CHARACTERIZATION OF SOME COMMERCIAL HIGH-PERFORMANCE SIZE-EXCLUSION CHROMATOGRAPHY COLUMNS FOR WATER-SOLUBLE POLYMERS

Test procedures for characterizing size-exclusion chromatography (SEC) columns utilizing molecular probes of various sizes are described. Studies with several commercially available SEC columns showed that under certain conditions they exhibited ion exclusion, cation exchange, and hydrophobic partitioning. These nonpermeation effects were minimized by using mobile phases with ionic strengths between 0. 10 and 0. 60 M at pH 7. 05. Deutrium oxide was found to be the most suitable probe for the determination of total permeation volume (V//T), while the UV absorbing nucleoside monophosphates and glycyltyrosine were also acceptable on most columns. An equation was derived which allowed determination of SEC resolution by using the slope (m) of the molecular weight calibration curve, pore volume (V//i), and theoretical plates (N) for a particular sample. Column characteristics and performance data are reported that serve as a guide to their optimization for use in the strict SEC mode and as a reference for further column development. 29 Refs.