Regulation on both pore structure and pressure-resistant property of uniform agarose microspheres for high-resolution chromatography

How to improve the performance of chromatographic media is very important in chromatography. Uniform agarose microspheres were successfully prepared using membrane emulsification method with a controllable particle size, followed by multi-step crosslinking and dextran-grafting, respectively. To obtain both fine pore structure and good pressure-resistant property, the effects of both dextran-grafting and crosslinking process were studied carefully and also, the preparation conditions were delicately adjusted. Inverse size-exclusion chromatography was used for determining the pore structure of these agarose microspheres. Uniform agarose microspheres with an average particle size of about 8 μm were obtained with regularly spherical, transparent and smooth appearance. By introducing a certain molecular weight of dextran or pentaerythritol glycidyl ether at different crosslinking steps, both the pressure-resistant and the chromatographic properties of microspheres were improved. Both the maximum flow velocity and the corresponding pressure drop increased with the decrease of the molecular weight of dextran, i.e., 99 cm/h and 3.22 MPa, respectively, using dextran T3 (3 kDa). The average pore size of agarose microspheres decreased from 6.04±0.56 nm to 2.50±0.12 nm with the increase of the molecular weight of dextran from dextran T3 (3 kDa) to dextran T100 (100 kDa), with a high resolution obtained for a certain molecular range of model proteins. Also, the pressure-resistant property was highly improved in multi-step crosslinking process, with a maximum flow velocity of 107 cm/h and a corresponding pressure drop of 3.62 MPa obtained after the whole crosslinking steps. The average pore size of agarose microspheres was 3.72±0.32, 3.90±0.21 and 3.60±0.27 nm for the introduction of pentaerythritol glycidyl ether as the crosslinking agent at different steps, respectively. These uniform dextran-grafted agarose microspheres have a finely controllable molecular range with a high resolution compared with traditional ones, which are beneficial for chromatographic selectivity. Therefore, they are very useful for high-resolution chromatography and have wide applications in downstream process.