A precolumn derivatization procedure for the analysis of marine amino acids with 9-fluorenylmethyl chloroformate and high performance liquid chromatography

Master Thesis


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University of Cape Town

The separation of 20 amino acids has been achieved by gradient elution and reversed phase high performance liquid chromatography employing 9-fluorenylmethyl chloroformate as the precolumn derivatizing reagent. The application of this technique to assess the extent of marine bacterial uptake of amino acids released from kelp has also been determined. The problem of excess reagent reacting with water to form a hydrolysis product has largely been overcome. Pentane extraction of the reagent after amino acid reaction caused the loss of less polar amino acid derivatives. Other factors such as the formation of dilabelled products and the pH dependance of the derivatization reaction have been investigated. The reproducibility between-analyses had a percentage error of 2 - 6%. The stability of the derivatives is about 2 weeks at room temperature. The application to physiological samples and seawater has been demonstrated. The method was applied to the study of kelp release and bacterial uptake of marine amino acids. Other chemical profiles of ammonia, nitrate, total N, particulate C, together with bacterial activity and bacterial density (biomass) were determined to provide correlative profiles to the amino acid values. The experiment was set up with kelp fronds in buckets, some containing antibiotics to halt bacterial activity, and a control bucket with untreated seawater. Alanine is the most dominant amino acid (concentration between 5 and 100 nmol.dm⁻³). Values of glycine, aspartic acid, glutamic acid and arginine have much lower levels. Traces, of histidine, asparagine, cystine, serine and tyrosine appeared near the end of the experiment. It was found that the amino acid concentrations were low compared with the inorganic nitrogen species. The flux of these species was found to be too low to create a substantial response, as the activities are also low compared with normal in-shore regions. In order to infer more from the processes occurring in this study, we would have to increase the experimental time to the order of days.

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