Studies of lipophilic dextran gels for the chromatographic analysis of steroids

Anderson, Robert Arthur (1973) Studies of lipophilic dextran gels for the chromatographic analysis of steroids. PhD thesis, University of Glasgow.

Abstract

The work described in this thesis is concerned with the preparation of gel stationary phases for liquid chromatography in organic solvents. Sephadex dextran is a crosslinked polymer of D-glucose molecules. It contains many free hydroxyl groups which make it hydrophilic. Substitution of these groups with a suitable organic residue gives a lipophilic .gel which swells in organic solvents and which is well suited for use as a chromatographic stationary phase. Gels were prepared with optically active substituents with the intention that selective phases would be produced which might permit the separation of enantiomers and phytosterols containing epimeric alkyl substituents on the side chain. No satisfactory chromatographic method of analyzing such sterols has yet been reported. A suitable method of attaching substituents (first reported by Sjoali and co-workers) was the BF3-catalysed reaction of an olefin oxide with LE-20, a hydroxypropyl derivative of Sephadex G-25. A chiral oxide, 25,24-oxido-5 (choline was prepared from naturally occurring bile acids. The conditions of the substitution reaction were optimised such that stoichiometric amounts of oxide w'ere incorporated efficiently into the gel, A product containing 405S by weight of hydroxy-cholanyl residues (hydroxy-5 P-cholanyl IiH-20) was obtained. The physical properties of the gel were examined. Solvent regain values, sample recovery from the column, and the effect of sample size on the chromatographic elution profile were determined. The gel was then examined with respect to chromatographic properties in two solvent systems (benzene and methanol-heptane 9tl). These gave rise to straight-phase or reversed-phase gel systems respectively. A model set of compounds, mainly steroids, was used in this evaluation. The gel proved satisfactory as a stationary phase. Quantitative recovery of samples, symmetrical peaks, and an absence of tailing were characteristic of gel chromatograms. The straight-phase system was useful for the analysis of hydroxylic steroids. Alcohols, amines and carboxylic acids were all retarded on the column. Hydrocarbons, esters and ketones all had similar elution volumes and were not markedly retarded on the column. In the reversed-phase system, the order of elution was inverted, and non-polar compounds were retarded on the column relative to polar materials. This system gave rise to separations of substances differing in the hydrocarbon substitution. However, no separations were observed, in either system, of enantiomers or of phytosterols containing diastereomeric side chains. A second derivative was prepared using cyclohexene oxide. This was a model compound for the reaction of more complex alicyclic oxides. The BP3-catalysed substitution reaction was successful and marked the first occasion when an non-terminal olefin oxide had been incorporated into a gel. While generally similar to hydroxy-5P-cholanyl IiH-20, this gel proved useful for the separation of epimeric steroids containing hydroxyl substituents and also 5ci/A-steroids. Pour other alicyclic oxides were reacted with LH-20. Two of these, 2a,5o-oxido-5o-cholestane and endo-2,5-oxidobornane were prepared from optically active starting materials. The gels were evaluated as chromatographic stationary phases. Improved separations of liydroxylic steroid epimers were obtained on hydroxycholestanyl LH-20.. The properties of the four gels were generally similar to hydroxycyclohexyl LH-20. Neither enantiomers nor phytosterol diastereoisomers could he separated on these phases. Some inferences are drawn on the mechanisms of separation on the gels, and suggestions are made for further work. The second section deals with the analysis of the metabolites of cholesterol (labelled with 4-C-cholesterol) in the plant pathogenic fungus, Phytophthora cactorum. The major products were esters of cholesterol and long chain fatty acids, The fatty acid pattern was determined and compared with that of the fungal triglycerides.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: C JW Brooks
• Keywords: Analytical chemistry
• Date of Award: 1973
• Depositing User: Enlighten Team
• Unique ID: glathesis:1973-72837
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 11 Jun 2019 11:06
• Last Modified: 11 Jun 2019 11:06
• URI: https://theses.gla.ac.uk/id/eprint/72837