反相高效液相色谱法同时测定鲜牛乳中的乳清蛋白

Determination of whey protein in fresh milk by reversed phase high performance liquid chromatography

通过对高效液相色谱条件中色谱柱、流动相、流速、检测波长、柱温优化的试验,建立了反相高效液相色谱对鲜牛乳中α-乳白蛋白、β-乳球蛋白、乳铁蛋白同时微量检测的分析方法,最终确定采用Agela Venusil ASB C8（5μm,4.6 mm×250 mm,150μm）色谱柱;三氟乙酸/乙腈/水流动相,流速0.8 m L/min,检测波长215 nm,柱温30℃的色谱条件,并利用外标法定量。结果表明,α-乳白蛋白、β-乳球蛋白、乳铁蛋白3种组分的线性关系良好,相关系数分别为0.9999、0.9991、0.9998,检出限为0.001~0.012 mg/m L,样品平均回收率在86.5%~109.08%之间,精密度和重现性试验RSD值均小于5%。该方法的前处理简单、耗时少,样品损失少,分离效果好,能够实现牛乳中3种蛋白的同时检测。

A analytical method was established for the determination of α-lactalbumin, β-lactoglobulin and lactoferrin by reverse phase high performance liquid chromatography, separation and quantifi cation was achieved by using a Kromasil C8 column（5 μm, 4.6 mm×250 mm, 150 μm）. This method consisted of a linear gradient of the two mobile phases of 0.1% trifl uoroacetic acid in water as mobile phase A and 0.1% trifl uoroacetic acid in acetonitrile as mobile phase B at a fl ow rate of 0.8 m L/min, wavelength of 215 nm and temperature 30 ℃. The quantifi ed analysis of external standard calibration curves was used, the results showed that the three components of whey protein had a good linear relationship. The correlation coeffi cients were 0.9999, 0.9991 and 0.9998 and the detection limit varied from 0.001 mg/m L to 0.012 mg/m L. The average recoveries for all three analytes ranged from 86.5% to 109.08%. The RSD value of precision and reproducibility tests were all less than 5%. The pretreatment method was simple, less time consuming, less loss of samples and the separation effect was better, the most important was that it could achieve trace detection of three proteins at the same time.