How to calibrate your GPC columns for best results
By Andrew Coffey and Richard Harmer Agilent GPC Applications
Calibrating GPC/SEC columns using narrow molecular weight standards is a well-established technique for the investigation of the molecular weight distribution of polymers. Standard operating procedures should always use best practice when calibration is an integral part of your GPC method development. Here are some helpful guidelines to ensure that you do just that.
Choose the right standards and solvents
Your choice of calibration standards depends on the solvent you are using. The four common standard/solvent combinations shown in Table 1 will cover most applications.
Table 1. GPC/SEC solvents and appropriate calibration standards
Agilent offers prepackaged GPC/SEC calibration kits that contain 0.5 g of ten different molecular weights and are used for column sets operating over a large molecular weight range. For a narrower molecular weight range, select individual standards to focus around the operating limitations of the column. Selecting the narrowest polydispersity is critical; a polydispersity index (D) of 1.20 is the minimum. Larger polydispersity standards lead to compromised reproducibility as the Mp will be less defined from injection to injection.
Correctly prepare calibration standards
Calibration standards must be correctly prepared to avoid spurious peak shapes, inaccurate calibration curves, and potential blockages in filters and column frits. Use the same mobile phase for standards, which should be filtered and of high purity. Filtering standard solutions is not required providing they are fully dissolved. Avoid vortexing, sonicating, or shaking because these high-shear activities will change peak shape, retention time, and molecular weight.
The concentration of standard solutions is also critical. Too high a concentration leads to reduced mass transfer and band broadening due to excessive sample viscosity; too low and the signal-to-noise ratio becomes too small to integrate reproducibly.
True sample solubility only exists when equilibrium of solvent inside and outside the random-coil polymer chain is achieved. Higher molecular weight standards will take longer to fully dissolve and may need to be heated to assist dissolution. However, you should always keep the temperature 10 °C below the boiling point of the solvent to avoid thermal degradation.
How many standards do you need?
A calibration curve is typically constructed from ten to twelve data points in order to provide an accurate polynomial regression. The standards you use should ideally cover the entire resolving range of the column set to reduce extrapolation errors during the molecular weight calculations, and to increase reproducibility compared to calibrations and unknown samples over time. Standards should bracket the full molecular weight distribution of the unknown compound. If it is not possible to calibrate the full molecular weight range, then you should use a column with a linear extended range, such as Agilent PLgel MIXED. You should also obtain baseline separation of all components for maximum reproducibility. This avoids the shifting retention times attributed to artificial band broadening. To help you achieve the baseline separations, the individual molecular weight standards should be separated by a 10x difference.
Molecular weight standards are often injected as cocktails consisting of three to four standards each, to reduce analysis time. Agilent EasiVial offers the quickest and most convenient method to deliver an accurate column calibration when using such a cocktail, for organic and aqueous GPC/SEC. The pre-prepared kit contains three, color-coded 2- or 4-mL autosampler vials, each with a mixture of four accurately weighed polymer standards to provide a twelve-point calibration in just three injections.
Standards can degrade once made up, and so it is good practice to make fresh ones every week. PEOs and PEGs should be refrigerated for storage but brought to room temperature before use.
Strategies for improving resolution and efficiency
Increasing temperature generally results in reduced viscosity of the mobile phase, which may either improve resolution or let you connect additional columns in series. Maximum operating temperature is dependent on eluent boiling point, sample stability, and other factors including column stability.
Figure 1. Increasing the number of Agilent PLgel columns improves baseline resolution and peak height.
Particle size (µm)
Number of GPC/SEC columns
1 to 2
5 to 6
2 to 3
8 to 10
2 to 4
13 to 20
3 to 4
Table 2. Particle sizes and corresponding number of GPC columns
Columns with smaller particle size offer increased efficiency. However, this option is dependent on the molecular weight of your sample. When using columns with very small particles, shear can occur as the molecular weight increases. In addition, optimal flow rates can vary with the particle size.
Increasing the number of columns in series is possible, as shown in Figure 1, though this will be limited by pressure restrictions. Table 2 is a useful starting point when assessing the most appropriate column set.
The work you do determines how often you should calibrate
For continuous work you should calibrate every day. However if you use internal verification, a weekly calibration should be performed. Recalibration is essential whenever part of the system or eluent is changed. Tip: you can guarantee optimum performance by bracketing calibrations for every column set. This will significantly boost your confidence in your results.
Proper column calibration instills confidence
GPC/SEC is the only established method for obtaining a comprehensive understanding of the molecular weight distribution of a polymer. This makes regular column calibration important for achieving the best results from instruments and columns. Polymer standards from Agilent are the ideal reference materials for generating accurate, reliable GPC/SEC column calibrations. Agilent has many useful GPC resources available to you, including application compendia, primers, and selection guides. Why not start by reading Calibrating GPC Columns – a Guide to Best Practice? It’s free and provides excellent advice on how to get the best from your GPC calibration.