Method development for high performance liquid chromatography: novel organic modifiers and column packing conditions

Neutral eluents were proved to be applicable for the separations of organic acids and bases on reversed phase high performance liquid chromatographic columns when a straight chain alcohol, diol or amino alcohol is used as the organic modifiers in the mobile phase. Upon incorporating 2% 1-butanol, 1% 1,2-hexanediol, or 0.25% 1,2-octanediol into the aqueous eluent, the separation of alkane carboxylic acids on a silica C-18 column was improved greatly in terms of both separation time and peak shape. When the same silica C-18 column was pre-coated with 1,2-decanediol, good separations of these acids were obtained with either water alone or 1% 1-butanol as the eluent. When 1.5% 1-hexanol, 0.09% 129 decanol or 0.01% 1-dodecanol was added into 30% acetonitrile/water, much sharper peaks and shorter retention times resuhed for a group of seven to eight aromatic bases on an underivatized polystyrene/divinylbenzene column. Columns packed with lightly sulfonated polystyrene/divinylbenzene pre-treated with amino alcohols such as ethanolamine, 3-amino-lpropanol or 4-amino-l-butanol allows much improved separations of alkane carboxylic acids when water or methanol/water mixture was used as the eluent. The retention times of analytes on the treated column were affected by the ion exchange capacity of the stationary phase and the hydrocarbon-chain length of amino alcohol used to treat the column. The effect of these alcohols, diols and amino alcohols on the separations was believed to be that these molecules coat onto the surface of the stationary phase by hydrophobic interactions or electrostatic attractions resulting in a more hydrophilic surface of the stationary phase and hence less hydrophobic interactions between analytes and stationary phase, and reduced retention times and improved separations. INTRODUCTION Ion exclusion chromatography is often used for the separation of hydrophilic molecules such as weak acids or weak bases. Such separations are usually performed on gel type ion exchange columns of fairly large dimensions with primarily aqueous solution as the eluent [1, 2]. The separation mechanism was long considered to be the partitioning of the solute acids or bases between the eluent and the water inside the resin gel. Hydrophobic interactions between the analyte molecules and polymeric resin matrix play a role in these separations [330 5]. Separations of weak acids or bases generally require eluents containing dilute acid or base to suppress the ionization of the analytes in order to obtain sharp peaks. However, Tanaka et al. were able to obtain good separations of alkane carboxylic acids using eluents containing no acid but only a neutral sugar-alcohol additive [6]. Separations of carboxylic acids and small polar compounds have been reported by Morris and Fritz [7] on smaller, lightly sulfonated non-gel polymeric resins. The same authors have also foxmd that small straightchain alcohols added to aqueous solvent had a dramatic effect on the chromatographic behavior of small polar compounds such as alkane carboxylic acids. The alcohols were believed to coat the surface of the polymeric resin due to a dynamic equilibrium in which the alcohol additives were distributed between the mobile and stationary phases [8]. The present work was performed in parallel with and in continuation of the work by Morris and Fritz. Here, typical reversed phase HPLC columns, such as silica CI8 and underivatized polystyrene/divinylbenzene columns were used for the separation of carboxylic acids and organic bases, respectively. Low percentage of 1-butanol or 1,2-diol present in aqueous eluent allows dramatic reduction in retention time of carboxylic acids and excellent separations of these compounds on a silica C-18 column. The same column pre-coated with 1,2-decanediol permits much improved separation of theses acids when water alone or 1% butanol in water was used as the eluent. Separation of organic bases on underivatized polystyrene/divinylbenzene resin column was also improved greatly when a straight-chain alcohol, such as 1-hexanol and 1-dodecanol, was incorporated in eluent containing 30%