Torre M, Cohen M E, Corzo N, Rodríguez M A, Diez-Masa J C
Instituto de Química Orgánica General (CSIC), Madrid, Spain.
J Chromatogr A. 1996 Apr 5;729(1-2):99-111. doi: 10.1016/0021-9673(95)00889-6.
A perfusion reversed-phase (RP) HPLC method was developed for the rapid separation of the main bovine whey proteins: alpha-lactalbumin (alpha-LA), serum albumin (BSA) and the genetic variants of beta-lactoglobulin (A and B) (beta-LG A and beta-LG B). For the method development, the influence of factors favouring structural changes of proteins (temperature and organic acid concentration in the mobile phase), gradient and other chromatographic conditions and the mass of protein injected was examined. The optimized method allowed the separation of proteins in about 1.5 min (cycle time 3.5 min) with resolution around 1.0 for the beta-lactoglobulins. The method was applied to the determination of proteins in a whey from raw bovine milk. The precision of the determinations was < or = 3.75 mg per 100 ml (S.D.). With respect to the accuracy, errors < or = 7.0% in the determination of alpha-LA, beta-LG A and beta-LG B were obtained, compared with an RP-HPLC reference method. However, higher errors in the quantification of BSA were found owing to the lack of purity of the peak assigned. In addition, the proposed method has proved to be very useful in the detection of homologous whey proteins from different species (cow, sheep and goat) in milk mixtures.
建立了一种灌注反相(RP)高效液相色谱法,用于快速分离主要的牛乳清蛋白:α-乳白蛋白(α-LA)、血清白蛋白(BSA)以及β-乳球蛋白的遗传变体(A和B)(β-LG A和β-LG B)。在方法开发过程中,考察了有利于蛋白质结构变化的因素(流动相中的温度和有机酸浓度)、梯度及其他色谱条件以及进样蛋白质的质量的影响。优化后的方法可在约1.5分钟内分离蛋白质(循环时间3.5分钟),β-乳球蛋白的分离度约为1.0。该方法应用于生鲜牛乳清中蛋白质的测定。测定的精密度为每100毫升≤3.75毫克(标准差)。在准确性方面,与RP-HPLC参考方法相比,α-LA、β-LG A和β-LG B测定中的误差≤7.0%。然而,由于所归属峰的纯度不足,发现BSA定量存在较高误差。此外,该方法已证明在检测牛奶混合物中不同物种(牛、绵羊和山羊)的同源乳清蛋白方面非常有用。