Separation of lactoferrin from bovine whey by dye affinity chromatography

Eight triazinic dyes were assayed as ligands for chromatographic affinity purification of lactoferrin from rennet whey, at pH 5.0, 7.0 and 9.0. Blue R-HE, Orange R-HE and Scarlet G-A did not adsorb lactoferrin at any pH, whereas Yellow FR adsorbed a small amount of lactoferrin and showed poor selectivity. Red HE-3B was most selective and lactoferrin was most adsorbed at pH 5.0 and 7.0. Blue F3-GA, Red HE-7B and Red F-5B adsorbed intermediate amounts of lactoferrin. At pH 9.0, Blue F3-GA, Red HE-3B and Red F-5B adsorbed intermediate amounts of lactoferrin. Red HE-7B maintained its low lactoferrin adsorption and good selectivity. At pH 5.0-7.0, lactoferrin shows high adsorption on Red HE-3B and at pH 7.0, selectivity was better than at pH 5.0, because lactoferrin was accompanied by only a small amount of β-lactoglobulin. Red HE-3B was selected for tests on ionic-strength gradient elution. At pH 7.0, lactoferrin eluted with NaCl at about 1 mol/L, and the contaminating β-lactoglobulin did so with NaCl at 0.3 mol/L. Treatment of immobilized Red HE-3B with sodium dithionite and sodium nitrite did not improve separation and considerably decreased recovery. A technique was developed for batch purification based on data from gradient elution. The procedure allowed extraction of 82% of the lactoferrin whey content with a purity of 98%.