Determination of the leakage from Phenyl-Sepharose Cl-4B, Phenyl-Sepharose FF and Phenyl-Superose in bulk and column experiments.

The release of ligands from Phenyl-Sepharose CL-4B, Phenyl-Sepharose FF and Phenyl-Superose has been studied in bulk and column experiments. The leakage products have been identified and quantified by liquid chromatography, fluorescence spectroscopy and proton NMR spectroscopy. It is demonstrated that the leakage occurs primarily through hydrolysis of the agarose support. However, leakage via ether cleavage of the spacer arm-ligand moiety is also observed especially for Phenyl-Superose. The release of ligands at acidic pH is in agreement with a first-order reaction and correspondingly the rate constants have been estimated for all three gels at pH 1 and 2. These show that 50% of the ligands are intact after 15 years of incubation at pH 2. Phenyl-Superose is the most stable gel at acidic pH, whereas Phenyl-Sepharose CL-4B and Phenyl-Sepharose FF are the most stable at neutral and basic pH.