Capillary electrochromatography of proteins on an anion-exchanger column.
Capillary electrochromatography (CEC) of proteins was carried out using 50-microm-i.d. fused-silica capillaries packed with 5-microm silica beads having strong anion-exchanger functions attached to hydrophilic spacers at the chromatographic surface. The siliceous microspheres and the capillary innerwall were treated first with a heterobifunctional silanizing agent and reacted subsequently with a vinyl monomer containing quaternary ammonium groups to form a "tentacular" anion exchanger. A mixture of bovine carbonic anhydrase, alpha-lactalbumin, soybean trypsin inhibitor, and ovalbumin was separated using CEC by isocratic elution in the codirectional mode with aqueous phosphate buffer, pH 7.0, containing sodium chloride. The retention mechanism of isocratic CEC for proteins on the anion-exchanger column was illustrated by the results of a study on the effect of salt concentration on the separation. The potential of CEC for protein separation with high resolution was also demonstrated by electrochromatograms of conalbumin and hemoglobin variants. The results shed light on the mechanism of protein separation by isocratic CEC, which is believed to be a combination of chromatographic retention by electrostatic interactions and electrophoretic migration. Assuming that the contributions of the two mechanisms to the overall migration velocity are additive, an electrochromatographic resolution equation was derived and compared to the resolution equation in HPLC to reveal the constituents responsible for the enhancement of resolution by CEC with respect to that in HPLC. The advantage of CEC was also examined by comparing peak capacities in CEC on an, isocratic platform with peak capacities obtained with isocratic and gradient elution HPLC.
Department of Chemical Engineering, Yale University, New Haven, Connecticut 06520, USA.
J
J
Zhang
X
X
Huang
S
S
Zhang
C
C
Horváth
D000838:Anions / Q000032:analysis; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002852:Chromatography, Ion Exchange; D020374:Chromatography, Micellar Electrokinetic Capillary / Q000295:instrumentation*; D004563:Electrochemistry; D011506:Proteins / Q000032:analysis*
D000838:Anions; D011506:Proteins