A novel approach to the analysis of dissolved free amino acids in seawater by high-performance liquid chromatography

Liu, Zhu (1993) A novel approach to the analysis of dissolved free amino acids in seawater by high-performance liquid chromatography. Masters thesis, Memorial University of Newfoundland.

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Abstract

Accurate determination of the dissolved free amino acids (DFAA) in seawater is critical in understanding the nitrogen cycle and marine food web in the ocean. – Traditional ion-exchange chromatographic analysis (IC) is very tedious requiring pre-concentration and desalting steps before analysis. Using conventional o-phthaldialdehyde (OPA) pre-column derivization high-performance liquid chromatography (HPLC), measurements of DFAA concentrations “at time of sampling” is not possible unless an HPLC is on board. An alternative approach to DFAA analysis is HPLC using 9-fluorenylmethyl chloroformate (FMOC) as a fluorescence labelling reagent whereby the amino acids are derivatized (and thus fixed) in the field and brought back for analysis. – The effects of the ionic medium pH, chemical nature and concentrations of the mobile phases on the separation of the FMOC derivatives were investigated. Under optimized HPLC conditions, the twelve of the sixteen amino acid derivative standards were well-resolved using a reversed-phase column. Interference from the FMOC hydrolysis product, FMOH, was significantly minimized. The newly-proposed FMOC derivatization approach was compared with that of the well-established OPA derivatization in terms of stability, precision, sensitivity and recovery. FMOC derivatives were found to be very stable and were more sensitive (lower detection limit) than OPA derivatives. – Comparisons were also made of DFAA measurements in seawater, obtained by FMOC derivatization at time of sampling, and, as the result of sample storage followed by OPA derivatization. Losses of DFAA in stored samples were found to be as high as 200%. – During 1992, various field studies were conducted using the FMOC method for the analysis of DFAA in real seawater. The variable levels of DFAA measured during an annual spring plankton bloom in a coastal region is studied.

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