Protein A Immobilized Monolithic Capillary Column For Affinity Chromatography

Capillary columns offer some distinct advantages over standard HPLC-columns, such as considerably reduced sample sizes, lower solvent consumption and low-volumeflow rates, which enhance the detection performance when using concentration sensitivedetection devices, such as mass spectrometers or flame-based detectors. The monolithiccapillary column can provide the advantages of good hydrodynamic characteristic, lowflow resistance for the biomacromolecules as well as can avoid the cumbersome and expensive procedure of the preparation and packing of beads. Affinity chromatography experiments were performed on a capillary electrophoresis instrument, using its pressure system as the driving force.Reactive monolithic capillary column based on macroporous poly(glycidyl methacrylate-co-ethylene dimethacrylate) and macroporous poly(glycidyl methacrylate-co- trimethylolpropane trimethacrylate ) had been prepared by "in-situ" copolymerization of the monomers in the presence of porogenic diluents, respectively. Protein A were immobilized on both two monoliths with a spacer arm or directly. These columns were characterized by scanning electron microscopy (SEM) and mercury intrusion porosimetry.The EDMA based capillary monolithic columns were applied for analysis standard human immunoglobulin G (HIgG) and human serum. The non-specific adsorption ofthe media had been studied and the results showed that the medium without space arm gave very low non-specific adsorption of BSA. The affinity column without spacer arm was used to determine the HIgG in human serum. The correlative coefficient of the calibration curve reached 0.9987. The total time of rapid analysis was less than 0.7 min.The consumption of eluent buffer and sample was much low than by conventional HPLC.The TRIM based capillary monolithic columns were also applied for analysis standard HIgG and human serum. Non-specific adsorption was also not observed on the TRIM based affinity column without spacer arm, as proved with BSA as a test protein. The affinity column prepared without spacer arm was then applied to determine the HIgG concentration in human serum. The correlative coefficient of the calibration curve reached 0.9942. Furthermore, the amount of adsorbed HIgG was unaffected by the flow rate of the loading buffer, which makes this method suitable for fast determination of biopolymers in micro liter samples.The mechanical stability of both the EDMA based and TRIM based monolithic capillary columns was compared by testing the reproducibility and the linearity of the flow velocity vs. the applied pressure of the column performance. The column prepared with GMA and TRIM proved to be mechanically more stable than the one prepared with GMA and EDMA, which could be explained by the difference of the porous properties and polymerization mechanism between the TRIM and EDMA based monoliths.