Monolithic poly(butyl methacrylate-co-ethylene dimethacrylate) capillary columns have been prepared via either thermally or photochemically initiated polymerization of the corresponding monomers and the repeatability of their preparation has been explored. Three separate batches of five columns each were prepared using thermal and photochemical initiation for a total of thirty columns. All thirty capillary columns were tested in liquid chromatography-electrospray ionizationmass spectrometry mode for the separation of a model mixture of three proteins -ribonuclease A, cytochrome c and myoglobin. Excellent repeatability of retention times was observed for the proteins as evidenced by relative standard deviation (RSD) values of less than 1.5%. Somewhat broader variations with RSD values of up to 10% were observed for the pressure drop in the columns. The stability of retention times was also monitored using a single monolithic column and no significant shifts in either retention times or back pressure was observed in a series of almost 2200 consecutive protein separations.
Preparation of monolithic capillary columns for separations in the CEC mode using UV-initiated polymerization of the plain monolith followed by functionalization of its pore surface by photografting has been studied. The first step enabled the preparation of generic poly(butyl methacrylate-co-ethylene dimethacrylate) monoliths with optimized porous properties, controlled by the percentages of porogens 1-decanol and cyclohexanol in the polymerization mixture, irradiation time, and UV light intensity. Ionizable monomers [2-(methacryloyloxy)ethyl]trimethylammonium chloride or 2-acryloamido-2-methyl-1-propanesulfonic acid were then photografted onto the monolithic matrix, allowing us to control the direction of the EOF in CEC. Different strategies were applied to control the grafting density and, thereby, the magnitude of the EOF. To control the hydrophobic properties, two approaches were tested: (i) cografting of a mixture of the ionizable and hydrophobic monomers and (ii) sequential grafting of the ionizable and hydrophobic monomers. Cografting resulted in similar retention but higher EOF. With sequential grafting, more than 50% increase in retention factors was obtained and a slight decrease in EOF was observed due to shielding of the ionizable moieties.
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