Activation of Sepharose with epichlorohydrin in water-free system for preparation of high capacity immobilized metal ion affinity adsorbent

Epoxy activation of agarose gel Sepharose 6 FF by epichlorohydrin (ECH) was carried out in a water-free medium with dimethyl sulfoxide as solvent. The activated agarose gel was subsequently coupled with iminodiacetic acid and Cu²⁺ ions for preparation of immobilized metal ions affinity chromatographic (IMAC) adsorbent. The results indicated that the epoxy activation efficiency was significantly enhanced in the water-free system, such that a epoxy density of agarose gels as high as 165 μmol/mL was achieved at the optimized conditions of 40%(ψ) ECH, 0.02 g/mL NaOH, 50°C and 4 h, which is about 50% higher than the highest value reported so far. The prepared IMAC adsorbent showed a high chelating capacity for Cu²⁺ ion up to 128.3 mol/mL, thus it ensured a high equilibrium adsorption capacity of 2.05 mmol/L for bovine serum albumin, and 90% of the adsorbed protein could be efficiently eluted when 0.5 mol/L imidazole was used as elute.