Journal
JOURNAL OF SEPARATION SCIENCE
Volume 32, Issue 15-16, Pages 2827-2834Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/jssc.200800741
Keywords
Amino acids; HPLC; Microalgae; Monolithic columns
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Funding
- Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)
- Conselho Nacional de Desenvolvintento Cientifico e Tecnologico (CNPq)
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This paper describes the development and application of an RP HPLC method using a C-18 monolithic stationary phase for the separation and quantification of extra- and intracellular amino acids in a batch cultivation of the marine alga Tetraselmis gracilis. Fluorimetric detection was made after separation of the o-phthaldialdehyde 2-mercaptoethanol (OPA-2MCE) derivatives using a binary gradient elution. Separation of 19 amino acids was achieved with resolution >1.5 in about 39 min at a flow rate of 1.5 mL/min. RSD of analyses in seawater medium ranged from 0.36% for Orn (0.50 mu mol/L) to 12% for Ile (0.10 mu mol/L). The main constituents of the intracellular dissolved free amino acids (DFAAs) in the exponential growth phase were arginine (Arg), asparagine (Asn), alanine (Ala), aspartic acid (Asp), glutamic acid (Glu), serine (Ser), glycine (Gly), glutamine (Gln), and leucine (Leu). The major amino acids excreted to the media were valine (Val), Ala, Ser, and Gly. The monolithic phase facilitates the analysis by shortening the separation time and saving solvents and instrumentation costs (indeed conventional HPLC instrumentation can be used, running at lower pressures than those ones used with packed particle columns).
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