The concentration of the organic modifier, acetonitrile and pH were altered to measure their effects on the retention of various hydrophilic proteins and vitamin B12using high performance size exclusion chromatography (HPSEC). The best separations were obtained with 0.1 or 0.2M sodium phosphate in the pH range of 6.0 to 7.0. In the absence of TFA, up to 25% acetonitrile did not dramatically influence the size exclusion profile of the protein components. Although 30 to 40% acetonitrile and 0.1% TFA for elution was not acceptable for the chromatography of hydrophilic proteins, hydrophobic apolipoproteins were nicely resolved under these conditions.
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