Tip: Getting the best value from your UHPLC analyses

Stephen Luke, LC Columns Product Manager

Ultrahigh performance liquid chromatography (UHPLC) is used to achieve two main objectives; fast separations typically using short columns, or high resolution separations using longer columns. This article offers tips and examples on maximizing your UHPLC analyses.

Figure 1. Ultra-fast separations using Agilent InfinityLab Poroshell column (1a) for increased productivity. Improved accuracy is achieved with high resolution separations (1b).

Why UHPLC?

Faster separations improve throughput and decrease cost-per-sample (Figure 1a). Improved resolution increases the accuracy and precision of analysis results, as shown in (Figure 1b), which is associated with a reduction in the amount of required rework.

Requirements for successful UHPLC

The key requirement for successful UHPLC is high chromatographic efficiency, however this drive for efficiency comes with consequences.

High efficiency peaks have by definition narrow peak widths—especially when narrow internal diameter (id) columns are used—with low peaks volumes. To maintain high efficiency peaks, it is necessary to use fast enough detector sampling rates and minimize instrument dispersion volume. Tip: with low volume (short, narrow id) capillaries, zero dead-volume connectors, and low volume flow cells, dispersion volume can be minimized.

Example: LC columns loaded with small (< 2 µm) diameter particles develop high efficiencies but can also generate high pressures. This means that both column and instrument must be rated to withstand high pressures, often 1000 bar (~15,000 psi) and above. At high pressures, frictional heating occurs in the column. Narrow id columns are used to mitigate the impact on separations as a result of the radial temperature gradients. Extra efficiency can be generated using superficially porous particles (SPP) rather than totally porous particles (TPP). A 1.9 µm SPP typically generates 120% the efficiency of a 1.8 µm TPP.

Figure 2. Lifetime test of SPP UHPLC columns under 1000 bar conditions.

Figure 3. Agilent InfinityLab Poroshell 120 columns provide value and efficiency in every day lab operations.

Column lifetime is key in UHPLC

Selection of the correct column is critical to achieve optimal value from your UHPLC analyses. Columns that cannot maintain high efficiency at high pressure, need to be replaced frequently.

Tip: the innovative particle and loading technology used in the new Agilent InfinityLab Poroshell 120 1.9 µm columns provides long lifetime under UHPLC conditions.

The plot in Figure 2 shows how the efficiency of four UHPLC columns, containing SPP, changes with the number of injections made under 1000 bar UHPLC conditions.

Extensive testing shows long column lifetime compared to other SPP columns, up to 10x fewer columns are required for the same amount of work. What’s more, long column lifetime reduces costs associated with the resulting disruption and rework that is required when columns fail. When evaluated long term, these factors can represent a significant economic value to your laboratory (Figure 3).

Quick and easy method development

Agilent InfinityLab Poroshell 120 1.9 µm columns are available in multiple chemistries, providing a range of selectivities to make method development quick and easy. One of the chemistries available is the HPH-C18, which provides extended lifetime under mobile phase pH conditions.

Full scalable family of particles

With the introduction of the 1.9 µm particle, InfinityLab Poroshell 120 columns are now available in a full scalable family of particles. This extended range means that you can choose a size that offers the best fit for your separation needs and the optimum compatibility with your LC instrumentation.

Get the next level of efficiency and confidence with the Agilent InfinityLab LC family

Agilent InfinityLab is an optimized portfolio of LC instruments, columns, and supplies designed to work together seamlessly. The InfinityLab integrated solution provides you with the highest efficiency in your liquid chromatography workflow—regardless of application area. For more details about Agilent InfinityLab and the new InfinityLab Poroshell 120 1.9 µm columns, explore our resource page, where you’ll find this informative brochure and a free information kit.

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Article Directory – January 2017