The Determination of a Small Amount of a Biological Constituent by the Use of Chemiluminescence. XV. A Zeolite Column-Chemiluminescence Detection System for the Separation and the Determination of a Protein Mixture

Abstract

Abstract To make use of zeolite as the packing material for the separation of proteins by means of adsorption chromatography, the adsorption behavior of protein onto the Na-X type of zeolite (Molecular Sieve 13X) in an aqueous solution or onto a column (3 mm i.d.×50 mm) packed with the same zeolite as above was examined using a ultraviolet absorption detector and model proteins, such as human-serum albumin and human-serum γ-globulin. The following results were obtained: 1) Human-serum γ-globulin was much more adsorbed onto the zeolite than was human-serum albumin and 2) in general, more protein was adsorbed in an acidic solution than in an alkaline solution. Moreover, a chemiluminescence detector using a 1,10-phenanthroline–hydrogen peroxide–copper(II) system, which has been developed by the present authors, was modified to improve its sensitivity for the determination of protein. Both the carrier solution and the eluting solution which were used in combination with a zeolite column with a chemiluminescence detector were optimized so as to give a smooth baseline on a recorder. A microbore zeolite column (1 mm i.d.×250 mm)-chemiluminescence detection system suitable for the separation and the determination of a small amount of a protein mixture was established on the basis of these experimental results. Since human-serum albumin passed through a microbore zeolite column, while human-serum γ-globulin was retained on the column, when a model sample containing human-serum albumin and human-serum γ-globulin was injected into the column, human-serum albumin was first determined, followed by the determination of human-serum γ-globulin after its desorption. Both human-serum albumin and human-serum γ-globulin in the concentration range of 2×10−5–5×10−3 g dm−3 could be determined economically and conveniently, with a detection limit of about 1 ng (injected sample volume: 50 mm3; S/N=2). The present method was, therefore, applicable to the separation and the determination of albumin and globulin in a serum sample.