The Determination of a Small Amount of Biological Constituent by the Use of Chemiluminescence. XIII. High Sensitive Metal Chelate Affinity Chromatography

Abstract

Abstract A chemiluminescence detector using a 1,10-phenanthroline–hydrogen peroxide–copper(II) system was successfully combined with a metal chelate affinity column. A mixture comprising bovine serum albumin, lysozyme, and bovine serum γ-globulin as model proteins was completely separated by the pH-gradient elution method on a TSK gel Chelate-5PW adsorbed with zinc ions, also, each protein could be quantitatively determined. According to the present method, using a 0.4 cm3 column of bed volume, 1) the determinable concentration range for bovine serum γ-globulin was formed to be 1.0×10−4–1.0×10−1 g dm−3 with a detection limit of 5 ng for a 50 mm3 of injection volume, 2) the recovery of 1.0×10−3 g dm−3 bovine serum γ-globulin was 85% with 7.9% of the coefficient of variation (n=5), and 3) the time necessary for an analysis was about 50 min. The present method was about 200 times more sensitive than that using an ultraviolet detector and was also applicable to a human serumn sample.