Comparison of the electrophoretic separation of proteins in capillaries with different inner diameter
Abstract
Two fused-silica capillaries of considerably different inner diameter (75 and 10 mum) were used for the separation of a set of five standard proteins. The separations were run in acid pH (50 mM phosphate buffer, pH 2.5).
Generally better separations (with minor tailing only) were obtained using a standard capillary [27 cm (20 cm effective length)x75 mum I.D.] in comparison with a narrow bore capillary [27 cm (20 cm effective length)x 10 mum I.D.]. The conditions of the electrophoretic separation were the same for both types of capillaries (25 degreesC; 10 W; positive polarity at the inlet).
The sequence of the proteins was cytochrome c, albumin, transferrin followed by a partly resolved peak of catalase and chymotrypsinogen A. In the narrow bore capillary severe tailing was observed - tailing factor ranged from 2.11 to 5.54 or 1.67 to 2.53 depending on the concentration of the analytes injected (2 or 0.2 mg/ml of each test compound injected).
The relative [Delta(DeltaG(0))] values of the interaction with the capillary wall in the small bore capillary (with cytochrome c taken as initial standard) ranged from -0.74 to - 1.04 kJ/mol. The problem of assaying the speed of the endoosmotic flow (EOF) in both capillary types was thoroughly investigated using thiourea and dithiothreitol as EOF markets.
It was revealed that if thiourea is used as the EOF marker, the obtained value was dependent on the concentration of the marker injected. Optimum conditions for the EOF determination in acid buffer were specified.
The higher speed of the EOF in the narrow bore capillary (10 mum) as compared to the 75 mum I.D. capillary is discussed.