RP-HPLC法分离和定量测定未变性的乳清蛋白成分

建立了一种基于反相高效液相色谱(RP-HPLC)来分离并定量测定脱脂乳、加热脱脂乳及脱脂乳粉中未变性的乳清蛋白的分析方法。采用Venusil ASB-C8色谱柱;流速0.8 mL/min;检测波长215 nm;柱温30℃;流动相A为体积分数0.1%的三氟乙酸水溶液;流动相B为体积分数0.1%的三氟乙酸乙腈溶液;洗脱方式:流动相B开始在38%下等度洗脱2 min,然后在5 min内上升到43%,最后在43%下等度洗脱13 min。此方法在20 min内将4种乳清蛋白组分分离且各蛋白线性关系良好,适合定性和定量检测未变性的乳清蛋白组分。

Isolation and quantification of main composition with undenatured whey protein with reverse-phase HPLC

Four main proteins(α-La,β-LgA,β-LgB,BSA) with the undenatured whey protein in skim milk,heat treatment skim milk and skim milk powder were isolated and determined by RP-HPLC.It was achieved by using a Venusil ASB-C8 column(250 mm×4.6 mm,5 μm,300■) at a flow-rate of 0.8 mL/min,column temperature 30 ℃ and monitored with 215 nm.A gradient of solvent A 0.1%(v/v) TFA dissolved in water and B consisting of 0.1%(v/v) TFA in acetonitrile was applied.The gradient started with 37% of solvent B keeping for 2min and was increased to 43% within 5min and then kept at 43% for 13 min.This method successfully separated these four proteins within twenty minutes.With good linear correlations about the four proteins,it was used to qualitative and quantitative analysis the composition of the undenatured whey proteins.