Absorption spectra and multicapillary imaging detection for capillary isoelectric focusing using a charge coupled device camera

Abstract

Two absorption imaging detectors using charge coupled device (CCD) cameras are designed for capillary isoelectric focusing (CIEF). In the first detector, a light beam passes through a 4 cm capillary and is dispersed by a grating onto a CCD camera. The two-dimensional CCD in the camera records the light absorption at different positions along the capillary in one dimension, and at different wavelengths in the second dimension, simultaneously. The resolution in wavelength is about 1 nm. Since the separation time in the 4 cm long capillary column is only 4 min, the complete analysis takes 4 min, which is much faster than conventional CIEF methods. In the second detector, a light beam passes through a capillary array and then onto a CCD camera. Isoelectric focusing separation and detection of several samples can be completed in about 4 min, and the focusing processes in all capillaries can be observed simultaneously by the real-time, on-line imaging detector. In both detectors, images are normalized by light intensity, recorded simultaneously with the images, to compensate for intensity fluctuation of the light source. The detection limit of the detector is 1.5 × 10–3 absorbance units. The pH resolution of the instrument with the 4 cm long capillaries is 0.01 which is the same or better than that of conventional CIEF instruments with much longer capillaries. The deviation in pH of replicate zone positions in different capillaries of the capillary array is less than 0.01 which is much better than capillary array IEF methods using the mobilization process.