Monolithic membrane-receptor columns: Optimization of column performance for frontal affinity chromatography/mass spectrometry applications

Capillary-scale monolithic silica columns containing an entrapped transmembrane receptor were prepared by a sol–gel processing method and evaluated by frontal affinity chromatography with tandem mass spectrometric detection (FAC/MS/MS). Monolithic stationary phases were made by infusing micro-bore capillaries with a mixture containing the water soluble polymer polyethylene glycol (PEG), the biocompatible silica precursor diglycerylsilane (DGS) and membranes containing the nicotinic acetylcholine receptor (nAChR) from Torpedo californica. Through multivariate factorial analysis, the conditions used to form the stationary phase materials were analyzed to determine which factors were the most significant in controlling the silica pore diameter, silica gelation time, column reproducibility and the apparent activity of the immobilized receptor. Of the variables studied it was found that altering the concentration of PEG and the additive 3-(aminopropyl)triethoxysilane had the largest effect on the silica pore diameter and gelation times, as well as the apparent receptor activity. The information gained through factorial analysis was used to prepare an optimized “second-generation” material that was suitable for the reproducible preparation of nAChR-based bioaffinity columns. Using FAC/MS/MS, it was determined that the immobilized receptor-doped columns retained 100% of the receptor in active form, and that the binding constant for the nanomolar agonist epibatidine was very close to that obtained in buffered aqueous media. These results indicate that monolithic receptor columns should be useful for screening compound mixtures and identifying small molecules with nanomolar binding affinities.