Abstract
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 7.0$. Deuterium oxide was found to be the most suitable probe for the determination of total permeation volume (VT), while the UV absorbing nucleoside monophosphatee and glycyltyroslne were also acceptable on most columns. An equation was derived which allowed the determination of SEC resolution by using the elope (m) of the molecular weight calibration curve, pore volume (Vi), and theoretical plates (N) for a particular sample. Resolution was expressed in terms of the minimum molecular weight ratio (RM) necessary for two components to be resolved on the column. A series of commercial SEC columns was subjected to the described protocol which compared support characteristics and column efficiency. 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.
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