Separation of β-lactoglobulin A, B and C variants of bovine whey using capillary zone electrophoresis

β-Lactoglobulin is a whey protein that affects milk composition and product functionality and which can be present in up to eight genetic variant forms. A free zone capillary electrophoresis method has been developed to separate and identify the β-lactoglobulin A, B and C variants. Three buffer systems borate, 2-(N-morpholino)-ethanesulphonic acid (MES) and bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane (Bistris)] were examined over a range of pH values and with the addition of the separation buffer modifiers Tween 20 and/or ethanolamine. The most successful combination of these was 50 mM MES at pH 8.0 with the addition of 0.1% Tween 20 which clearly resolved the three variants from both each other and from the other whey proteins even though the MES buffer was acting well outside its pK_a range (pH 5.3–7.3). The retention times and identification of the individual variants were verified by spiking with commercially purified β-lactoglobulin A and B proteins and a β-lactoglobulin AC whey. The method was then used to phenotype β-lactoglobulin in a sample population of New Zealand Jersey cows.