Crawford, Andrew John (1993) A study of carbohydrate stationary phases for the separation of enantiomers by high performance liquid chromatography. PhD thesis, University of Warwick.

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Abstract

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.

Item Type:	Thesis (PhD)
Subjects:	Q Science > QD Chemistry
Library of Congress Subject Headings (LCSH):	Silica -- Analysis, Stationary phase (Chromatography), Enantiomers, Carbamates -- Analysis
Official Date:	December 1993
Dates:	Date Event December 1993 Submitted
Institution:	University of Warwick
Theses Department:	Department of Chemistry
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Matlin, S. A.
Sponsors:	Science and Engineering Research Council (Great Britain) (SERC) ; Rhône-Poulenc Rorer Foundation
Extent:	xi, 239 leaves
Language:	eng

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