Daniela Held | Authors

This instalment describes the interplay between column length, pore size distribution, and particle size to optimize GPC/SEC separations.

Hydrolyzed collagens (collagen peptides) are water-soluble products obtained by hydrolysis of natural proteins and used for dietary supplements. A simple GPC/SEC method is described for molar mass determination of collagen peptides, allowing reliable molar mass determination using ultraviolet (UV) detection.

How to create a calibration curve when no chemically-matching reference materials with narrow molar mass distribution are available.

A review of alternative approaches to narrow standard calibration.

Gel permeation chromatography/size-exclusion chromatography (GPC/SEC) columns are filled with porous particles differing in particle and pore sizes. Typical particle sizes in analytical GPC/SEC range from sub-2-µm particles applied in oligomer and protein separations to approximately 20 µm for separations of ultrahigh molar mass macromolecules (1,2). While the effect of combining columns of different pore sizes has previously been discussed in GPC/SEC Tips & Tricks (3,4), the effect of combining columns of different particle sizes has not been addressed before.

The chemistry of samples analyzed using gel permeation chromatography/size-exclusion chromatography/gel filtration chromatography (GPC/SEC/GFC) is very diverse. Different chemistries of stationary phases are required to allow for true size separation. Several types of materials are available, all of which have their advantages and limitations. While silica‑based stationary phases are most common in high performance liquid chromatography (HPLC), for macromolecules polymer-based phases are popular.