Acid stable hyper crosslinked stationary phases for the reversed phase high performance liquid chromatographic separation of basic and biological analytes

Low pH mobile phases are very useful for the HPLC separation of a wide variety of silanophilic solutes such as basic drugs, peptides and proteins. Acidic mobile phases suppress deleterious interactions between positively charged solutes and the surface by protonating the surface silanol groups. The poor stability of conventional columns under low pH conditions seriously inhibits the use of pH as a mobile phase variable in separation optimization. This type of column degradation will profoundly affect the reproducibility of analyte analysis and is not acceptable.; The work presented here primarily describes the synthesis and chromatographic characterization of hyper crosslinked stationary phases. Hyper crosslinked silica-based stationary phases have been developed with dramatically improved acid stability compared to any currently available silica-based stationary phase. The hyper crosslinked aromatic network is formed by marrying silanization chemistry and Friedel-Crafts crosslinking. Superior low pH stability is achieved. However, the performance of basic analytes on these HC phases under acidic conditions was unusually poor compared to that of conventional silica-based C18 phases. The effects of the specific F-C catalysts used and of the type of silica substrate on the chromatographic properties of HC phases have been studied. Modified synthetic strategies that give both good observed plate counts for basic analytes under acidic conditions and very good low pH stability without compromising other chromatographic properties of the hyper-crosslinked phases have been developed. In formic acid buffered mobile phases, basic analytes showed much better performance on the new generation of HC phases than on other conventional commercial phases tested. This new type of HC phases were characterized in terms of acid stability, efficiency in separating basic analytes, flow dynamics, mass transfer properties, sample loading capacity, and selectivities for neutral and ionic compounds.; Additionally, acid stability of conventional RPLC stationary phases at elevated temperatures was systematically studied. Metal ions in acidic mobile phases were found to affect the acid stability of RPLC phases at elevated temperatures more than protons. Practical methods of improving acid stability of conventional RPLC stationary phases such as using titanium inlet frit or adding reductive reagents into the acidic mobile phases have been developed.