Browsing by Author "Stephen, A M"

Now showing 1 - 13 of 13
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    The application of gas-liquid chromatography to the structural investigation of Polysaccharides
    (1965) Kaplan, Morris; Stephen, A M; Leisegang, E C
    This work constitutes the first reported analysis of carbohydrate derivatives by gas-liquid chromatography, using the hydrogen-flame ionization detector. The conditions used compared favourably with conditions reported by other workers and were well suited to the quantitative analysis of methanolysis products from methylated polysaccharides. Excellent separations were achieved, and at the same time a wide range of sugars could be estimated quantitatively using a single column. The detector response was shown to be linearly related to sample size. Authenticated standards were converted to their methyl glycosides by heating with methanolic hydrogen chloride, and their relative molar responses (R ' values) to the hydrogen-flame ionization detector were determined. R values were also obtained by calculations based on recent theories accounting for the observed ionization of organic compounds in the hydrogen flame, but the calculated values were not in agreement with the R values found experimentally. The experimental results obtained suggest that, under the conditions used, the response of the sugar is related to its retention time but not to the mechanism of ionization of the sugar molecule in the flame. From the observed relationship between R values and relative retention times, relative molar responses were predicted for those sugars which were not available as standards. The possible sources of error in quantitative analysis have been discussed. Errors resulting from degradation during the methylation procedures and incomplete methylation of the polysaccharides, and from the demethylation and degradation of methylated sugars during methanolysis, were found to be greater than experimental errors arising from small variations in operating conditions and the measurement of peak areas. Using the R values already determined, the methanolysis products from twelve methylated polysaccharides were separated quantitatively by gas chromatography. The structural significance of the results obtained were discussed and possible model structures for ten of the polysaccharides investigated were presented. As far as possible, the structural models were based on the findings of previous investigations, and in those cases where published data were available, gas chromatography results were in good agreement with the published results. The hydrogen-flame ionization detector was found to be ideally suited to the analysis of methylated sugars. The greater resolving power, sensitivity of detection and, in particular, the speed with which quantitative analyses can be performed, are the major advantages which make gas-liquid chromatography superior to other techniques in this area of polysaccharide chemistry.
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    Chemical and spectroscopic studies of the capsular polysaccharides of some Klebsiella serotypes
    (1988) Ravenscroft, Neil; Stephen, A M
    As part of an international collaborative programme concerned with the elucidation of the molecular structures of capsular polysaccharides (the K-antigen) produced by strains of the bacterial genus Klebsiella, the capsular material of serotype K71 has been investigated, and that of serotypes K36 and K64 re-examined, by novel enzymic and spectroscopic methods. The cultivation and employment of bacteriophages which are capable of cleaving (by specific glycanase action) the isolated, cognate bacterial polysaccharide in vitro has yielded highly significant oligosaccharides. These may represent the repeating unit in the polysaccharide or be derivatives resulting from conversion of uronic acid to the 4,5-unsaturated analogue where, as found for serotype K64, the mode of cleavage is β-elimination not hydrolysis. The oligosaccharides thus generated have proved to be far more amenable to chemical and spectroscopic studies than their parent polymers, thereby facilitating complete characterisation of the molecular structures of the original polysaccharides. Chemical methods applied to these oligosaccharides included specific degradations by periodate oxidation and acid-, alkali- or enzyme- catalysed hydrolysis, products being identified by methylation analysis (involving the extensive use of gas-liquid chromatography coupled to mass spectrometry) and spectroscopic studies (mass and n.m.r.). During the course of these investigations it became apparent that the structures of the intact oligosaccharides (containing six or seven sugar residues) could be determined almost entirely from spectroscopic analysis, chiefly by detailed two-dimensional n.m.r. studies involving the use of high field spectrometers and the application of homo- and heteronuclear shift correlated spectroscopy, the sequence of sugar units being confirmed by mass spectrometric analysis of the permethylated derivatives. Methylation analysis of the oligosaccharides derived from Klebsiella serotype K36 proved that the glucuronic acid residue is linked through 0-2, and not 0-4 as published by others; this finding was corroborated during characterisation of the monomeric oligosaccharide by mass- and n.m.r. spectroscopy. Bacteriophage φ64 was shown to cleave the cognate K64 exopolysaccharide by a β-elimination process; the resulting hex-4-enuronic acid, present as a terminal group in the derived oligosaccharide was fully characterised by hydrogenation and g.l.c.-m.s. of acetylated products, and by detailed n.m.r. studies including long-range heteronuclear experiments. Finally the structure of the heptasaccharide repeating unit of the Klebsiella K71 capsular polysaccharide was established by spectroscopic analysis of the oligosaccharides derived by bacteriophage φ71 cleavage of the polymer; features of the proposed structure were confirmed by chemical degradation studies performed on the native polysaccharide.
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    Factors affecting the separation of organic compounds by gas liquid chromatography
    (1965) Hagens, Rodger Graham Robertson; Leisegang, E C; Stephen, A M
    The objects of this thesis have been threefold. In the first place it has been an investigation of the factors which influence retention time in an attempt to predict the retentions of new compounds. Secondly it has been an investigation into the conditions for the best separation of diastereoisomers, and thirdly it has been an application of gas chromatography in the study of the forces of intermolecular interaction in solution. To carry out these investigations, the retention times of twenty five monofunctional and bifunctional compounds have been determined on four stationary liquids at 130°C. It was found that by dividing the retention time of a solute by that of a hypothetical normal hydrocarbon of the same molar volume of the solute, a functional group constant was obtained. By means of this constant, termed the Group Retention Factor, it was found that it is possible to predict the retention of bifunctional compounds. The separation of diastereoisomeric pairs is discussed in terms of the Group Retention Factors of the functional groups on the diastereomers. The molar volume is also utilized in order to develop an equation by means of which the ratio of retention times on two solvents can be related to the forces of interaction between solute and solvent.
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    The separation of diastereoisomers by gas-liquid chromatography
    (1966) Nurok, David; Stephen, A M
    The preparation and the gas chromatographic behaviour of several series of diastereoisomeric compounds is described. The series are the following: (1) Alkyl lactates, {2) Afkyl á-alkanoyloxypropionates, (3) Dialkyl esters of 2,3-butanediol, (4) Di-(haloacetic) esters of 2 1 3-butanediol and (5) á,â-Diols. Chromatography was performed at various temperatures and a variety of stationary phases was used. Capillary columns were used for chromatography of series 1 and 2 whereas packed columns were used for series 3 to 5. It was found that for all of the above series nearly all of th compounds could be separated into diastereoisomers. The separability was found to be a function of the chemical structure of the compounds, of the polarity of the stationary phases and of the temperature at which chromatography was performed.
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    Some non-cellulosic b-D-Glycans from plant sources
    (1987) Mabusela, Wilfred Thozamile; Stephen, A M
    The structures of some non-cellulosic β D-Glycans from three plant sources have been investigated and each was found to be characterised by linked D-pyranosyl a main chain consisting of β -(1-44)- sugars. The polysaccharides were, however, different in structural features in a manner apparently related to their respective locations within the organs of the plants concerned. The polysaccharides were isolated and purified using standard fractionation methods including chromatographic techniques and selective precipitation methods. Structural information was obtained by employing techniques such as methylation analysis (involving use of gas liquid chromatography mass spectrometry), optical rotation measurements, mass spectrometry and n.m.r. spectroscopy on the original polysaccharides and on degraded products obtained by methods such as acid- or enzyme-catalysed hydrolysis and Smith degradation.
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    Steroidal saponins and sapogenins from Agapanthus praecox Willd
    (1970) Mathew, George Ernest Albert; Stephen, A M
    Agapanthin, C₅₁H₈₄O₂₄, a steroidal saponin, isolated from rhizomes of Agapanthus praecox Willd., yielded, on acid hydrolysis, the steroidal sapogenin agspanthagenin and galactose and rhamnose in the molecular ratio of 3:l. The structure assigned to agapanthagenin has been confirmed oh the basis of further synthetic and spectroscopic evidence. The structure of a closely associated steroidal sapogenin, praecoxigenin, C₂₇H₄₀O₄ , has been partially elucidated.
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    Structural Investigations of Acacia and Other Polysaccharides
    (1968) Woolard, Graham Robert; Stephen, A M
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    Structural investigations of Acacia and other polysaccharides
    (1968) Woolard, Graham Robert; Stephen, A M
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    Structural investigations of the polysaccharide gum exudates from Brabeiurn Stellatifoliurn and Grevillea Robusta
    (1971) Van der Bijl, Pieter; Stephen, A M
    The structures of the polysaccharide gum exudates from Brabeium stellatifolium and Grevillea robusta, both belonging to the family Proteaceae, have been investigated and compared. A general similarity between these two polysaccharide gums, both of high molecular weight, was apparent, although techniques such as methylation analysis, Smith-degradation and partial acid hydrolysis revealed small differences in fine structure. Brabeium stellatifolium gum was examined in somewhat more detail. In a study of hydrolysis of the gum by acid the degradation process was followed by gel chromatography. The elution curves obtained at different stages of hydrolysis demonstrated the production of fragments of preferred molecular size, and permitted analysis of the degradation pattern in terms of the fission of peripheral and of internal linkages. Hydrolysis of methylated Brabeium stellatifolium g·um, followed by separation of the resulting methyl ethers on a cellulose column, gave results in good agreement with those obtained on semi-quantitative GLC analysis of the methanolysate of the methylated gum. In Smith-degradation studies of both Brabeium stellatifolium and Grevillea robusta gums the conditions generally used for mild acid hydrolysis of the reduced, oxidised polysaccharides were modified to ensure complete cleavage of acetal linkages. Both gums yielded Smithdegradation products exhibiting a high degree of polyrnolecularity. The relative decreases in weight - average molecular weight M w, accompanying successive Smith-¬ degradations were similar for the two gums, A further point of resemblance was the unusual resistance to periodate oxidation of D-mannose and uronic acid residues in both gums.
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    Studies in molecular structure of plant polysaccharides : exudates from Encephalartos species
    (1990) Vogt, Daphne Constance; Stephen, A M
    This project forms part of a general programme in which the molecular structure of plant polysaccharides, particularly those that occur as gummy exudates from trees, are studied. Variations in the molecular structure of the polysaccharides are linked with the taxonomy of the plant concerned; this link with taxonomy is of particular interest where the cycads are concerned as these plants originated during the Mesozoic era and can therefore be considered as "living fossils". The exudate gums from six different species of the genus Encephalartos were surveyed, and the detailed molecular structures of the gums from two of the species, viz. E. friderici-guilielmi and E. longifolius were examined. Of particular interest in the gums from this genus is the glucuronomannoglycan core structure. The determination of the core structure is made particularly difficult in this case by its being surrounded by a complex structure of sugars and uronic acid residues.
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    Syntheses related to the preparation of substituted glycosyl derivates of pyrrolo (3,2-f) quinolines : spectroscopic studies of acetylated glycosylamine derivatives
    (1970) Magnin, Anthony Alan; Stephen, A M
    The synthesis of 25 acetylated glycopyranosylamine derivatives (15 of which are newly reported here) from substituted sugar derivatives and amines is described. Eleven different methods were employed in these syntheses. Of the compounds prepared, 15 are substituted glycosylindoline derivatives. The stereospecific synthesis of 1-(2,3,4,6-tetra-0-acetyl-β-D-glucopyranosyl)indoline from 3,4,6-tri-0-acetyl-α-D-glucopyranose l,2-(ethyl orthoacetate) and indoline is reported for the first time. The dehydrogenation of 10 substituted glycopyranosylindoline derivatives to the corresponding glycopyranosylindole derivatives (of which 7 are newly reported) by two methods, is described.
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    The chemistry of acacia gums
    (1954) Charlson, Alexander Jacob; Nunn, J R; Stephen, A M