Tips & Tricks GPC/SEC: The Art of Analyzing High Molar Mass Samples

Jun 06, 2014

Gel permeation/size-exclusion chromatography (GPC/SEC) can be performed for samples that range from a few hundred to several million daltons in molar mass. Analysis of low molar mass samples is relatively straightforward; however, it requires more skill to analyze high molar mass samples in the molar mass range of several million dalton.

The analysis of macromolecules requires patience, especially when analyzing high molar mass samples. The dissolution time required by macromolecules is significant and can take up to several weeks in a worst case scenario. Dissolution time is dependent on a number of parameters such as sample and solvent, molar mass, polydispersity, chain chemistry, crystallinity, composition, and stereochemistry. As a rule of thumb, the higher the molar masses and narrower the distributions the more time that is required.

It is not easy to speed up the polymer dissolution process. The use of ultrasonic devices is not recommended because it is likely to result in degradation of the sample, and for ultrahigh molar mass samples even the use of a magnetic stir bar might result in chain scission. Therefore, the only option is to wait until the sample is dissolved into single isolated chains. Stabilized solvent should be used if the dissolution process takes several days or weeks. Keeping the dissolution container in a dark environment is also often recommended. If needed, the dissolution container can be gently swirled to homogenize the solution.

Table 1: Recommended sample concentrations with respect to sample molar mass.
Lower concentrations of high molar mass samples should be used than otherwise recommended when analyzing low molar mass samples. High molar mass molecules need space to occupy their hydrodynamic volume without interference from other chains. Table 1 summarizes recommended sample concentrations based on molar mass, but please note that these recommendations are for samples with narrow molar mass distributions. For samples with a high polydispersity (broad molar mass distribution) higher concentrations are possible.

Figure 1: Overlay of chromatograms of two polystyrene reference materials for three different concentrations. While there is only a minor effect for the 5000 Da sample, the elution volume and even the peak shape of the 500,000 Da sample is highly affected.
The effect of sample concentration on elution volume increases with molar mass. Figure 1 shows the elution volume and peak shape for reference materials with molar masses of 5000 Da and 500,000 Da. There is a very small change seen for the low molar mass sample, compared to a significant change in the elution volume and the peak shape of the high molar mass reference standard. The change in elution volume is especially problematic when the GPC/SEC system is calibrated with narrow distributed molar mass standards where the peak position of a sample will be compared to that of a calibration standard. The change in peak shape (indicating the lack of separation) affects all kinds of calibrations, even those with on-line viscometers, light scattering detectors, or triple detection systems.

Filtering sample solutions through a membrane filter is usually recommended for solutions containing gels or particles, but care must be taken for high molar mass samples. It is advisable to adjust the pore size of the filter to avoid sample degradation, and if at all possible, filtration of high molar mass sample solutions should be avoided completely.