Separation and Quantification of Major Protein in Yak Milk by High Performance Liquid Chromatographic Method

A precise,sensitive and low cost method was developed for the determination of four casein(CN),e.g.αs1-CN,αs2-CN,β-CN and κ-CN,and four whey protein,e.g.α-lactalbumin(α-La),β-lactoglobulin A(β-Lg A),β-lactoglobulin B(β-Lg B) and serum albumin(SA) by reversed phase high performance liquid chromatography.Skimmed yak milk was diluted with denaturing solution,and separated on a Jupiter C4 column(250 mm×4.6 mm,300,5 μm i.d.) at 20 ℃.A gradient programme was carried out at a flow rate of 0.8 mL/min using 0.1% trifluoroacetic acid(TFA) water solution and 0.1% TFA acetonitrile solution as eluent.The detection wavelength was set at 220 nm.The results showed that eight protein fractions could be completely separated within 40 min.The calibration curves were linear in the ranges of 0.56-5.6 g/L for αs1-CN,0.14-1.4 g/L for αs2-CN,1.35-13.5 g/L for β-CN,0.35-3.5 g/L for κ-CN,0.17-1.7 g/L for α-La,0.036-0.36 g/L for β-Lg A and β-Lg B,and 0.98-9.8 g/L for SA,with correlation coefficients more than 0.99 except for α-La.The spiked recoveries of eight protein fractions in yak milk sample were in the range of 86%-103% with RSDs of 1.7%-8.7%.The limits of detection(LODs) of eight protein fractions were in the range of 10.7-39.2 mg/L,and the limits of quantitation(LOQs)were 35.7-130.7 mg/L.The developed method was simple and accurate,and could meet the demands for actual application.