螺旋槽圆盘柱高速逆流色谱及其在多肽和蛋白分离中的应用

Application of spiral disk column in high-speed counter-current chromatography for peptide and protein separation

摘要:

本研究重点考察了实验室自行设计研制的J型螺旋槽圆盘柱逆流色谱系统对正丁醇-醋酸-水体系和聚乙二醇(PEG1000)-磷酸盐-水双水相体系的固定相保留能力, 并研究了流动相流速(F)、柱转速(w)和温度(T)等因素对固定相保留率(Sf)的影响。结果表明, 该新型分离柱可使两种溶剂体系在L-I-T、U-O-H和L-I-H三种洗脱模式下都可获得较高的Sf, 即以下相为流动相, 采用由螺旋槽内端(I)向外端(O)的流通方式, 或以上相为流动相, 采用由螺旋槽外端(O)向内端(I)的流通方式可以获得较高的固定相保留。其保留能力较传统的螺旋管逆流色谱柱有显著提高。Sf随着w的增加而升高,随着F的增加而降低, 且Sf与F1/2/w线性相关。20oC~45oC之间温度对Sf影响不明显, 但低于20oC不利于双水相体系的保留。应用研究表明, 采用正丁醇-醋酸-水(4:1:5, V/V/V)和PEG1000-磷酸钾盐-水(12.5:12.5:75, W/W/W)(pH 9.0)体系可以在较高的流动相流速和较高的固定相保留下分别实现对亮氨酸-酪氨酸(Leu-Tyr)和缬氨酸-酪氨酸(Val-Tyr)二肽混合物以及细胞色素C与肌红蛋白、肌红蛋白与溶菌酶混合物及鸡蛋清样品中蛋白成分的成功分离。

Abstract:

In order to improve the stationary phase retention of polar solvent systems and aqueous two-phase systems (ATPSs), we designed a multiple spiral disk assembly for type-J high-speed counter-current chromatography (HSCCC). The stationary phase retention was studied under different elution modes by using two solvent systems that contained 1-butanol-acetic acid-water (4:1:5, V/V/V) and polyethylene glycol (PEG) 1000-K2HPO4-water (12.5:12.5:75, W/W/W). The best retention was obtained in L-I-T, U-O-H, L-I-H three modes by pumping lower mobile phase from inner terminal (I) to outer terminal (O), and upper mobile phase from outer terminal (O) to inner terminal (I) at a relatively high flow rate. Meanwhile, the relationship between retention percentage of the stationary phase retention (Sf) and various parameters such as flow-rate (F), rotation speed (w) and column temperature (T) was also studied. Sf increased with the increase of w and decreased with the increase of F. Regression analysis showed a linear relationship between Sf and F1/2/w. The influence of T on Sf was not obvious between 20°C and 40°C, lower temperature than 20°C was not suitable for viscous ATPSs. Acceptable resolutions were achieved when it was applied for the separation of dipeptides including Leu-Tyr and Val-Tyr by using 1-butanol-acetic acid-water (4:1:5, V/V/V) solvent system. The proteins including cytochrome C and myoglobin, lysozyme and myoglobin, and fresh chicken egg-white proteins were well separated by 12.5% PEG1000-12.5% K2HPO4-75% water (pH 9.0) and 16% PEG 1000-12.5% K2HPO4-71.5% water (pH 8.0) system.