珠蛋白水解产物的二维液相色谱分析

Two-dimensional Liquid Chromatography for the Analysis of the Peptides from Globin Peptic Hydrolysate

本文以强阳离子交换色谱(SCX)作第一维,反相色谱(RP)作第二维,以一个10通阀直接连接两支色谱柱,构建正交的SCX/RP二维液相色谱系统,并以珠蛋白水解产物的分析对其加以评价。样品首先由第一维阳离子交换色谱(HyperilSCX,100mm×4.6mmI.D.)在pH4.0的磷酸盐缓冲体系中以0.5mL/min的低流速洗脱分离,洗脱产物通过10通阀直接富集在反相分析柱(HypersilBDSC18,250mm×4.6mmI.D.)顶端,切换10通阀后进一步进行反相分析。阳离子交换色谱采用不连续的逐步增加盐浓度的19步台阶梯度方式洗脱,切割转移样品组份后再进行第二维分析。二维液相色谱系统的分辨率、峰容量都得到提高,出峰数量达到690多个,系统峰容量达到5643。二维液相色谱系统均采用常规尺寸的色谱柱,可以处理大进样量的样品,对生物样品的制备纯化有一定的指导意义。

A two-dimensional liquid chromatography system is constructed using strong cation exchange chromatography (SCX) followed by reversed phase chromatography (RP) to separate the complex peptides from globin peptic hydrolysate. A 10-port valve was used to connect the two columns directly. After the sample was loaded on the SCX column(Hyperil SCX, 100mm×4.6mmI.D.), the phosphate buffers(pH4.0)were used to elute the peptides at a flow rate of 0.5mL/min. The fractions from the SCX were focused on the head of the RP analytical column(Hypersil BDS C18, 250mm × 4.6mmI.D.)and then were analyzed by the mobile phase of the second dimension after the valve was switched. The enriched peptides on the SCX column were eluted by 19 discontinuously steps with linear increasing salt concentrations and then were analyzed by reversed phase chromatography. The resolution and the peak capacity were greatly increased in the 2D SCX/RP system. The system resolved more than 690 peaks and the total peak capacity reached as high as 5643. The system was constructed with the conventional size columns that can handle large amount of samples, and it’s useful for preparation and purification of bio-samples.