ABSTRACT
Capillary isoelectric focusing was applied to determine the pI value of human rhinovirus serotype 2 (HRV 2), a picornavirus of about 8,500,000 Da in size. Using fused silica capillaries dynamically coated with hydroxypropylmethyl cellulose (added at 0.08% to the catholyte), the virus zone failed to reach the steady state position in the pH gradient within times usually employed in focusing experiments, as the electroosmotic flow (EOF) pushed the analyte zone past the detector. Therefore, the residence time of the zones in the separation capillary was extended by applying hydrodynamic pressure at the detector side during focusing, thus pneumatically counteracting the EOF. After completion of focusing, the zones were mobilized by pressure maintaining the high voltage. For calibration of the pH gradient, low molecular mass pI marker substances were employed. Using the relation between the apparent pI value of the virus and the focusing time under counter pressure, the actual pI of HRV2 was determined as 6.8 by extrapolating to infinite time.
