Liquid chromatography

Surface-modified alumina containing covalently-bonded p-aminophenyl groups was prepared by refluxing chromatographic grade alumina with p-aminophenylphosphonic acid in n-butanol. The resulting material, p-aminophenylalumina (PAPA), was evaluated for use as a stationary phase in high performance liquid chromatography (HPLC). When used with mobile phases consisting of acetonitrile and water, retention of solutes on the PAPA phase exhibited normal phase behavior, as evidenced by the increases in solute retention times with increasing percentages of acetonitrile. Excellent separations of carbohydrate mixtures were obtained on the PAPA stationary phase. Comparisons of the chromatographic properties the PAPA phase with those of commercial aminoalkyl silica stationary phases were also performed.

The performance of several alkyl-bonded alumina-based stationary phases was evaluated by comparing the separation of synthetic octapeptide and polypeptide mixtures and tryptic digests of larger proteins. These phases were of differing pore diameter, alkyl chain length modification and particle shape and size. The separations were compared to standard silica phases. The narrow pore octadecyl bonded alumina phase outperformed the other alumina and silica phases in terms of separation efficiency and mobile phase resistance. Superior performance is attributed to the enhanced solute mass transfer properties and the unique morphology of the microplatelet alumina particles. The mechanism of separation gradually changes with increasing size of the peptide.

Sphingolipids (SPLs) are important structural components of membranes in some types of cells and are involved in numerous signaling processes. Sphingomyelin (SPM) and dihydrosphingomyelin (DHS) are the two major SPLs in membranes. Very little is known about the molecular species and role of DHS in biological systems. In this work, we employed high performance liquid chromatography with detection by atmospheric pressure chemical ionization and electrospray ionization (ESI) mass spectrometry to elucidate the SPL composition in biological extracts. No common dietary source of DHS is known to exist. A novel analytical method developed to analyze complex SPL mixtures was used to show that bovine milk contained substantial amounts of DHS. Also, the human lens is the only known system in which DHS is the most abundant SPL. The molecular species of DHS in cataractous lenses has never been reported. It was shown that there was a preference for monounsaturated species of DHS and SPM in all ages and in cataractous lenses. It was also discovered that SPLs were the primary PLs remaining in cataractous lenses. Finally, the formation of sodium adducts and dimers in the ESI source of the ion trap mass spectrometer prevented the accurate quantitative analysis of PLs. A new method was developed to eliminate these undesirable ions.