Fluorimetric determination of intra- and extracellular free amino acids in the microalgae Tetraselmis gracilis (Prasinophyceae) using monolithic column in reversed phase mode.
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 micromol/L) to 12% for Ile (0.10 micromol/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).
Penteado
José Carlo Pires
JC
Departamento de Química Fundamental, Instituto de Química, Universidade de São Paulo, São Paulo, SP, Brazil.
Rigobello-Masini
Marilda
M
Liria
Cleber Wanderlei
CW
Miranda
M Terêsa Machini
MT
Masini
Jorge Cesar
JC
D000596:Amino Acids / Q000032:analysis*; D000837:Anion Exchange Resins / Q000737:chemistry; D000460:Chlorophyta / Q000737:chemistry / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D005470:Fluorometry / Q000295:instrumentation / Q000379:methods*; D012623:Seawater
D000596:Amino Acids; D000837:Anion Exchange Resins