Rapid separation of milk whey proteins by anion exchange chromatography

A fast protein liquid chromatography system was used to fractionate the major proteins of sweet and acid wheys. Fifty to 500 microliter of whey were fractionated with a stepwise ionic strength gradient using water (buffer A) and increasing concentrations of .7 M sodium acetate (buffer B). Six well-resolved peaks were obtained: 1) amino acids (tentative identification), 2) low molecular weight peptides (tentative identification), 3) highly enriched alpha-lactalbumin, 4) highly enriched serum albumin 5) electrophoretically pure beta-lactoglobulin B, and electrophoretically pure beta-lactoglobulin A. A poor baseline or unresolved peaks resulted when .02 M bis-tris or .02 M histidine was used for buffer A and .7 M sodium acetate in .02 M bis-tris or histidine was used for buffer B. When sodium chloride was used in place of sodium acetate, beta-lactoglobulins A and B were poorly resolved. The column was cleaned after each run by injecting 2 ml of the following reagents: glacial acetic acid, 2 N sodium chloride, 2 N sodium hydroxide, 2 N sodium chloride, 2% detergent, and 100% acetonitrile. Time required to run each sample including column cleanup was 40 min.