Analysis of amino acids by gas-liquid chromatography: The development of a method and its application

Crichton, Robert R (1966) Analysis of amino acids by gas-liquid chromatography: The development of a method and its application. PhD thesis, University of Glasgow.

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Ion exchange chromatography is the most widely used method for the analysis of amino acids. However, in respect of economy, speed, sensitivity and resolving power, gas liquid chromatography is potentially better than the conventional amino acid analyser. The technique requires that the amino acids be converted into some suitable volatile derivative prior to their separation by GLC. N-acetyl amino acid methyl esters were examined with regard to their suitability for gas chromatographic analysis, but were found to be not sufficiently volatile. When the N-trifluoroacetyl methyl esters were investigated no single column was found on which all of the amine acids could be resolved. However, it was found possible to separate all of the N-trifluoroacetyl methyl esters of the twenty amino acids which occur in proteins by using two columns - one coated with NPGS (neopentyl glycol succinate polyester) and the other coated with a mixture of DEGS (diethylene glycol suecinate) EGSS-X (ethylene glycol succinate polyenter chemically combined with a methyl silicone) and EGSP-Z (ethylene glycol suceinate polyester chemically combined with a methyl silicone). No better resolution of the N-trifluoroacetyl n-propyl or n-butyl esters of the amino acids was obtained when these derivatives were chromatographed on a number of columns than had seen achieved with the N-trifluoroacetyl methyl esters. Taking the response of the flame ionisation detector to N-trifluoroacetyl leucine methyl estor as unity, the response of the detector to the corresponding derivatives of the other amino acids was determined. Good reproducibility in the response factors was observed on both of the columns used for all of the amino acid derivatives with the exception of histidine, tryptophan and cystine. In order to assess the potential of the method, it was applied to hydrolysates of a number of pure peptides and also to mothanolysates of these peptides, prepared with methanol/HCI. Good agreement was obtained by both procedures. However, when the method wasapplied to poptides which had been elated from peptide maps, the results were poor. This was found to be due to the fact that a considerable amount of material was elated from the chromatography cower which produced spurious peaks on the chromatograms. When the method was applied to the analysis of hydrolysates of cytochrome c, insulin and bovine serua albumen, the results obtained were in poor agreement with the results of amino acid analysis carried out on the amino acid analyser. The chromatogram obtained from a methanolysate of cytochrome c gave reduced values for a number of amine acids such as valine and isoleucine. It appears that under the conditions of mothanolysis used, cleavage of bonds involving these amino acids does not proceed to completion. Butanolysis of leucylglyeylglycine was found to be much more rapid than methanolysis of this peptide, and so the N-trifluoroacetyl n-butyl esters of the amino acids map be more suitable derivatives for GLC studies.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: George Leaf
Keywords: Analytical chemistry
Date of Award: 1966
Depositing User: Enlighten Team
Unique ID: glathesis:1966-72250
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 24 May 2019 15:12
Last Modified: 24 May 2019 15:12

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