Mobile phase additives for separation improvement in reversed-phase liquid chromatography and capillary electrophoresis

The effect of mobile phase additives in reversed-phase liquid chromatography and capillary electrophoresis has been investigated. The effect of reversed-phase stationary phase solvation on surface ordering and shape selectivity has been investigated using the non-polar mobile phase additive n-hexanol on C{dollar}sb{lcub}18{rcub}{dollar} stationary phases of known bonding density. No conclusive evidence of a relationship between C{dollar}sb{lcub}18{rcub}{dollar} solvation and shape selectivity was observed.; Cholesterol was evaluated as a mobile phase additive and provided dramatically improved shape selectivity by increasing the level of C{dollar}sb{lcub}18{rcub}{dollar} stationary phase ordering. The system selectivity could be controlled by varying the mobile phase cholesterol concentration or the system temperature. The coating was stable when using mobile phases of up to 70% methanol.; Mobile phase additives were also investigated to improve the separation of peptides. The amine-containing additive cyclohexylamine (CHA), which diminished the effects of residual surface silanols, proved extremely useful in improving the chromatographic efficiency of tryptic digests of bovine cytochrome c. When compared to the commonly used triethylamine and trifluoroacetic acid additives, CHA had significantly lower detection background, and did not require purification prior to use. Cholesterol enriched stationary phases were also evaluated for peptide separations, and provided greater separation selectivity for certain peptide pairs.; The surface adsorbing additives CTAB/Brij 35, Jeffamine ED-900, Jeffamine M-1000, and Emcol CC-9 were evaluated to improve peptide and protein separations in capillary electrophoresis. The results showed that excellent control of electroosmotic flow was possible by regulating the concentration of the Jeffamine additives. Peptide separations were dramatically improved by the addition of 1-2% Jeffamine ED-900 or M-1000 at pH 7.0, while protein separations at pH 3.0 showed improved recovery and efficiency when Jeffamines were used. The more hydrophobic additive Emcol CC-9 proved to have excellent surface coating properties and improved protein recovery 2-fold. By choosing additives with structural features that produce a hydrophilic coating greater separation control was achieved.