Improvements in the Separation Capabilities of Sequential Injection Chromatography: Determination of Intracellular Dissolved Free Amino Acid Profiles in Three Taxonomic Groups of Microalgae

Abstract

Dissolved free amino acids (DFAA) in intracellular extracts of marine microalgae can be determined by sequential injection chromatography (SIC). This technique uses portable, low-cost instrumentation but its applications have been limited to short monolithic columns because of components not resistant to high pressures. To develop a SIC method for determination of DFAA by exploring an instrument modified to handle pressures of 1000 psi. The method was based on pre-column derivatisation of the amino acids with o-phthalaldehyde and 2-mercaptoethanol in borate buffer (pH 9.4), separation and fluorimetric detection (λ(excitation)= 340 and λ(emission)= 450 nm). Separation was achieved by stepwise gradient elution using six mobile phases. The first elution step used a mobile phase composed of methanol:tetrahydrofuran:10 mm phosphate buffer (pH 7.2) at a volumetric ratio of 8:1:91. Additional elution steps used mobile phases containing methanol and 10 mM phosphate buffer at ratios of 17.5:82.5, 25:75, 35:65, 50:50 and 65:35. Nineteen chromatographic peaks were observed in a mixture of 20 amino acids. The only complete co-elution was between tryptophan and methionine. Detection limits varied from 0.10 µm for isoleucine to 1.5 µm for lysine. Recoveries from spiked extracts were between 84 and 131%. Resolutions of the amino acid pairs glutamine and histidine, valine and phenylalanine, and isoleucine and leucine were 1.5, 0.75 and 1.3, respectively. The proposed method found different profiles of DFAA among the three species of algae, suggesting its adequacy for metabolic studies.