An On-Line Electrophoretic Concentration Method for Capillary Electrophoresis of Proteins

Abstract

A simple on-line electrophoretic concentration method for capillary electrophoresis of proteins with a semipermeable hollow fiber is proposed. A short, semipermeable hollow fiber is connected to the inlet end of a capillary. An injection electric field is applied across the hollow fiber. Protein samples are electromigrated into the hollow fiber from a sample vial. Since the protein ions cannot pass through wall of the hollow fiber, protein ions are concentrated in the hollow fiber. After a certain period of injection (concentration) time, an electric field is applied across the hollow fiber and the capillary, and a conventional CE is carried out. The concentration effect of the method has been demonstrated successfully with model protein samples, cytochrome c, lysozyme, ribonuclease A, and r-chymotrypsinogen A. Experimental results show that the concentration factor is increased with the injection (concentration) time and the electrophoretic mobility of the proteins. When the injection (concentration) time is 60 s, CE detection limits of the model proteins can be lowered by 1000-fold for the present method. Factors affecting the concentration factors and the further improvements of the method are discussed.

An Improvement of the On-line Electrophoretic Concentration Method for Capillary Electrophoresis of Proteins and Experimental Factors Affecting the Concentration Effect

“…16 On the other hand, CE peaks of the proteins on the level of 10 -2 mg/ml would be overlapped for the present electrophoretic concentration method because of the great concentration factor up to 1000-fold. 16 Therefore, more dilute protein samples were used to demonstrate the concentration effect in this experiment. Figure 2 shows CE electropherograms of the model proteins of 10 -4 mg/ml for a gravity flow injection method and the present on-line electrophoretic concentration method.…”

“…16 A high-voltage power supply (Matsusada Precision Inc., Japan) provided injection and separation voltages. An UV absorbance detector (CE-970, Japan Spectroscopic Co. LTD) was used for detection of the proteins, its detection wavelength was 215 nm.…”

“…16 In the sample injection (concentration), the V-shaped glass tube filled with the buffer solution was inserted into a sample vial, and an injection voltage (below 100 V) was applied between the sample vial and the glass tube. As shown in Fig.…”

“…[4][5][6][7][8][9][10][11][12][13][14][15] Recently, we have reported a novel on-line electrophoretic concentration method for CE of proteins. 16 In our proposed method, a short semipermeable hollow fiber is connected to the inlet end of a capillary. An injection electric field is applied across the hollow fiber.…”

“…16 On the other hand, experimental results show that a coaxial and close connection between the capillary inlet end and the hollow fiber is necessary and critical to the success of the method. In our previous work, 16 the inlet end of the capillary and the hollow fiber were fixed into a Teflon tube with internal diameter of 0.5 mm. It was not easy to keep the coaxial and close connection between the inlet end and the fiber in this manner.…”