Abstract: Immobilized copper ion affinity membrane was prepared by using cellulose filter paper as matrix. The membrane was loaded into a chromatographic column designed in the laboratory and the immobilized copper ion affinity membrane chromatographic column was prepared. Hemoglobin (Hb) was adsorbed by the column and its adsorption rate was above 90%. The experimental process was as follows: a set of 5 immobilized copper ion affinity membranes were loaded into a column, uncombined copper ion was removed with 5 mL of sodium phosphate-citric acid buffer (pH 7); the Hb sample volume was 2 mL, and uncombined Hb was eluted with 10 mL of the buffer. The Hb concentration was determined by ultraviolet spectrophotometry at 405 nm. The major influence factors on hemoglobin adsorption rate such as Hb sample amount (1.58-31.6 mg/g (2 mL of sample volume)), pH value range (3.6-10.6), temperature range (25-50 ℃), and sample flow-rate (0.5-2.5 mL/min) were investigated. The optimal operation conditions were found as follows: the Hb sample amount of 3.16-7.90 mg/g, sample flow-rate of 0.5-1.0 mL/min, test temperature of room temperature, pH value range of 6-8. Compared with aqueous two-phase system, liquid-solid extraction system and the conventional column method, the membrane medium is cheaper and easier to be scaled up.

Key words: adsorption , affinity chromatography, hemoglobin, immobilized copper ion, affinity membrane