Monolithic protein stationary phases prepared by the sol-gel method for use in affinity chromatography
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Capillary monolithic silica columns used for normal and reversed phase separations offer the advantage of lower backpressure and higher efficiency compared to packed columns. However, capillary monolithic columns have not been applied to affinity chromatography. We used the sol-gel method to encapsulate proteins in a silica matrix for capillary affinity chromatography. A monolithic protein stationary phase was prepared by the tetramethyl orthosilicate procedure. The protein containing sol was injected into a 75um i.d. capillary that had been pretreated with sodium hydroxide. The sol gelled into a rigid micro-porous and macro-porous stationary phase. The porosity of the gel was characterized by scanning electron microscopy and Brunauer-Emmett-Teller adsorption/desorption isotherms. Ubiquitin, bovine serum albumin (BSA), and catalase at different loadings were used in preliminary tests to determine the effects of protein size and concentration on the stationary phase properties. Preliminary separations were performed using the pumps from a capillary electrophoresis instrument with BSA doped monolithic columns.