Hu Guanghui, Cao Xueli
Beijing Key Laboratory of Plant Resources Research and Development, School of Chemical and Environmental Engineering, Beijing Technology and Business University, Beijing 100048, China.
Sheng Wu Gong Cheng Xue Bao. 2009 Apr;25(4):618-25.
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 (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 degrees C and 40 degrees C, lower temperature than 20 degrees 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.
为了提高极性溶剂系统和双水相系统（ATPS）的固定相保留率，我们设计了一种用于J型高速逆流色谱（HSCCC）的多重螺旋盘组件。通过使用包含正丁醇 - 乙酸 - 水（4:1:5，V/V/V）和聚乙二醇（PEG）1000 - K2HPO4 - 水（12.5:12.5:75，W/W/W）的两种溶剂系统，研究了不同洗脱模式下的固定相保留情况。通过以相对较高的流速从内端（I）向外端（O）泵送下层流动相，同时从外端（O）向内端（I）泵送上层流动相，在L - I - T、U - O - H、L - I - H三种模式下获得了最佳保留效果。同时，还研究了固定相保留率（Sf）与流速（F）、转速（w）和柱温（T）等各种参数之间的关系。Sf随w的增加而增加，随F的增加而降低。回归分析表明Sf与F1/2/w之间存在线性关系。在20℃至40℃之间，T对Sf的影响不明显，低于20℃的温度不适用于粘性双水相系统。当使用正丁醇 - 乙酸 - 水（4:1:5，V/V/V）溶剂系统用于分离包括亮氨酸 - 酪氨酸和缬氨酸 - 酪氨酸的二肽时，获得了可接受的分离度。细胞色素C和肌红蛋白、溶菌酶和肌红蛋白以及新鲜鸡蛋白蛋白等蛋白质通过12.5% PEG1000 - 12.5% K2HPO4 - 75%水（pH 9.0）和16% PEG 1000 - 12.5% K2HPO4 - 71.5%水（pH 8.0）系统得到了良好的分离。
