The procedure for reverse phase chromatography used by the
AAGL is described by Kaufman and Manley (1998), and modified for microfossils by Kaufman (2000). The method uses pre-column derivatization with o-phthaldialdehyde (
OPA) together with the chiral thiol, N-isobutyryl-L-cysteine (
IBLC), to yield fluorescent diastereomeric derivatives of chiral primary amino acids. Sample derivatization is performed on-line prior to each injection using an auto-injector. Separation is by C18 stationary phase (Hypersil
BDS, 5 µm) using a linear gradient of aqueous sodium acetate, methanol, and acetonitrile. Detection is by fluorescence (Ex = 230 nm, Em = 445 nm). The procedure for ion exchange liquid chromatography was originated by Hare et al. (1985). The method uses step-wise addition of sodium-citrate buffers of increasing pH and post-column derivitization by
OPA, with detection by fluorescence. Both methods rely on internal spikes of non-protein amino acids to calibrate the concentration of amino acids in each sample.
References
Hare, P.E., St. John, P.A., and Engel, M.H., 1985, Ion-exchange separation of amino acids, in Garrett, G.C., ed., Chemistry and Biochemistry of the Amino Acids: Chapman and Hall, London, 415-443..
Kaufman, D.S., and Manley, W.F., 1998, A new procedure for determining enantiomeric (D/L) amino acid ratios in fossils using reverse phase liquid chromatography: Quaternary Science Reviews (Quaternary Geochronology) 17, 987-1000.
Kaufman, D.S., 2000, Amino acid racemization in ostracodes, in Goodfriend, G., Collins, M., Fogel, M., Macko, S., and Wehmiller, J., eds., Perspectives in Amino Acid and Protein Geochemistry: Oxford University Press, New York, 145-160.
