DUAL DETECTION FOR CAPILLARY ISOELECTRIC-FOCUSING WITH REFRACTIVE-INDEX GRADIENT AND ABSORPTION IMAGING DETECTORS

A unique imaging detector was constructed for capillary isoelectric fo cusing (CIEF) by combining a selective optical absorption imaging dete ctor with a universal refractive index gradient imaging detector. Ah s ample zones focused inside a 100-mu m-i.d., 4-cm-long capillary by iso electric focusing were detected in a real-time fashion by the universa l imaging detector, and their spectroscopic properties were measured b y the absorption imaging detector almost simultaneously. The refractiv e index gradient imaging is based on a dark-field Toepler-Schlieren sy stem. The optical alignment for both detection modes was optimized. Th e sensitivity of the detector was limited by noise due to optical defe cts in the capillary wan. Peptides without tyrosine and/or tryptophan can be detected by the universal made of the detector. These peptides are difficult to detect by commercial CIEF instruments which use a UV- visible on-column absorption detector operated at a 280-nm wavelength. The instrument can be used for fast analysis of protein samples, such as high-resolution separation and detection of human hemoglobin varia nts, and to investigate dynamic processes occurring inside capillary.