Daniela Held

After developing a GPC/SEC method by selecting the optimum stationary and mobile phase an optimization of the default conditions is a good idea.

The requirements for GPC/SEC data analysis to obtain precise, reproducible and accurate results are very specialized. Therefore, often dedicated software tools offering flow-rate correction or different calibration options need to be employed. This instalment of Tips & Tricks looks at these requirements.

The complexity of method development for GPC/SEC is often underestimated. Although only isocratic separation is applied, the development of a robust and stable method can be challenging. It is important that measures are taken to develop a suitable method that will deliver long-term reproducible results, especially when biomolecules, ions or functional groups are present.

GPC/SEC (gel permeation chromatography/size exclusion chromatography) methods generally contain multiple detectors in order to gather more information about the molecular structure of macromolecules.¹. Up until now, however, the practice and the impact of detector combination has not been discussed.

Traditionally GPC/SEC is a slow technique. A typical, non-optimized GPC/SEC run with 3 analytical columns and a precolumn needs about 45 to 60 min to be completed. This limits the number of samples that can be analysed.

A stable baseline is a prerequisite for precise, accurate and reproducible GPC/SEC analysis, especially for samples with a broad molar mass distribution. Baseline problems can include drift, wander (long-term noise), noise/spikes (short-term noise), as well as positive or negative system peaks.

In comparison to other GPC/SEC detectors such as light-scattering detectors or UV detectors, viscometers need more care. It takes a longer time to get a stable baseline after changing from one solvent to another and the quality of the LC components plays an important role. Nevertheless, a viscometer is worth these efforts as it provides unique information that is not available with any other detector.

GPC/SEC samples often show broad and/or multimodal peaks that are not baseline separated and look strange to users of other chromatographic techniques. However, although many weird looking peaks are possible in macromolecular characterization, some peaks are stranger than others and it is worth checking if the observed signal truly describes the sample.

GPC/SEC samples often show broad and/or multimodal peaks that are not baseline separated and look strange to users of other chromatographic techniques. However, although many weird looking peaks are possible in macromolecular characterization, some peaks are stranger than others and it is worth checking if the observed signal truly describes the sample.