Amino acid analysis with o-phthalaldehyde detection: effects of reaction temperature and thiol on fluorescence yields
Cronin, J.R.; Pizzarello, S.; Gandy, W.E.
Analytical Biochemistry 93(1): 174-179
1979
ISSN/ISBN: 0003-2697 PMID: 434461 Document Number: 144562
Chromatographic separation of amino acids with fluorometric detection of the o-phthalaldehyde-thiol reaction product offers an analytical system of high sensitivity for most amino acids. The fluorescence yield for amino acids having a fully substituted C atom in the .alpha.-position with respect to the amino group can be substantially improved by increasing the temperature of the reaction with o-phthalaldehyde-thiol and by substituting either ethanethiol or methanethiol for the more commonly used .beta.-mercaptoethanol. The analytical sensitivity for these .alpha.-branched amino acids is thus brought into line with that for the other primary amino acids. A comparison of chromatograms obtained at reaction temperatures of 25 and 100.degree. C allows recognition of amino acids of this type in complex mixtures by the substantial increase in fluorescence yield at 100.degree. C.