase: | A: 0.1% TFA B: 0.01% TFA + 80% ACN
Flow Rate: | 1.0 mL/min
Gradient: | 33 to 50% B, 0 to 4 min
Detector: | 1290 Infinity LC with diode array detector at 280 nm
Sample: | Insulin, insulin chain A and chain B, oxidized (bovinesigma, 1 mg/mL)

It is evident that the oxidized insulin chains are resolved from insulin in under 2 minutes using the Agilent ZORBAX RRHD 300SB-C18 2.1 x 50 mm, 1.8 μm column.

Intact MAb monomer and dimer separation

Column: | Bio SEC-3, 300Å 5190-2511 7.8 x 300 mm, 3 μm
Buffer: | Sodium phosphate buffer, pH 7.0, 150 mM
Isocratic: | 0-100% Buffer A from 0-30 min
Flow Rate: | 1.0 mL/min
Sample: | CHO-humanized MAb, 5 mg/mL – intact
Injection: | 5 μL
Detector: | UV 220 nm
Temperature: | Ambient

Separation of charge variants of human IgG1
with pH gradient

Column: | Agilent Bio MAb 5190-2411 2.1 x 250 mm, 5 μm
Mobile Phase: | A: 10 mM Na₂HPO₄, pH 6.0 B: A + 0.5 M NaCl or just 0.5 M Na₂HPO₄, pH 6.0
Flow Rate: | 2 mL/min

Gradient:

0.5 min hold with mobile phase A followed by a linear gradient to 45% B in 15 min (elapsed time 15.5 min); then 60% B at 15.6 min continued to 20 min. Column flushed with 100% B for 15 min before re-equilibration for the next run. pH Gradient: A: 5 mM Na₂HPO₄, buffer pH 5.5 and B: 40 mM Na₂HPO₄ (not buffered, pH 8.9). 2% B/min at 1 mL/min for 15 min, followed by a column wash with 90% B for 5 min.

Detector: | UV at 220 nm
Sample: | One mg each/mL in mobile phase A Monoclonal antibodies (MAb) -human IgG1 (5 mg/mL stock solution) derived from CHO cells
Instrument: | Agilent 1200 SL system with diode array detector

MAb c-terminal cleavage: Human IgG1 MAb, 1 mg/mL in 25 mM Na₂HPO₄ buffer, pH 7.5, was incubated with approximately 25 units of the carboxypeptidase B for 18 hours and 10 μL samples were injected.

Peptide Separations

Peptide Mapping

Peptide mapping is required for the characterization of proteins. It is used to confirm the identity of a protein and to identify and quantify post-translational modifications.

The purified protein is first digested using an enzyme, such as trypsin, yielding a range of peptide fragments. The specificity of the enzyme cleavage produces a fingerprint of peptides which is characteristic of that protein. Identification of the peptide fragments confirms the identity of the protein, and changes in the profile of the peptide digest can be used to identify post-translational modifications to that protein that may have occurred during the manufacturing or purification processes.

Reversed-phase UHPLC/HPLC is the preferred technique for the analysis of peptide digests with either MS or UV detection. LC/MS is used for the identification of the peptide fragments and determination of sequence coverage whereas LC/UV is more commonly used for peptide map comparisons in the monitoring/QC segments. To achieve sufficient resolution for quantification and identification, longer column lengths or higher efficiency particles such as the sub-2 μm ZORBAX RRHD, or superficially porous Poroshell are recommended.

Peptide digests are complex mixtures, and for complete coverage, i.e. resolution of the individual peptides, a high efficiency/high resolution column is required. The peptide fragments can range in size and hydrophobicity, so Agilent offers several columns for peptide mapping. There are three options: pore sizes, particle sizes, and superficially porous and fully porous for UHPLC separations.

| TIPS & TOOLS

Capillary electrophoresis is an alternative technique to liquid chromatography for the separation of complex peptide mixtures. Further information can be found in the following Case Study:

An orthogonal view of peptide mapping – analysis of bovine serum albumin digest using capillary electrophoresis and quadrupole time-of-flight mass spectrometry (publication # 5990-7631EN)

www.agilent.com/chem/library

Peptide Mapping Column Selection

Recommended column choices determined by system/column pressure maximum and peptide size/hydrophobicity.

Increased resolution for peptide mapping

Column: | ZORBAX 300SB-C18 858750-902 2.1 x 100 mm, 1.8 μm
Mobile Phase: | A: 0.1% TFA B: 0.01% TFA + 80% ACN
Flow Rate: | 0.5 mL/min
Gradient: | 2% B for 1 min, 2 to 45% B for 8.8 min, 45 to 95% B for 0.2 min, 95% B for 2 min, 95 to 2% B for 0.2 min
Temperature: | 50 °C
Detector: | 1290 Infinity LC with diode array detector at 280 nm
Sample: | Enzymatic protein digest (MAb)

The longer 100 mm Agilent ZORBAX RRHD 300SB-C18 column provides maximum resolution for protein digests – in this sample the total run time, including washing and equilibration, is under fifteen minutes.

Separation of Natural and Synthetic Peptides

Purification columns and media are required for the isolation and analysis of natural and synthetic peptides. Purity and recovery determination of the isolated or purified peptide requires the use of high efficiency columns. The primary technique used for the isolation and purification, and analysis, is reversed-phase HPLC.

The fractions from a purification or isolation workflow and the final peptide product are analyzed for purity using high efficiency columns. The peptides will vary in size, charge and hydrophobicity and so, as with peptide mapping applications, Agilent offers a range of columns to provide optimum separations of the full range of peptides. For small peptides, typically less than 10 amino acid residues, the smaller pore UHPLC materials are used, but if the peptide is larger, contains more amino acid residues, or exists in a dimeric or multimeric form, then the larger pore size 300Å columns provide better separations due to improved mass transfer.

Natural and Synthetic Peptides
Column Selection

Recommended column choices as determined by system/column pressure maximum for the analysis of natural and synthetic peptides.

Reversed-phase columns are also the first choice for purifying large numbers of individual peptides or larger amounts of a particular peptide. High efficiency, small particle pre-packed prep columns are available for the high efficiency purification of small amounts of peptides, and larger particle columns and bulk media for the larger scale purifications, as shown in Table 1.

After solid phase synthesis (SPS) using a polystyrene resin such as one of the Agilent StratoSpheres products, the peptide is cleaved from the support and the resultant mixture is separated to obtain the target peptide. A high efficiency column is needed for the purification as the candidate peptide must be resolved from peptides that are very similar in structure. Check www.agilent.com for further information.

Preparative scale purification of Leuprolide by concentration overload

Column: | PLRP-S 100Å, 10 μm PL1412-4100
Bulk Media: | Load & Lock 4001 Column PCG93LL500X25
Mobile Phase: | Isocratic separation using 0.1% TFA in 28% ACN:72% water
Flow Rate: | Linear velocity 360 cm/hr

Crude leuprolide separation of 30 mg on-column load.

Fraction analysis – the concentration overload purification.

DNA and RNA Oligonucleotide Separations

There is a renewed interest in oligonucleotides (oligos) as they are used in more and more applications, including potential therapeutics. The synthesis workflow is similar to that used for the more established synthetic peptide production, i.e. an activated solid phase synthesis resin is used with sequential addition of specific nucleotides to build the desired sequence.

The nucleotide building blocks are protected at the 5’ hydroxyl end with a dimethoxytrityl (DMT) group and the cleaved target oligo will have this protected group still attached. As DMT is hydrophobic, it is a useful handle that can be used for the first stage step. To increase the stability of the oligonucleotide, particularly to enzyme degradation, it may be chemically modified, for example by replacing oxygen with sulfur to produce phosphorothioates.

When using chemical synthesis to produce biomolecules, the coupling efficiency of each additional cycle is never 100%. The sample, after cleavage from the solid phase synthesis support, will contain deletion sequences, oligos where one or more residues are missing, and some amount of larger oligos produced by double coupling or branching. The sample mixture is complex and high efficiency techniques are required for analysis.

There are three UHPLC/HPLC techniques that are routinely used for oligonucleotide separations:

Trityl-on: This procedure is relatively simple to perform and separates the full-length target oligo, which still has the DMT group attached, from the deprotected failure sequences. The analytical information obtained is limited and this is generally considered to be a purification method.

Ion-exchange separations of the trityl-off, deprotected oligos: This method uses the negative charge on the backbone of the oligo to facilitate the separation. Resolution is good for the shorter oligos but decreases with increasing chain length. Aqueous eluents are used but oligos are highly charged, and high concentrations of salt are needed to achieve elution from the column.

Ion-pair reversed-phase separation of the trityl-off, deprotected oligos: This technique uses organic solvents and volatile ion-pairing agents and is suitable for LC/MS. The technique is best performed with high efficiency particles. Conditions that fully denature the oligos and prevent association with complimentary sequences are required. Thus, the separation is best performed at elevated temperatures.

DNA and RNA Oligonucleotide Column Selection

TIPS & TOOLS |

Further information can be found in the following publications:
Agilent PLRP-S 100Å HPLC Columns and Media (publication # 5990-8187EN)
Agilent PL-SAX 1000Å HPLC Columns and Media (publication # 5990-8200EN)
www.agilent.com/chem/library

Amino Acid Analysis

Agilent offers several good options for separation of amino acids, including the Agilent ZORBAX Eclipse AAA column which uses an updated protocol and is specially tested using amino acids. The ZORBAX Eclipse AAA high efficiency column rapidly separates amino acids following an updated and improved protocol. Total analysis from injection to injection can be achieved in as little as 14 min (9 min analysis time) on shorter, 7.5 cm columns and 24 min (18 min analysis time) on the 15 cm column. Exceptional sensitivity (5 to 50 pmol with diode array or fluorescence detectors) and reliability are achieved using both OPA-and FMOC-derivatization chemistries in one fully automated procedure using the Agilent 1200 Infinity LC. The newer ZORBAX Eclipse Plus C18 column is also an excellent choice for amino acid separations.

ZORBAX Eclipse AAA Column Selection
Application | Diameter x Length (mm) | Particle Size (μm)
Analytical routine sensitivity | 4.6 x 150 | 5.0
Analytical routine sensitivity, high-resolution using FLD | 4.6 x 150 | 3.5
Analytical routine sensitivity, high-throughput | 4.6 x 75 | 3.5
Solvent Saver high sensitivity, high-resolution | 3.0 x 150 | 3.5

For more information on the ZORBAX Eclipse Plus C18 column, View Here.

Broad Distribution Biomolecules

Carbohydrates, Lipids and PEGs

Aqueous size exclusion chromatography employing columns packed with polymeric media can be extremely useful when investigating biomolecules and their derived species with broad molecular weight distributions. Examples include PEGylated proteins and complex polysaccharides which find use in biopharma applications. The wide pore size distribution of polymeric SEC columns compared to silica-based material are excellent for samples with polydispersities greater than one.

Broad Distribution Biomolecule Column Selection
Low MW polymers and oligomers, oligosaccharides, PEGs, lignosulfonates | 2 or 3 PL aquagel-OH columns | The PL aquagel-OH analytical series has a pH range of 2-10, compatible with organic solvents (up to 50% methanol), mechanical stability up to 140 bar (2030 psi) and low column operating pressures.
• PL aquagel-OH 8 μm
• PL aquagel-OH 20 5 μm
• PL aquagel-OH MIXED-M 8 μm
Polydisperse biopolymers, polysaccharides, cellulose derivatives | 2 or 3 PL aquagel-OH columns |
• PL aquagel-OH MIXED-H 8 μm
• PL aquagel-OH 60/50/40 8 μm |
Very high MW polymers, hyaluronic acids, starches, gums | PL aquagel-OH 60/50/40 15 μm in series |

UHPLC/HPLC Techniques

High-performance liquid chromatography, HPLC, is a chromatographic technique that can separate a mixture of compounds and is used in biochemistry and analytical chemistry to identify, quantify and purify the individual components of the mixture. There has been an evolution toward ultra high-performance liquid chromatography (UHPLC) which is widely accepted for high-efficiency separations of small- to medium-sized molecules, and has been used to reduce analysis time and/or to increase resolution. The use of UHPLC has been extended to large biomolecules with the introduction of wide pore chromatographic media in columns that can withstand pressures of 600 to 1200 bar.

On the following pages you will see the wide range of columns that Agilent offers for the HPLC and UHPLC separation of proteins and other biomolecules.

UHPLC/HPLC Techniques for Biomolecule Analysis
Technique | Advantages | Disadvantages
Reversed-Phase | • High resolution | • Denaturing conditions
| • High capacity | • High efficiency silica columns cannot be cleaned using aggressive solvents when performing purifications
| • Relatively simple |
| • Sample concentrated on-column |
| • Small particle, 1.8 μm, for UHPLC separations |
| • Polymeric media for unsurpassed chemically and thermally stable |
Ion-Exchange | • Good recovery of biological activity | • Limited MS compatibility due to presence of salts
| • High capacity |
| • Sample concentrated on-column |
Size Exclusion | • Good recovery of biological activity | • No sample concentration
| • Non-interactive technique with good sample recovery | • Limited capacity
Affinity | • Highly selective | • No sample concentration
| • Good recovery of biological activity | • Limited capacity
| • Sample concentrated on-column |
| • Often single step isolation |

Reversed-Phase HPLC

Confidently perform high-resolution separations

Reversed-phase UHPLC/HPLC separates solutes based on differences in hydrophobicity, with the least hydrophobic peak eluting first. This high-resolution technique is capable of separating peptides, proteins and oligonucleotides that differ by only one amino acid or nucleotide residue.

Because HPLC uses organic solvents (such as acetonitrile, methanol, ethanol and propanol) it is also a denaturing technique that disrupts a biomolecules’s three-dimensional structure. This allows you to obtain information about a molecule’s primary structure and sequence, as well as variations in the sequence to be identified.

Agilent offers the industry’s broadest range of wide-pore reversed-phase columns, all backed by technical support experts and application chemists around the globe. This section features the following column innovations:

• ZORBAX 300Å pore silica columns – an industry first for reversed-phase protein and biomolecule separations – are available in 6 phases, along with a broad array of sizes. For fast UHPLC separations, we also offer a 1.8 μm particle size option that withstands pressures up to 1200 bar, and can be used with high-pressure instruments, such as Agilent’s 1290 Infinity LC.

• Agilent Poroshell columns feature the industry’s first solid core/porous shell particle. Our wide-pore Poroshell 300 columns are ideal for fast chromatography, and are available in a variety of phases.

• Agilent PLRP-S columns contain polymer particles, and can be used to separate peptides and proteins of various sizes and DNA/macromolecular complexes. These columns are unique in that they are 100% organic, can withstand temperatures as high as 200 °C, and can be used under conditions from pH 1 to pH 14.

• Choose from a range of column sizes, particle sizes (3-8 μm for analytical separations) and pore sizes (100Å to 4000Å). Preparative columns (10-50 μm) are also available, either prepacked in columns or as bulk material.

ZORBAX 300Å StableBond

Agilent ZORBAX 300Å StableBond columns are an ideal choice for the reproducible separations of proteins and peptides for two key reasons. First, wide-pore, 300Å columns are necessary for an efficient separation of proteins and peptides, or other large molecules, in order to allow these analytes to completely access the bonded phase. Second, 300StableBond columns are unmatched in their durability at low pH, such as with TFA-containing mobile phases typically used for protein and peptide separations. For LC/MS separations at low pH, 300StableBond columns can also be used with formic acid and acetic acid mobile phase modifiers. These columns are available in five different bonded phases (C18, C8, C3, CN, and Diphenyl*) for selectivity and recovery optimization of proteins and polypeptides. To further increase sample recovery and improve efficiency for difficult proteins, 300StableBond columns can be used up to 80 °C. 300SB-C18 and 300SB-C8 columns are an ideal choice for complex protein and protein digest separations. These columns are also available in capillary (0.3 and 0.5 mm id) and nano (0.075 and 0.10 mm id) dimensions for reversed-phase LC/MS separations of protein digests. Capillary and nano columns can be used for either 1-D or 2-D proteomics separations.

*Diphenyl is available in a 1.8 μm particle size only.

Sterically Protected 300StableBond Bonded Phase

TIPS & TOOLS |

Further information can be found in the following publication:

Comparison of ZORBAX StableBond 300Å LC Columns to Optimize Selectivity for Antibody Separations
Using HPLC and LC/MS (publication # 5989-6840EN)
www.agilent.com/chem/library

Higher resolution of intact monoclonal antibody

Column: | ZORBAX RRHD 300SB-C8 857750-906 2.1 x 50 mm, 1.8 μm
Mobile Phase: | A: H₂0:IPA (98:2) + 0.1% TFA (v/v) B: IPA:ACN:H₂0 (70:20:10) + 0.1% TFA (v/v)
Flow Rate: | Between 0.5 mL/min and 1.0 mL/min
Gradient: | Multi-segmented and linear elution
Temperature: | 80 °C
Detector: | Agilent 1290 Infinity LC with auto injector (ALS), binary pump and thermostatted oven and diode array detector (DAD), UV, 225 nm

Higher resolution of oxidation study

Column: | ZORBAX RRHD 300SB-C18 857750-902 2.1 x 50 mm, 1.8 μm
Mobile Phase: | A: 0.1% TFA B: 0.01% TFA + 80% ACN
Flow Rate: | 1.0 mL/min
Gradient: | 33 to 50% B, 0 to 4 min
Detector: | 1290 Infinity LC with diode array detector at 280 nm
Sample: | Insulin, insulin chain A and chain B, oxidized (bovinesigma, 1 mg/mL)

It is evident that the oxidized insulin chains are resolved from insulin in under 2 minutes using the Agilent ZORBAX RRHD 300SB-C18 2.1 x 50 mm, 1.8 μm column.

| TIPS & TOOLS
[picture] | Typical mobile phases for protein and peptide separations combine a very low pH with TFA (or other acids) to solubilize proteins. StableBond columns have extremely long lifetimes under these conditions. They are available in 300Å pore size for proteins up to 100-500 kDa.

Improved reproducibility of monoclonal antibodies

Column: | ZORBAX RRHD 300SB-C8 857750-906 2.1 x 50 mm, 1.8 μm
Mobile Phase: | A: H₂0:IPA (98.2), 0.1% TFA B: IPA:ACN:H₂0 (70:20:10), 0.1% TFA
Flow Rate: | 1.0 mL/min
Temperature: | 80 °C
Detector: | 1290 Infinity LC with diode array detector at 225 nm
Sample: | MAb

Gradient timescale

Time (min) | % Solvent B
0.00 | 25
3.00 | 35
4.00 | 90
5.00 | 25

Excellent column reproducibility and protein recovery using Agilent ZORBAX 300SB-C8.

Increased resolution for peptide mapping

Column: | ZORBAX 300SB-C18 858750-902 2.1 x 100 mm, 1.8 μm
Mobile Phase: | A: 0.1% TFA B: 0.01% TFA + 80% ACN
Flow Rate: | 0.5 mL/min
Gradient: | 2% B for 1 min, 2 to 45% B for 8.8 min, 45 to 95% B for 0.2 min, 95% B for 2 min, 95 to 2% B for 0.2 min
Temperature: | 50 °C
Detector: | 1290 Infinity LC with diode array detector at 280 nm
Sample: | Enzymatic protein digest (MAb)

The longer 100 mm Agilent ZORBAX RRHD 300SB-C18 column provides maximum resolution for protein digests – in this sample the total run time, including washing and equilibration, is under fifteen minutes.

Peptides: Effect of TFA concentration

Column: | ZORBAX 300SB-C8 883995-906 4.6 x 150 mm, 5 μm
Mobile Phase: | A: Water and TFA B: ACN and TFA
Flow Rate: | 1.0 mL/min
Gradient: | 0 min 0% B 30 min 30% B
Temperature: | 40 °C
Detector: | UV 254 nm
Sample: | Peptide Standards S1-S5, decapeptides differing slightly in hydrophobicity, 6 μL

Peptides/proteins: Effect of elevated temperature

Column: | ZORBAX 300SB-C3 883995-909 4.6 x 150 mm, 5 μm

Mobile Phase:

A: 5:95
ACN:Water with 0.10% TFA (v/v%)
B: 95:5
ACN:Water with 0.085% TFA (v/v%)

Flow Rate: | 1.0 mL/min
Gradient: | 15-53% in 20 min, posttime 12 min
Temperature: | Ambient – 60 °C
Detector: | UV 215 nm
Sample: | Polypeptides

Ambient

45 °C

35 °C

60 °C

1. Leucine Enkephalin

2. Angiotensin II

3. RNase A

4. Insulin (BOV)

5. Cytochrome c

6. Lysozyme

7. Myoglobin

8. Carbonic anhydrase

| TIPS & TOOLS

The Agilent 1290 Infinity LC delivers significantly faster results and higher data quality – enabling more informed decisions in shorter time. This higher productivity gives you competitive advantages and provides you a higher return on investment. Calculate for yourself how much you can save by deploying the 1290 Infinity technology. The online method translator and cost savings calculator helps you to transfer your HPLC methods and calculate your cost savings, at www.agilent.com/chem/hplc2uhplc

Short-chain ZORBAX 300SB-C3 is stable at low pH, high temperature

Column: | ZORBAX 300SB-C3 883995-909 4.6 x 150 mm, 5 μm

Mobile Phase:

Gradients 0-100% B in 80 min
A: 0.5% TFA in Water
B: 0.5% TFA in Acetonitrile
Isocratic Retention Test Conditions:
1-phenylheptane 50% A, 50% B

Flow Rate: | 1.0 mL/min
Temperature: | 60 °C

Four different 300SB bonded phases optimize separation of large polypeptides

Column A: | ZORBAX RRHD 300SB-C18 883995-902 4.6 x 150 mm, 5 μm
Column B: | ZORBAX 300SB-C8 883995-906 4.6 x 150 mm, 5 μm
Column C: | ZORBAX 300SB-C3 883995-909 4.6 x 150 mm, 5 μm
Column D: | ZORBAX 300SB-CN 883995-905 4.6 x 150 mm, 5 μm
Mobile Phase: | Linear Gradient, 25-70% B in 40 min A: 0.1% TFA in Water B: 0.09% TFA in 80% Acetonitrile/20% Water
Flow Rate: | 1.0 mL/min
Temperature: | 60 °C
Sample: | 3 μg each protein

1. RNase

2. Insulin

3. Cytochrome c

4. Lysozyme

5. Parvalbumin

6. CDR

7. Myoglobin

8. Carbonic Anhydrase

9. S-100β

10. S-100α

The 300SB-C18, C8, C3, and CN bonded phases all provide a different separation of this group of polypeptides. This adds an important parameter for quickly optimizing protein separations. The 300SB-CN column offers unique selectivity for more hydrophilic polypeptides.

ZORBAX RRHD 300-Diphenyl

Utilizing the same unique chemistry as the Pursuit 3.5 μm and 5 μm Diphenyl columns, the unique wide pore 300Å Diphenyl phase offers additional selectivity through pi-pi interactions with aromatic amino acids in the primary sequence. Agilent ZORBAX 1.8 μm 300Å Rapid Resolution High Definition (RRHD) columns bring UHPLC performance to the reversed-phase separation of intact proteins and protein digests.

The diphenyl column can be used for:

• Analysis of intact and modified proteins and polypeptides including protein structural analysis

• Detection of post-translational modifications

• Impurity analysis

• Confirming protein identity

The ZORBAX RRHD 300-Diphenyl provides:

• Stability at low pH – allowing you to run your protein and peptide separations down to pH 1 using trifluoroacetic acid (TFA), and formic acid eluents with complete confidence

• Temperature stability – you can run your separations up to 80 °C to improve efficiency and reduce eluent viscosity, without compromising column lifetime

• UHPLC compatible – enabling higher order characterization with reduced analysis time

Fast separation of reduced monoclonal antibody

Column: | Agilent ZORBAX RRHD 300-Diphenyl 858750-944 2.1 x 100 mm, 1.8 μm
Mobile Phase: | A: 0.1% TFA in water B: 80% n-propyl alcohol,      10% ACN, 9.9% water, and 0.1% TFA
Sample: | Reduced monoclonal antibody (IgG1) (1.0 mg/mL)
Sample Injection: | 2 μL
Flow Rate: | 0.5 mL/min
Gradient: | 0 min-1%B, 2 min-20% B, 5 min-50% B
Temperature: | 74 °C
Detector: | UV, 280

ZORBAX 300Å Extend-C18

• Rugged, high and low pH separations of polypeptides and peptides from pH 2-11.5

• Different selectivity possible at high and low pH

• High efficiency and good recovery of hydrophobic peptides at high pH

• Ideal for LC/MS with ammonium-hydroxide-modified mobile phase

Agilent ZORBAX 300Å Extend-C18 is a wide-pore HPLC column for high efficiency separations of peptides from pH 2-11.5. The unique, bidentate bonded phase provides excellent lifetime and reproducibility at high and low pH. At high pH, retention and selectivity of peptides and polypeptides can change dramatically as a result of changes in charge on molecules. Excellent recoveries of hydrophobic polypeptides have been achieved at room temperature and high pH. LC/MS sensitivity of peptides and polypeptides can also be improved at high pH using a simple ammonium-hydroxide-containing mobile phase.

Novel Bidentate C18-C18 Bonding for Extend-C18 Bonded Phase

| TIPS & TOOLS
[picture] | Selecting the right column is only part of the total solution. Don't forget key supplies such as our wide range of LC lamps. View Here.

LC/MS analysis of angiotensin on Extend-C18

Column: | ZORBAX Extend-C18 773700-902 2.1 x 150 mm, 5 μm

Mobile Phase:

Acidic Conditions:
A: 0.1% TFA in water
B: 0.085% TFA in 80% acetonitrile (ACN)
Basic Conditions:
A: 10 mM NH₄OH in water
B: 10 mM NH₄OH in 80% ACN

Flow Rate: | 0.2 mL/min
Gradient: | 15-50% B in 15 min
Temperature: | 35 °C
MS Conditions: | Pos. Ion ESI- Vf 70 V, Vcap 4.5 kV, N₂ – 35 psi, 12 L/min., 325 °C
Sample: | 2.5 μL sample (50 pmol each) Angiotensin I, II, III

Both small and large peptides demonstrate selectivity changes at high and low pH. At high pH, due to a change in charge, all three Angiotensins can be resolved. In addition, the spectral clarity of Angiotensin I is dramatically improved at high pH with the ammonium hydroxide mobile phase. The Extend-C18 column can be used for the analysis of small peptides at high pH as well.

Reference: B.E. Boyes. Separation and Analysis of Peptides at High pH Using RP-HPLC/ESI-MS, 4th WCBP, San Francisco, CA, Jan. 2000.

Long life at high pH with 300Extend-C18

Column: | ZorBAX extend-C18 773450-902 4.6 x 150 mm, 5 μm
Mobile Phase: | 20% 20 mM NH₄OH, pH 10.5 80% Methanol
Flow Rate: | 1.5 mL/min
Temperature: | Aging 24 °C Tests 40 °C

Each 10,000 column volume is approximately one working month.

Use ZORBAX Extend-C18 for alternate selectivity at high pH

Column: | ZORBAX Extend-C18 773700-902 2.1 x 150 mm, 5 μm
Mobile Phase: | A: 0.1% TFA in Water B: 0.085% TFA in 80% ACN
| A: 20 mM NH₄OH in Water B: 20 mM NH₄OH in 80% ACN
Flow Rate: | 0.25 mL/min
Gradient: | 5-60% B in 20 min
Temperature: | 25 °C
MS Conditions: | Pos. Ion ESI-Vf 70V, Vcap 4.5 kV N₂- 35 psi, 12 L/min, 300 °C 4 μL (50 ng each peptide)

The Extend column can be used for high pH separations of peptides. At high and low pH, very different selectivity can result. Just by changing pH, a complimentary method can be developed and it is possible to determine if all peaks are resolved. The Extend column can be used at high and low pH, so the complimentary separation can be investigated with one column. Better MS sensitivity for this sample is also achieved at high pH.

Poroshell 300

• UHPLC separations of biomolecules with superficially porous particles

• 300Å pore provide high efficiency and recovery with proteins (up to 1,000 kDa) and monoclonal antibodies

• Achieve long lifetime at low pH with Poroshell 300SB; at high pH with 300Extend-C18

• Optimize recovery and selectivity with four different bonded phases – 300SB-C18, 300SB-C8, 300SB-C3, and 300Extend-C18

Agilent Poroshell 300 columns are ideal for fast separations of proteins and peptides because the superficially porous particle allows for fast flow rates to be used while maintaining sharp, efficient peaks. Peptides and proteins are typically separated slowly to reduce the potential peak broadening of these slow diffusing analytes. However, Poroshell columns use a superficially porous particle made with a thin layer of porous silica, 0.25 μm thick, on a solid core of silica. This reduces the diffusion distance for proteins making practical rapid HPLC separations of peptides and proteins up to 500-1,000 kDA possible with 400/600 bar HPLC systems, including the Agilent 1260 Infinity Bio-inert. Poroshell columns bonded with StableBond bonded phases provide excellent stability and selectivity choices with TFA and formic acid mobile phases. The Poroshell 300Extend-C18 column can be used from pH 2-11 for unique separations. These columns can be used for analytical protein separations as well as LC/MS separations.

Poroshell 300 Columns

Poroshell 300 columns separate proteins and peptides in seconds

Column: | Poroshell 300SB-C18 660750-902 2.1 x 75 mm, 5 μm
Mobile Phase: | A: 0.1% TFA in H₂0 B: 0.07% TFA in ACN
Flow Rate: | 3.0 mL/min
Gradient: | 5-100% B in 1.0 min
Temperature: | 70 °C, 260 bar pressure
Detector: | 215 nm
Sample: | Proteins and Peptides

1. Angiotensin II

2. Neurotensin

3. RNase

4. Insulin

5. Lysozyme

6. Myoglobin

7. Carbonic Anhydrase

8. Ovalbumin

This separation of eight polypeptides and proteins is completed in less than 60 seconds. Each peak is sharp and efficient.

TIPS & TOOLS |

Further information can be found in the following publications:

Poroshell 300SB-C18 (publication # 5988-2100ENUS)

Rapid HPLC Analysis of Monoclonal Antibody IgG₁ Heavy Chains Using ZORBAX Poroshell 300SB-C8 (publication # 5989-0070EN)

Use of Temperature to Increase Resolution in the Ultrafast HPLC Separation of Proteins with ZORBAX Poroshell 300SB- C8 HPLC Columns (publication # 5989-0589EN)

Using the High-pH Stability of ZORBAX Poroshell 300Extend-C18 to Increase Signal-to-Noise in LC/MS (publication # 5989-0683EN)

www.agilent.com/chem/library

Reduce peptide map analysis time by 90% with Poroshell 300SB

Column A: | Poroshell 300SB-C18 660750-902 2.1 x 75 mm, 5 μm
Column B: | ZORBAX 300SB-C18 883750-902 2.1 x 150 mm, 5 μm
Mobile Phase: | A: 95% H2O, 5% ACN, 0.1% TFA B: 5% H2O, 95% ACN, 0.07% TFA
Flow Rate: | 1 mL/min 0.208 mL/min
Gradient: | 0-100% B = 12 min 0-100% B = 120 min
Temperature: | 70 °C
Sample: | 20 μL (0.22 μg/1 μL) BSA Tryptic Digest (15 hours, 70 pmol)

A single chromatographic run of a protein tryptic digest can require one hour or more to complete. With Poroshell columns, the same complex separation can be completed in 1/10th the time.

MicroBore Poroshell 300 columns provide maximum sensitivity for LC/MS

Column: | Poroshell 300SB-C18 661750-902 1.0 x 75 mm, 5 μm
Mobile Phase: | A: Water + 0.1% Formic Acid B: ACN + 0.1% Formic Acid
Flow Rate: | 600 μL/min
Gradient: | 20-100% B in 5.5 min
Temperature: | 80 °C

MS Conditions:

LC/MS: Pos. Ion ESI – Vcap 6000 V
Drying Gas Flow: 12 L/min
Drying Gas Temperature: 350 °C
Nebulizer: 45 psi
Fragmentor Volatage: 140 V
Scan: 600-2500
Stepsize: 0.15 amu
Peak width: 0.06 min

Sample: | 1 μL

With narrow bore diameters of 2.1 mm, 1.0 mm, and 0.5 mm, Poroshell columns make an ideal LC/MS partner. When the sample is very limited, the 1.0 mm or 0.5 mm id Poroshell columns are an excellent choice for high sensitivity LC/MS analyses. Sensitive MS molecular weight determinations are possible with as little as 0.5 to 5 pmole of protein on Poroshell columns. Poroshell columns have also been used for rapid MS identification of intact proteins, even in the presence of stabilizers and tissue culture media.

Monoclonal igG1 chains: Separation on Poroshell 300SB-C8

Column: | Poroshell 300SB-C8 660750-906 2.1 x 75 mm, 5 μm

Mobile Phase:

A: 90% water:
10% ACN + 3 mL/L of MW 300 PEG
B: 10% water:
90% ACN + 3 mL/L of MW 300 PEG

Flow Rate: | 1.0 mL/min

Gradient:

0 min 25% B
10 min 40% B
10.1 min 25% B
12 min 25% B

Temperature: | 70 °C
Sample: | Monoclonal IgG1

Courtesy of: Novartis Pharma, Biotechnology, Basel Dr. Kurt Forrer Patrik Roethlisberger

TIPS & TOOLS |
Agilent offers an extensive selection of certified chromatography sample vials including polypropylene and deactivated and siliconized glass. For more information see (publication # 5990-9022EN). www.agilent.com/chem/library | [picture]

Protein elution pattern on ZORBAX Poroshell 300SB-C8

Column: | Poroshell 300SB-C8 660750-906 2.1 x 75 mm, 5 μm
Mobile Phase: | A: 0.1% TFA in H₂0 B: 0.1% TFA in ACN
Flow Rate: | 1.0 mL/min
Gradient: | B: 20 to 70% in 3 min
Detector: | UV (214 nm)

1. Glycoprotein X, MW ~ 22 kDa

2. Protein I, MW ~ 4 kDa

3. Glucagon, MW ~ 3.5 kDa

4. Biosynthetic human insulin, MW ~ 6 kDa

5. Protein J, MW ~ 3 kDa

6. Protein K, MW ~ 6 kDa

7. Glycoprotein Y, MW ~ 45 kDa

8. Glycoprotein Z, MW ~ 30 kDa

Poroshell 120

• 120Å pore size for shorter chain peptide mapping

• UHPLC performance on 600 bar systems

• Up to 90% of the efficiency of sub-2 μm

• 2X the efficiency of 3.5 μm

• Up to 50% less pressure than sub-2 μm columns

Agilent Poroshell 120 columns are a 2.7 μm particle with a 1.7 μm solid core and 0.5 μm porous outer layer. This small particle size provides high efficiency, similar to sub-2 μm columns, but with 40-50% less pressure. These high efficiency, high resolution columns can be used on any type of LC. The porous outer layer and solid core limit diffusion distance and improve separation speed while the narrow particle size distribution improves efficiency and resolution. The columns can support high pressure and multiple columns can be used for the highest resolution and efficiency possible. The smaller 120Å pore size is ideal for fast high resolution analysis of small hydrophilic peptides in protein digests.

For information on the full family of Poroshell 120 phases, see here.

PLRP-S

• Contain durable and resilient polymer particles that deliver reproducible results over longer lifetimes

• Thermally and chemically stable

• Comply with USP L21 designation

• Used in bioscience, chemical, clinical research, energy, environmental, food and agriculture, material science and pharmaceutical industries

• Pore sizes (100Å-4000Å) for separations of small molecules to large complexes and polynucleotides

The PLRP-S family of columns consists of a range of pore sizes and particle sizes, all with identical chemistry and fundamental adsorptive characteristics. The particles are inherently hydrophobic, therefore no bonded phase, alkyl ligand is required for reversed-phase separations. This gives a highly reproducible material that is free from silanols and heavy metal ions. Columns within the extensive product range are suitable for nano/capillary separations, including both bottom-up and top-down proteomics, analytical separations, and preparative purifications. In addition, process columns can be packed with bulk media.

Column Specifications
pH Range | 1-14
Buffer Content | Unlimited
Organic Modifier | 1-100%
Temperature Limits | 200 °C
Maximum Pressure | 5-8 μm: 3000 psi (210 bar)
| 3 μm: 4000 psi (300 bar)

PLRP-S Applications
Pore Size | Application
100Å | Small molecules/peptides/oligonucleotides
300Å | Recombinant peptides/proteins
1000Å | Large proteins
4000Å | DNA/high speed

HPLC of 25 bp DNA ladder

Column: | PLRP-S, 2.1 x 150 mm
Mobile Phase: | A: 0.1 M TEAA B: 0.1 M TEAA in 50% water:50% ACN
Flow Rate: | 200 μL/min
Gradient: | 12.5-50% B in 150 min

Polyethylene glycols

Column: | PLRP-S 100Å PL1111-3500 4.6 x 150 mm, 5 μm
Mobile Phase: | A: Water B: ACN
Gradient: | 10-30% B in 12 min, held at 30% B for 3 min
Flow Rate: | 1.0 mL/min
Injection Volume: | 10 μL
Sample Conc: | 1 mg/mL
Detector: | ELS (neb=50 °C, evap=70 °C, gas=1.6 SLM)

Exploiting chemical stability – TFA concentration

Column: | PLRP-S 100Å PL1512-5500 4.6 x 250 mm, 5 μm
Mobile Phase: | A: TFA (various %) in water B: TFA (various %) in ACN
Gradient: | Linear 12-40% B in 15 min
Flow Rate: | 1.0 mL/min
Detector: | ELS (neb=75 °C, evap=85 °C, gas=1.0 SLM)

1. Angiotensin III

2. Angiotensin II

3. Angiotensin I

Selectivity in peptide RP-LC

Column: | PLrP-S 100Å PL1512-5500 4.6 x 250 mm, 5 μm
Mobile Phase: | A: 0.1% TFA/1% 2-Propanol/Water B: 0.1% TFA/1% 2-Propanol/ACN
Flow Rate: | 1.0 mL/min
Gradient: | 95% A (0-3 min) to 50% A (13 min)
Detector: | UV, 220 nm

Good separation of peptide standards on Agilent PLRP-S

1. YG

2. GYG

3. PY

4. YV

5. YY

6. GLY

7. YF

8. GFL

9. YGGFM

10. Oxalic acid (marker)

11. Benzoic acid (marker)

(see J.Chromatography 512 (1990) 315-23)

Exploiting chemical stability – NH₄OH concentration

Column: | PLRP-S 100Å PL1512-5500 4.6 x 250 mm, 5 μm
Mobile Phase: | A: NH₄OH (various mM) in water B: NH₄OH (various mM) in ACN
Gradient: | Linear 10-100% B in 15 min
Flow Rate: | 1.0 mL/min
Detector: | ELS (neb=80 °C, evap=85 °C, gas=1.0 SLM)

1. Angiotensin II

2. Angiotensin I

3. Angiotensin III

Alberta Peptide institute test mix

Column: | PLRP-S 100Å PL1512-5500 4.6 x 250 mm, 5 μm
Mobile Phase: | A: 0.1% TFA in 99% water:1% ACN B: 0.1% TFA in 70% water:30% ACN
Gradient: | 0-100% B in 30 min
Flow Rate: | 1.0 mL/min
Detector: | UV, 220 nm

1. Ala³-Gly⁴ (free amino)

2. Gly³-Gly⁴ (Nα-acetylated)

3. Ala³-Gly⁴ (Nα-acetylated)

4. Val³-Gly⁴ (Nα-acetylated)

5. Val³-Val⁴ (Nα-acetylated)

Whey proteins in dairy samples – milk

Column: | PLRP-S 300Å PL1512-3801 4.6 x 150 mm, 8 μm
Mobile Phase: | A: 0.1% TFA in 99% water:1% ACN B: 0.1% TFA in 1% water:99% ACN
Gradient: | 36-48% B, 0-24 min, 48-100% B, 24-30 min 100% B, 30-35 min, 100-36% B, 35-40 min
Flow Rate: | 1.0 mL/min
Injection Volume: | 10 μL
Detector: | UV, 220 nm

1. α-Lactalbumin

2. β-Lactoglobulin (B chain)

3. β-Lactoglobulin (A chain)

Temperature as a tool to enhance mass transfer and improve resolution of oligonucleotides in ion-pair reversed-phase HPLC

Column: | PLRP-S 100Å PL1512-1300 4.6 x 50 mm, 3 μm
Mobile Phase: | A: 100 mM TEAA B: 100 mM TEAA in 25% ACN
Gradient: | 5% change in buffer B over 5 min
Flow Rate: | 1.0 mL/min
Temperature: | 35 °C, 60 °C, or 80 °C
Detector: | UV, 254 nm

1. 29-mer

2. 30-mer

Large fibrous proteins

Column: | PLRP-S 300Å PL1512-3801 4.6 x 150 mm, 8 μm
Column: | PLRP-S 1000Å PL1512-3802 4.6 x 150 mm, 8 μm
Mobile Phase: | A: 0.25% TFA in water B: 0.25% TFA in 5% water:95% ACN
Flow Rate: | 1.0 mL/min
Gradient: | 20-60% B in 15 min
Detector: | UV, 220 nm

1. Collagen (120,000 MW)

2. Fibrinogen (340,000 MW)

TIPS & TOOLS |
For prep columns and media ordering information, View Here. For microbore columns ordering information, View Here. | [picture]

Amino Acid Analysis (AAA) Columns and Supplies

ZORBAX Eclipse Amino Acid Analysis (AAA) Columns

• High resolution and rapid analysis of 24 amino acids

• Tested for amino acid analysis

• Uses well-known OPA and FMOC precolumn derivatization chemistry

• Easily automated using a detailed online, derivatization protocol available for use with Agilent 1100/1200 autosampler

The Agilent ZORBAX Eclipse AAA high efficiency column rapidly separates amino acids following an updated and improved protocol. Total analysis from injection-to-injection can be achieved in as little as 8 min (7 min analysis time) on a 50 mm 1.8 μm column, 14 min (9 min analysis time) on shorter, 75 mm length columns and 24 min (18 min analysis time) on the 150 mm column length. Exceptional sensitivity (5-50 pmol with DAD, FLD) and reliability are achieved using both OPA and FMOC derivatization chemistries in one fully automated procedure using the Agilent 1100/1200 HPLC instrument.

ZORBAX Eclipse Plus C18 columns are another excellent choice for Amino Acid Analysis. For more information about ZORBAX Eclipse Plus Columns, see here.

| TIPS & TOOLS
[picture] | Further information can be found in the following publication: High-Speed Amino Acid Analysis (AAA) on 1.8 μm Reversed-Phase (RP) Columns (publication # 5989-6297EN) www.agilent.com/chem/library

Amino Acid Standards

Each amino acid standard contains the following amino acids:

• Glycine | • L-serine | • L-arginine
• L-cysteine | • L-alanine | • L-threonine
• L-histidine | • L-phenylalanine | • L-valine
• L-tyrosine | • L-glutamic acid | • L-lysine
• L-leucine | • L-proline | • L-aspartic acid
• L-methionine | • L-isoleucine |

Amino Acid Standards, 10 x 1 mL ampoules*

Description | Part No.
1 nmol/μL | 5061-3330
250 pmol/μL | 5061-3331
100 pmol/μL | 5061-3332
25 pmol/μL | 5061-3333
10 pmol/μL | 5061-3334
Amino acids supplement kit | 5062-2478
Includes 1 g each of norvaline, sarcosine, asparagine, glutamine, tryptophan, and 4-hydroxyproline |

*Consider shelf-life and buy limited quantities, P/N 5062-2478 ships as 1 g vials

Amino Acid Separations Reagents

Description | Part No.
OPA reagent, 10 mg/mL each in 0.4 M borate buffer o-phthalaldehyde (OPA) and 3-mercaptopropionic acid, 6 x 1 mL ampoules | 5061-3335
FMOC reagent, 2.5 mg/mL in acetonitrile, 9-fluorenylmethylchloroformate, 1 mL, 10 ampoules | 5061-3337
Borate buffer, 100 mL | 5061-3339
DTDPA (Dithiodiproprionic) reagent, for analysis of cysteine, 5 g | 5062-2479

High resolution of 24 amino acids using ZORBAX Eclipse-AAA protocol

Column: | ZORBAX Eclipse AAA 963400-902 4.6 x 150 mm, 3.5 μm
Mobile Phase: | A: 40 mM Na₂HPO₄, pH 7.8 B: ACN:MeOH:Water, 45:45:10 v/v
Flow Rate: | 2 mL/min
Temperature: | 40 °C
Detector: | Fluorescence
Sample: | 24 Amino Acids

1. Asp

2. Glu

3. Asn

4. Ser

5. Gln

6. His

7. Gly

8. Thr

9. Cit

10. Arg

11. Ala

12. Tyr

13. Cys

14. Val

15. Met

16. Nva

17. Trp

18. Phe

19. Ile

20. Leu

21. Lys

22. Hyp

23. Sar

24. Pro

This high resolution separation of 24 amino acids is done in 18 minutes. If the Rapid Resolution 4.6 x 75 mm Eclipse AAA column is selected, these amino acids are resolved in 9 minutes.

Ion-Exchange Chromatography

Purify proteins and other charged molecules

Ion-exchange chromatography (IEX) is a highly sensitive technique that allows you to separate ions and polar molecules based on their charge. Like SEC, IEX can be used to separate proteins in their native state.

Applying IEX to charge variant analysis

During production and purification, antibodies can exhibit changes in charge heterogeneity as a result of amino acid substitutions, glycosylation, phosphorylation, and other post-translational or chemical modifications. Because these changes can impact stability and activity – or cause immunologically adverse reactions – the analysis of charge heterogeneity in monoclonal antibody (MAb) preparations is critical to biopharmaceuticals.

In protein analysis, charge variations at a given pH indicate a change in the primary molecular structure – resulting in additional forms of the protein in question. These are called isoforms (or charge variants), and can be resolved by IEX chromatography. IEX is also useful as a preparative technique.

The pages that follow describe Agilent’s family of weak and strong ion-exchangers – both anionic and cationic.

• Agilent non-porous Bio IEX columns are designed for high-resolution, high-efficiency, and high-recovery separations.

• Agilent Bio MAb columns are optimized for separating charge isoforms of monoclonal antibodies.

• Agilent porous IEX columns (PL-SAX and PL-SCX) are chemically stable, and are available in two pore sizes – allowing you to separate peptides, oligonucleotides, and very large proteins.

• Bio-Monolith IEX columns are uniquely suited to separating antibodies, viruses, and DNA.

Ion-Exchange Column Selection
Application | Agilent Columns | Notes
Monoclonal antibodies | Agilent Bio MAb | Thorough characterization of monoclonal antibodies includes the identification and monitoring of acidic and basic isoforms. Agilent Bio MAb HPLC columns feature a unique resin specifically designed for high-resolution charge-based separations of monoclonal antibodies.
Peptides and proteins | Agilent Bio IEX | Agilent Bio Ion-Exchange columns are packed with polymeric, nonporous, ion-exchange particles. Bio IEX columns are designed for high resolution, high recovery and highly efficient separations.

Proteins, peptides and deprotected synthetic oligonucleotides

PL-SAX

The strong anion-exchange functionality, covalently linked to a fully porous chemically stable polymer, extends the operating pH range. In addition, the anion-exchange capacity is independent of pH. For synthetic oligonucleotides, separations using denaturing conditions of temperature, organic solvent, and high pH are all possible. The 5 μm media delivers separations at high resolution with the 30 μm media used for medium pressure liquid chromatography.

• 1000Å
• 4000Å
Globular proteins and peptides Very large biomolecules/high speed | PL-SAX 1000Å |
Very large biomolecules/high speed | PL-SAX 4000Å |

Small peptides to large proteins

PL-SCX

PL-SCX is a macroporous PS/DVB matrix with a very hydrophilic coating and strong cation-exchange functionality. This process is controlled to provide the optimum density of strong cation-exchange moieties for the analysis, separation and purification of a wide range of biomolecules. The 5 μm media delivers separations at higher resolution with the 30 μm media used for medium pressure liquid chromatography.

• 1000Å
• 4000Å
Globular proteins | PL-SCX 1000Å |
Very large biomolecules/high speed | PL-SCX 4000Å |
Antibodies (IgG, IgM), plasmid DNA, viruses, phages and other macro biomolecules | Bio-Monolith | Strong cation-exchange, strong and weak anion-exchange, and Protein A phases. Bio-Monolith HPLC columns are compatible with preparative LC systems, including Agilent 1100 and 1200 HPLC systems.
• Bio-Monolith QA
• Bio-Monolith DEAE
• Bio-Monolith SO₃
• Bio-Monolith Protein A
Viruses, DNA, large proteins | Bio-Monolith QA |
Plasmid DNS, bacteriophages | Bio-Monolith DEAE |
Proteins, antibodies | Bio-Monolith SO₃ |

Agilent Bio MAb HPLC Columns

• A packing support composed of a rigid, spherical, highly cross-linked polystyrene divinylbenzene (PS/DVB) non-porous bead

• Particles grafted with a hydrophilic, polymeric layer, virtually eliminating non-specific binding of antibody proteins

• A different process is used to layer the weak cation-exchange phase to the particle making it a higher density than the Agilent Bio WCX column particles

• Specifically designed for the separation of charge isoforms of monoclonal antibodies

Thorough characterization of monoclonal antibodies includes the identification and monitoring of acidic and basic isoforms. Agilent Bio MAb HPLC columns feature a unique resin specifically designed for high-resolution, charge-based separations of monoclonal antibodies. Compatible with aqueous solution buffers, acetonitrile/acetone/methanol and water mixtures. Commonly used buffers: phosphate, tris, MES and acetate.

Bio MAb columns are available in 1.7, 3, 5 and 10 μm sizes, providing higher resolution with smaller particles.

TIPS & TOOLS |

Capillary electrophoresis is an alternative technique to liquid chromatography for the separation of charged isoforms. Further information can be found in the following Technical Note:

Capillary electrophoreseis focusing on the Agilent Capillary Electrophoresis system (publication # 5989-9852EN)

www.agilent.com/chem/library

Consistent ion-exchange MAb separation

Column: | Bio MAb, PEEK 5190-2411 2.1 x 250 mm, 5 μm
Buffer: | A: Sodium phosphate buffer, 20 mM B: Buffer A + 400 mM NaCl
Gradient: | 15-35% Buffer B from 0-30 min
Flow Rate: | 0.65 mL/min
Sample: | CHO-humanized MAb, 1 mg/mL
Injection: | 2.5 μL
Detector: | UV 220 nm
Temperature: | Ambient

To provide a metal free flow path, Bio MAb PEEK columns are available.

Virtually eliminate retention time variations

Column: | Bio MAb, stainless steel 5190-2413 4.6 x 250 mm, 10 μm
Mobile Phase: | A: 10 mM phosphate, pH 6.0 B: A + 1.0 M NaCl
Flow Rate: | 1.0 mL/min
Gradient: | 0-100% B in 42 min
Temperature: | 25 °C
Detector: | UV 214 nm

1. Cytochrome c

2. Lysozyme

3. Ribonuclease A

The combination of well-controlled resin production, column surface chemistry, and column packing virtually eliminates retention time variations from column-to-column and lot-to-lot.

Charge isoform analysis of monoclonal antibodies

Column: | Bio MAb, PEEK 5190-2407 4.6 x 250 mm, 5 μm
Mobile Phase: | A: 10 mM Sodium Phosphate, pH 7.50 B: A + 100 mM NaCl, pH 7.50
Flow Rate: | 0.8 mL/min
Gradient: | 15-95% B in 60 min
Sample: | 5 μL, 5 mg/mL, MAb

High resolution separation of acidic and basic charge variants using the Agilent Bio MAb NP5 column

Agilent Bio IEX HPLC Columns

• Highly cross-linked and rigid nonporous poly(styrene divinylbenzene) (PS/DVB) particles are grafted with a hydrophilic, polymeric layer, eliminating nonspecific binding

• Uniform, densely packed ion-exchange functional groups are chemically bonded to the hydrophilic layer (multiple ion-exchange groups per anchoring) to increase column capacity

• Particles, coating and bonding are resistant to high pressures, promoting higher resolution and faster separations

• Multiple ion-exchange groups are captured on one anchoring to increase capacity

Agilent Bio IEX HPLC columns are packed with polymeric, nonporous, ion-exchange particles and are designed for high resolution, high recovery and highly efficient separations of peptides, oligonucleotides and proteins.

The Bio IEX family offers strong cation-exchange (SCX), weak cation-exchange (WCX), strong anion-exchange (SAX) and weak anion-exchange (WAX) phases. All phases are available in 1.7, 3, 5 and 10 μm non-porous particles sizes.

| TIPS & TOOLS
[picture] | More information is a click away. We have a variety of educational primers, application notes, maintenance guides, and literature available from Agilent for free. To learn more, visit www.agilent.com/chem/library

Exceptional separating power

Column: | Agilent Bio SCX, stainless steel 5190-2423 4.6 x 50 mm, 3 μm
Buffer: | 10 mM Phosphate, pH 6.0
Flow Rate: | 0.5 mL/min
Gradient: | 0-1.0 M NaCl, 15 min
Detector: | 280 nm

1. Ovalbumin, 4.6 pl

2. Ribonuclease A, 8.7 pl

3. Cytochrome c, 9.6 pl

4. Aprotinin, 10.0 pl

5. Lysozyme, 11.0 pl

N > 100,000/50 mm for Lysozyme

The hydrophilic, polymeric layer and densely packed ion-exchange functional groups provide extremely sharp peak shapes and high resolution of a mixture of proteins with a broad range of isoelectric points (pI).

Separation of protein standards on Agilent 3 μm ion-exchange columns by cation-exchange chromatography

Column A: | Agilent Bio SCX, NP 3, 4.6 x 50 mm, SS
Column B: | Agilent Bio WCX, NP 3, 4.6 x 50 mm, SS
Column C: | Agilent Bio MAb, NP 3, 4.6 x 50 mm, SS
Mobile Phase: | A: 10 mM NaH₂PO₄.2H₂O, pH 5.70 B: A + 1 M NaCl
Flow Rate: | 0.5 mL/min
Gradient: | 0 min - 100% A : 0% B 25 min - 0% A : 100% B
Temperature: | Ambient
Detector: | Agilent 1260 Infinity Bio-inert Quaternary LC with diode array detector at 220 nm
Sample: | Cytochrome c, ribonuclease A, lysozyme and protein mix

Weak cation-exchange chromatography for P128 therapeutic protein sample on the Agilent 1260 Bio-inert Quaternary LC system using different cation-exchange columns

Column A: | Bio MAb, PEEK 5190-2407 4.6 x 250 mm, 5 μm
Column B: | Bio MAb, PEEK 5190-2415 4.6 x 250 mm, 10 μm
Column C: | Brand B WCX-10 4.0 x 250 mm, 10 μm
Mobile Phase: | A: 20 mM sodium phosphate (pH = 6.0) B:20 mM sodium phosphate (pH = 6.0)     containing 1.0 M sodium chloride
Flow Rate: | 0.5 mL/min
Gradient: | 10% B 0 min, 35% B 35 min, 10% B 36 min, 10% B 45 min
Detector: | UV, 220 nm/4 nm, Reference: Off (data also acquired at 220, 230, 240, and 280 nm)
Sample: | P128

Sample was desalted by ultrafiltration and extracted into 20 mM sodium phosphate.

PL-SAX Strong Anion-Exchange Columns

• Small particles deliver excellent chromatographic performance

• Wide range of particle sizes and 2 pore sizes for flexible analysis to scale-up purification

• Exceptional stability for long column lifetime

PL-SAX -N(CH₃)₃⁺ is ideal for the anion-exchange HPLC separations of proteins, peptides and deprotected synthetic oligonucleotides under denaturing conditions. The strong anion-exchange functionality, covalently linked to a chemically stable fully porous polymer, extends the operating pH range. In addition, the anion-exchange capacity is independent of pH. For synthetic oligonucleotides, separations using denaturing conditions of temperature, organic solvent, and high pH are all possible. PL-SAX delivers improved chromatography for self-complementary or G-rich sequences that may associate to form aggregates or hairpin structures. The 5 μm material provides high efficiency separations of n and n-1 sequences. A wide range of particle sizes and column geometries permits analysis scale-up to purification. The strong anion-exchange functionality provides a material with exceptional chemical and thermal stability, even with sodium hydroxide eluents, leading to long column lifetime.

Standard ion-exchange protein separation

Column: | PL-SAX 1000Å PL1551-1502 4.6 x 50 mm, 5 μm
Mobile Phase: | A: 10 mM Tris HCl pH 8 B: A+0.35 M NaCl pH 8
Gradient: | 0-100% B in 20 min
Flow Rate: | 1.0 mL/min
Detector: | UV, 220 nm

1. Myoglobin

2. Bovine carbonic anhydrase

3. Ovalbumin

4. Soybean trypsin inhibitor

Analysis of choline kinase on PL-SAX 4000Å

Column: | PL-SAX PL1551-1803 4.6 x 50 mm, 8 μm

Mobile Phase:

A: 20 mM Tris 5% ethylene glycol, pH 7.5
(The following are required to retain enzyme activity)
1.0 mM Ethylene glycol tetraacetic acid
2.0 mM ß-Mercaptoethanol
0.2 mM
Phenylmethylsulfonyl fluoride
B: A + 1 M KCI

Flow Rate: | 3.0 mL/min
Detector: | UV, 280 nm

Separation courtesy of T Porter, Purdue University, USA

Analysis of representative whey proteins

Column: | PL-SAX 1000Å PL1551-1802 4.6 x 50 mm, 8 μm
Mobile Phase: | A: 0.02 M Tris HCI, pH 7 B: A + 0.5 M CH3COONa, pH 7
Flow Rate: | 1.0 mL/min
Gradient: | Linear 0-50% B in 10 min
Detector: | UV, 280 nm

1. Carbonic anhydrase

2. α-lactalbumin

3. β-lactoglobulin B

4. β-lactoglobulin A

High resolution separation of a Poly-T-Oligonucleotide size standard spiked with 10-mer, 15-mer, 30-mer and 50-mer (main peaks)

Column: | PL-SAX 1000Å PL1551-1802 4.6 x 50 mm, 8 μm
Mobile Phase: | A: 7:93 v/v ACN: 0.1 M TEAA, pH 8.5 B: 7:93 v/v ACN: 0.1 M TEAA, 1 M ammonium chloride, pH 8.5
Gradient: | 0-40% B in 10 min, followed by 40-70% B in 14 min and 70-100% B in 25 min
Flow Rate: | 1.5 mL/min
Temperature: | 60 °C
Detector: | UV, 220 nm

PL-SCX Strong Cation-Exchange Columns

• Optimal design for effective separation of biomolecules

• Pore sizes allow use of a range of solute sizes

• Exceptional stability for long column lifetime

PL-SCX -SO₃ - is a macroporous PS/DVB matrix with a very hydrophilic coating and strong cation-exchange functionality. This process is controlled to provide the optimum density of strong cation-exchange moieties for the analysis, separation and purification of a wide range of biomolecules. from small peptides to large proteins. Two pore sizes are available. 1000Å and 4000Å. to provide good mass transfer characteristics for a range of solute sizes. The 5 μm media delivers separations at higher resolution with the 30 μm media used for medium pressure liquid chromatography.

Standard protein separation

Column: | PL-SCX 1000Å PL1545-1502 4.6 x 50 mm, 5 μm
Mobile Phase: | A: 20 mM KH2PO4, pH 6.0 B: A + 1 M NaCl
Gradient: | 0-100% B in 20 min
Flow Rate: | 1.0 mL/min
Detector: | UV, 280 nm

1. Myoglobin

2. Chymotrypsinogen A

3. Cytochrome c

4. Lysozyme

Agilent Bio-Monolith Ion-Exchange HPLC Columns

Bio-Monolith Ion-Exchange HPLC Column

• Polymer-based, monolith HPLC columns designed for macro biomolecule separations

• Flow-rate independent separations; no diffusion, no pores and no void volume make transport between mobile and stationary phase very rapid

• Monolith disk is 5.2 mm x 4.95 mm (100 μL column volume) with continuous channels, eliminating diffusion mass transfer

• Extremely fast separations speed up method development time and decrease costs; locking in method parameters takes significantly less time and buffer

Agilent Bio-Monolith Ion-Exchange HPLC columns provide high resolution and rapid separations of antibodies (IgG, IgM), plasmid DNA, viruses, phages and other macro biomolecules. The product family offers strong cation-exchange, strong and weak anion-exchange and Protein A phases. Bio-Monolith HPLC columns are compatible with HPLC and preparative LC systems, including Agilent 1100 and 1200 HPLC systems.

| TIPS & TOOLS
[picture] | Agilent also offers a Protein A Bio-Monolith column for affinity chromatography. For more information, see here.

Column Specifications
Dimensions | 5.2 mm x 4.95 mm
Column volume | 100 μL
Maximum pressure | 150 bar (15 MPa, 2200 psi)
Temperature min/max | Working: 4-40 °C
| Storage: 4-30 °C
Recommended pH | Working range: 2-13
| Cleaning-in-place: 1-14
Materials of construction | Hardware: Stainless steel
| Packing: poly(glycidyl methacrylate-co-ethylene dimethacrylate) highly porous monolith
Color ring identifier | Bio-Monolith QA: Blue
| Bio-Monolith DEAE: Green
| Bio-Monolith SO₃: Red
Shelf life/expiration date | SO₃, QA, DEAE: 24-36 months

Baseline expansion of a separation of protein standards

Column: | Agilent Bio-Monolith CM15, 5.5 x 15 mm
Mobile Phase: | A: 10 mM Na₂HPO₄, pH 6.0 B: A + 0.5 M NaCl or just 0.5 M Na₂HPO₄, pH 6.0
Flow Rate: | 2 mL/min

Gradient:

0.5 min hold with mobile phase A followed by a linear gradient to 45% B in 15 min (elapsed time 15.5 min); then 60% B at 15.6 min continued to 20 min. Column flushed with 100% B for 15 min before reequilibration for the next run.
pH Gradient: A: 5 mM Na₂HPO₄, buffer pH 5.5 and B: 40 mM Na₂HPO₄ (not buffered, pH 8.9). 2% B/min at 1 mL/min for 15 min, followed by a column wash with 90% B for 5 min.

Detector: | UV at 220 nm

Sample:

One mg each/mL in mobile phase A.

1. RNAse from bovine pancreas (pI 9.6)

2. Cytochrome c from bovine heart (pI 10.37-10.8)

3. Lysozyme from chicken egg (pI 11.35) (0.5 mg)

Instrument: | Agilent 1200 SL with diode array detector

Bio-Monolith DEAE column monitors phage production during fermentation

Column: | DEAE 5069-3636 5.2 x 4.95 mm
Mobile Phase: | A: 125 mM Phosphate buffer, pH 7.0 B: 125 mM Phosphate buffer + 1 M NaCl, pH 7.0
Flow Rate: | 1 mL/min
Gradient: | 100% buffer A (2.5 min) 0-100% buffer B (10 min) 100% buffer A (2 min)
Detector: | UV at 280 nm
Instrument: | High pressure gradient HPLC system, Agilent 1200 Infinity LC

As phage proliferation progresses, the genomic DNA (gDNA) concentration increases as the host cells are being lysed. In the late stages of fermentation, gDNA begins to degrade into fragments. These gDNA fragments cannot be easily removed by purification media, therefore it is critical to stop the fermentation cycle prior to the degradation of the genomic DNA. The chromatogram above represents three samples taken from the bioreactor at 36, 158 and 191 minutes. Peak 1 represents phage, media and host cells, peak 2 the intact gDNA and peak 3 the fragmented gDNA.

Size Exclusion Chromatography (SEC)

Accurately determine biomolecule aggregation, fragmentation, and chemical ligation/modification

Size exclusion chromatography (SEC) is a technique for separating proteins, oligonucleotides, and other complex biopolymers by size using aqueous eluents.

Applying SEC to aggregation studies

The size, type, and content of aggregates present in protein biopharmaceuticals can affect both efficacy and formulation – or worse, induce an immunogenic response. Aggregation formations occur through a variety of mechanisms, including disulfide bond formation and non-covalent interactions.

Because the size of protein aggregates, including dimers, is sufficiently different from the protein monomer, you can separate the various forms using SEC. In fact, SEC with UV or light scattering is a standard technique for quantifying protein aggregation.

Applying SEC to quantitation and molecular weight determination

For proteins and other molecules of discreet molecular weight, SEC can be used to detect and quantitate monomers, dimers, aggregates and fragments. SEC can also separate oligonucleotide mixtures.

For biopolymers of varying sizes, like starches and other polysaccharides, SEC can provide data on molecular weight distribution and branching (with the proper detectors).

As a leading manufacturer of SEC columns and instruments for over 30 years, Agilent is continually developing new SEC products that will provide even higher resolution and quicker separations. This section highlights Agilent’s broad family of SEC columns for protein biopolymer analysis:

• Bio SEC-3 and Bio SEC-5 columns are available in a variety of pore sizes, and are well suited for protein analysis – especially when determining the presence of dimers and aggregates in therapeutic biologicals. Note that 3 μm Bio SEC-3 columns provide higher resolution than our industry-standard 5 μm Bio SEC-5 columns.

• ProSec 300S columns work well with globular proteins under high salt conditions.

• ZORBAX GF-250 and GF-450 columns are best for preparative SEC of proteins, because of their larger column size and higher flow rates.

• PL aquagel-OH columns can be used to analyze biopolymers of broad molecular weights, such as PEGs, oligo- and polysaccharides, starches, and gums.

Size Exclusion Chromatography (SEC)
Application | Agilent Columns | Notes
Peptides, proteins | Agilent Bio SEC-3 | Higher resolution and faster separations from 3 μm particles, with 100Å, 150Å, and 300Å pore sizes.
Large biomolecules and samples with multiple molecular weight components | Agilent Bio SEC-5 | More pore size options (100Å, 150Å, 300Å, 500Å, 1000Å, and 2000Å) to cover a wider range of analytes.
Globular proteins, antibodies | ProSEC 300S | Single column option for protein analysis in high salt conditions.
Proteins, globular proteins | ZORBAX GF-250/450 | Higher flow rate capabilities and larger column size for SEC semi-prep and prep.
Low MW polymers and oligomers, oligosaccharides, PEGs, lignosulfonates | 2 or 3 PL aquagel-OH | The PL aquagel-OH analytical series has a pH range of 2-10, compatibility with organic solvent (up to 50% methanol), mechanical stability up to 140 bar (2030 psi), and low column operating pressures.
• PL aquagel-OH 8 μm
• PL aquagel-OH 20 5 μm
• PL aquagel-OH MIXED-M 8 μm
Polydisperse biopolymers, polysaccharides, cellulose derivatives | 2 or 3 PL aquagel-OH |
• PL aquagel-OH MIXED-H 8 μm
• PL aquagel-OH 60/50/40 8 μm |
Very high MW polymers, hyaluronic acids, starches, gums | PL aquagel-OH 60/50/40 15 μm in series |

Agilent Bio SEC-3

• Exceptional loading capacity, stability, and reproducibility for size-based biomolecule separations

• Sharper peaks, higher resolution, and better protein recovery

• Faster separations than large-particle SEC columns

• Compatibility with most aqueous buffers

• Excellent stability in high-salt and low-salt conditions

Agilent Bio SEC-3 HPLC columns are a breakthrough technology for size exclusion chromatography (SEC). They are packed with spherical, narrowly dispersed 3 μm silica particles coated with a proprietary hydrophilic layer. This thin polymeric layer is chemically bonded to pure, mechanically stable silica under controlled conditions, ensuring a highly efficient size exclusion particle.

Agilent Bio SEC-3 HPLC columns are available in 100Å, 150Å and 300Å pore sizes to accommodate most peptide and protein size exclusion separations.

| TIPS & TOOLS
[picture] | Deactivated/silanized vials have inert surfaces that will not interact with metals, biologicals or proteins, and will not cause pH shifts. Avoid standard polypropylene vials for biological or light-sensitive compounds.

Calibration curves – Bio SEC-3

Column: | Bio SEC-3 7.8 x 300 mm, 3 μm
Mobile Phase: | 150 mM Na phosphate, pH 7.0
Flow Rate: | 1.0 mL/min
Detector: | UV

Intact MAb monomer and dimer separation

Column: | Bio SEC-3, 300Å 5190-2511 7.8 x 300 mm, 3 μm
Buffer: | Sodium phosphate buffer, pH 7.0, 150 mM
Isocratic: | 0-100% Buffer A from 0-30 min
Flow Rate: | 1.0 mL/min
Sample: | CHO-humanized MAb, 5 mg/mL – intact
Injection: | 5 μL
Detector: | UV 220 nm
Temperature: | Ambient

Comparison of Agilent Bio SEC-3 and competitor column in the analysis of a monoclonal antibody

Column: | Bio SEC-3, 300Å 5190-2511 7.8 x 300 mm, 3 μm
Column: | Competitor 7.8 x 300 mm
Mobile Phase: | 150 mM sodium phosphate + 100 mM Na sulfate (with salt) 150 mM sodium phosphate (without salt)
Flow Rate: | 1.0 mL/min
Detector: | UV, 220 nm
Sample: | MAb (2 mg/mL)

The Agilent Bio SEC-3 column reveals the presence of smaller MW species missed by the competitor column.

Pore Size Choice

The choice of media pore size will influence the resolution in SEC. As the separation is based on differences in molecular size in solution, the sample must be able to permeate the porous structure of the particles – if the pore size is too small, the samples will be excluded from the pores and elute in the void volume of the column, and if too large then, all will be able to fully permeate the particles and so there will be very little separation.

Pore size choice: Proteins

Column A: | Bio SEC-3, 100Å 5190-2503 4.6 x 300 mm, 3 μm
Column B: | Bio SEC-3, 150Å 5190-2508 4.6 x 300 mm, 3 μm
Column C: | Bio SEC-3, 300Å 5190-2513 4.6 x 300 mm, 3 μm
Mobile Phase: | 50 mM Na₂HPO₄, 50 mM NaH₂PO₄+ 0.15 M NaCl, pH 6.8
Flow Rate: | 0.35 mL/min
Detector: | UV, 220 nm
Sample: | BioRad Gel Filtration Standards Mix

1. Thyroglobulin Aggregates

2. Thyroglobulin

3. IgA

4. γ-globulin

5. Ovalbumin

6. Myoglobin

7. Vitamin B12

Pore size choice: Mouse IgG

Column A: | Bio SEC-3, 100Å 5190-2503 4.6 x 300 mm, 3 μm
Column B: | Bio SEC-3, 150Å 5190-2508 4.6 x 300 mm, 3 μm
Column C: | Bio SEC-3, 300Å 5190-2513 4.6 x 300 mm, 3 μm
Mobile Phase: | 50 mM Na₂HPO₄, 50 mM NaH₂PO₄+ 0.15 M NaCl, pH 6.8
Flow Rate: | 0.35 mL/min
Detector: | UV, 220 nm
Sample: | Mouse IgG

1. Dimer

2. Monomer

3. Monomer Fragment

4. Azide

Column Length

Where the separation time is a critical parameter, shorter columns packed with the higher efficiency, 3 μm media are used. With the shorter columns, higher flow rates are used to reduce the analysis time but without compromising the quality of the data – quantitation of monoclonal antibody monomer and dimer.

Agilent Bio SEC-3 column length comparison, 150 mm

Column: | Bio SEC-3, 300Å 5190-2512 7.8 x 150 mm, 3 μm
Mobile Phase: | 150 mM sodium phosphate
Flow Rate: | 1.0 mL/min (56 bar), 1.5 mL/min (75 bar)
Detector: | UV, 220 nm
Sample: | MAb (2 mg/mL)

Agilent Bio SEC-3 column length comparison, 300 mm

Column: | Bio SEC-3, 300Å 5190-2511 7.8 x 300 mm, 3 μm
Mobile Phase: | 150 mM sodium phosphate + 100 mM Na sulfate (with salt) 150 mM sodium phosphate (without salt)
Flow Rate: | 1.0 mL/min
Detector: | UV, 220 nm
Sample: | MAb (2 mg/mL)

Agilent Bio SEC-5

• Maximum recovery for a broad range of size-based, biomolecule separations

• Outstanding reproducibility and column lifetime

• Excellent stability, even under high-pH, high-salt, and low-salt conditions

• Compatibility with most aqueous buffers

Agilent Bio SEC-5 HPLC columns are packed with 5 μm silica particles coated with a proprietary, neutral, hydrophilic layer for maximum efficiency and stability. Our specially designed packing also provides high pore volume, improving both peak capacity and resolution.

Bio SEC-5 columns are available in 5 μm particles with 100Å, 150Å, 300Å, 500Å, 1000Å, and 2000Å nominal pore sizes.

Calibration curves – Bio SEC-5

Column: | Bio SEC-5 7.8 x 300 mm, 5 μm
Mobile Phase: | 150 mM Na phosphate, pH 7.0
Flow Rate: | 1.0 mL/min
Detector: | UV, 214 nm

Side-by-side comparison

Column: | Bio SEC-5 5190-2521 7.8 x 300 mm, 5 μm
Mobile Phase: | 150 mM Na phosphate, pH 7.0
Flow Rate: | 1.0 mL/min
Detector: | UV, 214 nm

1. Thyroglobulin, 5.43 min

2. BSA dimer, 6.19 min

3. BSA monomer, 6.93 min

4. Ribonuclease A, 8.74 min

5. Poly-DL-alanine (1-5 kDa), 9.90 min

6. Uracil, 12.13 min

1. Thyroglobulin, 5.64 min

2. BSA dimer, 6.23 min

3. BSA monomer, 7.02 min

4. Ribonuclease A, 9.22 min

5. Poly-DL-alanine (1-5 kDa), 10.02 min

6. Uracil, 11.81 min

Separation of a protein mixture on an Agilent Bio SEC-5 HPLC column and a Tosoh TSK-Gel column. Notice the sharper peaks and better resolution on the Agilent Bio SEC-5 HPLC column.

Exceptional lifetime, and lot-to-lot reproducibility

Column: | Bio SEC-5, 150Å 5190-2521 7.8 x 300 mm, 5 μm
Mobile Phase: | 150 mM Phosphate Buffer, pH 7.0

1. Thyroglobulin

2. BSA

3. Ribonuclease A

4. Uracil

The four protein mixture shows excellent retention time reproducibility over 300 injections and on three columns from different manufacturing lots.

Comparison between Agilent Bio SEC-3 and Agilent Bio SEC-5

Analysis of monoclonal antibody

Column: | Bio SEC-3, 300Å 5190-2511 7.8 x 300 mm, 3 μm
Column: | Bio SEC-5, 300Å 5190-2526 7.8 x 300 mm, 5 μm
Mobile Phase: | 150 mM Sodium Phosphate, pH 7
Flow Rate: | 1 mL/min
Detector: | UV @ 220 nm
Sample: | Humanized monoclonal antibody

1. Dimer

2. Monomer

3. Monomer fragment

The 3 μm column gives better separation

TIPS & TOOLS |
The Agilent rack can be used to optimize your 1290 Infinity LC for ultra-low dispersion, which can enhance performance of high-efficiency columns. Further information can be found in application note 5990-9502EN at www.agilent.com/chem/library | [picture]

ProSEC 300S

• Mechanically robust silica particles that do not bleed during use

• Single column with extended linear resolving range

• Column dimensions for use with multi-detector systems

The Agilent ProSEC 300S column is specifically designed as a single column solution for globular protein analysis. The pore size selection and optimization provides an extended linear resolving range so that this single column can be used for analysis across the full range of globular proteins.

The particles are extremely robust and do not fragment during use to leach particulates. This gives exceptionally stable baselines making this column an ideal choice for use with light scattering detectors.

Two column dimensions, 7.5 mm id and 4.6 mm id, to suit multi-detector size exclusion chromatography provide an option for the analysis of small masses.

ProSEC 300S

Dimensions | Particle Size (μm) | Part No.
4.6 x 250 | 5 | PL1547-5501
7.5 x 300 | 5 | PL1147-6501
Guard Columns |  |
4.6 x 50 | 5 | PL1547-1501
7.5 x 50 | 5 | PL1147-1501

Calibration of the ProSEC 300S column with globular proteins

Mobile Phase: | 50 mM KH2PO4-K2HPO4 (@ pH 6.8) containing 0.3 M NaCl
Flow Rate: | 1.0 mL/min
Detector: | UV, 280 nm
Sample: | Protein samples

  Molecular weights of the proteins

Mw/Daltons | Protein
670,000 | Thyroglobulin
155,000 | g-Globulin
66,430 | Bovine serum albumin
44,287 | Ovalbumin
29,000 | Carbonic anhydrase
16,700 | Myoglobulin
12,384 | Cytochrome c
1,423 | Bacitracin

Analysis of Bovine Serum Albumin by light scattering using ProSEC 300S columns

Column: | ProSEC 300S PL1147-6501 7.5 x 300 mm, 5 μm
Mobile Phase: | Water + 120 mM NaCl, 2.7 mM KCI, 10 mM NaH₂PO₄
Flow Rate: | 1.0 mL/min
Detector: | Differential refractive index + PL-GPC 50 Dual Angle Light Scattering Detector
Sample: | Bovine serum albumin

  Molecular Weights

Monomer | 66,900 Daltons, 88.5%
Dimer | 34,900 Daltons (2.02 x monomer molecular weight), 9.8%
Trimer | 197,000 Daltons (2.94 x monomer molecular weight), 1.2%
Tetramer | 279,300 Daltons (5.17 x monomer molecular weight), 0.5%

Overlay of differential refractive index and dual angle light scattering sample.

Overlay of UV and light scattering 90°
for a sample of γ-globulins, illustrating monomer,
dimer, and trimer peaks

Column: | ProSEC 300S PL1147-6501 7.5 x 300 mm, 5 μm
Mobile Phase: | 0.1 M KH2PO4 containing 0.3 M NaCl, pH 8.0
Flow Rate: | 1.0 mL/min
Temperature: | 5 °C
Detector: | UV at 310 nm + PL-GPC 50 Dual Angle Light Scattering Detector
Sample: | Proteins

Overlay of UV and light scattering 90° for a sample of BSA, illustrating monomer, dimer, trimer and aggregate peaks

Column: | ProSEC 300S PL1147-6501 7.5 x 300 mm, 5 μm
Mobile Phase: | 0.1 M KH2PO4 containing 0.3 M NaCl, pH 8.0
Flow Rate: | 1.0 mL/min
Temperature: | 5 °C
Detector: | UV at 310 nm + PL-GPC 50 Dual Angle Light Scattering Detector
Sample: | Proteins

ZORBAX GF-250 and GF-450 Gel Filtration Columns

• High efficiency and reproducibility with short analysis time

• Semi-prep and prep column dimensions

• Compatible with organic modifiers and denaturants

• Wide usable pH range (3-8)

GF-250 Gel Filtration Columns

Agilent ZORBAX GF-250 and GF-450 size exclusion (gel filtration) columns are ideal for size separations of proteins and other biomolecules. The separation range is 4,000-900,000 for globular proteins when using GF-250 and GF-450 columns in series. The GF-250/GF-450 size exclusion columns have a hydrophilic diol bonded phase for high recovery of proteins (typically >90%) and a unique zirconia modification of the silica for a pH operating range from 3-8. The GF-250 and GF-450 columns are packed with precisely sized porous silica microspheres with narrow pore size and particle size distributions. The result is a highly efficient, rugged and reproducible size exclusion column that can be used for both analytical and preparative separations of proteins with flow rates of up to 3 mL/min. These columns are compatible with organic modifiers (<25%) and denaturants in the mobile phase to reduce protein aggregation. Some common applications include separations of protein monomers, dimers and aggregates, desalting, protein molecular weight estimation and separations of modified proteins.

Retention volume versus log (MW) for the Bio-Rad standards separated on an Agilent ZORBAX GF-250 column

Column: | ZORBAX GF-250 884973-901 9.4 x 250 mm, 4 μm
Mobile Phase: | 200 mM Sodium phosphate, pH 7.0
Temperature: | Ambient
Detector: | UV, 254 nm

Separations of proteins on preparative columns

Column: | ZORBAX GF-250 884973-901 9.4 x 250 mm, 4 μm
Column: | ZORBAX GF-450 884973-902 9.4 x 250 mm, 6 μm
Mobile Phase: | 0.2 M Na₂HPO₄, pH 7.0
Flow Rate: | 5.0 mL/min
Detector: | UV, 280 nm
Sample: | 200 μL

1. Thyroglobulin

2. Catalase

3. BSA

4. Ii-Lactoglobulin B

5. Myoglobin

6. Tyr-Gly-Gly

Affinity Chromatography

Bio-Monolith Protein A Column, 5069-3639

Affinity chromatography is a powerful technique which takes advantage of highly specific molecular interactions, frequently between specific proteins (e.g. antigen/antibody). Agilent offers several specialty affinity products, a monolithic Protein A column for the isolation and quantitation of IgG and a series of Multiple Affinity Removal Systems for the elimination of high abundance proteins in biological samples.

Agilent Bio-Monolith Protein A HPLC Columns

• Designed for the analytical separation of all IgG (human and mouse), except for IgG class3

• Flow-rate independent separations; no diffusion, no pores and no void volume make transport between mobile and stationary phase very rapid

• Extremely fast separations speed up method development time and decrease costs

• Locking in method parameters takes significantly less time and buffer

Agilent Bio-Monolith Protein A HPLC columns are part of the Agilent Bio-Monolith column family. Protein A Bio-Monolith columns are compatible with HPLC and preparative LC systems, including Agilent 1100 and 1200 HPLC systems.

| TIPS & TOOLS
[picture] | For information on Ion-Exchange Bio-Monolith columns, View Here.

Column Specifications
Dimensions | 5.2 mm x 4.95 mm
Column volume | 100 μL
Maximum pressure | 150 bar (15 MPa, 2200 psi)
Temperature min/max | Working: 4-40 °C
| Storage: 4-30 °C
Recommended pH | Working range: 2-13
| Cleaning-in-place: 1-14
Materials of construction | Hardware: Stainless steel
| Packing: poly(glycidyl methacrylate-co-ethylene dimethacrylate) highly porous monolith
Color ring identifier | Bio-Monolith Protein A: White
Shelf life/expiration date | Protein A: 12 months

TIPS & TOOLS |
Further information can be found in the following application note: Rapid Human Polyclonal IgG Quantification Using the Agilent Bio-Monolith Protein A HPLC Column (publication # 5989-9733EN) www.agilent.com/chem/library | [picture]

Rapid human polyclonal IgG quantification using the Agilent Bio-Monolith Protein A HPLC column

Column: | Protein A 5069-3639 5.2 x 4.95 mm
Mobile Phase: | PBS buffer, pH 7.4 0.5 M acetic acid, pH 2.6
Flow Rate: | 1 mL/min
Gradient: | Stepwise gradient: 100% buffer A-100% buffer B-100% buffer A (0.5 min each step)
Detector: | A high pressure gradient HPLC system, Agilent 1200 Infinity LC - UV at 280 nm
Sample: | Human Plasma diluted with binding buffer (PBS buffer, pH 7.4)

The selectivity of the Bio-Monolith Protein A column for the IgG from human plasma. IgG binds to protein A, a 100% buffer B step gradient is applied, and IgG elutes at 1.5 min.

SDS PAGE analysis of fractions from the separation.

Agilent Protein Fractionation System and Proteomics Reagents

• LC/MS analysis of biological samples

• Preparation for electrophoretic analysis

• Sample preparation for biomarker discovery

• Instrument and workflow validation

• Cost-effective immunodepletion

• Sample desalting, concentration, and fractionation

In order to more easily isolate and identify proteins in biological samples, such as serum, plasma, and cerebro-spinal fluid (CSF), the Agilent Multiple Affinity Removal System is designed to chromatographically eliminate interfering high-abundance proteins from biological samples. Removal of these abundant proteins improves the subsequent LC/MS and electrophoretic analysis of the sample by effectively expanding the dynamic range.

For sample fractionation and desalting, the Agilent mRP-C18 High-Recovery Protein column is designed to simultaneously desalt, concentrate, and fractionate in one easy step with extremely high recovery of samples as compared to conventional RP HPLC columns that are fully compatible with LC/MS analysis.

In addition, validated reagents for sample preparation in biomarker discovery and other proteomics applications are also available, including a complex standard, and proteomics grade trypsin. For your convenience, these reagents are fully compatible with Agilent LC/MS methods and require no additional sample pretreatments.

Large volume requirements and custom column dimensions can also be addressed with our custom configurations.

Multiple Affinity Removal System

The Multiple Affinity Removal System from Agilent enables the identification and characterization of high-value, low abundant proteins and biomarkers found in serum, plasma, and other biological fluids.

Multiple Affinity Removal System

The Multiple Affinity Removal System reproducibly and specifically removes up to 14 high abundant proteins found in human biological fluids and 3 high abundant proteins found in mouse biological fluids.

The Multiple Affinity Removal System is available in a variety of LC column dimensions and in spin cartridge format. When combined with Agilent's optimized buffers, convenient spin filters and concentrators, the Agilent Multiple Affinity Removal System creates an automated, integrated depletion solution compatible with most LC instruments (columns), and bench top centrifuges (spin cartridges).

Samples depleted using the Multiple Affinity Removal System are ready for downstream analyses such as 2-D gel electrophoresis, LC/MS, and other analytical techniques.

Illustration of high abundance proteins removed by Agilent Multiple Affinity Removal Columns and Spin Cartridges

| TIPS & TOOLS
[picture] | Learn more about Agilent’s complete services portfolio at www.agilent.com/chem/services

Multiple Affinity Removal System Starter Kits

The LC Column and Spin Cartridge Reagent Starter Kits include all the required supplies to use with Multiple Affinity Removal System. These buffers provide optimal conditions for column longevity and sample reproducibility.

• The kits provide enough Buffer A and Buffer B for approximately 200 sample depletions using the 4.6 x 50 mm LC columns, approximately 100 sample depletions using the 4.6 x 100 mm LC columns and 200 spin cartridge uses.

• Buffer A, the loading buffer, minimizes protein-protein interactions, allowing low abundant proteins often bound to high abundant proteins to pass through the column, while the targeted high abundant proteins bind to their associated antibodies.

• Buffer B, the elution buffer, then disrupts the antibody-protein interaction eluting the high abundant proteins off the column.

LC Column Reagent Starter Kit, 5185-5986

Luer-Lok adapters, 5188-5249

Luer-Lok syringe, 5188-5250

Luer-Lok needles, 5188-5253

Multiple Affinity Removal System Starter Kits

Description | Part No.
LC Column Reagent Starter Kit | 5185-5986
Includes: |
Buffer A, 1 L, for loading, washing, and equilibrating, qty 2 | 5185-5987
Buffer B, 1 L, for eluting | 5185-5988
0.22 μm cellulose acetate, 25/pk, qty 2 | 5185-5990
Spin concentrators, 5K MWCO, 4 mL, 25/pk | 5185-5991
Multiple Affinity Removal Spin Cartridge Reagent Kit Includes: | 5188-5254
Buffer A, 1 L, for loading, washing, and equilibrating | 5185-5987
Buffer B, 1 L, for eluting | 5185-5988
Spin filters, 0.22 μm cellulose acetate, 25/pk, qty 2 | 5185-5990
Spin concentrators, 5K MWCO, 4 mL, 25/pk | 5185-5991
Luer-Lok adapters, 2/pk | 5188-5249
Plastic syringe, 5 mL, Luer-Lok, 2/pk | 5188-5250
Microtube, 1.5 mL, screw top, 100/pk, qty 6 | 5188-5251
Caps and plugs, 6/pk | 5188-5252
PTFE needles, Luer-Lok, 10/pk | 5188-5253
High concentration sample dilution buffer, 50 mL | 5188-8283

mRP-C18 High-Recovery Protein Column, 4.6 x 50 mm, 5188-5231

The mRP (macroporous reversed-phase) C18 High-Recovery Protein column is designed for high recovery, high resolution separation, fractionation, and simultaneous desalting of complex protein samples (like immunodepleted serum or plasma proteins).

mRP-C18 High-Recovery Protein Columns

• Greater than 95-99% protein sample recovery has been observed with immunodepleted serum using the Agilent Multiple Affinity Removal System – LC column

• Can load up to 380 μg of total protein mass without reducing chromatographic resolution of the proteins

• Column packed with macroporous C18-bonded ultrapure 5 μm particle silica designed to reduce or eliminate strong adsorption of proteins

• Maximum operating pressure of 250 bar (4000 psi)

• Compatible with water and all common organic solvents

mRP-C18 High-Recovery Protein Columns

Description | Protein Load Capacity | Part No.
mRP-C18, 0.5 x 100 mm | 10 ng - 5 μg | 5188-6510
mRP-C18, 2.1 x 75 mm | 8 - 85 μg | 5188-6511
mRP-C18, 4.6 x 50 mm | 40 - 380 μg | 5188-5231

Proteomics Reagents for LC/MS Analysis

The Agilent Complex Proteomics Standard is a soluble Pfu protein extract containing over 1,500 proteins. Together with our TPCK-treated proteomics grade trypsin this is an ideal combination for workflow validation in LC/MS biomarker discovery and other proteomic studies.

Proteomics Reagents for LC/MS Analysis

Description | Part No.
Complex Proteomics Standard | 400510
Proteomics Grade Trypsin | 204310

Protein Fractionation of Complex Samples on the mRP Column

mRP-C18 High-Recovery Protein Column, 0.5 x 100 mm, 5188-6510

Method Development

ZORBAX Column Methods

This ZORBAX Column Selection Strategy for Proteins and Peptides provides some critical details on method development for proteins or polypeptides.

Reversed-Phase LC/MS Methods

LC/MS of proteins and peptides is used to provide information for protein characterization, to accurately identify post-translational modifications of proteins, and to determine the molecular weight of synthetic and natural peptides. LC/MS is also used to provide protein identification in 2-D separations for proteomics applications. Therefore, LC/MS of proteins and peptides is a critical separation area, which requires some special column and mobile phase recommendations. In general, smaller column sizes are used for LC/MS and TFA is generally not used in mobile phase because of reduced sensitivity in the MS with this mobile phase additive.

Bio Ion-Exchange Column Methods

SEC Column Methods

After the initial chromatogram, additional changes may be needed to improve the separation, maintain protein solubility, or to decrease sample interaction with the chromatographic media. The ionic strength of the mobile phase can be adjusted up or down in strength to attain an optimized separation. pH can also be adjusted usually + 0.2 units. If further optimization is necessary, the upward or downward range should be expanded. A change of temperature or addition of an organic solvent can also be used.

For protocols requiring additional salt, these buffers are typical:

100-150 mM sodium chloride in 50 mM sodium phosphate, pH 7.0

100-150 mM sodium sulfate in 50 mM sodium phosphate, pH 7.0

50-100 mM urea in 50 mM sodium phosphate, pH 7.0

Other similar salts (e.g. KCl) and guanidine hydrochloride can also be used

pH Range:

2.0-8.5

Potential organic solvent additions include:

5-10% ethanol (or other similar solvents) in 50 mM sodium phosphate, pH 7.0

5% DMSO in 50 mM sodium phosphate, pH 7.0

Temperature:

Typically, SEC separations are run at 20-30 °C. Separation of proteins and peptides may require higher temperature to improve both resolution and recovery of proteins and hydrophobic peptides.

Maximum temperature of Bio SEC columns is 80 °C

High Sensitivity Capillary Column Methods

Capillary and Nano Columns

• Highest sensitivity for your smallest sample sizes

• Compatible with all LC/MS interfaces

• Internal diameters of 0.5, 0.3, 0.1, and 0.075 mm

• Packings/phases for both small and large molecules (80Å and 300Å pore sizes, respectively)

• Ideal for 1-D and 2-D (proteomics) applications

Agilent ZORBAX Capillary (0.5 and 0.3 mm id) and Nano (0.1 and 0.075 mm id) columns are now available in a wide variety of phases, pore sizes, and dimensions. These columns are ideal for very sample-limited applications because they provide enhanced sensitivity by reducing on-column sample dilution. This high sensitivity can be provided with exceptional reproducibility using Agilent columns and low dispersion HPLC instruments. The fastest growing application for capillary and nano columns is 2-D LC/MS for complex proteomics samples. Agilent provides all the columns needed for the 2-D separation – the SCX columns for the first dimension, the reversed-phase trapping column, and the reversed-phase column for the second dimension.

Nano Columns

[picture] | TIPS & TOOLS Agilent offers a variety of e-Seminars and on-site training to help you learn how to be a more effective chromatographer. For more information, visit www.agilent.com/chem/education

ZORBAX Nano columns for high sensitivity protein digest analysis by LC/MS

Column: | ZORBAX 300SB-C18 5065-9911 0.075 x 150 mm, 3.5 μm
Mobile Phase: | A: Water + 0.1% Formic acid, B: ACN + 0.1% Formic acid
Flow Rate: | 600 nL/min
Gradient: | 2% B to 52% B in 25 min
Detector: | Positive Ion Nano Electrospray MS
Sample: | 100 fm (1 μL) Digest of 8 Proteins

A ZORBAX Nano HPLC column, 0.075 mm id, is used for high sensitivity LC/MS analysis of a protein digest sample.

High sensitivity with capillary columns

Column: | ZORBAX SB-C18 5064-8255 0.3 x 150 mm, 5 μm
Column: | ZORBAX SB-C18 5064-8256 0.5 x 150 mm, 5 μm
Column: | ZORBAX SB-C18 863600-902 1.0 x 150 mm, 3.5 μm
Column: | ZORBAX SB-C18 883975-902 4.6 x 150 mm, 5 μm
Sample: | 200 ng Biphenyl

Sample-limited applications require capillary column dimensions to minimize on-column sample dilution and to enhance sensitivity. The 0.3 mm capillary in this example provides 100 times more sensitivity than the standard 4.6 mm column. Agilent Nanobore (0.1 mm to 0.075 mm id) columns can provide up to 2,000 times more sensitivity for your most limited sample applications.

Human serum: Low abundance protein isolation and identification from 1-D gel band by LC/MS

Column: | ZORBAX 300SB-C18 Trap: 0.3 x 5 mm, 5 μm, 5065-9913 Analytical: 0.3 x 150 mm, 5 μm, 5064-8263

Mobile Phase: | A: Water + 0.1% Formic acid B: Acetonitrile + 0.1% Formic acid
Flow Rate: | 6 μL/min

Gradient:

0 min 3% B
5 min 3% B (loading)
50 min 45% B
52 min 80% B
57 min 80% B
60 min 3% B

Sample: | Band from 1-D in gel digest

Base Peak Chromatogram

Proteins Identified

1. α-1-Antichymotrypsin

2. Antithrombin-III Precursor

3. Complement Factor B Precursor

Sample Preparation of Human Serum:

Major serum proteins removed using Multiple Affinity Removal Column:

4.6×100 mm, P/N 5185-5985

Followed by 1-D gel digest

Peptide phosphorylation sites LC and LC/MS using Capillary LC columns

Column: | ZORBAX 300SB-C18 5064-8268 0.5 x 150 mm, 3.5 μm
Mobile Phase: | A: Water + 0.1% Formic acid B: Acetonitrile + 0.1% Formic acid
Flow Rate: | 5.5 μL/min
Gradient: | 5-55% B in 50 min, to 85% B from 55-57 min
Detector: | UV, 206 nm
MS Conditions: | LC/MS: | Pos. Ion ESI with LC/MSD trap
| Vcap: | 4000 V
| Drying gas flow: | 7 L/min
| Drying gas |
| temperature: | 250 °C
| Nebulizer: | 15 psi
| Capillary Exit Volt: | 50 V Max
| Accum Time: | 300 ms
| Total Averages: | 3
| Isolation Width: | 3 m/z
| Frag Ampitude: | 1.0 V
Sample: | Beta case in digest, 100 nL (4 pmol)

Capillary columns for HPLC analyses with UV and MS detection

Column: | ZORBAX 300SB-C18 5064-8263 0.3 x 150 mm, 5 μm
Mobile Phase: | 5-55% B in 50 min, to 85% B from 55-57 min A: Water + 0.1% Formic acid B: Acetonitrile + 0.1% Formic acid
Flow Rate: | 5.5 μL/min
Detector: | UV, 206 nm
MS Conditions: | LC/MS: | Pos. Ion ESI with LC/MSD trap-Vcap 4000 V
| Drying Gas Flow: | 7 L/min
| Drying Gas Temperature: | 250 °C
| Nebulizer: | 15 psi
| Capillary Exit Volt: | 50 V
| Max Accum Time: | 300 ms
| Total Averages: | 3
| Isolation Width: | 3 m/z
| Frag Ampitude: | 1.0 V
Sample: | 100 nL Beta Casein Digest (4 pmol)

A ZORBAX 300SB-C18 capillary column (0.3 mm id) is used for the separation of the protein digest. Detection is by both UV and Electrospray MS. MS detection can be used for identification of peptide fragments.

2-D LC/MS Analyses Using ZORBAX Capillary and Nano LC Columns

Typical Column Configuration for 2-D HPLC

Flow path of an Agilent customized Nanoflow Proteomics Solution system.

1. Sample loading, elution from SCX and trapping on enrichment column

2. Valve switch in column compartment, elution from enrichment column; separation on RP, and MS anaylsis

Proteins in a complex sample by 2-D HPLC with Nano HPLC columns

Column: | ZORBAX 300SB-C18 5065-9913 0.3 x 5 mm, 5 μm
Column: | ZORBAX 300SB-C18 5065-9911 0.075 x 150 mm, 3.5 μm
Mobile Phase: | Quaternary Pump: 3% Acetonitrile/0.1% Formic acid Nanopump: A = Water, 0.1% Formic acid, B = ACN, 0.1% Formic acid
Flow Rate: | Quaternary Pump: 30 μL/min Nanopump: 300 nL/min
Gradient: | Quaternary Pump: Isocratic Nanopump: 6 min = 3% B, 120 min = 60% B, 125 min = 80% B, 130 min = 80% B, 131 min = 3% B, 140 min = 3% B
MS Conditions: | Source: Nano ESI, drying gas flow: 5 L/min, drying gas temp: 225 °C Ion Trap: Skim: 1:35 V, cap exit offset: 115 V, octupole 1:12 V, octupole 2:3.5 V, trap drive: 80 V. ICC: on, averages: 4, max accu time: 150 ms; target 60.000, ion mode positive, MS/MS mode.

Sample:

Tryptic Digest of bovine serum albumin
Volume: 1 to 8 μL
Salt Step Elution: 8 mL of 10 mM-100 mM KCI (10 mM increments),
125 mM, 150 mM, 200 mM, 300 mM, 500 mM, 1 M.

Tryptic digest of bovine serum albumin (BSA). The base peak chromatograms show a selection of fractions from a 2-D HPLC separation. Single chromatograms represent peptides from BSA eluting at a given salt concentration followed by enrichment and reversed-phase chromatography.

ZORBAX Bio-SCX Series II

ZORBAX has Bio-SCX Series II columns designed for optimized 2-D separations of peptides and proteins using LC/MS. This packing is based on ultra-pure 3.5 μm ZORBAX silica particles, bonded with a bio-friendly polymer that is functionalized with sulfonic acid groups. This gives strong retention and good peak shape in the ion-exchange step of 2-D analysis of peptides and proteins.

Nano Columns

ZORBAX Bio-SCX Series II provides more retention of small peptides

Column: | ZORBAX Bio SCX Series II 5065-9912 0.3 x 35 mm, 3.5 μm
Mobile Phase: | 95% 40 mM NaCl: 5% ACN, 0.3% Formic acid
Flow Rate: | 5 μL/min
Detector: | 230 nm
Sample: | Synthetic Dipeptides

The new ZORBAX Bio-SCX Series II column retains smaller peptides more strongly than some other SCX columns. The result is increased resolution of more hydrophilic peptides fragments and more accurate identification when these columns are used in 2-D HPLC analysis.

ZORBAX 300SB-C18 trap/guard, 5065-9913

MicroBore (1.0 mm id) Columns

• High sensitivity for small sample sizes

• Compatible with LC/MS interfaces

• Wide variety of bonded phases

• Silica and polymeric particles

Agilent MicroBore (1.0 mm id) columns are a good choice when sample sizes are limited. They can improve detection limits 5 times over 2.1 mm id columns when the same sample mass is used. This increase in sensitivity can be critical. MicroBore columns use low flow rates (typically ~ 50 μL/min). Therefore, these columns are ideal for use with detectors requiring low flow rates such as some mass spectrometers and with capillary LC systems.

Optimum performance is achieved when MicroBore columns are used with UHPLC/HPLC Microbore systems. A wide variety of bonded phases is available for use up to 400 bar including StableBond, 300SB-C18, 300SB-C8, and Poroshell columns. Polymeric reversed-phase, PLRP-S, and ion-exchange PL-SAX and PL-SCX are also available for applications requiring exceptionally stable wide pore particles. Guard columns are also now available with an adjustable tube stop depth to provide a perfect zero dead volume connection every time.

Sterically Protected 300StableBond Bonded Phase

Separation of a tryptic digest on ZORBAX MicroBore 300SB-C18

Column: | ZORBAX 300SB-C18 863630-902 1.0 x 150 mm, 3.5 μm
Mobile Phase: | Gradient: 2-60% B in 60 min A: 0.1% TFA B: 0.075% TFA/80% ACN
Flow Rate: | 50 μL/min
Temperature: | 50 °C
Detector: | UV, 215 nm
Sample: | 2 μL Tryptic Digest of rhGH

This example of a tryptic digest separated on a MicroBore column demonstrates the high sensitivity and resolution possible with 1.0 mm id columns.

Microbore HPLC for sensitive peptide analysis

Column: | PLRP-S 100Å 5 μm, 150 mm x various id
Mobile Phase: | A: 0.01 M Tris HCI, pH 8 B: A + 0.35 M NaCl, pH 8
Flow Rate: | 1 mL/min
Gradient: | Linear 20% ACN, 0.1% TFA to 50% ACN, 0.1% TFA over 15 min
Injection Volume: | 0.5 μL
Sample Conc: | 0.25 mg/mL
Detector: | UV, 220 nm

Peak Identification

A. 1.0 mm id (flow rate 47 μL/min)

B. 2.1 mm id (flow rate 200 μL/min)

C. 4.6 mm id (flow rate 1 mL/min)

1. Oxytocin

2. Angiotensin II

3. Angiotensin I

4. Insulin

Peptide separation on Agilent PLRP-S 100Å 5 μm columns

Peak Identification

  1.0 mm

  2.1 mm

  4.6 mm

Standard curve data-point graphs on Agilent PLRP-S columns

Purification – Prep HPLC

Agilent has a comprehensive range of silica and polymeric HPLC columns and media designed for biomolecule purification. There are high efficiency small particle prep columns optimized for the purification of μg and mg amounts of a biopharmaceutical drug candidate and fully porous bulk media, to pack development and process columns to purify multiple 100 g, kg and multi-kg of API.

Some columns are specifically designed to address the needs of high efficiency purification, while other products provide easy scale-up from small particle analytical columns to full scale API production. Table 1 shows prep column/media options and the quantity of product that can be purified.

Polymeric Prep HPLC Columns

TIPS & TOOLS

Further information can be found in the following publication:

Biomolecule Purification (publication # 5990-8335EN)

www.agilent.com/chem/library

ZORBAX PrepHT

High purity, high recovery, and high throughput can be easily achieved with Agilent ZORBAX PrepHT columns. These are available in a variety of bonded phases – StableBond 300Å, C18, C8, C3, and CN – for optimized resolution and loadability under any conditions.

ZORBAX PrepHT columns are packed with 5 and 7 μm particle sizes for very high resolution. The high resolution allows high loadability, high yield, and high purity of compounds. The larger diameter columns and mechanically stronger ZORBAX particles allow for flow rates up to 100 mL/min, thus increasing throughput.

ZORBAX PrepHT columns are designed for rapid scale-up from analytical to preparative scale without losing resolution. For complex separations on larger columns (21.2 mm id, 150 mm length and longer), Agilent has carefully chosen the 7 μm particle size to achieve a balance between high efficiency and high loadability.

ZORBAX 300Å StableBond Prep HT Cartridge Columns

PLRP-S for Prep to Process

• Discovery stage to multi-kg cGMP production reduces method development time

• Chemical stability for separations, optimization, sanitation, and regeneration increases selectivity and column lifetime

• Single batch packing of multiple columns reduces system downtime and validation costs

The PLRP-S media, rigid poly(styrene/divinylbenzene) particles, are available in a range of pore sizes for small molecule, synthetic biomolecule and macromolecule purification. Their thermal and chemical stability makes them ideal for purifications that require extreme conditions for sample preparation, compound elution, and column regeneration.

Capacity and resolution are two key parameters for maximizing the throughput of a purification. With a wide choice of pore sizes and extended range of operating conditions, PLRP-S provides more options to achieve the optimum process. Particle sizes range from 3 μm to 50 μm for scale-up from the μg/mg discovery stage to multi-kg cGMP production. Excellent chemical stability, up to 1 M NaOH, permits sanitation and regeneration that increase column lifetime. PLRP-S media batch sizes of up to 600 L are available, providing single batch packing of multiple columns.

As part of our commitment to quality and continuity of supply, all manufacturing is carried out under a fully documented process. A Type II Drug Master File and regulatory support files are available for process materials, and facility audits are routinely conducted.

Column Specifications
pH Range | 1-14
Buffer Content | Unlimited
Organic Modifier | 1-100%
Temperature Limits | 200 °C
Maximum Pressure | 5-8 μm: 3000 psi (210 bar)
| 3 μm: 4000 psi (300 bar)

Purification of a 25-mer trityl-off oligonucleotide and analytical quantitation of the fraction using PLRP-S 100Å, 4.6 x 50 mm

Column: | PLRP-S 100Å PL1512-1300 4.6 x 50 mm, 3 μm
Mobile Phase: | A: 100 mM Triethylammonium acetate (TEAA) B: 100 mM TEAA in 25:75 Acetonitrile:water
Flow Rate: | 1 mL/min
Gradient: | 25% B 0 min, 35% B 2 min, 45% B 22.5 min, 45% B 23 min, 25% B 23.05 min, 25% B 26 min
Temperature: | 80 °C

Crude bradykinin prep load

Column: | PLRP-S 100Å PL1512-5100 4.6 x 250 mm, 10 μm
Sample: | 30 μL containing 1.5 mg of crude peptide
Mobile Phase: | 0.1% TFA in 21% ACN:79% water
Flow Rate: | 1 mL/min (360 cm/hr)

Fraction analysis – the concentration overload purification

HPLC analysis of the fractions collected across the peak showed that fractions 1 to 4 contained only the peptide of interest and that the level of the critical impurity increased with increasing fraction number. Using the high efficiency PLRP-S column it was possible to obtain from the crude, 91.7% pure, a recovery of 97% with 100% purity. For more information, see application note 5990-7736EN.

PL-SAX and PL-SCX for Prep to Process

• Ion-exchange purifications over a wider pH range extend applications

• HPLC flow rates and rapid equilibration reduce purification cycle times

• Large pore size for improved mass transfer delivers high speed, high resolution purifications

These rigid, strong ion-exchange materials are extremely hydrophilic and are designed for purification of biomolecules. The PL-SAX and PL-SCX materials are totally polymeric and are chemically and thermally stable over a full range of HPLC conditions. The strong ion-exchange functionalities, covalently linked to a chemically stable polymer, facilitate ion-exchange purifications over a wider pH range. This stability can be exploited for column sanitation and clean-up. Thermal stability also enables the use of denaturing conditions and stabilizing/solubilizing agents for the purification of target compounds, as encountered in the purification of synthetic oligonucleotides with self-complementary sequences.

Both the 1000Å and 4000Å wide-pore materials are mechanically stable and robust and can be operated over a wide range of linear velocities, with fast loading of dilute solutions and wash cycles. HPLC flow rates and rapid equilibration reduces purification cycle times.

Packing in dynamic axial compression (DAC) column hardware is straightforward and high efficiency columns are achieved with excellent reproducibility and lifetimes. The 1000Å pore size is for high-capacity purifications and the 4000Å gigaporous particles with improved mass transfer are intended for large biomolecules and high-speed, high-resolution purifications.

Column Specifications
| PL-SAX | PL-SCX
Matrix | Fully polymeric | Fully polymeric
Pore Sizes | 1000Å, 4000Å | 1000Å, 4000Å
Particle Sizes | 10 μm, 30 μm | 10 μm, 30 μm
Bead Form | Rigid spherical | Rigid spherical
Functionality | Quaternary amine | Sulfonic acid
Pressure Stability | 3000 psi | 3000 psi
Temperature Stability | 80 °C | 80 °C
pH Range | 1-14 | 1-14
Eluent Compatibility | All anion-exchange buffers | All cation-exchange buffers
Packed Bed Density | 0.39 g/mL | 0.39 g/mL

Purification of a large oligonucleotide

Column: | PL-SAX 1000Å, 8 μm
Mobile Phase: | A: 93% 0.1 M TEAA, pH 7:7% ACN B: 93% 0.1 M TEAA, 3.24 M ammonium acetate, pH 7:7% ACN
Gradient: | 0-100% B in 20 min
Flow Rate: | 1.5 mL/min
Temperature: | 60 °C
Detector: | UV, 290 nm

Preparative fractionation of a culture filtrate containing amyloglucosidases on Agilent PL-SAX 4000Å

Column: | PL-SAX PL1551-1803 4.6 x 50 mm, 8 μm
Mobile Phase: | A: 0.01 M Tris HCI, pH 8 B: A + 0.5 M NaCl, pH 8
Flow Rate: | 4.0 mL/min
Gradient: | Linear 0-100% B in 2 min
Detector: | UV, 280 nm

Prep to Process PL-SAX and PL-SCX Columns and Bulk Media

Peptide Purification

VariTide is a cost-effective solution for the production of synthetic peptides. This column lets you manage the cost and efficiency of high-volume synthetic peptide purification, from μg to g scale. VariTide provides a solution for peptide houses that manufacture small quantities of hundreds or thousands of peptides where manufacturing time is the economic driving force.

VariTide RPC Columns

VariTide RPC Columns for Synthetic Peptides

• A single column to cover the full range of synthetic peptides

• Small particle size for maximum efficiency, even with 1 and 2 in prep columns

• Bulk media to pack 1 and 2 in prep columns for the purification of mg to g quantities

VariTide RPC columns and media are part of the VariPep Peptide Solution. This is the recommended option for cost-effective separation and purification of synthetic peptides using generic methods.

VariTide RPC Columns for Synthetic Peptides

Size (mm) | Part No.
21.2 x 250 | PL1E12-5A05
10.0 x 250 | PL1012-5A05
4.6 x 250 | PL1512-5A05

VariTide RPC Bulk Media

Description | Part No.
100 g | PL1412-4A05
1 kg | PL1412-6A05

Crude peptide screen

Column: | VariTide RPC PL1512-5A05 4.6 x 250

Mobile Phase:

Acidic
A: 0.1% TFA in 95% water: 5% ACN
B: 0.1% TFA in 50% water: 50% ACN
Basic
A: 5% ACN, 95% 20 mm ammonium
carbonate pH 9.5
B: 50% ACN, 50% 20 mm ammonium
carbonate pH 9.5

Flow Rate: | 1.0 mL/min (360 cm/h)
Gradient: | 0-100% B in 30 min
Detector: | UV, 220 nm

VariPure IPE

• Pre-packed for convenience

• Removal of ion-pairing agents for improved productivity

• High performance and economy for excellent efficiency

VariPure IPE is a polymer-supported quaternary-amine resin with a bicarbonate counter ion, designed for removing acidic ion-pair reagents, such as trifluoroacetic acid (TFA), formic acid or acetic acid. VariPure IPE is a high performance and economical acid removal material conveniently supplied as pre-packed SPE type devices. The particle size, capacity and device geometry are matched to provide sufficient residence time to achieve effective ion-air extraction under gravity flow. For acid labile peptides, removal of the ion-pairing agent prevents acid degradation of the peptide during post-HPLC work-up, and increases the yield of purified product.

[picture] | TIPS & TOOLS For the latest application notes and new product information, go to www.agilent.com/chem/library

Citations

ZORBAX 300 Citations

Portelius, E. et al. (2007) Characterization of Amyloid β Peptides in Cerebrospinal Fluid by an Automated Immunoprecipitation Procedure Followed by Mass Spectrometry. Journal of Proteome Research, 6 (11): 4433-4439

Montes-Bayon, M. et al. (2006) Direct comparison of capillary electrophoresis and capillary liquid chromatography hyphenated to collision-cell inductively coupled plasma mass spectrometry for the investigation of Cd-, Cu- and Zn-containing metalloproteins. Journal of Chromatography A, 1114(1): 138-144

Zahariev, S. et al. (2006) Synthesis of ‘difficult’ peptides free of aspartimide and related products, using peptoid methodology. Tetrahedron Letters, 47(25): 4121-4124

Kohler, M. et al. (2009) Identification of Human Pituitary Growth Hormone Variants by Mass Spectrometry. Journal of Proteome Research, 8(2): 1071-1076

Berg, M. et al. (2006) Reproducibility of LC-MS-based protein identification. Journal of Experimental Botany, 57(7): 1509-1514

Esteban-Fernández, D. et al. (2008) Atomic (HPLC-ICP-MS) and molecular mass spectrometry (ESI-Q-TOF) to study cis-platin interactions with serum proteins. Journal of Analytical Atomic Spectrometry, 23: 378-384

Everberg, H. et al. (2006) Aqueous Two-Phase Partitioning for Proteomic Monitoring of Cell Surface Biomarkers in Human Peripheral Blood Mononuclear Cells. Journal of Proteome Research, 5(5): 1168-1175

Portelius, E. et al. (2010) Identification of novel N-terminal fragments of amyloid precursor protein in cerebrospinal fluid. Experimental Neurology, 223(2): 351-358

Ahrends, R. et al. (2009) Metal-Coded Affinity Tag Labeling: A Demonstration of Analytical Robustness and Suitability for Biological Applications. Analytical Chemistry, 81(6): 2176-2184

Bíliková, K. et al. (2009) Towards functional proteomics of minority component of honeybee royal jelly: The effect of post-translational modifications on the antimicrobial activity of apalbumin2. Proteomics, 9(8): 2131-2138

Schwab, K. et al. (2011) Adaptation of proteomic techniques for the identification and characterization of protein species from murine heart. Amino Acids, 41(2): 401-414

Perreault, A. et al. (2009) A Methyltransferase-independent Function for Rmt3 in Ribosomal Subunit Homeostasis. The Journal of Biological Chemistry, 284: 15026-15037

Magherini, F. et al. (2009) Different carbon sources affect lifespan and protein redox state during Saccharomyces cerevisiae chronological ageing. Cellular and Molecular Life Sciences, 66(5): 933-947

Cao H. & Xu S-Y. (2008) Purification and characterization of type II collagen from chick sternal cartilage. Food Chemistry, 108(2): 439-445

Chen, X. et al. (2010) Quantitative Proteomics Analysis of Cell Cycle-regulated Golgi Disassembly and Reassembly. The Journal of Biological Chemistry, 285: 7197-7207

Trusch, M. et al. (2010) Application of displacement chromatography for the analysis of a lipid raft proteome. Journal of Chromatography B, 878(3-4): 309-314

Amoresano, A. et al. (2008) A Rapid and Selective Mass Spectrometric Method for the Identification of Nitrated Proteins. Methods in Molecular Biology, 477(I): 15-29

Kodali, VP., Das, S. & Sen, R. (2009) An exopolysaccharide from a probiotic: Biosynthesis dynamics, composition and emulsifying activity. Food Research International, 42(5-6): 695-699

Loeber, R. et al. (2008) Cross-Linking of the DNA Repair Protein O⁶-Alkylguanine DNA Alkyltransferase to DNA in the Presence of Antitumor Nitrogen Mustards. Chemical Research in Toxicology, 21(4): 787-795

Millette, M. et al. (2008) Purification and identification of the pediocin produced by Pediococcus acidilactici MM33, a new human intestinal strain. Journal of Applied Microbiology, 104(1): 269-275

Chen, X. et al. (2009) Quantitative Organellar Proteomics Analysis of Rough Endoplasmic Reticulum from Normal and Acute Pancreatitis Rat Pancreas. Journal of Proteome Research, 9(2): 885-896

Ameri, M., Daddona, P. & Maa, YF. (2009) Demonstrated Solid-State Stability of Parathyroid Hormone PTH(1–34) Coated on a Novel Transdermal Microprojection Delivery System. Pharmaceutical Research, 26(11): 2454-2463

Gianazza, E. et al. (2010) Different expression of Fibrinopeptide A and related fragments in serum of type 1 diabetic patients with nephropathy. Journal of Proteomics, 73(3): 593-601

Yuan, W. et al. (2008) Two New Holostan-Type Triterpene Glycosides from the Sea Cucumber Bohadschia marmorata JAEGER. Chemical & Pharmaceutical Bulletin, 56(8): 1207

Poroshell 300 Citations

Matilainen, L. et al. (2008) The effect of cyclodextrins on chemical and physical stability of glucagon and characterization of glucagon/g-CD inclusion complexes. Journal of Pharmaceutical Sciences, 97(7): 2720-2729

Astarita, G., Ahmed, F. & Piomelli, D. (2008) Identification of biosynthetic precursors for the endocannabinoid anandamide in the rat brain. The Journal of Lipid Research, 49: 48-57

Matilainen, L. et al. (2008) The stability and dissolution properties of solid glucagon/g-cyclodextrin powder. European Journal of Pharmaceutical Sciences, 36(4-5): 412-420

Wang, Y. et al. (2009) Comparison of Methods for Extracting Kafirin Proteins from Sorghum Distillers Dried Grains with Solubles. Journal of Agricultural & Food Chemistry, 57(18): 8366-8372

Guerrera, IC. et al. (2009) A Novel Lipidomic Strategy Reveals Plasma Phospholipid Signatures Associated with Respiratory Disease Severity in Cystic Fibrosis Patients. PLoS ONE, 4(11): e7735

Yung, KM. et al. (2011) An amyloid β₄₂-dependent deficit in anandamide mobilization is associated with cognitive dysfunction in Alzheimer’s disease. Neurobiology of Aging

Reid, CQ. et al. (2010) Rapid whole monoclonal antibody analysis by mass spectrometry: An Ultra scale-down study of the effect of harvesting by centrifugation on the post-translational modification profile. Biotechnology and Bioengineering, 107(1): 85-95

Heinig, K. & Wirz, T. (2009) Determination of Taspoglutide in Human and Animal Plasma Using Liquid Chromatography - Tandem Mass Spectrometry with Orthogonal Column-Switching. Analytical Chemistry, 81(10): 3705-3713

Rossi, EA. et al. (2010) A Bispecific Antibody-IFNa2b Immunocytokine Targeting CD20 and HLA-DR Is Highly Toxic to Human Lymphoma and Multiple Myeloma Cells. Cancer Research, 70: 7600

Murphy, MP. et al. (2010) Changes in Cognition and Amyloid-β Processing with Long Term Cholesterol Reduction using Atorvastatin in Aged Dogs. Journal of Alzheimer’s Disease, 22(1): 135-150

PLRP-S Citations

Nakagawa, M., Tojo, H. & Fujii, S. (2011) A Glycan of Y-Factor from Dictyostelium discoideum Contains a Bisecting-GlcNAc, an Intersecting-GlcNAc, and a Core a-1,6-Fucose. Bioscience, Biotechnology, and Biochemistry, 75(10):1964-1970

Della Donna, L. et al. (2011) Mass spectrometric measurements of the apolipoproteins of bovine (Bos taurus) HDL. Comparative Biochemistry and Physiology Part D: Genomics and Proteomics

Vondenhoff, GHM. et al. (2011) Characterization of Peptide Chain Length and Constituency Requirements for YejABEF-Mediated Uptake of Microcin C Analogues. The Journal of Bacteriology, 193(14): 3618-3623

Šotkovský, P. et al. (2011) A new approach to the isolation and characterization of wheat flour allergens. Clinical & Experimental Allergy, 41(7): 1031-1043

Laabs, E. et al. (2011) Optical Imaging of CCK2/Gastrin Receptor-Positive Tumors With a Minigastrin Near-Infrared Probe. Investigative Radiology, 46(3): 196-201

Rickert, KW. et al. (2011) Structural Basis for Selective Small Molecule Kinase Inhibition of Activated c-Met. The Journal of Biological Chemistry, 286: 11218-11225

Stuknyte, M. et al. (2011) Potential immunomodulatory activity of bovine casein hydrolysates produced after digestion with proteinases of lactic acid bacteria. International Dairy Journal, 21(10): 63-769

Nakano, E. et al. (2011) Riboflavin Depletion Impairs Cell Proliferation in Adult Human Duodenum: Identification of Potential Effectors. Digestive Diseases and Sciences, 56(4): 1007-1019

Webb, K. et al. (2011) The ribosomal L1 protuberance in yeast is methylated on a lysine residue catalyzed by a seven beta-strand methyltransferase. The Journal of Biological Chemistry, M110. 200410

Uehata, T. et al. (2011) Serum hepcidin-25 levels and anemia in non-dialysis chronic kidney disease patients: a cross-sectional study. Nephrology Dialysis Transplantation, 27(3): 1076-1083

Lohman, G, Chen, L. & Evans Jr, T. (2011) Kinetic characterization of single strand break ligation in duplex DNA by T4 DNA Ligase. The Journal of Biological Chemistry, 286: 44187-44196

Hudak, J., Yu, H. & Bertozzi, C. (2011) Protein Glycoengineering Enabled by the Versatile Synthesis of Aminooxy Glycans and the Genetically Encoded Aldehyde Tag. Journal of the American Chemical Society, 133(40): 16127-16135

Schneider, N. et al. (20111) Prevalence and stability of lysozyme in cheese. Food Chemistry, 128(1): 145-151

Yan, B. & Boyd, D. (2011) Breaking the Light and Heavy Chain Linkage of Human Immunoglobulin G1 (IgG1) by Radical Reactions. The Journal of Biological Chemistry, 286: 24674-24684

Landau, M. et al. (2011) Towards a Pharmacophore for Amyloid. PLoS Biology, 9(6): e1001080

Kerkaert, B. et al. (2011) Use of lysozyme as an indicator of protein cross-contact in fresh-cut vegetables via wash waters. Food Research International, 45(1): 39-44

Schneider, N., Werkmeister, K. & Pischetsrieder, M. (2011) Analysis of nisin A, nisin Z and their degradation products by LCMS/MS. Food Chemistry, 127(2): 847-854

Quenee, L. et al. (2011) Prevention of pneumonic plague in mice, rats, guinea pigs and non-human primates with clinical grade rV10, rV10-2 or F1-V vaccines. Vaccine, 29(38): 6572-6583

PL-SAX Citations

Pratto, F. et al. (2008) Streptococcus pyogenes pSM19035 requires dynamic assembly of ATP-bound ParA and ParB on parS DNA during plasmid segregation. Nucleic Acids Research, 3 (11): 3676-3689

Sendovski, M. et al. (2010) Crystallization and preliminary X-ray crystallographic analysis of a bacterial tyrosinase from Bacillus megaterium. Acta Crystallographica, 66(9): 1101-1103

Bunger, MK. et al. (2008) Automated Proteomics of E. coli via Top-Down Electron-Transfer Dissociation Mass Spectrometry. Analytical Chemistry, 80(5): 1459-1467

Vantourout, P. et al. (2009) Specific Requirements for Vγ9Vδ2 T Cell Stimulation by a Natural Adenylated Phosphoantigen. The Journal of Immunology, 183(6): 3848-3857

Scaboo, AM. et al. (2009) Confirmation of Molecular Markers and Agronomic Traits Associated with Seed Phytate Content in Two Soybean RIL Populations. Crop Science, 49(2): 426-432

PL-SCX Citations

Zhang, W. & Czupryn, M. (2003) Analysis of isoaspartate in a recombinant monoclonal antibody and its charge isoforms. Journal of Pharmaceutical and Biomedical Analysis, 30(5): 1479-1490

Collinge, J. et al. (2005) Differential Proteomics via Probabilistic Peptide Identification Scores. Analytical Chemistry, 77(2): 596-606

Schönleben, S. et al. (2007) Proteome analysis of Apis mellifera royal jelly. Analytical and Bioanalytical Chemistry, 389(4): 1087-1093

Lohaus, C. et al. (2007) Multidimensional Chromatography: a Powerful Tool for the Analysis of Membrane Proteins in Mouse Brain. Journal of Proteome Research, 6(1): 105-113

Zahedi, RP. et al. (2007) Phosphoproteome of Resting Human Platelets. Journal of Proteome Research, 7(2): 526-534

Boehm, A. et al. (2007) Precise protein quantification based on peptide quantification using iTRAQ™. BMC Bioinformatics, 8: 214

Heller, M. et al. (2003) Trypsin catalyzed ¹⁶O-to-¹⁸O exchange for comparative proteomics: tandem mass spectrometry comparison using MALDI-TOF, ESI-QTOF, and ESI-ion trap mass spectrometers. Journal of the American Society for Mass Spectrometry, 14(7): 704-718

GPC/SEC COLUMNS AND STANDARDS

GPC/SEC Columns and Standards

• A full portfolio of products for analysis of synthetic and natural polymers

• A wide selection of polymer standards to cover the range of applications in organic and water based solvents

• PL aquagel-OH-series, for aqueous SEC separations, and PLgel, for organic polymer applications, are available in mixed and individual pore sizes across a range of particle sizes, to cover the full spectrum of molecular weights (MW)

• Prep scale columns are available, along with narrow bore columns and columns designed for specific applications

Gel permeation chromatography (GPC) and size exclusion chromatography (SEC) are names applied to the most popular technique for measuring the molecular weight distribution (MWD) of natural and synthetic polymers, a property that affects many of the physical parameters of materials such as strength, toughness and chemical resistance. GPC and SEC are liquid chromatographic techniques that separate individual polymer chains on the basis of their size in solution and not on their chemistry. Gel permeation chromatography (GPC) is the name used to describe the analysis of polymers in organic solvents, such as tetrahydrofuran. Size exclusion chromatography (SEC) is the name used to describe the analysis of polymers in water and water-based solvents, such as buffer solutions. GPC/SEC is the only established method for obtaining a comprehensive understanding of a polymer’s molecular weight distribution.

[picture] | TIPS & TOOLS For information on SEC columns for proteins, View Here.

Table of Contents

LC and LC/MS Columns for GPC/SEC

GPC/SEC Columns

PLgel GPC

PLgel MIXED

PLgel MIXED-LS

PLgel MiniMIX

PLgel Individual Pore Size

PLgel Preparative

EnviroPrep

PLgel Olexis

PL HFIPgel

PL Rapide

PolarGel

PlusPore

PolyPore

ResiPore

MesoPore

OligoPore

PL aquagel-OH SEC

PL aquagel-OH SEC Analytical

PL aquagel-OH SEC Preparative

GPC Column Accessories

Polymer Standards for GPC/SEC

EasiVial

EasiCal

Polystyrene

Polymethylmethacrylate

Polyethylene Glycol/Oxide

Polysaccharides

Polyacrylic Acid

GPC/SEC Columns

The key to successful GPC/SEC separations is the correct choice of columns. The comprehensive range of Agilent products for GPC/SEC has been designed to cover virtually all polymer analysis application areas, and to make selection for the correct column, solvent, and calibration standard fast and reliable.

Agilent’s PLgel GPC series of columns are for polymer applications using organic solvents. PLgel is a highly cross-linked, porous polystyrene/divinylbenzene matrix, which is recognized as a market leader in GPC column technology. PLgel materials have high pore volume and high-efficiency to maximize resolution. Their unequalled solvent compatability makes for easy transfer between polar and non-polar eluents, and outstanding physical rigidity provides extended lifetimes that maximize downtime. For more information and full ordering details, see here.

Agilent’s PL aquagel-OH series of columns provide a chemically and physically stable matrix for reliable aqueous SEC separations. The columns are packed with macroporous copolymer beads with an extremely hydrophilic polyhydroxyl functionality. The “neutral” surface and the capability to operate across a wide range of eluent conditions provide for high performance analyses of compounds with neutral, ionic, and hydrophobic moieties, alone or in combination. PL aquagel-OH is available for analytical and preparative applications. For more information and full ordering details, see here.

Polymer standards for GPC/SEC

Agilent manufactures the highest quality polymer standards with extremely narrow polydispersity and the widest molecular weight range commercially available. These quality polymer standards are supplied with extensive characterization data utilizing a variety of independent techniques (e.g. light scattering and viscometry) and high performance GPC to verify polydispersity and assign the peak molecular weight (Mp).

EasiVial – for organic and aqueous calibration. EasiVial is the fastest and most convenient method to deliver an accurate 12-point column calibration. EasiVial eliminates tedious weight procedures for improved calibration accuracy and reduces solvent dispensing to limit risks associated with handling solvents.

EasiCal – for organic solvents. EasiCal packs are pre-prepared for a no-fuss process. Two different combs, each with ten detachable spatulas, support a mixture of five polymer standards. The cost-effective format is designed to save money.

Individual standards and kits – an extensive range of polymer standard kits of different chemistries designed to match specific column sets are available, as well as individual standards in various pack sizes. For more details about Agilent’s calibration standards for GPC/SEC, see here.

How GPC/SEC works:

• Polymer molecules dissolve in solution to form spherical coils with size dependent on molecular weight

• The polymer coils are introduced to eluent flowing through the column

• Columns are packed with insoluble porous beads with well-defined pore structure

• The size of pores is similar to that of the polymer coils

• The polymer coils diffuse in and out of the pores

• Result is elution based on size – large coils first, smaller coils last

• Size separation converted to molecular weight separation by use of a calibration curve constructed by the use of polymer standards

Recommendations for setting up a GPC/SEC system

The following questions will help you find the recommended columns and standards for any given application, as well as system parameters such as injection volumes.

[picture] | TIPS & TOOLS More information on GPC/SEC instrumentation and systems is a click away. We have a variety of application notes, data sheets and brochures available from Agilent for free. To learn more, visit www.agilent.com/chem/gpc

Typical polymer molecular weights
If you are unsure of the molecular weight of your sample, the table below shows some approximate molecular weight ranges for common polymers, which will help you select the right column for your application.
Polymer Type | Typical molecular weight of polymer | Typical polydispersity¹ of polymer
Polymers from free radical synthesis | High (up to several million) | ~ 2
Intermediate (up to hundreds of thousands)
Polymers from ionic synthesis | Intermediate (up to hundreds of thousands) | ~ 1.01
Low (up to tens of thousands)
Polymers from addition synthesis | Intermediate (up to hundreds of thousands) | ~ 2
Low (up to tens of thousands)
Polymers from controlled radical polymerization | Low (up to tens of thousands) | ~ 1.1 to 1.5
Very low (a few thousand)
Polyolefins | Intermediate (up to hundreds of thousands) | ~ 2 to 200
High (up to several million)
Acrylates | Intermediate (up to hundreds of thousands) | ~ 2
High (up to several million)
Small molecule additives | Very low (a few thousand) | 1
Pre-polymers | Low (up to tens of thousands) | ~ 2 to 10
Very low (a few thousand)
Resins | Low (up to tens of thousands) | ~ 2 to 10
Very low (a few thousand)
Natural biopolymers such as polysaccharides | Intermediate (up to hundreds of thousands) | ~ 2 to 10
High (up to several million)
Rubbers | Intermediate (up to hundreds of thousands) | ~ 2 to 10
High (up to several million)
Biodegradable polymers | Intermediate (up to hundreds of thousands) | ~ 1.1 to 2
Low (up to tens of thousands)

¹ Polydispersity is a measure of the distribution of molecular mass of a polymer. Polydispersity index (PDI) = Mw/Mn.

Organic GPC

PLgel GPC Columns

• Robust performance under the most exacting conditions

• Temperature stability up to 220 °C

• Solvent compatibility allows easy and rapid transfer between solvents of varying polarity

PLgel materials have high pore volume and high efficiency to maximize resolution. Their unequalled solvent compatibility makes for easy transfer between polar and non-polar eluents, and outstanding physical rigidity provides extended lifetimes that minimize downtime.

The key to successful GPC separations is the correct choice of columns. The comprehensive range of PLgel products has been designed to cover virtually all organic solvent-based polymer analysis application areas, and to make selection of the correct column, solvent, and calibration standard fast and reliable.

PLgel is a highly cross-linked, porous polystyrene/divinylbenzene matrix, which is recognized as a market leader in GPC column technology. PLgel is manufactured to ISO 9001:2000 and benefits from comprehensive QC/QA for total reproducibility, batch-to-batch and column-to-column.

Solvent Compatibility

PLgel columns are routinely supplied in ethyl benzene* but you can easily and rapidly transfer between solvents of varying polarity. In organic GPC, sample to column interaction may occur occasionally and eluent modification can be used to eliminate these effects. PLgel columns are the ideal choice for such analyses, as they easily tolerate eluents in the pH range 1-14, as well as up to 10% water in a miscible organic solvent.

PLgel is compatible with all of these solvents
Solvent Polarity | Solvent
6.0 | Perfluoroalkane
7.3 | Hexane
8.2 | Cyclohexane
8.9 | Toluene
9.1 | Ethyl acetate
9.1 | Tetrahydrofuran (THF)
9.3 | Chloroform
9.3 | Methyl ethyl ketone (MEK)
9.7 | Dichloromethane
9.8 | Dichloroethene
9.9 | Acetone
10.0 | o-Dichlorobenzene (o-DCB)
10.0 | Trichlorobenzene (TCB)
10.2 | m-Cresol
10.2 | o-Chlorophenol (o-CP)
10.7 | Pyridine
10.8 | Dimethyl acetamide (DMAc)
11.3 | n-Methyl pyrolidone (NMP)
12.0 | Dimethyl sulfoxide (DMSO)
12.1 | Dimethyl formamide (DMF)

*We also provide a custom packing service in which columns can be shipped in specific solvents to provide extra convenience to our customers.

PLgel Frit Porosity
Media Type | Porosity (μm)
PLgel 3 μm | 2
PLgel 5 μm | 2
PLgel 10 μm | 5
PLgel 20 μm | 10

For PLgel column accessories ordering information please see here

PLgel MIXED Columns

The PLgel MIXED range greatly simplifies column selection for easy decision making. By using these mixed columns, you can eliminate mismatched column sets and spurious peaks for more reliable results. Every column contains a mixture of individual pore size materials, accurately blended to cover a specified broad range of molecular weight with a linear calibration to eliminate column mismatch. Simply add extra columns for even greater resolution.

PLgel MIXED Column Selection Guide

PLgel MIXED Gel Calibration Curves

MIXED gel calibration curves are designed to be linear over a specified molecular weight range, ensuring that the same degree of resolution is achieved across the full operating range of the column. The particle size of the packing and porosity of a particular MIXED gel column are carefully matched to the MW range and application, thus optimizing performance and eliminating the effects of shear degradation. Resolution in GPC is controlled by the slope of the calibration curve and the particle size of the packing material. Agilent has scientifically determined the minimum number of MIXED gel columns required to perform accurate MWD determinations based on specific resolution (Rsp). Thus you can have complete confidence in the accuracy and precision of the calculated data.

PLgel MIXED Columns

Description | Size (mm) | Part No.
PLgel 20 μm MIXED-A | 7.5 x 300 | PL1110-6200
PLgel 10 μm MIXED-B | 7.5 x 300 | PL1110-6100
PLgel 5 μm MIXED-C | 7.5 x 300 | PL1110-6500
PLgel 5 μm MIXED-D | 7.5 x 300 | PL1110-6504
PLgel 3 μm MIXED-E | 7.5 x 300 | PL1110-6300

PLgel MIXED Guards

Size (mm) | Particle Size (μm) | Part No.
7.5 x 50 | 20 | PL1110-1220
7.5 x 50 | 10 | PL1110-1120
7.5 x 50 | 5 | PL1110-1520
7.5 x 50 | 3 | PL1110-1320

Starches

Column: | 4 x PLgel 20 μm MIXED-A PL1110-6200 7.5 x 300 mm
Mobile Phase: | DMSO + 5 mM NaNO₃
Flow Rate: | 1.0 mL/min
Temperature: | 80 °C
Detector: | RI

Polyphenylene Sulfides

Column: | 3 x PLgel 10 μm MIXED-B PL1110-6100 7.5 x 300 mm
Mobile Phase: | o-Chloronaphthalene
Flow Rate: | 1.0 mL/min
Temperature: | 210 °C
Detector: | RI

Plasticized PVC

Column: | 3 x PLgel 5 μm MIXED-C PL1110-6500 7.5 x 300 mm
Mobile Phase: | THF
Flow Rate: | 1.0 mL/min
Detector: | RI

Epoxy Resin

Column: | 3 x PLgel 5 μm MIXED-D PL1110-6504 7.5 x 300 mm
Mobile Phase: | THF
Flow Rate: | 1.0 mL/min
Detector: | RI

Polyol

Column: | PLgel 3 μm MIXED-E PL1110-6300 7.5 x 300 mm
Mobile Phase: | THF
Flow Rate: | 1.0 mL/min
Detector: | UV, 254 nm

PLgel MIXED-LS Columns

• Obtain an instant improvement in data quality

• No need for conditioning, saving time and solvent costs

• Maximize the potential of light scattering detectors

The PLgel MIXED-LS series is a PS/DVB packing using an innovative proprietary suspension polymerization technique to virtually eliminate nano-particle leakage. A startling improvement is achieved immediately in the quality of light scattering data obtained with PLgel MIXED-LS columns in place of conventional GPC columns. The light scattering chromatograms shown here were obtained after flushing the columns for one hour in THF at 1 mL/min. A polystyrene standard (Mp 210,000) was injected at 1 mg/mL in order to illustrate the dramatic improvement in signal-to-noise with the PLgel MIXED-LS column.

The performance of PLgel MIXED-LS columns has been matched to PLgel 20 μm MIXED-A and PLgel 10 μm MIXED-B columns in terms of calibration, column efficiency, wide solvent compatibility, and operating temperature. MIXED-LS are also ideal for online viscosity detection, minimizing the risk of capillary blockage, and can be used with regular PLgel guard columns that are packed with rigid low pore size gels with no particle bleed.

Conventional GPC column

Column: | Conventional GPC column
Mobile Phase: | THF
Flow Rate: | 1.0 mL/min
Detector: | LS

Light scattering detection with a conventional GPC column – noise due to particulate bleed.

PLgel LS column

Column: | PLgel 10 μm MIXED-B LS PL1110-6100LS 7.5 x 300 mm, 10 μm
Mobile Phase: | THF
Flow Rate: | 1.0 mL/min
Detector: | LS

Light scattering detection with a PLgel LS column – minimal particulate bleed gives greatly improved baseline.

PLgel MiniMIX Columns

• Use about 70% less solvent and save money

• Store less solvent and increase operator safety

• High performance comparable to Agilent’s conventional id columns

For reduced solvent cost and consumption, use industry standard PLgel MiniMIX mixed gel columns in 250 x 4.6 mm narrow bore dimensions. These narrow bore columns offer high performance, excellent solvent compatibility and mechanical stability. PLgel MiniMIX columns can be used with conventional GPC equipment.

To maintain the same linear velocity through the column, the volumetric flow rate must be reduced to 0.3 mL/min in line with the column cross sectional area, resulting in significantly lower solvent consumption. Sample loading should also be scaled down in line with reduced column volume, and system dead volume should be minimized to avoid excessive band broadening.

PLgel Individual Pore Size Columns

• Very high efficiency improves productivity

• Choose the optimum column for a perfect match of performance and application

• Fast analysis with fewer columns saves time and money

Individual pore size GPC columns offer high resolution over a specific molecular weight range. The linear portion of the calibration curve, where the slope is at its shallowest, defines the MW region over which optimum resolution will be achieved.

Calibration curves

Calibrant: | Polystyrene
Mobile Phase: | THF
Flow Rate: | 1.0 mL/min

PLgel Preparative Columns

• Excellent column efficiency provides optimum resolution

• High loading can isolate mg amounts for further study

• Over 10 times scale up permits efficient quantification

Preparative GPC is generally employed to fractionate polymers, isolate components in a polymer formulation or simplify mixtures of relatively small molecules in complex matrices. Mixtures of materials are easily separated on the basis of size, preferably in a low boiling organic solvent. They are then collected as a series of discrete fractions and isolated by simple evaporation of the solvent.

PLgel preparative columns are packed with the same rigid, high performance media as the analytical columns. The 10 μm particle provides high column efficiency (> 25,000 p/m) for optimum resolution and loading characteristics. PLgel 25 mm id preparative columns offer over 10 times scale-up compared to the 7.5 mm analytical columns. The increased id and column volume permit even higher loading. With low molecular weight materials, sample concentration can also be significantly increased, enabling production of milligram quantities of very pure material. The actual loading is ultimately controlled by the sample and its molecular weight.

Columns for Special GPC/SEC Applications

EnviroPrep

• High sample loading ensures effective trace analysis

• Simple clean-up procedure saves sample preparation costs

• Optimized particle size distribution provides high resolution

EnviroPrep columns permit a simple, one stage clean-up as part of a methodology to determine pesticides in many organic matrices. The higher molecular weight fractions such as lipids, polymers, natural resins and dispersed high molecular weight components are easily eliminated in the GPC analysis.

Preparative GPC for soil extract clean-up is described in EPA Method 3640A using 300 x 25 mm and 150 x 25 mm columns to give higher sample loading and fraction yields, which is particularly useful for low levels of pollutants. Low pore size EnviroPrep columns are ideal for this method. The columns have 10 μm particles with 100Å pore sizes for high resolution, with an exclusion limit of 4000 g/mol. The preparative columns offer good resolution and high loading through optimization of the particle size distribution.

EnviroPrep

Size (mm) | Part No.
21.2 x 150 | PL1E10-3120EPA
25 x 150 | PL1210-3120EPA
21.2 x 300 | PL1E10-6120EPA
25 x 300 | PL1210-6120EPA

Columns for sample clean-up

Column: | EnviroPrep PL1210-6120EPA 25 x 300 mm
Column: | EnviroPrep PL1210-3120EPA 25 x 150 mm
Mobile Phase: | DCM
Flow Rate: | 10 mL/min
Detector: | UV, 254 nm

1. Corn oil, 25,000 mg/L

2. Bis(2-ethylhexyl) phthalate, 1,000 mg/L

3. Methoxychlor, 200 mg/L

4. Perylene, 20 mg/L

5. Sulfur, 80 mg/L

PLgel Olexis

• Optimized design for polyolefin analysis

• High temperature capability

• High resolution with no damage from sample shear provides clean separations

PLgel Olexis is designed for the analysis of very high molecular weight polymers, specifically polyolefins. The column resolves up to 100,000,000 g/mol (polystyrene in THF), and is packed with 13 pm particles to optimize efficiency and resolution without the risk of sample shear degradation during analysis. The packing of PLgel Olexis has the mechanical stability and robustness expected from a PLgel column, and so it is able to operate up to 220 °C for the analysis of highly crystalline materials.

PLgel Olexis

Description | Size (mm) | Part No.
PLgel Olexis | 7.5 x 300 | PL1110-6400
PLgel Olexis guard | 7.5 x 50 | PL1110-1400

PLgel Olexis reveals true modalities across the range of polyolefins

Column: | 3 x PLgel Olexis PL1110-6400 7.5 x 300 mm
Mobile Phase: | Trichlorobenzene + 0.0125% BHT
Flow Rate: | 1.0 mL/min
Injection Volume: | 200 μL
Temperature: | 160 °C
Detector: | PL-GPC 220 (RI)

PL HFIPgel

• Optimized separation range delivers high performance with no artifacts

• Highly durable packing prolongs column lifetime

• Low operating pressure reduces system wear and unnecessary downtimes

Hexafluoroisopropanol (HFIP) is used as a solvent in GPC for the analysis of important industrial polymers such as polyesters, polyamides and polylactide/glycolide copolymers. For greatly improved performance in extremely polar solvents such as HFIP and trifluoroethanol, we have developed novel “multipore” technology to produce PL HFIPgel, a PS/DVB packing featuring a monodisperse particle size, high pore volume, and high resolution.

Using PL HFIPgel avoids issues associated with conventional packings and HFIP, such as excessive curvature of calibration curves, dislocations/shoulders on peaks for polydisperse samples, and poor resolution in the low MW region.

Column efficiency is guaranteed > 30,000 p/m and the columns are very durable, with a maximum operating pressure of 145 bar (2030 psi). They are packed and tested in methanol but shipped ready-to-use in HFIP.

PL HFIPgel columns with 7.5 mm id normally operate at 1 mL/min. However, the 4.6 mm id columns run at 0.3 mL/min, providing a 70% reduction in solvent consumption with consequent savings in the cost of buying and disposing of solvents.

MW range for PL HFIPgel columns is 2,000,000 g/mol (PMMA in THF).

PL HFIPgel

Description | Size (mm) | Part No.
PL HFIPgel | 4.6 x 250 | PL1514-5900HFIP
PL HFIPgel | 7.5 x 300 | PL1114-6900HFIP
PL HFIPgel guard | 7.5 x 50 | PL1114-1900HFIP
PL HFIPgel guard | 4.6 x 50 | PL1514-1900HFIP

Polyamides

Column: | 2 x PL HFIPgel PL1114-6900HFIP 7.5 x 300 mm
Mobile Phase: | HFIP + 20mM NaTFAc
Flow Rate: | 1.0 mL/min
Temperature: | 40 °C
Detector: | PL-GPC 50 (RI)

PL Rapide

• Analysis in less than ten minutes saves time

• Significantly increased sample throughput improves efficiency

• Reduced solvent consumption and disposal costs save money

• Available in L, M, and H versions for low, medium, and high molecular weights; available in F version for flow injection analysis

Rapid GPC is an excellent tool for screening polymer MWD for trend analysis. Short PL Rapide columns reduce analysis times while maintaining the excellent solvent compatibility and mechanical stability of all GPC columns from Agilent.

PL Rapide columns are ideal for high speed applications such as high throughput screening, process monitoring, or tracking changes in MW distributions, where time is the most critical factor in the analysis. Packed with high quality gels, these columns cover the complete spectrum of molecular weights and are available for the analysis of both organic and water soluble polymers. Key features include high pore volume, high resolution packing materials, no special system requirements, choice of molecular weight resolving range, wide solvent compatibility, and excellent mechanical stability.

Resin analysis by rapid GPC

Column: | PL Rapide L PL1013-2300 10 x 100 mm
Sample: | Epoxy resin
Mobile Phase: | THF
Flow Rate: | 1.0 , 2.0 and 3.0 mL/min
Detector: | UV, 254 nm

PolarGel

• Medium polarity surface and high mechanical stability

• Operate in a wide range of solvents and solvent combinations

• Available in two resolving ranges, PolarGel-L and PolarGel-M

The PolarGel range is ideal for use with polar solvents, such as dimethyl formamide (DMF) and dimethyl sulfoxide (DMSO), and for solvent combinations such as tetrahydrofuran with water. These eluents are very useful in GPC/SEC to separate polar materials, such as polar resins, modified polysaccharides or complex polar polymers that are difficult to analyze in traditional SEC solvents, such as tetrahydrofuran alone. PolarGel-L is used for low molecular weight polar polymers and PolarGel-M for high MW polar polymers.

With polar polymers, highly polar groups can lead to non-specific interactions and secondary separation mechanisms when using polar solvents and traditional non-polar styrene/divinylbenzene columns. Additives and/or column conditioning are normally required to reduce these interactions. PolarGel has no need for these interventions, and also avoids the interactions and secondary effects that produce chromatogram distortions.

These PolarGel “mixed bed” columns have a medium polarity surface and high mechanical stability. They are capable of operating in a wide range of solvents and solvent combinations, greatly enhancing your ability to analyze polar polymers that are not necessarily water soluble. PolarGel is available in two resolving ranges to meet your precise requirements.

Two samples of melamine resin analyzed by PolarGel-L

Column: | 2 x PolarGel-L, 300 x 7.5 mm PL1117-6830
Mobile Phase: | Dimethyl acetamide + 0.1% LiBr
Flow Rate: | 1.0 mL/min
Injection Volume: | 100 μL
Detector: | Agilent PL-GPC 220 (RI)

Excellent separation of two phenol formaldehyde resins with PolarGel-M

Column: | 2 x PolarGel-M, 300 x 7.5 mm PL1117-6800
Mobile Phase: | 0.2% (w/v) DMF & 0.1% LiBr to reduce sample aggregation
Flow Rate: | 1.0 mL/min
Injection Volume: | 100 μL
Detector: | Agilent PL-GPC 50 (RI)

PlusPore

The PlusPore range has an increased pore volume that provides high resolution for specific applications. The high stability media permits the use of a wide range of organic solvents with accuracy and precision so that there is no distortion of the MW distribution shape.

The PlusPore series of columns has been specifically designed for high resolution GPC, and represents the very latest in GPC column technology. These novel packing materials are based on the industry standard, highly cross-linked polystyrene/divinylbenzene (PS/DVB), for the widest applicability and solvent compatibility. Each is made using a novel polymerization process to produce particles that exhibit a specific, controlled pore structure for optimum GPC performance. Typical applications include resins, condensation polymers, prepolymers, and oligomers.

For high resolution polymer analysis, the PolyPore, ResiPore, MesoPore, and OligoPore columns of the PlusPore product series exhibit a wide pore size distribution with near linear calibration curves covering an extended molecular weight range. These so-called “multipore” structures have increased pore volume compared to regular PS/DVB packing materials. This results in very high resolution GPC columns designed for specific application areas. The highly cross-linked porous particles provide excellent chemical and physical stability and permit easy transfer across the full range of organic solvents with little change in the shape of the calibration curve or the efficiency of the columns. As this multipore column technology does not require the combination of individual pore size packing materials, the result is high accuracy and precision without any artifacts in the shape of the molecular weight distribution.

PlusPore calibration curves

    Key

PolyPore

ResiPore

MesoPore

OligoPore

PolyPore

• Routine polymer analysis with very high resolution

• Wide operating range simplifies column choice

• Low particle size extracts maximum information from the analyte

PolyPore columns have been specifically developed to give unrivaled resolution for the analysis of polymers with broad molecular weight distributions. With a wide operating range covering many decades of molecular weight, PolyPore columns combine a 5 μm particle size with extremely high pore volume to give the highest possible resolution, ensuring the most detailed information possible from your analysis.

PolyPore

Description | Size (mm) | Part No.
PolyPore | 7.5 x 300 | PL1113-6500
PolyPore guard | 7.5 x 50 | PL1113-1500

Comparison of PolyPore with conventional individual pore size GPC columns

Column A: | 2 x PolyPore PL1113-6500 7.5 x 300 mm
Column B: | PLgel 10³Å 7.5 x 300 mm, 5 μm
Column C: | PLgel 10⁵Å 7.5 x 300 mm, 5 μm
Sample: | High MW Resin |
Mobile Phase: | THF
Flow Rate: | 1.0 mL/min
Injection Volume | 100 μL
Detector: | Agilent PL-GPC 50 (RI)

Polymethylmethacrylate in DMF

Column: | 2 x PolyPore PL1113-6500 7.5 x 300 mm
Sample: | Commercial PMMA
Mobile Phase: | DMF + 0.1% LiBr
Flow Rate: | 1.0 mL/min
Temperature: | 80 °C
Injection Volume: | 100 μL
Detector: | Agilent PL-GPC 50 (RI)

ResiPore

• Efficient separation of complex molecular weight distributions

• Reveals oligomer content to provide a true representation of the sample

• High pore volume extracts maximum information from the analyte

ResiPore columns are the ideal choice for the analysis of resins and condensation polymers with complex molecular weight distributions that include oligomer content. By combining a 3 μm particle size and high pore volume, high efficiency ResiPore columns offer maximum resolution of these intermediate molecular weight polymers.

ResiPore

Description | Size (mm) | Part No.
ResiPore | 7.5 x 300 | PL1113-6300
ResiPore guard | 7.5 x 50 | PL1113-1300

Alkyd resin

Column: | 2 x ResiPore PL1113-6300 7.5 x 300 mm
Mobile Phase: | THF
Flow Rate: | 1.0 mL/min
Injection Volume: | 20 μL
Detector: | UV, 254 nm

Polyester

Column: | 2 x ResiPore PL1113-6300 7.5 x 300 mm
Mobile Phase: | THF
Flow Rate: | 1.0 mL/min
Injection Volume: | 20 μL
Detector: | UV, 254 nm

MesoPore

• Full solvent compatibility with no detrimental effect on efficiency

• Low particle size extracts maximum information from the analyte

• No MWD dislocations so the distribution is an accurate representation of the sample

MesoPore columns have been specifically designed to provide optimum results in the analysis of prepolymers, i.e. polymeric materials with a large oligomeric component. By combining a 3 μm particle size with high pore volume, MesoPore columns give the highest resolution separations for the analysis of low molecular weight polymers, such as prepolymers, resins, polyols, and siloxanes.

MesoPore

Description | Size (mm) | Part No.
MesoPore | 7.5 x 300 | PL1113-6325
MesoPore guard | 7.5 x 50 | PL1113-1325

Polyurethanes

Column: | 2 x MesoPore PL1113-6325 7.5 x 300 mm
Mobile Phase: | THF
Flow Rate: | 1.0 mL/min
Injection Volume: | 20 μL
Detector: | Agilent PL-GPC 50 (RI)

Polyesterimide

Column: | 2 x MesoPore PL1113-6325 7.5 x 300 mm
Mobile Phase: | THF
Flow Rate: | 1.0 mL/min
Injection Volume: | 20 μL
Detector: | Agilent PL-GPC 50 (RI)

OligoPore

• Near linear calibration curve for best accuracy and precision

• Very stable media allows for a wide choice of solvents

• Isolation of individual fractions reveals more information from whole samples

OligoPore columns have been developed from an innovative new media that exhibits significantly increased pore volumes compared to conventional low pore size GPC columns. The outcome is higher resolution in the oligomeric region. The 300 x 25 mm preparative column offers high resolution at greatly increased loading for effective isolation of individual components. Oligomer fractions collected from the OligoPore preparative column can then be re-injected on analytical columns to check for the purity of the fractions and for comparison with the whole sample.

OligoPore

Description | Size (mm) | Part No.
OligoPore | 25 x 300 | PL1213-6520
OligoPore | 7.5 x 300 | PL1113-6520
OligoPore guard | 7.5 x 50 | PL1113-1320

Analysis of low molecular weight polystyrene and oligomer fractions collected from OligoPore preparative columns

Column: | 2 x OligoPore PL1113-6520 7.5 x 300 mm
Mobile Phase: | THF
Flow Rate: | 1.0 mL/min
Detector: | UV

Analytical separation of low molecular weight polystyrene

Column: | 2 x OligoPore PL1213-6520 7.5 x 300 mm
Mobile Phase: | THF
Flow Rate: | 1.0 mL/min
Loading: | 0.2%, 100 mL
Detector: | UV

Preparative separation of low molecular weight polystyrene

Column: | 2 x OligoPore PL1213-6520 25 x 300 mm
Mobile Phase: | THF
Flow Rate: | 10.0 mL/min
Loading: | 2.0%, 2 mL
Detector: | UV

Aqueous SEC of Polymers

PL aquagel-OH SEC

Aqueous size exclusion chromatography (SEC) is widely used for the determination of molecular weight distributions of a variety of synthetic and naturally occurring water-soluble polymers, and separations of oligomers and small molecules. The requirement to eliminate ionic and hydrophobic effects makes aqueous SEC very demanding.

The PL aquagel-OH series provides a chemically and physically stable matrix for reliable aqueous SEC separations. The columns are packed with macroporous copolymer beads with an extremely hydrophilic polyhydroxyl functionality. The “neutral” surface and the capability to operate across a wide range of eluent conditions provide for high performance analyses of compounds with neutral, ionic, and hydrophobic moieties, alone or in combination. PL aquagel-OH is available for analytical and preparative applications.

Optimizing Conditions for Aqueous SEC with PL aquagel-OH Columns

Due to the complex nature of water-soluble polymers, it is often necessary to modify the eluent in order to avoid sample-to-sample and sample-to-column interactions which can result in poor aqueous SEC separations. The excellent stability of the PL aquagel-OH packing material allows the eluent to be tailored to suit the polymer, while retaining the high column efficiency. For ionic interactions, the eluent can be modified by the addition of salt and/or the adjustment of pH. For water soluble polymers with a hydrophobic character, only the addition of a weak organic solvent (methanol) is required to inhibit hydrophobic interactions.

Guide to eluent selection for PL aquagel-OH applications

PL aquagel-OH Column Selection Guide

Sample Type | Typical Applications | Recommended Column Sets

Low MW polymers and oligomers

Surfactants, oligosaccharides, PEGs, lignosulfonates, polyacrylates

2 or 3,30,20
PL aquagel-OH 8 μm. or
PL aquagel-OH 20 5 μm, or
PL aquagel-OH MIXED-M 8 μm

Polydisperse synthetic or naturally occurring polymers | Polysaccharides, PVA, cellulose derivatives, PEO, polyacrylic acid | 2 or 3 PL aquagel-OH MIXED-H 8 μm. or PL aquagel-OH 60/50/40 8 μm
Very high MW polymers | Polyacrylamides, hyaluronic acids, CMC, starches, gums | PL aquagel-OH 60/50/40 15 μm in series

PL aquagel-OH Analytical

• Highly stable matrix ensures reliable separations, even with modified eluents

• MIXED columns cover a wide range of molecular weights, simplifying column selection

• Highly versatile for neutral, polar, anionic and cationic samples

The PL aquagel-OH analytical series has a pH range of 2-10, compatibility with organic solvent (up to 50% methanol), mechanical stability up to 140 bar (2030 psi) and low column operating pressures.

TIPS & TOOLS Buffers in a stored column may crystallize and cause damage. Flush the column with water containing a small amount of sodium azide to prevent biological growth. | [picture]

Polyvinyl alcohol

Column: | 3 x PL aquagel-OH MIXED-H 8μm PL1149-6800 7.5 x 300 mm
Mobile Phase: | 0.2 M NaNO₃, 0.01 M NaH₂PO₄, pH 7
Flow Rate: | 1.0 mL/min
Detector: | Agilent PL-GPC 50 (RI)

Heparin

Column: | 2 x PL aquagel-OH 30 8 μm PL1120-6830 7.5 x 300 mm
Mobile Phase: | 0.2 M NaNO₃, 0.01 M NaH₂PO₄, pH 7
Flow Rate: | 1.0 mL/min
Detector: | Agilent PL-GPC 50 (RI)

Hyaluronic acid

Column: | PL aquagel-OH 60 15 μm PL1149-6260 7.5 x 300 mm
| And
| PL aquagel-OH 40 15 μm PL1149-6240 7.5 x 300 mm
Mobile Phase: | 0.2 M NaNO₃, 0.01 M NaH₂PO₄, pH 7
Flow Rate: | 1.0 mL/min
Detector: | Agilent PL-GPC 50 (RI)

Differences in composition of two alkyl naphthalene sulfonates

Column: | 2 x PL aquagel-OH 20 5μm PL1120-6520 7.5 x 300 mm
Mobile Phase: | 0.25 M ammonium formate in water
Flow Rate: | 1.0 mL/min
Injection Volume: | 20 μL
Detector: | ELS (neb=30 °C, evap=30 °C, gas=1.4 SLM)

PL aquagel-OH Preparative

• Up to 10 times scale-up maximizes yield

• High loading maximizes sample throughput

• Carefully chosen particle size provides optimum resolution

Preparative SEC is used for the fractionation of a wide variety of water-soluble samples based on their size in solution. The technique is applied to the fractionation of disperse polymers or to isolate components in a polymer formulation.

Preparative PL aquagel-OH columns and associated guard columns enable rapid and convenient scale-up from analytical separations. The 25 mm id prep column offers at least a 10 times scale-up in loading from the 7.5 mm id analytical columns. Typically, a 10 mL/min flow rate results in a separation time of ten minutes with a 300 mm column. The columns are packed with the same robust macroporous particles as the analytical column range. The 8 μm particle size provides optimum resolution and loading characteristics with column efficiency > 20,000 plates/m.

Polyvinyl alcohol

Column: | PL aquagel-OH 40 8 μm PL1249-6140 25 x 300 mm
Mobile Phase: | 0.2 M NaNO₃, 0.01 M NaH₂PO₄, pH 7
Flow Rate: | 10.0 mL/min
Loading: | 10 mg/mL, 2 mL
Detector: | Agilent PL-GPC 50 (RI)

GPC Column Accessories

Description | Unit | Part No.
Frit removal tool for threaded columns only | 1/pk | PL1310-0001
2 μm frit kit for threaded columns, 7.5 mm id | 5/pk | PL1310-0002
5 μm frit kit for threaded columns, 7.5 mm id | 5/pk | PL1310-0012
10 μm frit kit for threaded columns, 7.5 mm id | 5/pk | PL1310-0036
PLgel column repair gel, 10 μm | 1/pk | PL1410-0101
PLgel column repair gel, 5 μm | 1/pk | PL1410-0501
Column connecting nuts, 1/16 in tube | 5/pk | PL1310-0007
Tubing ferrules, 1/16 in tube | 5/pk | PL1310-0008
Connecting tubing, 10 cm length, 0.01 in id | 10/pk | PL1310-0048
LDV intercolumn stainless steel connector | 1/pk | PL1310-0005
PLgel column repair gel, 3 μm | 1/pk | PL1410-0301
PLgel Olexis column repair gel | 1/pk | PL1410-0200

Polymer Standards for GPC/SEC

Polymer standards from Agilent are the ideal reference materials for generating accurate, reliable GPC/SEC column calibrations, with the assurance of the ISO 9001:2000 quality standard. Additional applications for our highly characterized homopolymers exhibiting unique characteristics are used as model polymers for research and analytical method development.

Agilent manufactures the highest quality polymer standards with extremely narrow polydispersity and the widest molecular weight range commercially available. These quality polymer standards are supplied with extensive characterization data utilizing a variety of independent techniques (e.g. light scattering and viscometry) and high performance GPC to verify polydispersity and assign that all important peak molecular weight (Mp).

Our comprehensive range of EasiVial, EasiCal, and traditional calibration kits has been specifically designed to cover all molecular weight ranges for organic and aqueous GPC/SEC applications. We provide you with the widest choice to maximize your specific characterization needs. In addition, we supply other polymers as individual molecular weights, and broad distribution polymers for system validation or broad standard calibration procedures.

Calibration Kits

Agilent offers a wide range of polymer standards kits for conventional GPC/SEC column calibration or for calibrating light scattering and viscometry detectors. The kits are in boxed sets of ten different polymer standards covering a particular molecular weight range, to be used with organic and aqueous, medium polarity, and polar solvents. Every individual polymer has its own Certificate of Analysis of the analytical conditions and values, such as Mp needed for constructing a calibration plot. The polymers are chosen to give equidistant calibration points on a logarithmic MW scale, providing a more uniform calibration curve.

Individual Polymer Molecular Weights

We design our individual standards to have the narrowest molecular weight distribution commercially available. Additionally, they cover the widest molecular weight range, from 162-15 million MW. The current polystyrene nominal molecular weight of 15 million MW has a polydispersity ≤1.10. These standards are generally available in 1, 5 and 10 g quantities, and each comes with its own Certificate of Analysis detailing analysis conditions and relevant data.

EasiVial

• Eliminates tedious weighing procedures to improve calibration accuracy

• Reduces solvent dispensing to limit risks associated with handling solvents

• For conventional and multi detector GPC to maximize applicability

For organic and aqueous GPC/SEC column calibration, this premier product is the quickest and most convenient method to deliver an accurate 12-point column calibration.

The key to achieving baseline separation from polymer mixtures, and therefore eliminating doubt and errors, is in selecting only the narrowest polydispersity polymers. This is where Agilent polymer standards excel and deliver, as shown in the chromatograms.

The EasiVial standards kit is a pre-prepared, time saving product for rapid and reliable GPC column calibration. EasiVial kits contain three vials, each with a mixture of four accurately pre-weighed polymer standards, providing a 12-point GPC calibration in just three injections. The mass of each polymer in the vial is accurately known, so that upon addition of a fixed volume of eluent, the solution is prepared at a precise concentration. EasiVial is ideal for both conventional and multi detector GPC calibration. Simply prepare and manually inject, or transfer to autosampler vials, or place directly into a compatible autosampler.

Every EasiVial kit contains 30 vials (ten of each type) that are color-coded for easy identification and are available in 4 or 2 mL vials making them suitable for most autosamplers. The kits are available for polystyrene (PS), polymethylmethacrylate (PMMA), polyethylene glycol/oxide (PEG/PEO) and polyethylene glycol (PEG). For added value, a Tri-Pack (90 vials) is offered, extending reproducibility.

EasiVial PS-H

Column: | 3 x PLgel 10 μm MIXED-B PL1110-6100 7.5 x 300 mm
Mobile Phase: | THF
Flow Rate: | 1.0 mL/min
Temperature: | 40 °C
Detector: | PL-GPC 220 (RI)

1. 6,035,000

2. 483,000

3. 19,720

4. 1,260

5. 3,053,000

6. 84,900

7. 8,450

8. 580

9. 915,000

10. 60,450

11. 3,370

12. 162

Polystyrene calibration generated with EasiVials

Description Key

PS: Polystyrene |
PM: Polymethylmethacrylate |
PEG/PEO: Polyethylene Glycol/Oxide |
H: High |
M: Medium |
L: Low |

EasiCal

• Easy three-step process with no fuss

• Cost-effective format saves money

• Only two injections for improved productivity

The EasiCal system for organic solvents consists of two different combs, each with ten detachable spatulas, supporting a mixture of five polymer standards. The thin film of polymer (approximately 5 mg) on the tip of the PTFE spatulas rapidly dissolves when immersed in eluent to provide two GPC/SEC calibration solutions. A single pack provides ten spatulas of each type, with MWs selected to provide equidistant calibration points for greater accuracy.

Column calibration for GPC/SEC is as easy as 1, 2, 3...

1. Place one spatula of each type into appropriate volume of solvent.

2. Chromatograph each solution; only two injections required

3. Generate a 10-point calibration

Polystyrene

• Compatible with most organic solvents

• Certificate of Analysis meets international protocols

• Calibration capability for virtually all applications

Polystyrene standards are the first choice for many organic solvents, either for conventional GPC column calibration or for calibrating light scattering and viscosity detectors. Our organic polymers cover a range from 162-15 million MW, with MWs selected to provide equidistant calibration points for greater accuracy. Every kit contains 0.5 g of ten different molecular weight standards.

Description Key

H: High |
M: Medium |
L: Low |

Polystyrene standards

Column: | 2 x OligoPore PL1113-6520 7.5 x 300 mm
Mobile Phase: | THF
Flow Rate: | 1.0 mL/min
Detector: | Agilent PL-GPC 50 (RI)

Polymethylmethacrylate

• Many solvent options increase applicability

• Stringent quality control improves performance

• Proprietary manufacturing methods ensure consistent supply

Polymethylmethacrylate (PMMA) standards are extremely versatile as they can be used for organic GPC with a wide range of medium polarity eluents, such as tetrahydrofuran, toluene, methyl ethyl ketone, and ethyl acetate. They also work well with more polar organic eluents, for example dimethylformamide, dimethylacetamide, and hexafluoroisopropanol. The MWs are selected to provide equidistant calibration points for greater accuracy, covering from 500-1.5 million MW. Every kit contains 0.5 g of ten different molecular weight standards.

Calibration Kits, (All Kits 10 x 0.5 g)

M-L-10 Part No. PL2010-0100 | M-M-10 Part No. PL2020-0101
Constituent Polymer Nominal Mp (g/mol) |
600 | 1,000
840 | 2,200
1,400 | 5,000
2,350 | 11,200
3,900 | 25,500
6,400 | 58,000
10,800 | 130,000
18,000 | 290,000
30,000 | 660,000
50,000 | 1,500,000

Description Key

M: Medium |
L: Low |

Polymethylmethacrylate standards

Column: | 2 x PL HFIPgel PL1114-6900HFIP 7.5 x 300 mm
Mobile Phase: | HFIP + 20 mM NaTFAc
Flow Rate: | 1.0 mL/min
Temperature: | 40 °C
Detector: | Agilent PL-GPC 50 (RI)

1. 790,000

2. 144,000

3. 28,900

4. 5,720

5. 1,020

Polyethylene Glycol/Oxide

• Simple-to-use kit form

• Combines glycols and oxides to extend the MW range and cover more applications

• MWs selected to provide equidistant calibration points for greater accuracy

These hydrophilic polymers are suitable for both aqueous SEC and organic GPC using the majority of polar organic solvents. The oxides are available in high molecular weights, while the glycols cover the lower molecular weight range. The two types are chemically similar so they can be used together across a wider molecular weight range, with aqueous and organic polymers from 106-1 million MW. Every kit contains 0.2 g or 0.5 g of ten different molecular weight standards.

Calibration Kits

PEG-10 (10 x 0.5 g) Part No. PL2070-0100 | PEO-10 (10 x 0.2 g) Part No. PL2080-0101
Constituent Polymer Nominal Mp (g/mol) |
106 | 20,000
194 | 30,000
400 | 50,000
600 | 70,000
1,000 | 100,000
2,000 | 200,000
4,000 | 300,000
7,000 | 400,000
13,000 | 700,000
20,000 | 1,000,000

Polyethylene Glycol/Oxide standards

Column: | PL aquagel-OH MIXED-H 8 μm PL1149-6800 7.5 x 300 mm
Mobile Phase: | Water
Flow Rate: | 1.0 mL/min
Detector: | Agilent PL-GPC 50 (RI)

1. 1,702,000

2. 120,000

3. 12,600

4. 1,470

5. 106

Polysaccharides

• Comprehensive format provides full MW range in one handy kit

• Also available as individual standards

The pullulan polysaccharides kit consists of several simple sugars with relatively narrow polydispersity linear macromolecules of maltotriose units.

Calibration Kits

AC-10 (10 x 0.2 g) Part No. PL2090-0100 |
Constituent Polymer Nominal Mp (g/mol) |
180 |
738 |
5,000 |
10,000 |
20,000 |
50,000 |
100,000 |
200,000 |
400,000 |
700,000 |

Individual Polymer Molecular Weights

Polymer Nominal Mp (g/mol) | Unit | Part No.
1,500 | 0.2 g | PL2091-2000
2,000 | 0.2 g | PL2091-3000
3,000 | 0.2 g | PL2091-4000
5,000 | 0.5 g | PL2090-1000
20,000 | 0.5 g | PL2090-3000
50,000 | 0.5 g | PL2090-4000
100,000 | 0.5 g | PL2090-5000
200,000 | 0.5 g | PL2090-6000
700,000 | 0.5 g | PL2090-8000
1,660,000 | 0.2 g | PL2091-1000

Pullulan polysaccharide standards

Column: | 3 x PL aquagel-OH MIXED-H 8 μm PL1149-6800 7.5 x 300 mm
Mobile Phase: | 0.2 M NaNO₃, 0.01 M NaH₂PO₄, pH 7
Flow Rate: | 1.0 mL/min
Detector: | Agilent PL-GPC 50 (RI)

1. 788,000

2. 212,000

3. 47,300

4. 11,800

5. 667

Polyacrylic Acid

• Compatible with all aqueous columns for wide applicability

• Aqueous polymers 1,000-2 million MW

• Well-characterized Mp values ensure wide utility

Calibration Kits

PAA-10 (10 x 0.2 g) Part No. PL2140-0100 |
Constituent Polymer Nominal Mp (g/mol) |
1,000 |
3,000 |
7,000 |
13,000 |
30,000 |
70,000 |
100,000 |
300,000 |
700,000 |
1,000,000 |

Individual Polymer Molecular Weights

Polymer Nominal Mp (g/mol) | 0.2 g Part No. | 1 g Part No.
1,000 | PL2142-3000 | PL2142-3001
2,000 | PL2142-5000 |
3,000 | PL2142-6000 | PL2142-6001
5,000 | PL2142-7000 | PL2142-7001
7,000 | PL2142-8000 | PL2142-8001
13,000 | PL2143-0000 | PL2143-0101
30,000 | PL2143-2000 | PL2143-2001
50,000 | PL2143-3000 | PL2143-3001
70,000 | PL2143-4000 | PL2143-4001
100,000 | PL2143-5000 | PL2143-5001
130,000 | PL2143-6000 | PL2143-6001
200,000 | PL2143-7000 | PL2143-7001
300,000 | PL2143-8000 | PL2143-8001
500,000 | PL2143-9000 | PL2143-9001
700,000 | PL2144-0000 | PL2144-0101
1,000,000 | PL2144-1000 | PL2144-1001
1,500,000 | PL2144-2000 | PL2144-2001
2,000,000 | PL2144-3000 | PL2144-3001

LC AND LC/MS TROUBLESHOOTING

LC and LC/MS Troubleshooting

HPLC Troubleshooting

Symptom Type | Possible Cause | Solution
Baseline disturbance at void time | Positive/negative – Difference in refractive index of injection solvent | Use mobile phase for sample solvent
Detector leaks | Plugged inlet frit | Replace seals/gaskets
Drifting baseline | Positive direction – Contaminant buildup/elution | Flush column, clean up sample, use pure solvents
| Positive/negative – Difference in refractive index of injection solvent | Use mobile phase for sample solvent
| Negative direction (gradient) – Absorbance of “A” mobile phase solvent | Use non-absorbing or HPLC-grade or better solvent
| Positive direction (gradient) – Absorbance of “B” mobile phase solvent | Use non-absorbing or HPLC-grade or better solvent
| Random – Temperature changes | Insulate column and tubing
| Random – Temperature changes | Thermostat column and tubing
| Wavy or undulating – Temperature changes in room | Monitor room temperature and control
Ghost peaks | Peaks from previous injection | Flush column to remove contaminants
| Contamination | Sample cleanup or pre-fractionation
| Unknown interferences in samples | Sample cleanup or pre-fractionation
| Ion-pair – Upset equilibrium | Prepare sample in actual mobile phase to minimize disturbance
| Peptide mapping – Oxidation of TFA | Prepare fresh daily; use anti-oxidant
| Reversed-phase – Contaminated water | Check suitability of water by running different amount through reversed-phase column and measure peak height with elution; use HPLC grade solvents
| Spikes – Bubbles in solvent | De-gas solvents
High column backpressure | Column blockage, adsorbed sample | Better sample cleanup; use guard column
| Mobile phase viscosity too high | Use lower viscosity solvents or higher temperature
| Particle size too small | Use larger dp packing
| Plugged inlet frit | Replace column
| Plugged inlet frit | Reverse solvent flow
Leaks | Subtle – White powder at fitting/loose fitting | Tighten fittings, cut tubing, or replace ferrules
Leaks, injection valve | Catastrophic – Worn valve rotor | Replace rotor in valve
Leaks, column or other fittings | Catastrophic – Loose fittings | Tighten or replace fittings
Leak, pump | Catastrophic – Pump seal failure | Replace pump seal
Negative peaks | RI detector – solute refractive index less than solvent | No problem; reverse polarity to make positive
| UV detector – solute absorbance less than mobile phase | Use mobile phase with lower UV absorbance; do not recycle solvent too long
Noisy baseline | Random – Contaminant buildup | Flush column; clean up sample; use HPLC-grade solvent
| Continuous – Detector lamp problem | Replace detector lamp
| Occasional – External electrical interference | Use voltage stabilizer for LC system
Peak doubling | Sample volume too large | Reduce the volume e.g. by half and re-inject
| Injection solvent too strong | Use weaker injection solvent or mobile phase
| Blocked frit | Replace and use 0.5 μm porosity in-line filter
| Column void or channeling | Replace column; for some columns, fill in void with packing
| Unswept injector flowpath | Replace injector rotor
| Void at head of column | Replace column, top off column with packing
| Column overloaded with sample | Use higher capacity stationary phase Increase column diameter Decrease sample size
| Single peak – interfering components | Sample cleanup; pre-fractionation
Peak tailing | Beginning of peak doubling | See “peak doubling”
| Unswept dead volumes | Minimize number of connections Ensure injector seal is tight Ensure fittings are properly seated
| Basic compounds – Silanol interactions | Choose endcapped bonded phase Switch to polymeric phase
| Basic substances – Silanol interactions | Use stronger mobile phase or add competing base (e.g. TMA)
| Silica-based – Column degradation | Use specialty column; polymeric column or sterically protected
Peaks are broad | Injection volume too large | Decrease solvent strength of injection solvent to focus solute
| Peak dispersion in injector valve | Introduce air bubble in front/back of sample to decrease dispersion
| Sampling rate of data system too slow | Increase frequency of sampling
| Slow detector time constant | Adjust time constant to match peak width
| Mobile phase viscosity too high | Increase column temperature
| Detector cell volume too large | Use smallest possible cell volume with no heat exchanger in system
| Injector volume too large | Decrease injection volume
| Long retention times | Use gradient elution or stronger mobile phase
Pressure fluctuation | Leaky check valve | Replace check valve
| Pump seal leaks | Replace pump seals
| Buildup of particulates | Filter sample; in-line filter; filter mobile phase
Pressure increasing | Buildup of particulates | Filter sample; in-line filter; filter mobile phase
| Water/organic systems – buffer precipitation | Test buffer-organic mixtures; ensure compatibility
Retention beyond total permeation volume | Size exclusion – Specific interactions | Add mobile phase modifiers or change solvent
Retention times changing | Column temperature varying | Thermostat column; insulate column; ensure lab temperature constant
| Equilibration time insufficient with gradient run or changes in isocratic mobile phase | Make sure at least 10 column volumes pass through column after solvent change or gradient conclusion
| Selective evaporation of mobile phase component | Less vigorous helium sparging; keep solvent reservoirs covered; prepare fresh mobile phase
| Buffer capacity insufficient | Use >20 mM concentration of buffer
| Inconsistent on-line mobile phase mixing | Ensure gradient system delivering constant composition; check vs. manual prep of mobile phase
| Contamination buildup | Occasionally flush column with strong solvent to remove contaminants
| First few injections – Adsorption on active sites | Condition column by initial injection of concentrated sample
Retention times decreasing | Flow rate increasing | Check pump to make sure correct; if not, reset
| Column overloaded with sample | Decrease sample size
| Loss of bonded stationary phase | Keep mobile phase pH between 2 and 8.5
Retention times increasing | Flow rate is slowing | Fix leaks in liquid lines, replace pump seals, check for pump cavitation or air bubbles
| Active sites on silica packing | Use mobile phase modifier
| Loss of bonded stationary phase | Keep mobile phase pH between 2 and 8.5
| Mobile phase composition changing | Make sure mobile phase container is covered
| Active sites on silica packing | Add competing base to mobile phase
| Active sites on silica packing | Use higher coverage packing for stationary phase
Sensitivity problem | Peaks are outside of linear range of detector | Dilute/concentrate to bring into linear region
| First few sample injections – Absorption of sample in loop or column | Condition loop/column with concentrated sample
| Autosampler flow lines blocked | Check flow and make sure there are no blockages
| Injector sample loop underfilled | Make sure that loop is overfilled with sample
| Sample-related losses during preparation | Use internal standard during sample prep; optimize sample prep method
Slow column equilibration times (ion-pairing) | Equilibration time slow for long-chain ion-pairing reagents | Use shorter alkyl chain ion-pair reagent

LC/MS Troubleshooting

Symptom Type | Solution
No peaks | Spray from the nebulizer
| Make sure capillary voltage is set correctly
| Make sure LC/MSD is tuned correctly
| Make sure LC/MSD pressures are within normal ranges
| Check drying gas flow and temperature
| Make sure fragmentor is set correctly
Poor mass accuracy | Recalibrate the mass axis
| Make sure ions used for tuning span mass range of sample ions and show strong stable signals
Low signal | Check the solution chemistry; make sure solvent is appropriate for sample
| Make sure sample is fresh and has been stored correctly
| Make sure LC/MSD is tuned correctly
| Check the nebulizer condition
| Clean the capillary entrance
| Check the capillary for damage and contamination
Unstable signal | Make sure drying gas flow and temperature are correct for the solvent flow
| Make sure solvent is throroughly degassed
| Make sure LC backpressure is steady; this indicates a steady solvent flow
High spectral noise | Use appropriate mass filter values
| Check spray shape; nebulizer may be damaged or set incorrectly
| Make sure drying gas flow and temperature are correct for the solvent flow
| Make sure solvent is throroughly degassed
| Make sure LC backpressure is steady; this indicates a steady solvent flow
| If you are using water as part of the mobile phase, make sure it is deionized (> 18 MΩ cm)
Droplets, not spray, exiting the nebulizer | Make sure nebulizing gas pressure is set high enough for the LC flow
| Check position of needle in nebulizer
| Stop solvent flow and remove nebulizer assembly
| Examine end of nebulizer for damage
No flow | Make sure LC is on and there is sufficient solvent in correct bottle
| Check for LC error messages
| Check for blockages
| Repair or replace any blocked components
| Check for leaks
| Make sure MS stream selector valve is set to LC to MSD
Undesired fragmentation | (APCI vs. Electrospray)
| APCI temperature is too high
| Fragmentor voltage is set too high

BIOPHARMACEUTICAL APPLICATIONS

BioPharmaceutical Applications

NEW!

Protein digest analysis

Column: | ZORBAX 300SB-C18 858750-902 2.1 × 100 mm, 1.8 μm
Mobile Phase: | A: 0.1% TFA in water B: 0.085% TFA in ACN
Flow Rate: | 0.5 mL/min
Pressure: | 640 bar
Gradient: | 2% B 1 min, 2-45% B 8.8 min, 45-95% B 0.2 min, 95% B 2 min, 98-2% B 0.2 min, 2% B 1.8 min
Temperature: | 50 °C
Detector: | Agilent 1290 Infinity LC
Injection: | 5 μL
Sample: | Protein digest
Sample Conc: | 1 mg/mL

Overlaid chromatograms of 30 runs of a protein digest on an Agilent ZORBAX RRHD 300SB-C18 column.

NEW!

Analysis of oxidized insulin chains

Column: | ZORBAX RRHD 300SB-C18 857750-902 2.1 × 50 mm, 1.8 μm
Mobile Phase: | A: 0.1% TFA in water B: 80% ACN + 0.01% TFA in water
Flow Rate: | 1.0 mL/min
Pressure: | 650-700 bar
Gradient: | 33-50% B, 0-4 min; 33% B, 4-5 min
Detector: | UV, 280 nm Agilent 1290 Infinity LC
Sample: | Insulin, oxidized insulin chain A and chain B from bovine pancreas (Sigma Aldrich, St. Louis, MO)
Sample Conc: | 1 mg/mL
Injection: | 2 μL

Insulin and oxidized insulin A and B chains are resolved quickly but insulin and oxidized chain B often co-elute.

NEW!

Fast separation of recombinant human erythropoietin

Column: | ZORBAX RRHD 300SB-C18 857750-902 2.1 × 50 mm, 1.8 μm
Mobile Phase: | A: 0.1% TFA in water B: 0.01% TFA in ACN
Flow Rate: | 1.0 mL/min
Pressure: | 650 bar
Gradient: | 5 to 100% B solvent from 0 to 2.5 min
Detector: | UV, 280 nm Agilent 1290 Infinty LC
Sample: | Recombinant human EPO protein (rEPO)
Sample Conc: | 1.0 mg/mL
Injection: | 3 μL

Heat-treated rEPO protein are well resolved by the Agilent ZORBAX RRHD 300SB-C18 column. The column separated these heat-treated rEPO proteins.

NEW!

Separation optimization for ultra fast analysis of reduced and alkylated monoclonal antibody

Column: | ZORBAX RRHD 300SB-C8 858750-906 2.1 × 100 mm, 1.8 μm
Mobile Phase: | (Various) A: H₂O + 0.1% TFA (v/v) B: n-propanol:ACN:H₂O (80:10:10) + 0.1% TFA (v/v)
Injection: | 1-3 μL
Flow Rate: | 0.5 mL/min
Gradient: | Multi-segmented
| A (optimized for speed): 0 min-20% B, 3 min-35% B, 4 min-40% B, 5 min-40% B, 5.1 min-90% B, 5.5 min-90% B, 6 min-25% B
| B (optimized for resolution): 0 min-25% B, 15 min-32% B, 16 min-32% B, 17 min-90% B, 17.5 min-90% B, 18 min-25% B
Temperature: | 75 °C
Detector: | UV, 225 nm Agilent 1290 Infinity LC

For consecutive chromatographic runs, a 2-minute post run was added to re-equilibrate the column.

Comparison of two optmimized gradients for the ultra fast separation of reduced and alkylated monoclonal antibodies on an Agilent ZORBAX RRHD 300SB-C8 column. The top panel details a rapid separation of the light and heavy chain variants in a shortened run time of less than 4 minutes. The bottom panel displays complete baseline resolution of the two heavy chain variants during a longer runtime using a shallower gradient profile. Both separations were performed at 75 °C and completed with a fast 90% 1-propanol wash step (UV not shown).

NEW!

Column reproducibility – 200 injections of reduced monoclonal antibody using an Agilent ZORBAX RRHD 300SB-C3 column

Column: | Agilent ZORBAX RRHD 300SB-C3 858750-909 2.1 × 100 mm, 1.8 μm
Mobile Phase: | A: 0.1% TFA in water
| B: 80% n-propyl alcohol, 10% ACN, 9.9% water and 0.1% TFA
Flow Rate: | 0.4 mL/min
Gradient: | 0 min-1% B, 2 min-20% B, 5 min-50% B, 7 min-50% B, 8.0 min-90% B, 8.3 min-1% hold for 2 min
Temperature: | 75 °C
Detector: | UV, 280 Agilent 1290 Infinity LC
Sample: | Reduced monoclonal antibody (IgG1) (1.0 mg/mL) - Agilent BL05 IgG1
Injection: | 2 μL

Reduced and alkylated mAb profiling during 200 repeated injections.

[picture] | For a comprehensive listing of chromatograms searchable by compound name, visit our online Chromatogram Library at www.agilent.com/chem/library

NEW!

Gradient optimizations for ultra fast analysis of reduced monoclonal antibody

Column:

Agilent ZORBAX RRHD
300SB-Diphenyl
858750-944
2.1 × 100 mm, 1.8 μm

Mobile Phase: | A: 0.1% TFA in water B: 80% propyl alcohol, 10% ACN, 9.9% water and 0.1% TFA
Flow Rate: | 0.5 mL/min

Gradient:

1st condition:   0 min-1% B,

2 min-20% B,
5 min-70% B

2nd condition:  0 min-1% B,

2 min-20% B,
5 min-50% B

Temperature: | 74 °C
Detector: | UV, 280 nm
Sample: | Reduced monoclonal antibody (IgG1) (1.0 mg/mL) - BioCreative IgG1
Injection: | 2 μL

Comparison of two ultra-fast separations of reduced monoclonal antibodies was achieved on a Agilent ZORBAX RRHD 300SB-Diphenyl under different optimized conditions. The top panel separation delivered narrow peak widths with shorter retention times. The bottom panel separation displays higher resolution between the two heavy chain peaks, but with less efficiency.

[picture] | For a comprehensive listing of chromatograms searchable by compound name, visit our online Chromatogram Library at www.agilent.com/chem/library

NEW!

Ultra high speed and high resolution of intact monoclonal antibodies

Column: | Agilent ZORBAX RRHD 300-Diphenyl 858750-944 2.1 × 100 mm, 1.8 μm

Mobile Phase:

A: 0.1% TFA in water

B: 80% n-propyl alcohol,
10% ACN,
9.9% water and 0.1% TFA

Flow Rate: | 1.0 mL/min
Temperature: | 74 °C
Detector: | UV, 280 nm
Sample: | Monoclonal antibody (IgG1) (1.0 mg/mL) - BioCreative IgG1 and Agilent Standard IgG1
Injection: | 1 μL

Details of intact mAb profiling during 200 repeated injections. Intact mAb separations shown were collected at 1, 50, 150, and 200th run intervals. The bottom panel displays 5 UV blank run trace overlays collected every 20th run during the column evaluation (note: overlay traces are scaled to 2 mAu).

NEW!

Optimizing protein separations with Agilent weak cation-exchange columns

Column: | Agilent Bio WCX, stainless steel 5190-2453 4.6 × 250 mm, 10 μm
Column: | Agilent Bio WCX, stainless steel 5190-2445 4.6 × 250 mm, 5 μm

Mobile Phase:

A: water
B: 1.6 M NaCl
C: 40.0 mM NaH₂PO₄
D: 40.0 mM Na₂HPO₄
By combining predetermined proportions of C and D, 20 mM buffer solutions at the desired pH range were produced (proportions determined using Buffer Advisor software)

Flow Rate: | 1.0 mL/min
Gradient: | 0 to 50% B, 0 to 20 min 50% B, 20 to 25 min 0% B, 25 to 35 min
Temperature: | Ambient
Detector: | UV, 220 nm Agilent 1260 Infinity Bio-inert Quaternary LC
Sample: | Ovalbumin, Ribonuclease A, Cytochrome c, Lysozyme
Sample Conc: | 2 mg/mL (in 20 mM sodium phosphate buffer, pH 6.0)

Effect of pH on retention time of protein standards using a Agilent Bio WCX column.

NEW!

Improved resolution with smaller particle size with Agilent weak cation-exchange columns

Column: | Agilent Bio WCX, stainless steel 5190-2453 4.6 × 250 mm, 10 μm
Column: | Agilent Bio WCX, stainless steel 5190-2445 4.6 × 250 mm, 5 μm

Mobile Phase:

A: water
B: 1.6 M NaCl
C: 40.0 mM NaH₂PO₄
D: 40.0 mM Na₂HPO₄
By combining predetermined proportions of C and D, 20 mM buffer solutions at the desired pH range were produced (proportions determined using Buffer Advisor software)

Gradient: | 0 to 50% B, 0 to 20 min 50% B, 20 to 25 min 0% B, 25 to 35 min
Temperature: | Ambient
Detector: | UV, 220 nm Agilent 1260 Infinity Bio-inert Quaternary LC
Sample: | Ovalbumin, Ribonuclease A, Cytochrome c, Lysozyme
Sample Conc: | 2 mg/mL (in 20 mM sodium phosphate buffer, pH 6.0)

Separation of protein standards at pH 6.5 using an Agilent Bio WCX, NP10 column.

Separation of protein standards at pH 6.5 using an Agilent Bio WCX, NP5 column.

1. Ovalbumin

2. Ribonuclease A

3. Cytochrome c

4. Lysozyme

[picture] | For a comprehensive listing of chromatograms searchable by compound name, visit our online Chromatogram Library at www.agilent.com/chem/library

NEW!

Faster separations using Agilent weak cation-exchange columns

Column: | Agilent Bio WCX, stainless steel 5190-2445 4.6 × 250 mm, 5 μm
Column: | Agilent Bio WCX, stainless steel 5190-2443 4.6 × 50 mm, 3 μm
Column: | Agilent Bio WCX, stainless steel 5190-2441 4.6 × 50 mm, 1.7 μm
Mobile Phase: | A: 20 mM sodium phosphate, pH 6.5 B: A + 1.6 M NaCl
Flow Rate: | 1.0 mL/min
Gradient: | 0 to 50% B
Temperature: | Ambient
Detector: | UV, 220 nm Agilent 1260 Infinity Bio-inert Quaternary LC
Sample: | Ovalbumin, Ribonuclease A, Cytochrome c, Lysozyme
Sample Conc: | 0.5 mg/mL

1. Ovalbumin

2. Ribonuclease A

3. Cytochrome c

4. Lysozyme

Protein separation on Agilent Bio WCX NP5 versus Agilent Bio WCX NP3.

Comparison of Agilent Bio WCX NP3 versus Agilent Bio WCX NP1.7 (flow rate 1.0 mL/min).

Agilent Bio WCX NP1.7 for protein separations under 3 minutes (flow rate 1.7 mL/min).

NEW!

pH gradient elution for improved separation of monoclonal antibody charged variants

Column: | Bio MAb, stainless steel 5190-2405 4.6 × 250 mm, 5 μm

Mobile Phase:

A: water
B: 1.6 M NaCl
C: 100 mM NaH₂PO₄
D: 100 mM Na₂HPO₄
By combining predetermined proportions of C and D, buffer solutions at the desired pH range were produced at the selected buffer strengths.

Flow Rate: | 1.0 mL/min
Gradient: | pH 6.0 to 8.0, 0 to 20 minutes 0 to 800 mM NaCl, 20 to 25 minutes 800 mM NaCl, 25 to 30 minutes
Temperature: | Ambient
Detector: | UV, 220 nm Agilent 1260 Infinity Bio-inert Quaternary LC
Sample: | IgG monoclonal antibody
Sample Conc: | 2 mg/mL (in 20 mM sodium phosphate buffer, pH 6.0)

Chromatograms of IgG monoclonal antibody at different ionic strengths.

[picture] | For a comprehensive listing of chromatograms searchable by compound name, visit our online Chromatogram Library at www.agilent.com/chem/library

NEW!

Separation of recombinant human erythropoietin (rEPO) using Agilent Bio SEC-3

Column: | Bio SEC-3, 100Å 5190-2503 4.6 × 300 mm, 3 μm
Mobile Phase: | 150 mM sodium phosphate buffer, pH 7.0
Flow Rate: | 0.35 mL/min
Detector: | UV, 225 nm Agilent 1260 Infinity Bio-inert Quaternary LC
Sample: | Recombinant human EPO protein (rEPO)
Sample Conc: | 1.0 mg/mL

Consistent ion-exchange MAb separation

Column: | Bio MAb, PEEK 5190-2411 2.1 × 250 mm, 5 μm
Buffer: | A: Sodium phosphate buffer, 20 mM B: Buffer A + 400 mM NaCl
Gradient: | 15-35% Buffer B from 0-30 min
Flow Rate: | 0.65 mL/min
Sample: | CHO-humanized MAb, 1 mg/mL
Injection: | 2.5 μL
Detector: | UV, 220 nm
Temperature: | Ambient

Intact MAb monomer and dimer separation

Column: | Bio SEC-3, 300Å 5190-2511 7.8 × 300 mm, 3 μm
Buffer: | Sodium phosphate buffer, pH 7.0, 150 mM
Gradient: | 0-100% Buffer A from 0-30 min
Flow Rate: | 1.0 mL/min
Sample: | CHO-humanized MAb, 5 mg/mL – intact
Injection: | 5 μL
Detector: | UV, 220 nm
Temperature: | Ambient

Separation of heated, stressed MAb

Column: | Bio SEC-3, 300Å 5190-2511 7.8 × 300 mm, 3 μm
Buffer: | Sodium phosphate buffer, pH 7.0, 150 mM +150 mM sodium sulfate
Gradient: | 0-100% Buffer A from 0-30 min
Flow Rate: | 1.0 mL/min
Sample: | CHO-humanized MAb, 5 mg/mL – stressed at 60 °C
Injection: | 5 μL
Detector: | UV, 220 nm
Temperature: | Ambient

[picture] | For a comprehensive listing of chromatograms searchable by compound name, visit our online Chromatogram Library at www.agilent.com/chem/library

Nucleosides, purines and pyrimidines

Column: | Eclipse Plus Phenyl Hexyl 959993-912 4.6 × 150 mm, 5 μm
Mobile Phase: | 1% MeOH: 99% 20 mm Ammonium Acetate, pH 4.5
Flow Rate: | 1 mL/min
Detector: | UV, 254 nm

1. Cytosine

2. Uracil

3. Cytodine

4. Guanine

5. Uridine

6. Adenine

7. Thymine

8. Guanosine

9. Thymidine

10. Adenosine

Amino acid standard separation on Eclipse Plus C18

Column: | Eclipse Plus C18 959763-902 2.1 × 150 mm, 3.5 μm
Mobile Phase: | A: 10 mM Na₂HPO₄, 10 mM Na₂B₄O₇, 0.5 mM NaN₃, pH 8.2 B: acetonitrile: methanol: water (45:45:10) (v/v/v)
Flow Rate: | 0.42 mL/min
Temperature: | 40 °C
Detector: | UV, 338 nm, then switch to 280 nm at 15.7 min
Sample: | 900 pmol Amino Acids with extended Amino Acids and Internal Standards (500 pmol)
Derivatization: | Automated, online, OPA / FMOC

Gradient |
Time (min) | % B
0 | 2
0.5 | 2
20 | 57
20.1 | 100
23.5 | 100
23.6 | 2
25 | stop

1. ASP

2. GLU

3. ASN

4. SER

5. GLN

6. HIS

7. GLY

8. THR

9. ARG

10. ALA

11. TYR

12. CY2

13. VAL

14. MET

15. NVA

16. TRP

17. PHE

18. ILE

19. LEU

20. LYS

21. HYP

22. SAR

23. PRO

Antibodies: Fast separation of IgM and IgG antibodies

Column: | ZORBAX GF-250 884973-701 4.6 × 250 mm, 4 μm
Mobile Phase: | 200 mM Sodium Phosphate (pH 7), 0.01% Azide
Flow Rate: | 0.94 mL/min
Temperature: | Ambient
Detector: | UV, 230 nm
Sample: | 2.5 μL (1 mg/mL)

1. lgM, MOPC-104E

2. lgG2a, I HOPC-1

3. Buffer Solution

Glycosylated proteins:
Large molecules on Poroshell 300SB-C18 and 300SB-C8

Column A: | Poroshell 300SB-C18 661750-902 1.0 × 75 mm, 5 μm
Column B: | Poroshell 300SB-C8 661750-906 1.0 × 75 mm, 5 μm
Column C: | ZORBAX 300SB-C18 865630-902 1.0 × 50 mm, 3.5 μm
Mobile Phase: | A:       0.1% TFA in H₂O B:       0.07% TFA in ACN
Flow Rate: | A, B:  0.454 mL/min C:       0.071 mL/min

Gradient:

A, B:  0 min 5% B

10 min 100% B

C:       0 min 5% B

50 min 100% B

Temperature: | 70 °C
Detector: | DAD 212 nm, 1.7 μL flow cell, <0.01 min peak width
Sample: | Large glycosylated proteins

Courtesy of:

Novartis AG, Basel.

Dr. Kurt Forrer

Patrik Roethlisberger

[picture] | For a comprehensive listing of chromatograms searchable by compound name, visit our online Chromatogram Library at www.agilent.com/chem/library

HSA tryptic digest on ZORBAX Rapid Resolution HT 1.8 μm

Column A: | ZORBAX SB-C18 883700-922 2.1 × 150 mm, 5 μm
Column B: | ZORBAX SB-C18 822700-902 2.1 × 50 mm, 1.8 μm
Mobile Phase: | A: Water w/0.1% TFA B: ACN w/0.1% TFA
Flow Rate: | A: 0.2 mL/min B: 0.5mL/min
Gradient: | A: 2 to 50% B in 70min B: 2 to 50% B in 30min
Temperature: | 50 °C
Detector: | UV, 214 nm
Sample: | HSA tryptic digest, 8 μL of 15 pmol/μL (120 pmol on column)

Human serum: Low abundance protein isolation and identification from 1-D gel band by LC/MS

Column:

ZORBAX 300SB-C18
Trap: 0.3 × 5 mm, 5 μm, 5065-9913
Analytical: 0.3 × 150 mm,
5 μm, 5064-8263

Mobile Phase: | A: Water + 0.1% Formic acid B: Acetonitrile + 0.1% Formic acid
Flow Rate: | 6 μL/min |

Gradient:

0 min 3% B
5 min 3% B (loading)
50 min 45% B
52 min 80% B
57 min 80% B
60 min 3% B

Sample: | Band from 1-D in gel digest

Sample Preparation of Human Serum:

Major serum proteins removed using Multiple Affinity Removal

Column: 4.6 × 100 mm, P/N 5185-5985

Followed by 1-D gel digest

Base Peak Chromatogram

Proteins Identified

1. α-1-Antichymotrypsin

2. Antithrombin-III Precursor

3. Complement Factor B Precursor

Monoclonal IgG1 chains:
Separation on Poroshell 300SB-C8

Column: | Poroshell 300SB-C8 660750-906 2.1 × 75 mm, 5 μm
Mobile Phase: | A: 90% water: 10% ACN + 3 mL/L of MW 300 PEG B: 10% water: 90% ACN + 3 mL/L of MW 300 PEG
Flow Rate: | 1.0 mL/min

Gradient:

0 min 25% B
10 min 40% B
10.1 min 25% B
12 min 25% B

Temperature: | 70 °C
Sample: | Monoclonal IgG1

Courtesy of:

Novartis AG, Basel.

Dr. Kurt Forrer

Patrik Roethlisberger

Use ZORBAX Extend-C18 for alternate selectivity at high pH

Column: | ZORBAX Extend-C18 773700-902 2.1 × 150 mm, 5 μm
Mobile Phase: | A: 0.1% TFA in Water B: 0.085% TFA in 80% ACN
| A: 20 mM NH₄OH in Water B: 20 mM NH₄OH in 80% ACN
Flow Rate: | 0.25 mL/min
Gradient: | 5-60% B in 20 min
Temperature: | 25 °C
MS Conditions: | Pos. Ion ESI-Vf 70V, Vcap 4.5 kV N₂ – 35 psi, 12 L/min, 300 °C 4 μL (50 ng each peptide)

The Extend column can be used for high pH separations of peptides. At high and low pH, very different selectivity can result. Just by changing pH, a complementary method can be developed and it is possible to determine if all peaks are resolved. The Extend column can be used at high and low pH, so the complementary separation can be investigated with one column. Better MS sensitivity for this sample is also achieved at high pH.

Nucleosides: Separation of deoxy and ribonucleosides

Column: | ZORBAX SB-C3 883975-909 4.6 × 150 mm, 5 μm
Mobile Phase: | 4 mM Ammonium Phosphate (pH 4.0 with Phosphoric Acid)
Flow Rate: | 2.0 mL/min
Temperature: | 35 °C
Detector: | UV, 254 nm
Sample: | 2 μL (1.6 μg each)

1. Cytidine

2. 2’ Deoxycytidine

3. Inosine

4. Guanosine

5. 2’ Deoxyinosine

6. 2’ Deoxyguanosine

7. Adenosine

Nucleotides: Separation of mononucleotides

Column: | ZORBAX SAX 880952-703 4.6 × 250 mm, 5 μm
Mobile Phase: | 0.1 M NH₄H₂PO₄
Flow Rate: | 2.0 mL/min
Temperature: | Ambient
Detector: | UV, 254 nm
Sample: | Orotic Acid, UMP, GMP, XMP

1. Orotic Acid

2. UMP

3. GMP

4. XMP

Separation of basic peptides on Bonus-RP versus traditional Alkyl phase

Column A: | ZORBAX Bonus-RP 883668-901 4.6 × 150 mm, 5 μm
Column B: | Alkyl C8
Mobile Phase: | A: 0.010 M ammonium phosphate, pH 7/0.050 M sodium perchlorate B: 0.010 M ammonium phosphate/0.050 M sodium perchlorate in 50% ACN
Flow Rate: | 1.0 mL/min
Gradient: | 0-100% B in 50 min
Temperature: | 40 °C
Detector: | 215 nm
Sample: | Basic 11-residue peptides with net +1, +2, +3, +4 positive charges at neutral pH

C1: Ac-Gly-Gly-Gly-Leu-Gly-Gly-Ala-Gly-Gly-Leu-Lys-amide

C2: Ac-Lys-Tyr-Gly-Leu-Gly-Gly-Ala-Gly-Gly-Leu-Lys-amide

C3: Ac-Gly-Gly-Ala-Leu-Lys-Ala-Leu-Lys-Gly-Leu-Lys-amide

C4: Ac-Lys-Tyr-Ala-Leu-Lys-Ala-Leu-Lys-Gly-Leu-Lys-amide

Peptides: Effect of TFA concentration

Column: | ZORBAX 300SB-C8 883995-906 4.6 × 150 mm, 5 μm
Mobile Phase: | A: Water and TFA B: ACN and TFA
Flow Rate: | 1.0 mL/min
Gradient: | 0 min 0% B 30 min 30% B
Temperature: | 40 °C
Detector: | UV, 254 nm
Sample: | Peptide Standards S1-S5, decapeptides differing slightly in hydrophobicity, 6 μL

Exploiting chemical stability – TFA concentration

Column: | PLRP-S 100Å PL1512-5500 4.6 × 250 mm, 5 μm
Mobile Phase: | A: TFA (various %) in water B: TFA (various %) in ACN
Gradient: | Linear 12-40% B in 15 min
Flow Rate: | 1.0 mL/min
Detector: | ELS (neb=75 °C, evap=85 °C, gas=1.0 SLM)

1. Angiotensin III

2. Angiotensin II

3. Angiotensin I

Peptides:
Separation of Antiotensins I, II, III with TFA and NH₄OH

Column: | ZORBAX Extend-C18 773700-902 2.1 × 150 mm, 5 μm
Mobile Phase: | A: Acidic Conditions
| A: 0.1% TFA in water B: 0.085% TFA in 80% ACN
| B: Basic Conditions
| A: 10 mM NH₄OH in water B: 10 mM NH₄OH in 80% ACN
Flow Rate: | 0.2 mL/min
Gradient: | 15-50% B in 15 min
Temperature: | 35 °C
MS Conditions: | Pos. Ion ESI - Vf 70V, Vcap 4.5 kV N₂-35 psi, 12 L/min, 325 °C
Sample: | 2.5 μL sample (50 pmol each)

Peptides/proteins:
Equivalent gradient separations

Column: | ZORBAX 300SB-C8 883995-906 4.6 × 150 mm, 5 μm
Column: | ZORBAX 300SB-C8 883750-906 2.1 × 150 mm, 5 μm
Mobile Phase: | A: 95% Water: 5% ACN with 0.1% TFA B: 5% Water: 95% ACN with 0.085% TFA
Flow Rate: | A: Analytical 1 mL/min
| B: Narrow Bore 0.2 mL/min
Gradient: | 10-60% B in 30 min
Temperature: | 35 °C
Detector: | UV, 215 nm
Sample: | 10 μL injection, concentration 2-6 μg

1. Met-enkephalin

2. Leu-enkephalin

3. Angiotensin II

4. Neurotensin

5. RNase

6. Insulin (BOV)

7. Lysozyme

8. Calmodulin

9. Myoglobin

10. Carbonic anhydrase

Peptides/proteins:
Effect of elevated temperature

Column: | ZORBAX 300SB-C3 883995-909 4.6 × 150 mm, 5 μm
Mobile Phase: | A: 5:95 ACN:Water with 0.10% TFA (v/v%) B: 95:5 ACN:Water with 0.085% TFA (v/v%)
Flow Rate: | 1.0 mL/min
Gradient: | 15-53% in 20 min, post time 12 min
Temperature: | Ambient – 60 °C
Detector: | UV, 215 nm
Sample: | Polypeptides

1. Leucine Enkephalin

2. Angiotensin II

3. RNase A

4. Insulin (BOV)

5. Cytochrome c

6. Lysozyme

7. Myoglobin

8. Carbonic anhydrase

Separation of polypeptides in under 1 minute

Column: | Poroshell 300SB-C18 660750-902 2.1 × 75 mm, 5 μm
Mobile Phase: | A: 0.1% TFA, H₂O B: 0.07% TFA, ACN
Flow Rate: | 3 mL/min
Gradient: | 0-100% B in 1.33 min
Temperature: | 70 °C
Detector: | DAD 215/16 nm, ref = 310/10 nm
Sample: | Peptides/proteins, 0.5 μL

Mixer bypassed with P/N G1312-67301; Loop-bypass program

Sample (peptides/proteins)

1. gly-tyr | 0.125 mg/mL
2. Val-tyr-val | 0.5 mg/mL
3. Met-enkephalin | 0.5 mg/mL
4. Leu-enkephalin | 0.5 mg/mL
5. Angiotensin II | 0.5 mg/mL
6. RNase A | 1 mg/mL
7. Cytochrome C | 1 mg/mL
8. Holotransferrin | 1 mg/mL
9. Apomyoglobin | 1 mg/mL

Fast, high-resolution separation of peptides and proteins with Poroshell 300SB-C18

Column: | Poroshell 300SB-C18 660750-902 2.1 × 75 mm, 5 μm
Mobile Phase: | A: 0.1% TFA B: 0.07% TFA in ACN
Flow Rate: | 3.0 mL/min (360 bar pressure)
Gradient: | 5-100% B in 1.0 min
Temperature: | 70 °C
Detector: | UV, 215 nm

Spaces between solutes indicate good peak capacity for rapidly separating complex samples.

1. Angiotensin II

2. Neurotensin

3. RNase

4. Insulin

5. Lysozyme

6. Myoglobin

7. Carbonic Anhydrase

8. Ovalbumin

Peptide RP-HPLC/ESI-MS using NH₄OH mobile phase yields both positive and negative ion spectra

Column: | ZORBAX Extend-C18 773700-902 2.1 × 150 mm, 5 μm
Flow Rate: | 0.25 mL/min
Gradient: | 5-60% B in 20 min
Temperature: | 25 °C
MS Conditions: | Pos. Ion ESI – Vf 70 V, Vcap 4.5 kV, N₂ – 35 psi, 12 L/min, 300 °C TIC 150-1500 m/z
Sample: | 4 μL (50 ng each peptide)

Comparison of Aβ peptide RP-HPLC separations at low and high pH

Column: | ZORBAX Extend-C18 773700-902 2.1 × 150 mm, 5 μm
Mobile Phase: | A: 0.1% TFA in water B: 0.085% TFA in 80% ACN
Flow Rate: | 0.25 mL/min
Gradient: | 29-41% B in 30 min
Temperature: | 80 °C
Detector: | UV, 210 nm
Sample: | 5 μL sample (100 pmol each)
Mobile Phase: | A: 20 mM NH₄OH in water B: 20 mM NH₄OH in 80% ACN
Flow Rate: | 0.25 mL/min
Gradient: | 26-38% B in 30 min
Temperature: | 25 °C
Detector: | UV, 210 nm
Sample: | 5 μL sample (100 pmol each)

Selectivity comparison of TFA and NH₄OH for peptide RP-HPLC\ESI-MS analysis

Column: | ZORBAX Extend-C18 773700-902 2.1 × 150 mm, 5 μm

Mobile Phase:

TFA Conditions:

A: 0.1% TFA in water

B: 0.085% TFA in 80% ACN
NH₄OH Conditions:

A: 20 mM NH₄OH in water

B: 20 mM NH₄OH in 80% ACN

Flow Rate: | 0.25 mL/min
Gradient: | 5-60% B in 20 min
Temperature: | 25 °C
MS Conditions: | Pos. Ion ESI – Vf 70V, Vcap 4.5 kV, N₂ – 35 psi, 12 L/min., 300 °C TIC 150-1500 m/z
Sample: | 4 μL (50 ng each peptide)

Peptide phosphorylation sites LC and LC/MS using Capillary LC columns

Column: | ZORBAX 300SB-C18 5064-8268 0.5 × 150 mm, 3.5 μm
Mobile Phase: | A: Water + 0.1% Formic acid B: Acetonitrile + 0.1% Formic acid
Flow Rate: | 5.5 μL/min
Gradient: | 5-55% B in 50 min, to 85% B from 55-57 min
Detector: | UV, 206 nm

MS Conditions:

LC/MS: Pos. Ion ESI with LC/MSD trap
Vcap: 4000 V
Drying gas flow: 7 L/min
Drying gas temperature: 250 °C
Nebulizer: 15 psi
Capillary Exit Volt: 50 V Max
Accum Time: 300 ms
Total Averages: 3
Isolation Width: 3 m/z
Frag Ampitude: 1.0 V

Sample: | Beta case in digest, 100 nL (4 pmol)

Proteins: Effect of bonded phase, RP

Column A: | ZORBAX 300SB-C8 883995-906 4.6 × 150 mm, 5 μm
Column B: | ZORBAX 300SB-CN 883995-905 4.6 × 150 mm, 5 μm
Mobile Phase: | A: 0.1% TFA in Water, B: 0.1% TFA in 50/50 ACN/Water
Flow Rate: | 1.0 mL/min
Gradient: | 1. 46-96% B in 60 min 23-48% ACN 2. 50-86% B in 60 min 25-43% ACN
Temperature: | 50 °C
Detector: | UV, 210 nm
Sample: | Wheat proteins, including w-gliadins

Proteins: Effect of bonded phase

Column A: | ZORBAX RRHD 300SB-C18 883995-902 4.6 × 150 mm, 5 μm
Column B: | ZORBAX 300SB-C8 883995-906 4.6 × 150 mm, 5 μm
Column C: | ZORBAX 300SB-C3 883995-909 4.6 × 150 mm, 5 μm
Column D: | ZORBAX 300SB-CN 883995-905 4.6 × 150 mm, 5 μm
Mobile Phase: | A: 0.1% TFA in H₂O B: 0.09% TFA in 80% ACN/20% Water
Flow Rate: | 1.0 mL/min
Gradient: | 25-70% B in 40 min
Temperature: | 60 °C
Detector: | UV, 210 nm
Sample: | Polypeptides, 3 μg each

Standard proteins by reversed-phase

Column: | PLRP-S 4000Å PL1512-1803 4.6 × 50 mm, 8 μm
Mobile Phase: | A: 0.1% TFA in 95% water:5% ACN B: 0.1% TFA in 5% water:95% ACN
Gradient: | Linear 18-60% B in 1 min
Flow Rate: | 4.0 mL/min
Detector: | UV, 280 nm

1. Ribonuclease A

2. Cytochrome C

3. Lysozyme

4. BSA

5. Myoglobin

6. Ovalbumin

Standard ion-exchange protein separation

Column: | PL-SAX 1000Å PL1551-1502 4.6 × 50 mm, 5 μm
Mobile Phase: | A: 10 mM Tris HCl pH 8 B: A+0.35 M NaCl pH 8
Gradient: | 0-100% B in 20 min
Flow Rate: | 1.0 mL/min
Detector: | UV, 220 nm

1. Myoglobin

2. Bovine carbonic anhydrase

3. Ovalbumin

4. Soybean trypsin inhibitor

Deoxynucleosides:
Using rapid resolution 3.5 μm columns

Column A: | ZORBAX SB-CN 883975-905 4.6 × 150 mm, 5 μm
Column B: | ZORBAX SB-CN 835975-905 4.6 × 50 mm, 3.5 μm
Mobile Phase: | A: 0.1% TFA B: 90/10 v/v Methanol/Water (0.1% TFA) Isocratic, 97.5% A, 2.5% B
Flow Rate: | 1.0 mL/min
Temperature: | 30 °C
Detector: | UV, 254 nm

1. Cytosine

2. Guanine

3. Thymine

4. Adenine

BSA tryptic digest on RRHT

Column: | ZORBAX SB-C18 820700-902 2.1 × 150 mm, 1.8 μm
Mobile Phase: | A: 0.1% TFA, 5% ACN B: 0.08% TFA, 95% ACN
Flow Rate: | 0.5 mL/min
Gradient: | A: Time 0% B 5 min, Time 30% B 60 min B: Time 0% B 5 min, Time 45% B 60 min C: Time 0% B 5 min, Time 67.5% B 60 min
Temperature: | 80 °C
Detector: | UV, 214 nm
Sample: | BSA tryptic digest

Catecholamines

Column: | PLRP-S 100Å PL1111-3500 4.6 × 150 mm, 5 μm
Mobile Phase: | 95% 25 mM citric acid, 25 mM Na₂HPO₄, 1 mM heptane sulfonic acid:5% ACN, pH 2.85
Flow Rate: | 1.0 mL/min
Detector: | UV, 280 nm

1. Noradrenaline

2. Adrenaline

3. Dopamine

Whey proteins in dairy samples – milk

Column: | PLRP-S 300Å PL1512-3801 4.6 × 150 mm, 8 μm
Mobile Phase: | A: 0.1% TFA in 99% water:1% ACN B: 0.1% TFA in 1% water:99% ACN
Gradient: | 36-48% B, 0-24 min, 48-100% B, 24-30 min 100% B, 30-35 min, 100-36% B, 35-40 min
Flow Rate: | 1.0 mL/min
Injection Volume: | 10 μL
Detector: | UV, 220 nm

1. α-Lactalbumin

2. β-Lactoglobulin (B chain)

3. β-Lactoglobulin (A chain)

Temperature as a tool to enhance mass transfer and improve resolution of oligonucleotides in ion-pair reversed-phase HPLC

Column: | PLRP-S 100Å PL1512-1300 4.6 × 50 mm, 3 μm
Mobile Phase: | A: 100 mM TEAA B: 100 mM TEAA in 25% ACN
Gradient: | 5% change in buffer B over 5 min
Flow Rate: | 1.0 mL/min
Temperature: | 35 °C, 60 °C, or 80 °C
Detector: | UV, 254 nm

1. 29-mer

2. 30-mer

Hydrophilic purine/pyrimidine separation

Column: | ZORBAX SB-Aq 883975-914 4.6 × 150 mm, 5 μm
Mobile Phase: | 50 mM NaOAc, pH 4.6
Flow Rate: | 2.0 mL/min
Temperature: | 35 °C
Detector: | UV, 254 nm

1. Cytosine

2. Fluorocytosine

3. Uracil

4. Guanine

5. Thymine

6. Adenine

[picture] | For a comprehensive listing of chromatograms searchable by compound name, visit our online Chromatogram Library at www.agilent.com/chem/library

CHEMICAL/INDUSTRIAL APPLICATIONS

Chemical/Industrial Applications

Analysis of biocides in hand sanitizer

Column: | ZORBAX RRHD Eclipse Plus C18 959757-902 2.1 × 50 mm, 1.8 μm
Mobile Phase: | A: H₂O (0.5% TFA) B: ACN (0.04% TFA)
Flow Rate: | 1.7 mL/min
Gradient: | Time 0.0    95/5 A/B Time 1.0    55/45 A/B Time 1.1    0/100 A/B
Sample: | 1 μL injection of 50 ppm std.
Temperature: | 30 °C
DAD: | 275 nm (0 min) 225 nm (0.46 min) 255 nm (0.67 min)

Biocides Standards

Hand Sanitizer Sample - Injected Neat

1. Kathon 1A

2. Kathon 1B

3. Carbendazim

4. 1,2-Benzisothiazol-3(2H)-one

5. 2-Phenoxyethanol

6. Benzoic Acid

7. Methyl Paraben

Triton X-114: Decreasing run-time by changing bonded phase

Column A: | ZORBAX SB-C3 883975-909 4.6 × 150 mm, 5 μm
Column B: | ZORBAX SB-C18 883975-902 4.6 × 150 mm, 5 μm
Mobile Phase: | MeOH and H₂O (as indicated)
Flow Rate: | 1.0 mL/min
Temperature: | 50 °C
Detector: | UV, 225 nm
Sample: | Triton X-114

Organic acids separated on ZORBAX SB-Aq

Column: | ZORBAX SB-Aq 883975-914 4.6 × 150 mm, 5 μm
Mobile Phase: | 99% 20 mM NaH₂PO₄, pH 2, 1% ACN
Flow Rate: | 1.0 mL/min
Temperature: | 35 °C
Detector: | UV, 210 nm

1. Lactic Acid

2. Acetic Acid

3. Citric Acid

4. Fumaric Acid

5. Succinic Acid

Brij 35

Column: | PLRP-S 100Å PL1111-3500 4.6 × 150 mm, 5 μm
Mobile Phase: | A: Water B: ACN
Gradient: | 0-100% B in 40 min
Flow Rate: | 0.8 mL/min
Injection Volume: | 10 μL
Sample Conc: | 1 mg/mL
Detector: | ELS (neb=50 °C, evap=70 °C, gas=1.5 SLM)

Alcohols and aliphatic compounds

Column: | Hi-Plex H PL1170-6830 7.7 × 300 mm, 8 μm
Mobile Phase: | Water
Flow Rate: | 0.6 mL/min
Temperature: | 40 °C
Detector: | 356-LC RI

1. Methanol

2. Ethanol

3. Isopropanol

4. Tert-butanol

5. n-Propanol

1. Ethanol

2. Acetone

3. Tert-butanol

[picture] | For a comprehensive listing of chromatograms searchable by compound name, visit our online Chromatogram Library at www.agilent.com/chem/library

ENVIRONMENTAL APPLICATIONS

Environmental Applications

NEW!

Fast LC/MS/MS analysis of group 4 pharmaceuticals from EPA-1694

Column: | ZORBAX RRHD HILIC Plus 959758-901 2.1 × 100 mm, 1.8 μm
Mobile Phase: | A: 10 mM ammonium acetate in water, pH 6.7 B: acetonitrile
Flow Rate: | 1 mL/min
Detector: | Agilent 1290 Infinity LC with an Agilent 6410 Triple Quadrupole Mass Spectrometer

MS Conditions:

TCC: 25 °C
dMRM, ESI positive mode, cycle time 35 ms
Drying Gas: 9 L/min, 300 °C
Nebulizer Pressure: 40 psig
Capillary Voltage: 4000

Sample: | 0.1 μL injection of 0.1 mg/mL each in acetonitrile/water (3:1): cimetidine, albuterol, ranitidine and metformin

1. Cimetidine

2. Albuterol

3. Ranitidine

4. Metformin

NEW!

Separation of azo dye degradation products

Column A: | Poroshell 120 EC-C18 695775-902 2.1 × 100 mm, 2.7 μm
Column B: | Poroshell 120 SB-C18 685775-902 2.1 × 100 mm, 2.7 μm
Column C: | Poroshell 120 Phenyl-Hexyl 695775-912 2.1 × 100 mm, 2.7 μm
Column D: | Poroshell 120 Bonus RP 685775-901 2.1 × 100 mm, 2.7 μm
Flow Rate: | 0.4 mL/min
Gradient: | 15 to 100% MeOH over 10 min
Solvent: | 10 mM Ammonium acetate, pH 4.8

1. Aniline

2. o-Toluidine

3. Methoxyaniline

4. Chloroaniline

5. Benzidine

6. Dimethylbenzidine

7. 3,3’-Dimethoxybenzidine

8. Napthylamine

9. Dichlorobenzidine

Comparison of phenols separation with Poroshell 120

Column: | Poroshell 120 EC-C18 699975-902 4.6 × 50 mm, 2.7 μm
Mobile Phase: | A: Water with 0.1% Formic Acid B: Acetonitrile
Gradient: | Time     %B
| 0.8         5%
| 6.8         60%
| 1200 SL controlled temperature at 25 °C 2 mm flow cell

1. Hydroquinone

2. Resourcinol

3. Catechol

4. Phenol

5. 4-Nitrophenol

6. p-cresol

7. o-cresol

8. 2-Nitrophenol

9. 3,4 di methyl phenol

10. 2,3 di methyl phenol

11. 2,5 di methyl phenol

12. 1-napthol

Column: | Poroshell 120 EC-C18 695975-902 4.6 × 100 mm, 2.7 μm
Mobile Phase: | A: Water with 0.1% Formic Acid B: Acetonitrile
Gradient: | Time     %B
| 2.0         5%
| 17          60%
| 1200 RRLC SL controlled temperature at 25 °C 2 mm flow cell

DNPH: Derivatized Aldehydes obtained from air

Column: | ZORBAX ODS 884950-543 4.6 × 250 mm, 5 μm
Mobile Phase: | A: 100% Water B: 100% ACN
Flow Rate: | 1.0 mL/min
Gradient: | 60-75% B in 30 min; Wash: From 75-100% B in 5 min, after 5 min return to 60% B
Temperature: | 35 °C
Detector: | UV, 230 nm
Sample: | DNPH Derivatized Aldehydes

1. Formaldehyde – DNPH

2. Acetaldehyde – DNPH

3. Acetone – DNPH

4. Acrolein – DNPH

5. Propionaldehyde – DNPH

6. Crotonaldehyde – DNPH

7. 2-Butanone (MEK) – DNPH

8. Methacrolein – DNPH
n-Butyraldehyde – DNPH

9. Benzaldehyde – DNPH

10. Valeraldehyde – DNPH

11. m-Tolualdehyde – DNPH

12. Hexaldehyde – DNPH

Amitrol in water by LC/MS, 0.05 ppb

Column: | ZORBAX SB-C18 863954-302 3.0 × 150 mm, 3.5 μm |
Mobile Phase: | A: 10 mM ammonium acetate B: MeOH |
Flow Rate: | 0.4 mL/min |
Gradient: | 0 min, 65% B; 10 min, 65% B; 15 min, 100% B; 20 min, 65% B |
Temperature: | 30 °C |
MS Conditions: | Ionization Mode: | APCI, positive polarity
| SIM parameters: | Ion: 213 Amitrol
| Ion: | 259 IS
| Fragmentor: | 100 V
| SIM Resolution: | Low
| Vaporizer: | 325 °C
| Drying Gas (N₂): | 5.0 L/min
| Gas Temperature: | 350 °C
| Nebulizer pressure: | 60 psig
| Vcap: | 4000 V
| Corona: | 4.0 uA
Sample: | Amitrol in water, 100 μL |

Anilines, substituted: Rapid separation

Column: | ZORBAX Rx/SB-C8 866953-906 4.6 × 75 mm, 3.5 μm
Mobile Phase: | 20% ACN/80% 25 mM phosphate buffer, pH 2.5
Flow Rate: | 3.0 mL/min
Temperature: | 60 °C
Detector: | UV, 254 nm
Sample: | Anilines

1. p-Anisidine

2. m-Toluidine

3. 3-Amino-benzonitrile

4. p-Chloroaniline

5. m-Chloroaniline

6. o-Chloroaniline

Explosives and related compounds: Qualitative and quantitative analysis

Column A: | ZORBAX SB-C18 883700-922 2.1 × 150 mm, 5 μm
Column B: | ZORBAX SB-CN 883700-905 2.1 × 150 mm, 5 μm
Mobile Phase: | A = ACN + 5% H₂O + 5 mM CF₃COONH₄ B = H₂O + 5% ACN + 5 mM CF₃COONH₄, pH 2.7 (CF₃COOH)
Flow Rate: | 0.23 mL/min

Gradient:

A:
0 min 80% B
2 min 80% B
10 min 70% B
20 min 65% B
25 min 60% B
35 min 30% B
40 min 30% B
42 min 80% B

B:
0 min 80% B
1 min 80% B
15 min 70% B
30 min 20% B
35 min 20% B
37 min 80% B

Temperature: | 18 °C
Detector: | UV, 210, 240, 360 nm, wavelength switching for each compound
Sample: | 10 μL of 19 explosive compounds in ACN/H₂O (20/80)

1. Picric acid

2. 4-Amino-2-nitrotoluene

3. 2-Amino-6-nitrotoluene

4. RDX

5. 2-Amino-4-nitrotoluene

6. HMX

7. 1,3-Dinitrobenzene

8. 1,3,5-Trinitrobenzene

9. 2-Amino-4,6-dinitrotoluene

10. 2,4-Dinitrotoluene

11. 4-Amino-4,6-dinitrotoluene

12. 2-Nitrotoluene

13. 2,6-Dinitrotoluene

14. 4-Nitrotoluene

15. 3-Nitrotoluene

16. 2,4,6-Trinitrotoluene

17. Tetryl

18. Diphenylamine

19. Hexyl

[picture] | For a comprehensive listing of chromatograms searchable by compound name, visit our online Chromatogram Library at www.agilent.com/chem/library

Explosives from soil extract

Column: | ZORBAX SB-C18 880975-302 3.0 × 250 mm, 5 μm
Mobile Phase: | Methanol/Water (50/50) (v/v)
Flow Rate: | 0.3 mL/min
Temperature: | Ambient
Detector: | UV, 230 nm
Sample: | 10 μL explosives mix

1. Octogen (HMX)

2. Hexogen (RDX)

3. 2-Amino-6-nitrotoluene

4. 1,3,5-Trinitrobenzene

5. 2-Amino-4-nitrotoluene

6. 1,3-Dinitrobenzene

7. Tetryl

8. 2,4,6-Trinitrotoluene

9. 4-Amino-2,6-dinitrotoluene

10. 2-Amino-4,6-dinitrotoluene

11. 2,6-Dinitrotoluene

12. 2,4-Dinitrotoluene

13. 2-Nitrotoluene

14. 4-Nitrotoluene

15. 3-Nitrotoluene

Herbicides on different bonded phases

Column A: | ZORBAX SB-CN 883975-905 4.6 × 150 mm, 5 μm
Column B: | ZORBAX SB-Phenyl 883975-912 4.6 × 150 mm, 5 μm
Column C: | ZORBAX SB-C8 883975-906 4.6 × 150 mm, 5 μm
Mobile Phase: | 35% ACN, 65% Water
Flow Rate: | 1.0 mL/min
Temperature: | Ambient
Detector: | UV, 254 nm
Sample: | Herbicides

1. Bentazon

2. Tebuthiuron

3. Simazine

4. Atrazine

5. Prometon

6. Diuron

7. Propazine

8. Propanil

9. Prometryne

10. Metolachlor

Herbicide/pesticide standards: Effect of bonded phase

Column: | Eclipse XDB-C8 993967-906 4.6 × 150 mm, 5 μm
Mobile Phase: | Water/Acetonitrile
Flow Rate: | 1.0 mL/min
Gradient: | 20-60% in 15 min

Temperature:

50 °C
40 °C
30 °C
20 °C

Detector: | DAD 240
Sample: | Herbicide & pesticide standards

Column: | Eclipse XDB-C18 993967-902 4.6 × 150 mm, 5 μm
Mobile Phase: | Water/Acetonitrile
Flow Rate: | 1.0 mL/min
Gradient: | 20-60% in 15 min

Temperature:

50 °C
40 °C
30 °C
20 °C

Detector: | DAD 240
Sample: | Herbicide & pesticide standards

1. Desethyldesisopropylatrazine

2. Desethylatrazine

3. Benzthiazuron

4. Hexazinon

5. Metoxuron

6. Simazine

7. Methabenzthiazuron

8. Simazine

9. Atrazine

10. Isoproturon

11. Diuron

12. Monolonuron

13. Metobromuron

14. Metazachlor

15. Propazine

16. Sebutylazine

17. Terbutylazine

18. Linuron

19. Metolachlor

[picture] | For a comprehensive listing of chromatograms searchable by compound name, visit our online Chromatogram Library at www.agilent.com/chem/library

Separation of EPA 610 PAH Mix

Column: | Eclipse PAH 959990-318 3.0 × 250 mm, 5 μm |
Mobile Phase: | A: Water B: Acetonitrile Initial %B = 40 |
Flow Rate: | 0.85 mL/min |
Gradient: | Time (Min) | %B
| 0.00 | 45
| 17.5 | 100
| 24.0 | 100
| 25.5 | 40
| 27.5 | 40
| Stop Time = 25.0 |
Temperature: | 25 °C |
Detector: | 220, 4 nm No Ref.; Stop time = 26.0 min

1. Toluene

2. Naphthalene

3. Acenaphthylene

4. Acenaphthene

5. Fluorene

6. Phenanthrene

7. Anthracene

8. Fluoranthene

9.Pyrene

10. Benzo(a)anthracene

11. Chrysene

12. Benzo(b)fluoranthene

13. Benzo(k)fluoeanthene

14. Benzo(a)pyrene

15. Dibenzo(a,h)anthracene

16. Benzo(g,h,i)perylene

17. Indeno(1,2,3-c,d)pyrene

Polycyclic aromatic hydrocarbons according to EPA Method 610

Column: | Pursuit PAH A7001100X046 4.6 × 100 mm, 3 μm
Sample: | NIST 16473 Standard
Mobile Phase: | A: ACN:water, 25:75 B: ACN
Flow Rate: | 2.0 mL/min
Detector: | UV, 254 nm

1. Naphthalene

2. Acenaphthylene

3. Aceneaphthene

4. Fluorene

5. Phenanthrene

6. Anthracene

7. Fluoranthene

8. Pyrene

9. Benzo[a]anthracene

10. Chrysene

11. Benzo[b]fluoranthene

12. Benzo[k]fluoranthene

13. Benzo[a]pyrene

14. Dibenzo[a,h]anthracene

15. Benzo[ghi]perylene

16. Indeno[1,2,3-cd]pyrene

NEW!

Rapid method development for 18 PAH compounds with an Agilent RRHD Eclipse PAH column

Column: | ZORBAX RRHD Eclipse PAH 959758-918 2.1 × 100 mm, 1.8 μm
Mobile Phase: | A: Water B: Acetonitrile
Flow Rate: | 0.84 mL/min
Gradient: | 40-100% B, gradient time (tg) varies from 1 to 20 min; isocratic hold at 100% B for 2 min, re-equilibrate column at 40% B for 3 min
Temperature: | 25 °C
Detector: | Agilent 1290 Infinity LC
MS Conditions: | Sig = 220, 4 nm; Ref = Off
Sample: | 0.5 μL injection of diluted Agilent PAH Mixture (P/N 8500-6035) spiked with thiourea as a v0 marker

1. Thiourea (V0 marker)

2. Toluene

3. Naphthalene

4. Acenaphthylene

5. Acenaphthene

6. Fluorene

7. Phenanthrene

8. Anthracene

9. Fluoranthene

10. Pyrene

11. Benzo(a)anthracene

12. Chrysene

13. Benzo(b)fluoranthene

14. Benzo(k)fluoranthene

15. Benzo(a)pyrene

16. Dibenzo(a,h)anthracene

17. Benzo(g,h,i)perylene

18. Indeno(1,2,3-c,d)pyrene

Gradient times are rapidly screened for the separation of 18 compounds.

[picture] | For a comprehensive listing of chromatograms searchable by compound name, visit our online Chromatogram Library at www.agilent.com/chem/library

Separation of 20 PAHs on Eclipse PAH

Column: | Eclipse PAH 959964-918 4.6 × 100 mm, 1.8 μm |
Mobile Phase: | A: Water B: Acetonitrile |
Flow Rate: | 1.8 mL/min |
Gradient: | Time (Min) | % B
| 0 | 40
| 6 | 100
| 9.5 | 100
| 10 | 40
| Stop Time = 12 |
Temperature: | 25 °C |
Detector: | 230, 8 nm No Ref.; Data rate 0.2 s, micro flow cell

1. Toluene

2. Naphthalene

3. Acenaphthylene

4. 1-methyl naphthalene

5. 2-methyl naphthalene

6. Acenaphthene

7. Fluorene

8. Phenanthrene

9. Anthracene

10. Fluoranthene

11. Pyrene

12. Terphenyl-d14

13. Benzo(a)anthracene

14. Chrysene

15. Benz(e)pyrene

16. Benzo(b)fluoranthene

17. Benzo(k)fluoranthene

18. Benzo(a)pyrene

19. Dibenzo(a,h)anthracene

20. Benzo(g,h,i)perylene

21. Indeno(1,2,3-c,d)pyrene

Herbicides: Rapid separation

Column: | Eclipse XDB-C18 933975-902 4.6 × 30 mm, 3.5 μm
Mobile Phase: | MeOH:H₂O (60:40)
Flow Rate: | 2 mL/min
Temperature: | Ambient

1. Tébuthiuron

2. Atrazine

3. Diuron

4. Propazine

5. Propanil

6. Prométryne

Phenoxyacid herbicides

Column: | Pursuit XRs C8 A6010150X046 4.6 × 150 mm, 5 μm
Mobile Phase: | MeCN:water+0.1% HCOOH, 50:50
Flow Rate: | 1.0 mL/min
Temperature: | Ambient
Detector: | UV, 220 nm

1. Phenoxyacetic acid

2. o-Chlorophenoxyacetic acid

3. p-Chlorophenoxyacetic acid

4. 2,3-Dichlorophenoxyacetic acid

5. 2,4-Dichlorophenoxyacetic acid.

6. 2,4,5-Trichlorophenoxyacetic acid

7. 2,4,5-Trichlorophenoxypropionic acid (Silvex)

Triazine pesticides on Bonus-RP and Alkyl C8 phase

Column: | ZORBAX Bonus-RP 883668-901 4.6 × 150 mm, 5 μm
Mobile Phase: | MeOH: 0.1% TFA (70:30)*
Flow Rate: | 1.0 mL/min
Temperature: | Ambient
Detector: | 254 nm

Sample:

Triazine pesticides, 2 μL

1. Prometryne

2. Tebuthiuron

3. Atrazine

4. Propazine

5. Diuron

6. Propanil

7. Dacthal

* For low pH work with Bonus-RP, a TFA mobile phase is often preferred over phosphate, and is compatible with LC/MS.

Phenols, substituted

Column: | ZORBAX SB-C18 883975-902 4.6 × 150 mm, 5 μm
Mobile Phase: | 20% ACN/80% 0.01 M H₃PO₄ to 45% ACN in 7.5 min
Flow Rate: | 1.5 mL/min
Gradient: | 80% ACN in 2.0 min
Temperature: | 35 °C
Detector: | UV, 254 nm
Sample: | Phenols

1. Phenol

2. 4-Nitrophenol

3. m-Cresol

4. o-Cresol

5. 2-Chlorophenol

6. 2,4-Dinitrophenol

7. 2-Nitrophenol

8. 2,4-Dimethylphenol

9. 4-Chloro-3-methylphenol

10. 2,4-Dichlorophenol

11. 2-Methyl-4,6-dinitrophenol

12. 2,4,6-Trichlorophenol

13. 2,3,4,6-Tetrachlorophenol

14. Pentachlorophenol

Plant hormones:
Rapid gradient elution separation

Column: | ZORBAX Rx/SB-C8 866953-906 4.6 × 75 mm, 3.5 μm
Mobile Phase: | A: Water with 0.1% TFA B: Acetonitrile with 0.1% TFA
Flow Rate: | 3.0 mL/min
Temperature: | 60 °C
Detector: | UV, 245 nm
Sample: | Plant hormones

1. Kinetin

2. n-6-Benzyl adenine

3. 3-Indole acetic acid

4. 1-Naphthyl acetamide

5. 3-Indole proprionic acid

6. o-Chlorophenoxy acetic acid

7. p-Chlorophenoxy acetic acid

8. 3-Indole butyric acid

9. 1-Naphthyl acetic acid

10. o-Chlorophenoxy proprionic acid

11. 3,4,5-Trichlorophenoxy acetic acid

12. 3,4,5-Trichlorophenoxy proprionic acid

VX nerve agent metabolites by LC/MS-IS standard (C13 labeled)

Column: | ZORBAX NH2 860700-708 2.1 × 50 mm, 5 μm
Mobile Phase: | 1:1 (20 mM Ammonium Actetate pH 4.5/Acetonitrile)
Flow Rate: | 0.5 mL/min, 1 μL injection (prepared std in ACN)
Temperature: | 35 °C
Detector: | ESI-Negative Ion, Gas Flow 12 L/min, Nebulizer 60 psi
| Sample | MW
| 1. Cyclohexyl methylphosphonic acid | 178
| 2. Pinacolyl methylphosphonic acid | 180
| 3. Isopropyl methylphosphonic acid | 138
| 4. Ethyl methylphosphonic acid | 124

[picture] | For a comprehensive listing of chromatograms searchable by compound name, visit our online Chromatogram Library at www.agilent.com/chem/library

FOOD AND CONSUMER PRODUCT APPLICATIONS

Food and Consumer Product Applications

NEW!

Blueberry anthocyanin analysis

Column A: | Poroshell 120 SB-C18 687975-902 4.6 × 75 mm, 2.7 μm
Column B: | ZORBAX SB-C18 863953-902 4.6 × 150 mm, 3.5 μm
Column C: | ZORBAX SB-C18 880975-902 4.6 × 250 mm, 5 μm
Flow Rate: | 1 mL/min
Detector: | Agilent 1260 Rapid Infinity LC

Blueberry anthocyanin analysis on totally porous and superficially porous StableBond C18 columns. Overlay of anthocyanin method with 250 mm 5 μm, 150 mm 3.5 μm, and 75 mm 2.7 μm at 1 mL/min.

NEW!

Analysis of pesticide residues in green tea

Column: | Poroshell 120 EC-C18 695775-902 2.1 × 100 mm, 2.7 μm
Mobile Phase: | A: 5 mM FA in water B: 5 mM FA in ACN
Flow Rate: | 0.4 mL/min
Gradient: | 5% B in 1 min, 50% B in 3 min, 90% B in 7 min, 90% B in 8 min, 5% B in 8.2 min, 5% B in 9 min
Temperature: | 30 °C

MRM chromatograms of 50 ng/g fortified sample processed by EN method.

1. Acephate

2. Pymetrozine

3. Carbendazim

4. Thiabendazole

5. Imidacloprid

6. Imazalil

7. Propoxur

8. Carbaryl

9. Cyprodinil

10. Ethoprophos

11. Penconazole

12. Kresoxim-methyl

IS TPP

NEW!

An overlay of the original ZORBAX Eclipse Plus 5 μm method and Agilent Poroshell 120 method. All 11 peaks on Poroshell 120 are resolved by the time the first peak elutes on the original 5 μm ZORBAX Eclipse Plus method

Column: | Eclipse Plus C18 959990-902 4.6 x 250 mm, 5 μm
Mobile Phase: | A: 20 mM ammonium acetate, pH 4.80 B: acetonitrile
Flow Rate: | 1.000 mL/min
Gradient: | 14% B at to, ramp to 52% B in 12.0 min
Temperature: | 30 °C

• Analysis reduced from 13.1 to 2.6 min

• Post run reduced from 7 to 1.8 min

• Solvent and mobile phase consumption reduced by more than 80%

• Resolution of critical pair maintained on Poroshell 120

Column: | Poroshell 120 EC-C18 695975-302 3.0 x 100 mm, 2.7 μm
Mobile Phase: | A: 20 mM ammonium acetate, pH 4.80 B: acetonitrile
Flow Rate: | 0.851 mL/min
Gradient: | 14% B at to, ramp to 52% B in 2.1 min
Temperature: | 30 °C

[picture] | For a comprehensive listing of chromatograms searchable by compound name, visit our online Chromatogram Library at www.agilent.com/chem/library

NEW!

Fast analysis of sulfa drugs

Column: | Eclipse Plus C18 959990-902 4.6 x 250 mm, 5 μm
Column: | Poroshell 120 EC-C18 695975-902 4.6 x 100 mm, 2.7 μm
Gradient: | Formic acid/acetonitrile
Detector: | Agilent 1100 Series LC
Sample: | Ten sulfa drugs

A separation of ten sulfa drugs scaled from an Agilent ZORBAX Eclipse Plus C18 column to an Agilent Poroshell 120 EC-C18 column showing analysis time decreased from 30 min to 8 min using a formic acid/acetonitrile gradient.

• Faster analysis (shorter column, higher flow rate)

• More sensitive (smaller particle sharpens peak)

• Better optimized (shorter analysis allows faster tweaking)

• Both run on Agilent 1100 G1315B DAD (under 400 bar)

• No change in sample prep required (2 μm frit used on both columns)

[picture] | For a comprehensive listing of chromatograms searchable by compound name, visit our online Chromatogram Library at www.agilent.com/chem/library

NEW!

Determination of anthocyanins in blueberries

Column: | ZORBAX RRHD Eclipse Plus C18 959758-902 2.1 x 100 mm, 1.8 μm
Column: | ZORBAX RRHD Eclipse Plus Phenyl-Hexyl 959758-912 2.1 x 100 mm, 1.8 μm
Column: | ZORBAX RRHD SB-Aq 858700-914 2.1 x 100 mm, 1.8 μm
Column: | ZORBAX RRHD SB-Phenyl 858700-912 2.1 x 100 mm, 1.8 μm
Mobile Phase: | A: 5% HCOOH in H₂O B: CH₃CN
Flow Rate: | 0.65 mL
Gradient: | 10-50% B in 15 min
Detector: | Agilent 1290 Infinity LC

MS Conditions:

DAD: Sig = 525, 8 nm; Ref = Off
MS2 Scan: ESI + 200-1000
Scan time: 100 ms, 0.2 amu step
Fragmentor: 180 V
Drying gas: 10 L/min, 350 °C
Nebulizer Pressure: 50 psig
Capillary Voltage: 3500

Sample: | 5 μL injection of blueberry extract

1. Cyanidin, m/z 286

2. Peonidin, m/z 300

3. Delphinidin, m/z 302

4. Petunidin, m/z 316

5. Malvidin, m/z 330

Agilent ZORBAX RRHD Eclipse Plus C18

Agilent ZORBAX RRHD Eclipse Plus Phenyl-Hexyl

Agilent ZORBAX RRHD StableBond SB-Aq

Agilent ZORBAX RRHD StableBond SB-Phenyl

Separation of Azo Dyes

Column: | Eclipse Plus Phenyl Hexyl 959996-912 4.6 x 100 mm, 5 μm |
Mobile Phase: | A: 10 mM Ammonium Acetate, pH 4.7 B: MeOH |
Flow Rate: | 1.5 mL/min |
Gradient: | Time (Min): | %B:
| 0 | 25
| 5 | 50
Detector: | UV, 254 nm

1. Aniline

2. o-Toluidine

3. Anisidine

4. Benzidine

5. Chloroaniline

6. o-Tolidine

7. Dimethoxybenzidine

8. Naphthylamine

9. Dichlorobenzidine

Anthocyanins from blueberries: High-efficiency high-speed separation

Column A: | ZORBAX SB-C18 880975-902 4.6 x 250 mm, 5 μm
Column B: | ZORBAX SB-C18 863953-902 4.6 x 150 mm, 3.5 μm
Column C: | ZORBAX SB-C18 866953-902 4.6 x 75 mm, 3.5 μm
Mobile Phase: | A: 3% Phosphoric acid B: 100% MeOH
Flow Rate: | 1.0 mL/min
Gradient: | As shown
Temperature: | 30 °C
Detector: | UV, 525 nm
Sample: | Natural anthocyanins

Time | Percent B
0 min | 23% B
35 min | 26% B
97 min | 60% B

Time | Percent B
0 min | 23% B
21 min | 26% B
58.2 min | 60% B

Time | Percent B
0 min | 23% B
10.5 min | 26% B
29.1 min | 60% B

Aromatics II

Column: | Eclipse XDB-Phenyl 963967-912 4.6 x 150 mm, 3.5 μm
Mobile Phase: | H₂O: MeOH, 40:60
Flow Rate: | 1.0 mL/min
Temperature: | 35 °C
Detector: | UV, 254 nm
Sample: | Aromatic Sample

1. Acetophenone

2. Cinnamaldehyde

3. Eugenol

4. Cinnamaldehyde Impurity

5. Ethyl cinnamate

6. p-Cymene

Aspartame: Metabolites and applications

Column: | ZORBAX SB-C18 866953-902 4.6 x 75 mm, 3.5 μm
Mobile Phase: | 85/15, 0.1% TFA/ACN
Flow Rate: | 1.0 mL/min
Temperature: | 35 °C
Detector: | UV, 210 nm
Sample: | Aspartame

Aspartame and Its Metabolites

1. Phenylalanine

2. 5-benzyl-3,6-dioxo-2-piperazineacetic acid

3. Aspartic acid-phenylalanine dipeptide

4. Aspartame

Diet Coke

1. Caffeine

2. Aspartame

3. Unknown

Equal Sweetener

1. Aspartame

Carbohydrates: Carbohydrate standards

Column: | ZORBAX Carbohydrate Analysis 843300-908 4.6 x 150 mm, 5 μm
Mobile Phase: | 63% CH₃CN/H₂O
Flow Rate: | 0.5 mL/min
Detector: | Agilent RID
Sample: | Carbohydrate standard: A: 25 ng/L, 1 μL injected B: 500 pg/L, 50 μL injected

Carbohydrates: Separation showing high sensitivity

Sensitivity of high injection volume (50 μL)

1. Ribose

2. Rhamnose

3. Xylose

4. Fructose

5. Glucose

6. Sucrose

7. Maltose

8. Lactose

9. Raffinose

Carbohydrates: Effect of mobile phase strength

Column: | ZORBAX NH2 880952-708 4.6 x 250 mm, 5 μm
Mobile Phase: | ACN/Water, as indicated
Flow Rate: | 1.0 mL/min
Temperature: | Ambient
Detector: | RI
Sample: | Mono- and Disaccarides

1. Fructose

2. Glucose

3. Saccharose

4. Palatinose

5. Trehalulose

6. Isomaltose

Carbohydrates in colas

Column: | ZORBAX Carbohydrate Analysis 843300-908 4.6 x 150 mm, 5 μm
Mobile Phase: | 75% ACN:25% H₂O
Flow Rate: | 2.0 mL/min
Temperature: | 30 °C
Detector: | RID

Sample:

No dilution
A: COLA, Fountain
B: COLA, Can, Brand A
C: COLA, Brand B
D: COLA, Brand B, diet

Carbohydrates: Sugar alcohols

Column: | ZORBAX Carbohydrate Analysis 843300-908 4.6 x 150 mm, 5 μm
Mobile Phase: | 75% ACN:25% H₂O
Flow Rate: | 2.0 mL/min
Temperature: | 30 °C
Detector: | RID
Sample: | Chewing gum, sugar-free

Carbohydrates in juices

Column: | ZORBAX Carbohydrate Analysis 843300-908 4.6 x 150 mm, 5 μm
Mobile Phase: | 75% ACN/25% H₂O
Flow Rate: | 2.0 mL/min
Temperature: | 30 °C
Detector: | RID
Sample: | Diluted to 0.1X in 50:50 ACN:H₂O

Apple Drink

36.8% Fructose

24.9% Sucrose

38.3% Glucose

Apple Juice

58.7% Fructose

9.9% Sucrose

33.4% Glucose

Carbohydrates in milk

Column: | ZORBAX Carbohydrate Analysis 843300-908 4.6 x 150 mm, 5 μm
Mobile Phase: | 75% ACN/25% H₂O
Flow Rate: | 2.0 mL/min
Temperature: | 30 °C
Detector: | RID
Sample: | Partitioned between CH₃Cl₂: H₂O

Milk (2%)

100% Lactose-Free Milk

Flavoring agents

Column: | ZORBAX SB-Phenyl 860975-912 2.1 x 50 mm, 5 μm
Mobile Phase: | 0.3% TFA: ACN, 65:35
Flow Rate: | 0.3 mL/min
Temperature: | Ambient
Detector: | UV, 254 nm
Sample: | Cool mint Listerine sample

1. Unknown

2. Benzoic acid

3. Methyl salicylate

4. Carvone

5. Unknown

6. Thymol

7. Anethole

Food colors, FD&C

Column: | ZORBAX Eclipse XDB-C18 935967-902 4.6 x 50 mm, 3.5 μm
Mobile Phase: | A: 0.1% TFA, pH to 4.4 with TEA, B: MeOH
Flow Rate: | 1.0 mL/min
Gradient: | 17 to 100% B/4 min
Temperature: | Ambient
Detector: | UV, 254 nm

1. Yellow #5 | C16H9N4Na3O9S2 | MW=534
2. Red #40 | C18H14N2Na2O8S2 | MW=496
3. Blue #1 | C37H34N2Na2O9S3 | MW=760
4. Propylparaben | C10H12O3 | MW=180
5. Red #3 | C20H414Na2O5 | MW=878

Neutraceuticals: Extract from green tea

Column: | ZORBAX SB-C8 863953-906 4.6 x 150 mm, 3.5 μm
Mobile Phase: | 75% 0.1% Trifluoroacetic acid: 25% Methanol
Injection: | 1 mL/min
Temperature: | 40 °C
Detector: | UV, 280 nm
Sample: | Green tea extract, 5 μL

1. Epagallocatechin

2. Epicatechin

3. Epigallocatechin gallate

4. Catechol

5. Epicatechin gallate

Tocopherols by LC/MS with APPI

Column: | Eclipse XDB-C18 993967-302 3.0 x 150 mm, 5 μm
Mobile Phase: | 97% MeOH: 3% 10 mM CH₃COONH₄
Flow Rate: | 0.5 mL/min
Temperature: | 40 °C

MS Conditions: | MS: | Agilent 1100MSD SL
| Ionization: | APPI (Positive)
| Scan range: | m/z 100-500
| Vcap: | 1500 V
| SIM ion: | base peak
| Drying gas: | 7 L/min at 350 °C
| Nebulizer gas: | 60 psi
| Vaporizer temp: | 350 °C
| Fragmentor: | 140 V
| EM gain: | 4
Sample Volume: | 10 μL |

[picture] | For a comprehensive listing of chromatograms searchable by compound name, visit our online Chromatogram Library at www.agilent.com/chem/library

Sugars in plain and milk chocolate

Column: | Hi-Plex Pb PL1170-6820 7.7 x 300 mm, 8 μm
Mobile Phase: | Water
Flow Rate: | 0.6 mL/min
Temperature: | 80 °C
Detector: | RI

1. Sucrose

2. Lactose

Sugars

Column: | Hi-Plex K PL1170-6860 7.7 x 300 mm, 8 μm
Sample: | Sugars mixture (all 10 mg/mL), 20 μL injection
Mobile Phase: | Water
Flow Rate: | 0.6 mL/min
Temperature: | 85 °C
Detector: | 356-LC RI

1. Stachyose

2. Raffinose

3. Sucrose

4. Mannitol

5. Sorbitol

6. Glucose

7. Fructose

Parabens: High speed separation

Column:

ZORBAX SB-C18 Rapid Resolution
Cartridge
833975-902
4.6 x 30 mm, 3.5 μm

Mobile Phase: | 0.1% H₃PO₄:ACN, (50:50)
Flow Rate: | 2 mL/min
Temperature: | Top: ambient, bottom: 60 °C
Detector: | UV, 254 nm with standard flow cell (13 μL)
Sample: | Parabens, 1 μL

1. Methylparaben

2. Ethylparaben

3. Propylparaben

4. Butylparaben

Separation of vitamin D2/D3

Column: | Eclipse PAH 959941-918 4.6 x 50 mm, 1.8 μm
Mobile Phase: | 92% MeOH, 8% water
Flow Rate: | 2 mL/min
Temperature: | 40 °C
Detector: | 325 nm for VA/280 nm for VD and VE

1. Vitamin A

2. Vitamin D2

3. Vitamin D3

4. Vitamin E (a-VE)

Fat-soluble vitamins on ZORBAX Eclipse XDB-C8

Column: | Eclipse XDB-C8 993967-906 4.6 x 150 mm, 5 μm
Mobile Phase: | 5/95 Water/MeOH
Flow Rate: | 1.0 mL/min
Temperature: | A: Ambient B: 50 °C
Detector: | UV, 280 nm
Sample: | Fat-soluble vitamins

Ambient

50 °C

1. Retinol

2. Retinol acetate

3. Vitamin D3

4. γ-Tocopherol

5. α-Tocopherol

6. Tocopherol acetate

7. Retinol palmitate

Water-soluble vitamins

Column: | ZORBAX SB-C8 883975-906 4.6 x 150 mm, 5 μm
Mobile Phase: | A: 50 mM Sodium Phosphate, pH 2.5/MeOH (90/10) B: 50 mM Sodium Phosphate, pH 2.5/MeOH (10/90)
Flow Rate: | 1.0 mL/min
Gradient: | 0-70% B in 18 min
Temperature: | Ambient
Detector: | UV, 245 nm
Sample: | Water-soluble vitamins

1. B₁-Thiamine

2. Vitamin C

3. B₃-Niacin

4. B₆-Pyridoxine

5. Pantothenic acid

6. Folic acid

7. B₁₂-Cyanocobalamin

8. B₂-Riboflavin

Water-soluble vitamins:
High speed separation using ion-pairing

Column: | ZORBAX Rx/SB-C8 866953-906 4.6 x 75 mm, 3.5 μm
Mobile Phase: | 10 mM Hexane Sulfonate with 0.1% Phosphoric Acid: MeOH (74:26)
Flow Rate: | 1.0 mL/min
Temperature: | Ambient
Detector: | UV, 245 nm
Sample: | Water-soluble vitamins

1. Vitamin C

2. B₃-Niacin

3. B₆-Pyridoxine

4. Folic acid

5. B₂-Riboflavin

6. B₁-Thiamine

Water-soluble vitamins using the USP 23 method

Column: | ZORBAX SB-C18 880975-902 4.6 x 250 mm, 5 μm
Mobile Phase: | 7.2 mM Hexane Sulfonate/MeOH/Acetic Acid (73/27/1) (ratio to 101)
Flow Rate: | 1.0 mL/min
Temperature: | 30 °C
Detector: | UV, 280 nm
Sample: | Water-soluble vitamins

1. B₃-Niacin

2. B₆-Pyridoxine

3. B₂-Riboflavin

4. B₁-Thiamine

E. Excipent

Water-soluble B vitamins separated on ZORBAX SB-Aq

Column: | ZORBAX SB-Aq 883975-914 4.6 x 150 mm, 5 μm
Mobile Phase: | 5% MeOH/95% water (0.1% TFA)
Flow Rate: | 2.0 mL/min
Temperature: | 35 °C
Detector: | UV, 254 nm

1. Thiamine

2. Nicotinic Acid

3. Pyridoxine

4. Niacinamide

Sunscreen ingredients:
Perform conventional, fast and ultra-fast
separations on the same column family

Column A:

Eclipse XDB-C18
993967-902
4.6 x 150 mm, 5 μm
6 μL inj

Column B:

Eclipse XDB-C18
961967-902
4.6 x 100 mm, 3.5 μm
4 μL inj

Column C:

Eclipse XDB-C18
927975-902
4.6 x 50 mm, 1.8 μm
2 μL inj

Mobile Phase: | A: 15% water B: 85% MeOH
Flow Rate: | 1.0 mL/min
Temperature: | Ambient
Detector: | UV, 254 nm
Sample: | Sunscreens

1. 2-hydroxy-4-methoxybenzophenone

2. Padimate O

3. 2-ethylhexyl trans-4-methoxycinnamate

4. 2-ethylhexyl salicylate

Fast vitamin E analysis on Rapid Resolution HT

Column A: | Eclipse XDB-C18 927975-902 4.6 x 50 mm, 1.8 μm
Column B: | Eclipse XDB-C18 993967-902 4.6 x 150 mm, 5 μm
Mobile Phase: | A: 5% water B: 95% MeOH
Flow Rate: | 3 mL/min, 1 mL/min
Temperature: | Ambient

1. α-tocopherol

2. β-tocopherol

3. γ-tocopherol

Theobromine in beverages

Column: | ZORBAX SB-C18 827975-902 4.6 x 50 mm, 1.8 μm
Mobile Phase: | A: 92% 0.1% formic acid B: 8% 0.1% formic acid in ACN
Flow Rate: | 1.5 mL/min
Temperature: | Ambient
Detector: | UV, 254 nm, flow cell 2 μL, 3 mm flow path
Sample: | Theobromine

1. Theobromine

2. Caffeine

Benzoic acid/sorbic acid

Mobile Phase: | 0.1% formic acid in water: 0.1% formic acid in MeCN, 80:20
Flow Rate: | 0.7 mL/min
Detector: | UV, 254 nm

1. p-Aminobenzoic acid

2. Sorbic acid

3. Benzoic acid

Quantification and qualification of vitamin C and citric acid in fresh grapefruit juice

Column: | PLRP-S 100Å PL1512-5500 4.6 x 250 mm, 5 μm
Sample: | Diluted 1:50 in eluent
Mobile Phase: | 0.2M NaH₂PO₄, pH 2.14
Flow Rate: | 0.5 mL/min
Detector: | UV, 220 nm

1. Vitamin C

2. Citric acid

[picture] | For a comprehensive listing of chromatograms searchable by compound name, visit our online Chromatogram Library at www.agilent.com/chem/library

Rose wine

Column: | Hi-Plex H PL1170-6830 7.7 x 300 mm, 8 μm
Mobile Phase: | 0.004M H₂SO₄
Flow Rate: | 0.4 mL/min
Pressure: | 13 bar
Temperature: | 75 °C
Detector: | RI

1. Citric acid

2. Tartaric acid

3. Glucose

4. Malic acid

5. Fructose

6. Succinic acid

7. Lactic acid

8. Glycerol

9. Ethanol

Sports drink

Column: | Hi-Plex Na PL1171-6140 7.7 x 300 mm, 10 μm
Sample: | High energy orange flavor non-carbonated sports drink
Mobile Phase: | Water
Flow Rate: | 0.3 mL/min
Temperature: | 80 °C
Detector: | RI

1. Dp8

2. Dp7

3. Dp6

4. Dp5

5. Dp4

6. Dp3

7. Dp2

8. Dp1 (Glucose)

9. Fructose

Oligosaccharides

Column: | Hi-Plex Ca (Duo) PL1F70-6850 6.5 x 300 mm, 8 μm
Mobile Phase: | DI water
Flow Rate: | 0.5 mL/min
Temperature: | 90 °C
Detector: | RI

1. Higher MW sugars

2. DP5

3. DP4

4. DP3

5. DP2

6. DP1

7. Fructose

PHARMACEUTICAL APPLICATIONS

Pharmaceutical Applications

NEW!

Fast analysis of cefepime and related impurities

Column: | Poroshell 120 EC-C18 697975-902 4.6 x 75 mm, 2.7 μm
Column: | Eclipse Plus C18 959990-902 4.6 x 250 mm, 5 μm
Detector: | Agilent 1200 Infinity Series

NEW!

Naproxen analysis

Column A: | Eclipse Plus C18 959993-902 4.6 x 150 mm, 5 μm
Column B: | Poroshell 120 EC-C18 699975-902 4.6 x 50 mm, 2.7 μm
Mobile Phase: | 50:49:1 MeCN:H₂O:Glacial acetic acid
Flow Rate: | 1.2 mL/min
Injection: | Column A: 20 μL Column B: 6.7 μL
Injection: | Naproxen

4-fold reduction in analysis time for this method when transferring to Poroshell 120.

NEW!

Analysis of water soluble vitamins in multivitamin tablets

Column: | Poroshell 120 EC-C18 697975-902 4.6 x 75 mm, 2.7 μm
Flow Rate: | 1.5 mL/min
Gradient: | 0 min-1% B, 0.5 min-12% B, 0.52 min-30% B, 3.5 min-30% B, 4.5 min-1% B
Injection: | 5 μL

1. Thiamine (B₁)

2. Niacin (B₃₎

3. Ascorbic acid

4. Pyridoxyl phosphate (B₆)

5. Pantothenic acid (B₅)

6. Biotin (B₇₎

7. Folic acid (B₉)

8. Cobalamin (B₁₂)

9. Riboflavin (B₂₎

NEW!

Fast method for ginseng analyses scaled from a traditional method

Column: | Eclipse Plus C18 959993-902 4.6 x 150 mm, 5 μm
Column: | Poroshell 120 EC-C18 695975-302 3.0 x 100 mm, 2.7 μm
Detector: | 1200 Infinity Series
Sample: | Ginsenoside

NEW!

Separation of 8 steroids

Column A: | Poroshell 120 EC-C18 695775-902 2.1 x 100 mm, 2.7 μm
Column B: | Poroshell 120 SB-C18 685775-902 2.1 x 100 mm, 2.7 μm
Column C: | Poroshell 120 Phenyl-Hexyl 695775-912 2.1 x 100 mm, 2.7 μm
Column D: | Poroshell 120 Bonus RP 685775-901 2.1 x 100 mm, 2.7 μm
Mobile Phase: | 0.1% formic acid in both water and MeOH
Flow Rate: | 0.4 mL/min, 25 °C, 2.1 x 100 mm 40 °C
Gradient: | 40-80% MeOH in 14 min

1. Hydrocortisone

2. β-Estradiol

3. Androstadiene 3,17 dione

4. Testosterone

5. Estrone

6. Norethindrone acetate

7. Progesterone

* Naldolol is isobaric and elutes as two peaks.

NEW!

Mixture of beta blockers

Column A: | Poroshell 120 Bonus RP 685775-901 2.1 x 100 mm, 2.7 μm
Column B: | Poroshell 120 Phenyl-Hexyl 695775-912 2.1 x 100 mm, 2.7 μm
Column C: | Poroshell 120 EC-C18 695775-902 2.1 x 100 mm, 2.7 μm
Column D: | Poroshell 120 SB-C18 685775-902 2.1 x 100 mm, 2.7 μm
Mobile Phase: | 10 mM pH 3.8 NH₄HCO₂, methanol
Flow Rate: | 0.35 mL/min
Gradient: | 90% B to 30% B over 12 min

1. Atenolol

2. Pindolol

3. Naldolol

4. Metoprolol

5. Acebutolol

6. Propranolol

7. Alprenolol

NEW!

Several ZORBAX RRHD 1.8 μm selectivities facilitate method development

Column: | ZORBAX RRHD Eclipse Plus C18 959758-902 2.1 x 100 mm, 1.8 μm
Column: | ZORBAX RRHD Eclipse XDB-C18 981758-902 2.1 x 100 mm, 1.8 μm
Column: | ZORBAX RRHD SB-C18 858700-902 2.1 x 100 mm, 1.8 μm
Column: | ZORBAX RRHD Extend-C18 758700-902 2.1 x 100 mm, 1.8 μm
Mobile Phase: | A: H₂O B: CH₃CN, each with 0.1% HCOOH
Detector: | Agilent 1290 Infinity LC with an Agilent 6410 Triple Quadrupole Mass Spectrometer

MS Conditions:

TCC: 30 °C
MS Source: Electrospray AP-ESI
Drying-gas temperature and flow: 325 °C, 12 L/min
Nebulizer gas pressure: 35 psi
Capillary voltage: 3000 V

Sample:

Four endocannabinoid fatty amides:
Arachidonoylglycerol (AEA)
2-Arachidonoylglycerol (2-AG)
Palmitoylethanolamide (PEA)
Oleoylethanolamide (OEA)

1. AEA

2. PEA

3. 2-AG

4. *

5. OEA

* The second black peak is an impurity, believed to be 1,3-arachidonolyglycerol, a rearrangement of 2-AG

The selectivity of four Agilent ZORBAX RRHD C18 columns is compared using a method for endocannabinoids.

[picture] | For a comprehensive listing of chromatograms searchable by compound name, visit our online Chromatogram Library at www.agilent.com/chem/library

Fast analysis 11 common compounds found in analgesics

Column: | Poroshell 120 EC-C18 695975-902 4.6 x 100 mm, 2.7 μm
Mobile Phase: | A : Water + 0.1% formic acid B: ACN
Flow Rate: | 3.5 mL/min
Temperature: | 40 °C
Detector: | DAD 254 nm

1. Acetaminophen

2. Caffeine

3. 2-Acetamidophenol

4. Acetamide

5. Phenacetin

6. Sulindac

7. Piroxicam

8. Tolmetin

9. Ketoprofen

10. Diflusinal

11. Diclofenac

Faster analysis of USP Method for simvastatin tablet

Column A: | Eclipse Plus C18 959990-902 4.6 x 250 mm, 5 μm
Column B: | Poroshell 120 EC-C18 697975-902 4.6 x 75 mm, 2.7 μm
Mobile Phase: | 65% CH₃CN, 35% 3.9 g/L NaH₂PO₄ (pH 4.5)
Flow Rate: | 1.5 mL/min for 5 μm column 2.8 mL/min for 2.7 μm Poroshell 120 column
Temperature: | 45 °C
Detector: | DAD Sig = 238, 8 Ref = 360, 100 nm

    USP Requirement | 5 μm (1.5 mL/min) | 2.7 μm (2.8 mL/min)
TR   N/A | 9.907 | 1.457
k’   > 3.0 | 5.962 | 5.122
N   > 4500 | 16939 | 14439
Tf   < 2.0 | 1.09 | 1.10

Faster separation of sulfa drugs

Column A: | Eclipse Plus C18 959990-902 4.6 x 250 mm, 5 μm
Column B: | Poroshell 120 EC-C18 695975-902 4.6 x 100 mm, 2.7 μm
Mobile Phase: | A: 0.1% formic acid in Water B: 0.1% formic acid in ACN
Flow Rate: | 1 mL/min

Time | %B
0 | 8
33 | 33
35 | 33

Time | %B
0 | 8
12 | 33
13.2 | 33

Separation of pharmaceutical cardiac drugs

Column: | Eclipse Plus C18 959996-902 4.6 x 100 mm, 5 μm
Mobile Phase: | A: 20 mM Ammonium Acetate, pH 4.8 B: ACN
Flow Rate: | 1 mL/min
Gradient: | 10-90% in 10 min
Detector: | UV, 254 nm

1. Procainamide

2. Furosemide

3. Eletriptan

4. Buspirone

5. Diltiazen

6. Dipyridamole

7. Pioglitazone

Fast and ultra-fast analysis of basic compounds

Column: | Eclipse Plus C18 959941-902 4.6 x 50 mm, 1.8 μm
Mobile Phase: | A: 50% 8 mM K₂HPO₄, pH 7 B: 50% ACN
Flow Rate: | 1.0 mL/min
Temperature: | Ambient
Detector: | UV, 254 nm
Sample: | Berberine, 0.4 mg/mL, 2 μL

1. Berberine

Xanthines: Higher resolution, same selectivity with RRHT

Column A: | ZORBAX SB-C18 846975-902 4.6 x 50 mm, 5 μm
Column B: | ZORBAX SB-C18 827975-902 4.6 x 50 mm, 1.8 μm
Mobile Phase: | A: 92% 0.1% formic acid B: 8% 0.1% formic acid in ACN
Flow Rate: | 1.5 mL/min
Temperature: | Ambient
Detector: | UV, 254 nm
Sample: | Xanthines

1. 1-methylxanthine

2. 1,3-dimethyluric acid

3. 3,7-dimethylxanthine (theobromine)

4. 1,7-dimethylxanthine

Antihistamines:
Fast separations on RRHT Extend-C18

Column A: | ZORBAX Extend-C18 773450-902 4.6 x 150 mm, 5 μm
Column B: | ZORBAX Extend-C18 727975-902 4.6 x 50 mm, 1.8 μm
Mobile Phase: | A: 30% 50 mM pyrrolidine buffer B: 70% MeOH
Flow Rate: | 1.0 mL/min
Temperature: | Ambient
Detector: | UV, 220 nm
Sample: | Antihistamines

1. Scopolamine

2. Pseudoephedrine

3. Doxylamine

4. Chlorpheniramine

5. Diphenhydramine

6. Triprolidine

Ibuprofen:
Optimizing selectivity with RRHT Columns

Column A: | SB-C8 827975-906 4.6 x 50 mm, 1.8 μm
Column B: | Eclipse XDB-C8 927975-906 4.6 x 50 mm, 1.8 μm
Mobile Phase: | A: 63% water B: 37% acetonitrile + 1.8 mL H₃PO₄
Flow Rate: | 2.0 mL/min
Temperature: | Ambient
Detector: | UV, 254 nm
Sample: | Ibuprofen oral suspension

1. Benzophenone

2. Ibuprofen

Analgesics

Column: | Pursuit XRs Diphenyl A6020150X046 4.6 x 150 mm, 5 μm
Mobile Phase: | A: Water+0.1% HCOOH B: MeCN+0.1% HCCOH
Gradient: | 25-80% B in 20 min
Flow Rate: | 1.0 mL/min
Temperature: | Ambient
Detector: | UV, 254 nm

1. Acetaminophen

2. Acetanilide

3. Salicylic acid

4. Acetylsalicylic acid

5. Phenacetin

6. Carbamazepine

7. Tolmetin

8. Naproxen

9. Ibuprofen

10. Diclofenac

[picture] | For a comprehensive listing of chromatograms searchable by compound name, visit our online Chromatogram Library at www.agilent.com/chem/library

Anesthetics, local: Bonded phase selectivity

Column A: | ZORBAX SB-C18 883975-902 4.6 x 150 mm, 5 μm
Column B: | ZORBAX SB-C8 883975-906 4.6 x 150 mm, 5 μm
Column C: | ZORBAX SB-C3 883975-909 4.6 x 150 mm, 5 μm
Column D: | ZORBAX SB-Phenyl 883975-912 4.6 x 150 mm, 5 μm
Column E: | ZORBAX SB-CN 883975-905 4.6 x 150 mm, 5 μm
Mobile Phase: | A: 50 mM NaH₂PO₄ pH 2.5 in 95% H₂O/5% ACN B: 50 mM NaH₂PO₄ pH 2.5 in 47% H₂O/53% ACN
Flow Rate: | 1.5 mL/min
Gradient: | 0-100% B in 18.8 min
Temperature: | 26 °C
Detector: | UV, 254 nm
Sample: | 10 μL, 10 ug/mL

1. Procaine

2. Lidocaine

3. d-Cinchonine

4. Butacaine

5. Tetracaine

Local anesthetics

Column: | Pursuit XRs C8 A6010150X046 4.6 x 150 mm, 5 μm
Mobile Phase: | 65:35 MeOH:5 mM NH₄CO₃, pH 10
Flow Rate: | 1.0 mL/min
Temperature: | Ambient
Detector: | UV, 210 nm

1. Benzocaine

2. Procaine

3. Chlorocaine

4. Mepivacaine

5. 4-Hydroxyropivacaine

6. Cocaine

7. Lidocaine

8. Ropivacaine

Antibiotics: High speed separation

Column: | ZORBAX Rx/SB-C8 866953-906 4.6 x 75 mm, 3.5 μm
Mobile Phase: | 8.0% acetonitrile/92% 0.1% aqueous TFA
Flow Rate: | 3.0 mL/min
Gradient: | 45-70% B in 35 min
Temperature: | 60 °C
Detector: | UV, 260 nm
Sample: | 1 μL containing 0.40, 0.36, 0.10 and 0.37 μg each of 1-4 resp.

1. Ceftazidime

2. Cefotaxime

3. Ciprofloxacin

4. Cefazolin

Antibiotics: Lincomycin and Clindamycin
by LC-APCI-MS LC-TIC

Column: | ZORBAX SB-C18 cartridge 823700-902 2.1 x 30 mm, 1.8 μm
Mobile Phase: | Gradient: 15-50% B in 1 min, hold for 1.5 min, A: 0.2% formic acid pH 2.8 B: ACN + 0.2% formic acid
Flow Rate: | 0.5 mL/min
Gradient: | Post time: 1.5 min
Temperature: | Ambient
Detector: | APCI, Positive ion

MS Conditions: | Peak width: | 0.10 min
| Scan: | 150-600 Da, step 0.1
| Fragmentor: | 70
| Gas Temp: | 350 °C
| Vaporizer: | 350 °C
| Drying gas: | 12 L/min
| Nebulizer pres: | 50 psi
| Vcap: | +3000V
| Corona: | 4.0 μA
Sample: | Antibiotics, 1 μL |

1. Lincomycin

2. Clindamycin

Antifungal medications

Column: | ZORBAX Bonus-RP 883668-901 4.6 x 150 mm, 5 μm
Mobile Phase: | 35% 25 mM NaH₂PO₄, Dibasic (pH 6.5 with H₃PO₄): 65% ACN
Flow Rate: | 1 mL/min
Temperature: | Ambient
Detector: | UV, 220 nm
Sample: | Antifungals, 2 μL

1. Clotrimazole

2. Econazole

3. Miconazole

Antifungals

Column: | Pursuit XRs Diphenyl A6020150X046 4.6 x 150 mm, 5 μm
Mobile Phase: | Water+0.1% HCOOH: MeCN+0.1% HCOOH, 80:20
Flow Rate: | 1.0 mL/min
Temperature: | Ambient
Detector: | UV, 254 nm

1. 4-Aminobenzoic acid

2. Sorbic acid

3. Benzoic acid

4. Salicylic acid

[picture] | For a comprehensive listing of chromatograms searchable by compound name, visit our online Chromatogram Library at www.agilent.com/chem/library

Analgesics: Non-steroidal anti-inflammatory drugs: Narrow bore separation

Column: | Eclipse XDB-C8 993700-906 2.1 x 150 mm, 5 μm
Mobile Phase: | 50/50, 25 mM Sodium Phosphate (pH 7.0 with Phosphoric Acid), MeOH
Flow Rate: | 0.2 mL/min
Temperature: | 35 °C
Detector: | UV, 254 nm
Sample: | 2 μL, 10 ug/mL

NSAID | pKa
1. Phenacetin | 2.2
2. Tolmetin | 3.5
3. Phenylbutazone | 4.4
4. Fenoprofen | 4.5

Separation of small molecule anorectics

Column A: | ZORBAX Bonus-RP 883668-901 4.6 x 150 mm, 5 μm
Column B: | Traditional Alkyl C8 Phase
Mobile Phase: | 25 mM K₂HPO₄, pH 7.2/MeOH: ACN (50:50), 45/55
Flow Rate: | 1 mL/min
Temperature: | Ambient
Detector: | UV, 254 nm
Sample: | Anorectics “Fen-phen”, 5 μL

1. Phentermine

2. Fenfluramine

[picture] | For a comprehensive listing of chromatograms searchable by compound name, visit our online Chromatogram Library at www.agilent.com/chem/library

Aromatic acids/benzoic acids: Selectivity differences

Column A: | ZORBAX SB-C8 880975-906 4.6 x 250 mm, 5 μm
Column B: | ZORBAX SB-Phenyl 880975-912 4.6 x 250 mm, 5 μm
Column C: | ZORBAX SB-CN 880975-905 4.6 x 250 mm, 5 μm

Mobile Phase:

30-45% methanol in 25 mM Na Phosphate, pH 2.5
A: 45% Methanol
B: 40% Methanol
C: 30% Methanol

Flow Rate: | 1.0 mL/min
Temperature: | 35 °C
Detector: | UV, 254 nm
Sample: | Benzoic acids

1. Phthalic

2. 2-Nitrobenzoic

3. 2-Fluorobenzoic

4. 2-Chlorobenzoic

5. 3-Nitrobenzoic

6. 3-Fluorobenzoic

7. m-Toluic

Catecholamines/biogenic amines: Rapid separation using ion-pair reagents

Column: | ZORBAX Rx/SB-C8 866953-906 4.6 x 75 mm, 3.5 μm

Mobile Phase:

0.14 M sodium phosphate,
20 mM EDTA,
0.75 mM octyl sulfonate,
9% methanol pH 3.5

Flow Rate: | 1.5 mL/min
Temperature: | 26 °C
Detector: | 0.75 V vs Ag/AgCI with electro-chemical detection

Sample:

10 μg/mL each standard; volume
20 μL (2 g tissue sample)
A: Standards (2pmol; DHBA 5pmol)
B: Mouse Sriatum
C: Mouse Neocortex

1. DOPA-Dihydroxyphenylalanine

2. DHBA-Dihydroxybenzyl amine

3. DOPAC-Dihydroxyphenyl acetic acid

4. NE-Norepinephrine

5. DA-Dopamine

6. HIAA-Hydroxyindoleaacetic acid

7. EP-Epinephrine

8. HVA-Homovanillic acid

9. 5-HT-Hydroxytryptamine

10. 3-MT-Methaxytyrosine

Chiral ethiazide (diuretic drug) separation

Column: | Ultron ES-OVM Chiral 702111651 4.6 x 150 mm, 5 μm
Mobile Phase: | 20 mM KH₂PO₄(pH 4.6)
Flow Rate: | 1.0 mL/min
Temperature: | 25 °C
Detector: | UV, 220 nm
Sample: | 20 μL containing 0.35 μg Ethiazide

Chiral separation of fluoxetine enantioners (Prozac)

Column: | Ultron ES-OVM Chiral 702111651 4.6 x 150 mm, 5 μm
Mobile Phase: | 25/75 (v/v) EtOH / 20 mM KH₂ PO₄, pH 5.5 (adjusted with NaOH)
Flow Rate: | 0.8 mL/min
Temperature: | Ambient
Detector: | UV, 225 nm
Sample: | Mixture fluoxetine (Prozac) enantiomers

Courtesy of D.S. Ristry and V.S. Sharp, Eli Lilly and Co.

[picture] | For a comprehensive listing of chromatograms searchable by compound name, visit our online Chromatogram Library at www.agilent.com/chem/library

Goldenseal and related alkaloids on Rapid Resolution Eclipse XDB-C18

Column: | Eclipse XDB-C18 963967-902 4.6 x 150 mm, 3.5 μm
Mobile Phase: | 68% 30 mM ammonium acetate, 14 mM TEA, pH ~4.85 32% Acetonitrile
Flow Rate: | 1.0 mL/min
Temperature: | 30 °C
Detector: | 230 nm
Sample: | Goldenseal and related alkaloids

1. Palmatine

2. Barberine

3. Hydrastine

4. Canadine

Alkaloids, such as the active components in Goldenseal and other related plants, are quickly and accurately separated using isocratic conditions on an Eclipse XDB-C18 Rapid Resolution column.

Components of green tea separated on Rapid Resolution StableBond SB-C8

Column: | ZORBAX SB-C8 863953-906 4.6 x 150 mm, 3.5 μm
Mobile Phase: | 75% 0.1% TFA : 25% MeOH
Flow Rate: | 1.0 mL/min
Temperature: | 40 °C
Detector: | 280 nm
Sample: | Green tea

1. Epigallocatechin

2. Epicatechin

3. Epigallocatechin gallate

4. Catechol

5. Epicatechin gallate

Nutraceuticals, such as the components of green tea, are quickly separated on a StableBond SB-C8 Rapid Resolution column.

Chiral separation of hexobarbitol

Column: | Chiradex 79925CB-584 4.0 x 250 mm, 5 μm
Mobile Phase: | Methanol/water, 20:80
Flow Rate: | 1.0 mL/min
Detector: | UV, 220 nm
Sample: | Hexobarbitol

Chiral separation of S- and R-Norfluoxetine

Column: | Ultron ES-OVM Chiral 724111653 4.6 x 250 mm, 10 μm
Mobile Phase: | 6/94 (v/v) MeOH / 20 mM KH₂PO₄
Flow Rate: | 1.0 mL/min
Temperature: | Ambient
Detector: | UV, 225 nm
Sample: | 50 μg/mL of 2:3 mixture R- : S-Norfluoxetine

Courtesy of D.S. Ristry and V.S. Sharp, Eli Lilly and Co.

Chiral separation of salbutamol

Column: | Ultron ES-Pepsin 822111631A 4.6 x 150 mm, 5 μm
Mobile Phase: | 20 mM phosphate buffer, pH 6.0
Flow Rate: | 1.0 mL/min
Temperature: | 25 °C
Detector: | UV, 220 nm
Sample: | 20 μL containing 0.35 μg salbutamol mixture

[picture] | For a comprehensive listing of chromatograms searchable by compound name, visit our online Chromatogram Library at www.agilent.com/chem/library

Chiral separation of tolperison enantiomers

Column: | Ultron ES-OVM Chiral 702111651 4.6 x 150 mm, 5 μm
Mobile Phase: | 20 mM KH₂PO₄ (pH 5.5), C₂H₅OH (100/4 v/v)
Flow Rate: | 1.0 mL/min
Temperature: | Ambient
Detector: | UV, 220 nm, 0.04 AUFS
Sample: | Tolperison, 5 μL

Chiral separation of atenolol

Column: | Ultron ES-Pepsin 822111631A 4.6 x 150 mm, 5 μm
Mobile Phase: | 20 mM phosphate buffer, pH 6.0/Ethanol (99/1)
Flow Rate: | 1.0 mL/min
Temperature: | 25 °C
Detector: | UV, 220 nm, 0.04 AUFS
Sample: | 1.5 μL, 0.25 mg/mL, atenolol racemic mixture

Cocaine and metabolites

Column: | ZORBAX Rx-SIL 883975-901 4.6 x 150 mm, 5 μm
Mobile Phase: | MeOH: NH₄ Acetate, 25 mM, pH 6 (70:30)
Flow Rate: | 1.0 mL/min
Temperature: | 40 and 80 °C
Detector: | UV, 210 nm

1. Benzoylecgonine

2. Cocaine

3. Ecgonine methylester

Aspirin and cough remedy

Column: | Eclipse XDB-C8 993967-906 4.6 x 150 mm, 5 μm
Mobile Phase: | (75:25) 25 mM Na₂HPO₄ (pH 3.0): ACN
Flow Rate: | 1.0 mL/min
Temperature: | 40 °C
Detector: | UV, 254 nm
Sample: | 5 μL, 10 μg/mL

1. Acetylsalicylic acid

2. Dextromethorphan

Cough formula mixture: Fast and efficient separation

Column A: | ZORBAX SB-CN 866953-905 4.6 x 75 mm, 3.5 μm
Column B: | ZORBAX SB-CN 883975-905 4.6 x 150 mm, 5 μm
Mobile Phase: | 20/80, Acetonitrile/150 mM Na Citrate, pH 2.6
Flow Rate: | 1.5 mL/min, 1.0 mL/min
Temperature: | 35 °C
Detector: | UV, 270 nm
Sample: | 2 μL, cough formula

1. Maleic acid

2. Pseudo-ephedrine

3. Acetaminophen

4. Chlorpheniramine

5. Dextromethorphan

Guaifenesin: USP analysis of guaifenesin

Mobile Phase: | 40% Methanol:60% Water:1.5% Glacial Acetic Acid
Flow Rate: | 1.0 mL/min
Temperature: | 25 °C
Sample: | Guaifenesin A: 8 μL B: 2 mL

1. Guaifenesin: 0.04 mg/mL

2. Benzoic Acid: 0.10 mg/mL

Minimum Resolution Required = 3.0

Metronidazole: Updating USP methods

Column A: | ZORBAX C8 883952-706 4.6 x 150 mm, 5 μm
Column B: | Eclipse XDB-C8 993967-906 4.6 x 150 mm, 5 μm
Column C: | Eclipse XDB-C8 963967-906 4.6 x 150 mm, 3.5 μm
Mobile Phase: | 80/20, Water/Methanol
Flow Rate: | 1.0 mL/min
Temperature: | Ambient
Detector: | UV, 254 nm
Sample: | Metronidazole

ZORBAX C8
Plates | USP TF | Particle Size
9772 | 1.30 | 5 μm

ZORBAX Eclipse XDB-C8
Plates | USP TF | Particle Size
9850 | 0.98 | 5 μm

ZORBAX Rapid Resolution Eclipse XDB-C8
Plates | USP TF | Particle Size
21554 | 1.13 | 3.5 μm

Morphine and metabolites:
Extracted blood plasma sample separation

Column: | ZORBAX SB-C18 863953-902 4.6 x 150 mm, 3.5 μm
Mobile Phase: | 97/3 70 mM KH₂PO₄ + 1 mM EDTA/ACN, pH 4.5
Flow Rate: | 1.5 mL/min
Temperature: | Ambient
Detector: | A: Electrochemical, 720 mV B: Fluorescence, Ex = 285 nm, Em = 352 nm

Sample:

50 μL
Morphine-3-glucuronide 125 ng
Morphine-6-glucuronide 62.5 ng
Morphine 12.5 ng in Water

1. Morphine-3-glucuronide 125 ng

2. Morphine-6-glucuronide 62.5 ng

3. Morphine 12.5 ng in Water

Courtesy of J. Visser, Center for Pharmacy, Univ. Groningen, The Netherlands.

Opiates (drugs of abuse) by LC/MS

Column: | ZORBAX SB-AQ 830990-914 2.1 x 150 mm, 3.5 μm
Mobile Phase: | A: Acetonitrile with 0.1% formic acid B: Water with 0.1% formic acid
Flow Rate: | 0.25 mL/min
Gradient: | 0 min 10% B 5 min 35% B 5.1 min 100% B

MS Conditions:

Time of Flight (TOF)
Standard with calibrant delivery system
providing constant low flow of ~ 2 μM purine
and HP-921 calibrant to dual ESI for
continuous auto-calibration

Sample: | Opiates

1. Morphine

2. Codeine

3. 6-Acetylmorphine

[picture] | For a comprehensive listing of chromatograms searchable by compound name, visit our online Chromatogram Library at www.agilent.com/chem/library

NEW!

Comparing HILIC and RPLC of morphine
using Aglent ZORBAX RRHD columns with UHPLC/MS

Column: | Agilent ZORBAX Eclipse Plus C18 2.1 x 100mm, 5 μm (Custom column)
Column: | ZORBAX RRHD HILIC Plus 959758-901 2.1 x 100 mm, 1.8 μm

Mobile Phase:

A: 10 mM NH₄HCO₂, pH 3.2
B: CH₃CN/100 mM NH₄HCO₂, pH 3.2 (9:1)
Column A: 10% B isocratic
Column B: 70% B isocratic

Flow Rate: | Column A: 0.4 mL/min Column B: 1 mL/min
Pressure: | Column A: 90 bar Column B: 810 bar
Temperature: | 25 °C
Detector: | Agilent 1290 Infinity LC with an Agilent 6410A Triple Quadrupole Mass Spectrometer

MS Conditions: | MS Source: | Positive ESI, capillary 4000 V, drying gas temperature, flow rate and nebulizer pressure vary with mobile phase flow rate
| MS Acquisition: | Selected ion mode (SIM), delta EMV 200 V, MS dwell time caries with mobile phase flow rate
| Software: | Agilent MassHunter versions B.03.01, B.02.00 AND B.03.01 were used for data acquisition, qualitative, and quantitative analyses, respectively

Sample: | 2 μL injection of 1 μg/mL each of morphine, normorphine, morphine-3-ß-D-glucuronide: HILIC sample was prepared in CH₃CN; RPLC sample was prepared in H₂O

HILIC mode with UHPLC columns cuts analysis time in half, while improving sensitivity by more than a factor of 10, compared to traditional LC columns in RPLC mode with MS detection.

1. M3G

2. Normorphine

3. M6G

4. Morphine

Neutraceuticals:
Hypericin separation in St. John’s Wort

Column: | Eclipse XDB-C8 993967-906 4.6 x 150 mm, 5 μm
Mobile Phase: | 23% 25 mM Na₂HPO₄, Dibasic (pH 7.0 with H₃PO₄): 77% MeOH
Flow Rate: | 1.0 mL/min
Temperature: | 35 °C
Detector: | UV, 254 nm
Sample: | Neutraceuticals

Pharmaceuticals: Rapid,
high sensitivity LC and LC/MS of 11 drugs

Column: | Eclipse XDB-C18 925700-902 2.1 x 50 mm, 1.8 μm
Mobile Phase: | A: 10 mM NH₄ Formate (pH = 3.6) B: ACN with 10 mM NH₄ Formate
Flow Rate: | 0.6 mL/min
Gradient: | 5% B to 70% B in 7.5 min, to 95% B in 8.5 min
Temperature: | 65 °C
Detector: | UV, 230 nm and MSD Trap SL

MS Conditions: | Pos. Dry Gas: | 345 °C
| Neb.: | 45 psi
| HV Cap: | 3500 V
| Range: | 100-700
| Average: | 5 Spectra
| ICC: | 30000
| Charge Con: | On
| Smart Par. Settings: | Tar Mas: 250 m/z
| Comp. Stab.: | 100%
| Trap Drive: | 100%
| Frag. Options: | Smart Frag: On
| Frag. Width: | 10 m/z

1. Sulfamethoxazole

2. Tripelennamine

3. Prednisolone

4. Diphenhydramine

5. Carbamazepine

6. Promethazine

7. Protriptyline

8. Imipramine

9. Trimipramine

10. Perphenazine

11. Triflupromazine

Hormones/steroids

Column: | ZORBAX RRHT SB-C18 823975-902 4.6 x 30 mm, 1.8 μm
Mobile Phase: | 50% 20 mM NaH₂PO₄, pH 2.8: 50% ACN
Flow Rate: | 1.0 mL/min
Temperature: | RT
Detector: | UV, 230 nm
Sample: | Hormones/steroids

1. Estradiol

2. Ethinylestradiol

3. Dienestrol

4. Norethindrone

Steroids: Separation

Column: | Eclipse XDB-CN 993967-905 4.6 x 150 mm, 5 μm
Mobile Phase: | 40:60 ACN:Water
Flow Rate: | 1.0 mL/min
Temperature: | 25 °C
Detector: | UV, 205 nm
Sample: | 1. Norethindrone 0.514 mg/mL 2. Progesterone 0.407 mg/mL 3. Mestranol 0.057 mg/mL

[picture] | For a comprehensive listing of chromatograms searchable by compound name, visit our online Chromatogram Library at www.agilent.com/chem/library

Steroids

Column A: | Eclipse XDB-Phenyl 963967-912 4.6 x 150 mm, 3.5 μm
Column B: | Eclipse XDB-C18 993967-902 4.6 x 150 mm, 5 μm
Mobile Phase: | H₂O:ACN, 60:40
Flow Rate: | 1.0 mL/min
Temperature: | 35 °C
Detector: | UV, 254 nm

Sample:

1. Prednisolone
2. Corticosterone
3. 11 -hydroxyprogesterone
4. Cortisone acetate
5. Deoxycorticosterone
6. 17 hydroxyprogesterone
7. Progesterone

[picture] | For a comprehensive listing of chromatograms searchable by compound name, visit our online Chromatogram Library at www.agilent.com/chem/library

Triamcinolone – USP analysis of triamcinolone

Column: | Eclipse XDB-C18 923975-902 4.6 x 30 mm, 1.8 μm
Mobile Phase: | 47% Methanol:53% Water
Flow Rate: | 1.5 mL/min
Temperature: | 25 °C
Sample: | Triamcinolone, 1 μL

1. Triamcinolone: 0.2 mg/mL

2. Hydrocortisone: 0.3 mg/mL

Minimum Resolution Required = 3.0

Separation of highly basic antidepressants above their pKa in free base form (pKa 9.5-9.7)

Column: | ZORBAX Extend-C18 773450-902 4.6 x 150 mm, 5 μm
Mobile Phase: | 75% Methanol / 25% 50 mM Pyrrolidine Buffer, pH 11.5
Flow Rate: | 0.5 mL/min
Temperature: | 40 °C
Detector: | UV, 215 nm

1. Doxepin

2. Imipramine

3. Nortriptyline

4. Amitriptyline

5. Trimipramine

Basic drugs can often be separated in their charged form at low pH with StableBond or at mid-range pH with Eclipse XDB or Bonus -RP columns. With Extend-C18, you can separate at high pH to improve solubility, improve retention, or obtain different selectivity.

Antidepressants, tricyclic: Comparative separation

Column A: | ZORBAX Bonus-RP 883668-901 4.6 x 150 mm, 5 μm
Column B: | Brand A Polar-linked C8
Column C: | Brand B Polar-linked C18
Mobile Phase: | ACN: 20 mM Na Citrate, pH 6 (60:40)
Flow Rate: | 1.0 mL/min
Temperature: | Ambient
Detector: | UV, 254 nm
Sample: | Tricyclic antidepressants (u= uracil)

1. Propranolol

2. Doxepin

3. Nortriptyline

4. Amitriptyline

5. Trimipramine

Tricyclic antidepressants

Column: | Eclipse XDB-C8 993967-906 4.6 x 150 mm, 5 μm
Mobile Phase: | 38/62 THF/25 mM Potassium Phosphate, pH7
Flow Rate: | 1.0 mL/min
Temperature: | 23 °C
Detector: | UV, 254 nm
Sample: | 10 μL, Antidepressant mix, 10 μg/mL

1. Propanolol

2. Nortriptyline

3. Doxepin

4. Doxepin dimer

5. Amitriptyline

6. Trimipramine

Tricyclic antidepressants and metabolites: Effect of pore size

Column A: | ZORBAX SB-C18 863953-902 4.6 x 150 mm, 3.5 μm
Column B: | ZORBAX RRHD 300SB-C18 883995-902 4.6 x 150 mm, 5 μm
Mobile Phase: | 40/60, 25 mM Phosphate Buffer, 10 mM Triethylamine, pH 6.2/ACN
Flow Rate: | 1.2 mL/min
Temperature: | Ambient
Detector: | UV, 254 nm
Sample: | 10 μL, Antidepressant mix, 10 μg/mL

1. trans- 10-OH - Nortriptyline

2. trans- 10-OH - Amitriptyline

3. cis- 10-OH - Nortriptyline

4. cis- 10-OH - Amitriptyline

5. Nortriptyline

6. Amitriptyline

Ulcer treatment drugs at intermediate pH

Column: | ZORBAX Bonus-RP 883668-901 4.6 x 150 mm, 5 μm
Mobile Phase: | Na citrate, 20 mM, pH 6.1: MeOH, (80:20)
Flow Rate: | 1.0 mL/min
Temperature: | Ambient
Detector: | UV, 220 nm
Sample: | Ulcer treatment drugs

1. Famotidine

2. Cimetidine

3. Pirenzepine

[picture] | For a comprehensive listing of chromatograms searchable by compound name, visit our online Chromatogram Library at www.agilent.com/chem/library

Urine, LSD analysis by LC/MS

Column: | Eclipse XDB-C8 960967-906 2.1 x 50 mm, 5 μm
Mobile Phase: | 15 : 85, ACN : 10 mM Ammonium Formate, pH 3.7
Flow Rate: | 0.3 mL/min
Temperature: | 30 °C
Detector: | MS
MS Conditions: | SIM mode, Ions: 324.2, 223.1, 208.1 Fragmentor (dynamically ramped) 100V at 324.2, 148V at 223.1, 170V at 208.1
Sample: | LSD

Hughes, J.M., C.A. Miller and S.M. Fischer, “Development of a Method for the Forensic Analysis of LSD in Urine”, presented at the ASMS, Palm Springs, June 1997.

USP method:
Glyburide and internal standard, progesterone

Column: | Eclipse XDB-C8 990967-906 4.6 x 250 mm, 5 μm
Mobile Phase: | 45/55, 50 mM Ammonium Phosphate/ACN, Final pH 5.35
Flow Rate: | 1.5 mL/min
Temperature: | Ambient
Detector: | UV, 254 nm
Sample: | 5 μL, 10 ug/mL each of standard

1. Glyburide

2. Progesterone

Dexamethasone, USP method: Rapid analysis

Column A: | ZORBAX SB-C8 880975-906 4.6 x 250 mm, 5 μm
Column B: | ZORBAX Rx/SB-C8 866953-906 4.6 x 75 mm, 3.5 μm
Mobile Phase: | A = Water, B = ACN; Isocratic 30% B
Flow Rate: | 2.0 mL/min
Temperature: | Ambient
Detector: | UV, 254 nm
Sample: | Dexamethasone 10 μL and 5 μL, 10 ug/mL

USP analysis of tetracyclines

Column: | PLRP-S 100Å PL1512-5500 4.6 x 250 mm, 5 μm
Sample: | 20 mg tetracycline in 25 mL 0.01M HCl
Mobile Phase: | 60 g 2-Methyl-2-propanol + 200 mL UHP water + 400 mL 0.2 M K₂HPO₄ at pH 8 + 50 mL 10 g/L tetrabutylammonium hydrogen sulphate at pH 8 + 10 mL 40 g/L sodium edetate at pH 8, made up to 1000 mL with water (adjust pH with dilute NaOH)
Flow Rate: | 1.0 mL/min
Temperature: | 60 °C
Detector: | UV, 254 nm

1. Oxytetracycline

2. Metacycline

3. Doxycycline

[picture] | For a comprehensive listing of chromatograms searchable by compound name, visit our online Chromatogram Library at www.agilent.com/chem/library

Warfarin: USP chromatographic purity method using Eclipse XDB-CN

Column: | Eclipse XDB-CN 993967-905 4.6 x 150 mm, 5 μm
Mobile Phase: | 32:68:1 Acetonitrile:Water:Glacial Acetic Acid
Flow Rate: | 1.5 mL/min
Temperature: | 25 °C
Detector: | UV, 260 nm
Sample: | Warfarin, 2 μL

1. Warfarin (0.128 mg/mL)

2. Warfarin Related Compound A (0.118 mg/mL)

Ten cardiac drugs on Rapid Resolution HT SB-C18

Column: | SB-C18 829975-902 4.6 x 150 mm, 1.8 μm
Mobile Phase: | A: 0.1% TFA, 5% ACN B: 0.08% TFA, 95% ACN
Flow Rate: | 2 mL/min
Gradient: | 0.0 min 12.5% B 10.5 min 60% B 12.0 min 60% B
Temperature: | 70 °C
Detector: | UV, 230 nm
Sample: | Cardiac drugs

1. Procainamide

2. Procaine

3. Nadolol

4. Pindolol

5. Lidocaine

6. Disopyramide

7. Propranolol

8. Nifedipine

9. Nimodipine

10. Nisoldipine

Sulfonamides – Fast analysis with RRHT columns

Column: | SB-C18 824700-902 2.1 x 30 mm, 1.8 μm
Mobile Phase: | A: 90% 0.1% formic acid B: 10% 0.1% formic acid in MeOH
Flow Rate: | A: 0.2 mL/min B: 0.8 mL/min
Temperature: | 35 °C
Detector: | TIC, Single Quad
Sample: | Sulfonamides

1. Sulfanilamide

2. Sulfadiazine

3. Sulfathiazole

4. Sulfamerazine

5. Sulfamethazine

Sulfa drugs

Column: | Pursuit XRs Ultra C8 A7511100X020 2.0 x 100 mm, 3.0 μm
Mobile Phase: | A: Water+0.1% TFA B: MeCN+0.1% TFA
Gradient: | 10% B for 10 min, ramp to 45% B in 1 min and hold for 1 min, return to 10% B in 1 min and hold for 1 min
Flow Rate: | 0.65 mL/min
Temperature: | Ambient
Detector: | UV, 254 nm

1. Sulfanilamide

2. Sulfadiazine

3. Sulfamerazine

4. Sulfamethazine

5. Sulfamethizole

6. Sulfisoxazole

7. Sulfadimethoxine

Sulfa drugs

Column: | PLRP-S 100Å PL1111-3500 4.6 x 150 mm, 5 μm
Mobile Phase: | Potassium sulfate: ACN 7:1, pH 2.2
Flow Rate: | 1.0 mL/min
Detector: | UV, 254 nm

Column: | PLRP-S 100Å PL1111-3500 4.6 x 150 mm, 5 μm
Mobile Phase: | Disodium tetraborate: ACN 6:1, pH 9.3
Flow Rate: | 1.0 mL/min
Detector: | UV, 254 nm

1. Sulfanilic acid

2. Sulfadiazine

3. Sulfamethazine

4. Sulfanilamide

[picture] | For a comprehensive listing of chromatograms searchable by compound name, visit our online Chromatogram Library at www.agilent.com/chem/library

Fast analysis of Pindolol

Column A: | ZORBAX SB-CN 863953-905 4.6 x 150 mm, 3.5 μm
Column B: | ZORBAX SB-CN 827975-905 4.6 x 50 mm, 1.8 μm
Mobile Phase: | A: 70% 50 mM Na Acetate B: 30% ACN
Flow Rate: | 1 mL/min
Temperature: | Ambient
Detector: | UV, 219 nm
Sample: | Pindolol, 2 μL

1. Indole

2. Pindolol

Lamotrigine

Column: | Pursuit XRs Ultra C8 A7511100X020 2.0 x 100 mm, 3.0 μm
Mobile Phase: | ACN:water, 25:90 for 1 min
Flow Rate: | 0.2 mL/min
Injection Volume: | 5 μL, 50% MeOH
Detector: | MS

1. Lamotrigine

Barbiturates

Column: | PLRP-S 100Å PL1512-5500 4.6 x 250 mm, 5 μm
Mobile Phase: | Water
Flow Rate: | 1.0 mL/min
Temperature: | 200 °C
Detector: | UV, 220 nm

1. Barbitone

2. Phenobarbitone

3. Talbarbitone

4. Amylobarbitone

5. Heptabarbitone

Courtesy: Smith, RM, Burgess, RJ, Cheinthavorn, O and Stuttard, JR (1999) Superheated water: a new look at chromatographic eleunts for reversed-phase liquid chromatography. LCGC Europe, January 1999, 30-36. Used with permission.

Analysis of ciprofloxacin and ciprofloxacin metabolites

Column: | PLRP-S 100Å PL1111-3500 4.6 x 150 mm, 5 μm
Mobile Phase: | 74% 20 mM TCA:22% ACN:4% MeOH adjusted to pH 3
Flow Rate: | 1.0 mL/min
Detector: | UV, 277 nm

1. Blank urine sample containing known concentrations of internal standard, ciprofloxacin and its metabolites

2. Blank urine sample containing only internal standard

Krol GJ, Noe, AJ and Beerman, D (1986) Liquid chromatographic analysis of ciprofloxacin and ciprofloxacin metabolites in body fluids. Journal of Liquid Chromatography, 9(13), 2897-2919. Reprinted with permission of the publisher (Taylor & Francis Group, www.informaworld.com).

[picture] | For a comprehensive listing of chromatograms searchable by compound name, visit our online Chromatogram Library at www.agilent.com/chem/library