A study of carbohydrate stationary phases for the separation of enantiomers by high performance liquid chromatography
The relationship between the structure and chromatographic properties of silica is discussed and the preparation and properties of chemically-bonded stationary phases based on silica are reviewed. A detailed account is given of the history of the development of carbohydrate-based stationary phases, including microcrystalline cellulose, other polysaccharides and monosaccharides and their ester and carbamate derivatives, with emphasis on their utility for the chromatographic resolution of enantiomers. Mechanisms of chiral discrimination by these and other types of chiral HPLC phases are discussed in terms of the interactions between functionalitles in the stationary phase and solute enantiomers. An overview is given of literature methods for the preparation of oligosaccharide derivatives with suitable reactive groups, such as an isothlocyänate function, for linkage to the surface of aminopropylated silica. In the experimental part, the thesis describes work carried out to study the effects of stationary phase support properties on the chromatographic behaviour and enantiomer resolution capability of carbohydrate carbamate phases. These phases were prepared by the exhaustive reaction of free hydroxyl groups in carbohydrates, such as cellulose and amylose, with aryl isocyanates, such as phenyl isocyanate and 3,5-dimethylphenyl isocyanate. The resulting carbamates were characterised by 1H nmr and microanalysis and were coated onto aminopropyl silica supports by evaporation from organic solvents. It was shown that the retention of solutes by these phases correlated directly with the w/w phase loading, whilst both the separation factor and resolution for various enantiomers displayed a more complex relationship. The influence of changing pore diameter of the aminopropylated silica, with concomitant changes in pore volume and surface area, were evaluated for a series of materials at constant w/w phase loading. Whilst it is currently common practice to use very wide pore (up to 4000 Angstrom) silicas as supports for carbohydrate carbamate phases, it was concluded from the present work that there is little or no justification for using such supports and that good chromatographic performance and effective chiral discrimination can be achieved on much smaller pore (e. g. 500 Angstrom), higher surface area materials. Carbamate derivatives of cellulose were prepared a using phenyl, 3,5-dimethylphenyl and 1-naphthyl isocyanates and of amylose using the first two of these reagents and were shown to have close to the theoretical maximum levels of substitution. Their chiral discriminating abilities were investigated using a test panel of five racemic analytes: trans-stilbene oxide, 2,2,2-trifluoro-l-(9'- anthryl)-ethanol, 1-phenylethanol, benzoin and trogers base. The naphthyl carbamate phase showed no resolving ability for any of these racemates and this appeared to correlate with the presence of N-H bands in the it spectrum which were indicative of severe disruption of the organised, H-bonded 3-dimensional structure necessary for chiral discrimination. The other four carbamate phases all showed resolving ability, each with its own specific pattern of solute selectivity. The difference in behaviour of the corresponding, identically subsituted cellulose and amylose phases, differing only in the configeration of the C-O linkage at the anomeric carbon on each glucose ring, illustrates the importance of long-range stereochemical properties: chiral discrimination must result not only from local interactions with individual carbamate groups and the adjacent chiral centres on the glucose rings, but also from the influence of the organisation of the polymer chains at the macromolecular level, leading to the creation of "chiral ravines" which display an intrinsic shape selectivity. A series of malto-oligosaccharides with from 2 to '9 glucose units was obtained, the higher members of the series being separated from a commercially available oligosaccharide mixture by preparative HPLC on an aminopropyl silica column. Each oligosaccharide, and also the phenyl carbamate and 3,5- dimethylphenyl carbamate derivatives prepared from glucose, maltose and maltotriose, were characterised by FAB-MS and LSIMS and the fragmentation patterns of the carbamates were analysed In detail and shown to provide considerable structural information. The glucose penta(phenylcarbamate) was converted Into the 1-isothiocyanato-tetra(phenylcarbamate) by successive reactions with HBr and a thiocyanate salt. After spectroscopic characterisation to confirm its structure, it was reacted with aminopropyl silica and the conditions for achieving optimum surface coverage established. Use of a longer spacer chain was also investigated, but did not appear to offer advantages over aminopropyl silica. A model reaction with n-propylamine gave the expected N-n-propyl urea. The silica-bonded glucosyl carbamate phase was examined chromatographically using a test panel of racemic solutes and was found to give some resolution of certain racemates, but only when very low concentrations of polar modifier (isopropanol) were present in the hexane mobile phase. Some work was carried out to try to extend the above chemistry to enable carbamate derivatives of the malto-oligosaccharides to be linked to silica through the anomeric position of the first glucose ring. A satisfactory procedure has not yet been established, major experimental difficulties encountered being solubility problems with the higher homologues and a lack of reproducibility in the introduction of the anomeric bromine and its displacement by isothiocyanate. Once these problems have been overcome, the methodology developed should provide a novel series of immobilised oligosaccharide carbamates with potential utility for the resolution of enantiomers.