Hydrophobic interaction fast protein liquid chromatography of milk proteins

Bovine whey proteins and caseins were separated by hydrophobic interaction chromatography with the new Pharmacia fast protein liquid chromatography column, phenyl-Superose. Total casein was separated using a decreasing gradient of 0.8 to 0.05 M sodium phosphate and a constant 3.75 M urea concentration at pH 6.0. The order of elution of caseins was beta less than gamma, alpha s2 less than kappa less than alpha s1, and beta-casein was always eluted first. Whey proteins were separated with a decreasing salt gradient of 1.5 to 0 M ammonium sulphate in 0.05 M sodium phosphate at pH 7.0. The order of elution was beta-lactoglobulin less than bovine serum albumin less than immunoglobulin less than alpha-lactalbumin. The elution order of proteins from the column did not correlate with the calculated average hydrophobicities but the method was considered to be a measure of the "effective" hydrophobicity of proteins and therefore of more use for attempting to relate hydrophobicity to functional properties of proteins. The method shows significant advantages over conventional techniques allowing rapid optimization of elution conditions and reducing run times from 24 h or more to less than 2 h.