Chiral separation by open tubular capillary electrochromatography with adsorbed avidin as a stationary phase

Abstract

Chiral separations by open tubular capillary electrochromatography (OTCEC) were studied with avidin as a chiral stationary phase prepared by the physical adsorption method proposed previously. The amount of avidin adsorbed on the capillary wall was estimated by frontal analysis to be of the order of magnitude of 10–12 mol for a capillary of 50 cm effective length, corresponding to 10–8 mol/m2 or 10–6 mol/L. The effects of operating conditions such as buffer pH, organic modifier, applied voltage, and temperature on the separation were investigated. As expected, the buffer pH and the retention factor of the analyte proved to be key factors significantly influencing the separation. The buffer pH affected the amount of protein adsorbed as well as the magnitude of the electroosmotic flow, whereas the retention factor influenced both efficiency and resolution. The effects of applied voltage and temperature played a less important role in the separation. Separations were also performed on capillaries of 50 μm ID. Acceptable separations were obtained, but the speed and separation capability were lower as compared with narrower bore capillaries. To date, a total of 16 enantiomeric compounds have been separated. Due to its low phase ratio, however, OTCEC is only suitable for enantiomers having strong interactions with the stationary phase. The relative standard deviations for the run-to-run, day-to-day, and column-to-column reproducibility were 2.2% (n = 76), 2.3% (n = 5), and 1.1% (n = 6), respectively.