Optimization of Peak Separation and Broadening in Aqueous Gel Permeation Chromatography (GPC). Dextrans

Abstract

Herein is reported an experimental optimization of the aqueous size-exclusion chromatography of dextrans on untreated CPG-10 glass packings. The molecular weight calibration curve was independent of ionic strength and there was no evidence of polymer adsorption on the glass packings. However, in the absence of salt in the mobile phase, chromatograms did show a high molecular weight shoulder which is attributed to small negative charges on the dextran molecules resulting in ion exclusion from the pores of the negatively charged glass. These high molecular weight shoulders were completely eliminated with the addition of a small amount of salt (e.g. 0.05 M NaSO₄). A proper choice of pore sizes was essential to obtain good separation with minimal peak broadening giving a linear molecular weight calibration curve with a wide separation range and a small correction for imprefect resolution. Corrections to M_N and M_W were generally less than 5%. To establish an optimal column combination, it is recommended that single columns containing packing with one pore size be employed to establish the performance of a particular sized pore before a column combination is chosen.