An high-performance liquid chromatography matrix based on agarose cross-linked with divinyl sulphone

• Published in: Journal of Chromatography A 1987, Vol.396, p.101-113
• Main Authors: Hjertén, Stellan, Wu, Bo-Liang, Liao, Jia-Li
• Format: Article
• Language: English
• ISSN: 0021-9673
• DOI: 10.1016/S0021-9673(01)94046-4

By systematic investigation of some parameters [concentration of reducing agent (sodium borohydride), temperature, pH, reaction time, concentration of divinyl sulphonate (DVS), solvent for removal of excess of DVS, agents for reaction with free unreacted vinyl groups] that affect the cross-linking of agarose with DVS, an highly reproducible method has been obtained for the preparation of a chromatographic medium exhibiting low adsorption and high rigidity to allow high flow-rates: a 300 mm × 6 mm bed of 12% cross-linked agarose beads with diameters around 17 μm permitted flow-rates up to 5 ml/min (40 bar). The cross-linking procedure was performed at pH 12.0–12.5 at room temperature in the presence of appropriate concentrations of sodium borohydride. Above and below these concentrations the flow-rate decreases and below the risk of adsorption increases. To reinforce the hydrophilic character of the cross-linked gel and to make it somewhat more rigid, OH-rich substances were coupled to the cross-linked agarose via its unreacted vinyl groups. By varying the molecular weight of these substances the pore size of the gel can be varied. No decrease in the rigidity of the beads was observed upon exposure to pH 13 during a test period of 15 days. Additional evidence for the chemical stability of the beads is that no impairment of the performance of a molecular sieve chromatography column could be detected (except for a decrease in flow-rate which, however, returned to normal after repacking) during a 4-year period of use, alternating in conventional buffer media and buffers containing sodium dodecyl sulphate or guanidine.