Enhanced Separation Capability of Sequential Injection Chromatography for Fluorimetric Determination of Intracellular Dissolved Free Amino Acids in Marine Microalgae.

This chapter describes improvements in a sequential injection method to automate the fluorimetric determination of amino acids by pre-column derivatization with o-phthaldialdehyde in presence of 2-mercaptoethanol. Separation is achieved by reversed-phase liquid chromatography in a 50 × 4.6 mm C18 silica-based monolithic column. The method is low-priced, and the separation is performed by stepwise gradient elution using six mobile phases. The mobile phase used for the first elution step is composed of methanol/tetrahydrofuran/10 mM phosphate buffer (pH 7.2) at volumetric ratio 8:1:91. Additional elution steps use mobile phases containing methanol and 10 mM phosphate buffer at volumetric ratios of 17.5:82.5, 25:75, 35:65, 50:50, and 65:35. Nineteen chromatographic peaks are observed in a mixture of twenty amino acids. The only complete co-elution is between tryptophan and methionine. The entire cycle of amino acid derivatization, chromatographic separation, and column conditioning at the end of separation lasts for 30 min. The method is successfully applied to quantify the major intracellular dissolved free amino acids in the marine microalgae Tetraselmis gracilis, Phaeodactium tricornutum, and Synechococcus elongatus.

Improvements in the separation capabilities of sequential injection chromatography: determination of intracellular dissolved free amino acid profiles in three taxonomic groups of microalgae.

INTRODUCTION: 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. OBJECTIVE: To develop a SIC method for determination of DFAA by exploring an instrument modified to handle pressures of 1000 psi. METHOD: 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. RESULTS: 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%. CONCLUSION: 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.

Monolithic columns in plant proteomics and metabolomics.

Since "omics" techniques emerged, plant studies, from biochemistry to ecology, have become more comprehensive. Plant proteomics and metabolomics enable the construction of databases that, with the help of genomics and informatics, show the data obtained as a system. Thus, all the constituents of the system can be seen with their interactions in both space and time. For instance, perturbations in a plant ecosystem as a consequence of application of herbicides or exposure to pollutants can be predicted by using information gathered from these databases. Analytical chemistry has been involved in this scientific evolution. Proteomics and metabolomics are emerging fields that require separation, identification, and quantification of proteins, peptides, and small molecules of metabolites in complex biological samples. The success of this work relies on efficient chromatographic and electrophoretic techniques, and on mass spectrometric detection. This paper reviews recent developments in the use of monolithic columns, focusing on their applications in "top-down" and "bottom-up" approaches, including their use as supports for immobilization of proteolytic enzymes and their use in two-dimensional and multidimensional chromatography. Whereas polymeric columns have been predominantly used for separation of proteins and polypeptides, silica-based monoliths have been more extensively used for separation of small molecules of metabolites. Representative applications in proteomics and in analysis of plant metabolites are given and summarized in tables.

Study of photorespiration in marine microalgae through the determination of glycolic acid using hydrophilic interaction liquid chromatography.

Determination of organic acids in intracellular extracts and in the cultivation media of marine microalgae aid investigations about metabolic routes related to assimilation of atmospheric carbon by these organisms, which are known by their role in the carbon dioxide sink. The separation of these acids was investigated by hydrophilic interaction liquid chromatography (HILIC) using isocratic elution with a mobile phase composed of 70:30 v/v acetonitrile/20 mmol/L ammonium acetate buffer (pH 6.8) and detection at 220 nm. HILIC allowed the determinations of glycolic acid, the most important metabolite for the evaluation of the photorespiration process in algae, to be made with better selectivity than that achieved by reversed phase liquid chromatography, but with less detectability. The concentration of glycolic acid was determined in the cultivation media and in intracellular extracts of the algae Tetraselmis gracilis and Phaeodactylum tricornutum submitted to different conditions of aeration: (i) without forced aeration, (ii) aeration with atmospheric air, and (iii) bubbling with N(2). The concentration of glycolic acid had a higher increase as the cultures were aerated with nitrogen, showing higher photorespiratory flux than that occurring in the cultures aerated with atmospheric air.

Sequential injection chromatography for fluorimetric determination of intracellular amino acids in marine microalgae.

This chapter describes a sequential injection chromatography method to automate the fluorimetric determination of amino acids after precolumn derivatization with o-phthaldialdehyde in presence of 2-mercaptoethanol using reverse-phase liquid chromatography in C(18) silica-based monolithic column. The method is low-priced and based on six steps of isocratic elutions. At a flow rate of 30 µl/s, a 25 mm long-column coupled to 5-mm guard column is capable to separate aspartic acid (Asp), glutamic acid (Glu), asparagine (Asn), serine (Ser), glycine (Gly), threonine (Thr), citrulline (Ctr), arginine (Arg), alanine (Ala), tyrosine (Tyr), phenylalanine (Phe), ornithine (Orn), and lysine (Lys). Under these conditions, histidine (His) and glutamine (Gln), methionine (Met) and valine (Val), and isoleucine (Ile) and leucine (Leu) coelute. The entire cycle of amino acids derivatization, chromatographic separation, and column conditioning at the end of separation lasts 16 min. The method was successfully applied to the determination of the major intracellular free amino acids in the marine green alga Tetraselmis gracilis.

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).

Implementing stepwise solvent elution in sequential injection chromatography for fluorimetric determination of intracellular free amino acids in the microalgae Tetraselmis gracilis.

The concept of sequential injection chromatography (SIC) was exploited to automate the fluorimetric determination of amino acids after pre-column derivatization with o-phthaldialdehyde (OPA) in presence of 2-mercaptoethanol (2MCE) using a reverse phase monolithic C(18) stationary phase. The method is low-priced and based on five steps of isocratic elutions. The first step employs the mixture methanol: tetrahydrofuran: 10 mmol L(-1) phosphate buffer (pH 7.2) at the volumetric ratio of 8:1:91; the other steps use methanol: 10 mmol L(-1) phosphate buffer (pH 7.2) at volumetric ratios of 20:80, 35:65, 50:50 and 65:35. At a flow rate of 10 microL s(-1) a 25 mm long-column was able to separate aspartic acid (Asp), glutamic acid (Glu), asparagine (Asn), serine (Ser), glutamine (Gln), glycine (Gly), threonine (Thr), citruline (Ctr), arginine (Arg), alanine (Ala), tyrosine (Tyr), phenylalanine (Phe), ornithine (Orn) and lysine (Lys) with resolution >1.2 as well as methionine (Met) and valine (Val) with resolution of 0.6. Under these conditions isoleucine (Ile) and leucine (Leu) co-eluted. The entire cycle of amino acids derivatization, chromatographic separation and column conditioning at the end of separation lasted 25 min. At a flow rate of 40 microL s(-1) such time was reduced to 10 min at the cost of resolution worsening for the pairs Ctr/Arg and Orn/Lys. The detection limits varied from 0.092 micromol L(-1) for Tyr to 0.51 micromol L(-1) for Orn. The method was successfully applied to the determination of intracellular free amino acids in the green alga Tetraselmis gracilis during a period of seven days of cultivation. Samples spiked with known amounts of amino acids resulted in recoveries between 94 and 112%.

The profiles of nitrate reductase and carbonic anhydrase activity in batch cultivation of the marine microalgae Tetraselmis gracilis growing under different aeration conditions.

Tetraselmis gracilis, a Prasinophycean alga found in estuaries and in the open ocean, was cultivated under different conditions of aeration, which resulted in variations of inorganic carbon in the medium. Relative growth rates, nitrate reductase and carbonic anhydrase activities were daily determined and correlated to the concentration of nitrate, nitrite, phosphate, inorganic and organic carbon in the media. Nitrate reductase catalyzes the reversible carbon dioxide hydration reaction. The activity profiles of both enzymes during 10 days of cultivation under aeration with air showed an inverse relationship: the maximum in the activity of nitrate reductase coincided with the minimum of carbonic anhydrase activity. An ionizable organic carbon species with pKa in the range of metabolites of the photorespiratory path was found parallel with the increase of carbonic anhydrase activity and the decrease of nitrate reductase activity. The onset of photorespiration is probably one of the factors involved in the simultaneous regulation of these enzymatic processes. Cultures aerated with air containing 5% CO2 showed different profiles for nitrate reductase activity and nitrate uptake.

Extra and intracelular activities of carbonic anhydrase of the marine microalga Tetraselmis gracilis (Chlorophyta)

As atividades da Anidrase Carbônica (AC) extra e intracelular foram estudadas na microalga marinha Tetraselmis gracilis (Kylin) Butcher (Chlorophyta, Prasinophyceae) crescendo em cultivos laboratoriais. Durante dez dias de cultivo, determinacões diárias do pH, número de células, atividades enzimáticas, carbono inorgânico total dissolvido (CID) e suas principais espécies CO2 e HCO3- foram feitas. A atividade enzimática aumentou na medida em que a populacão celular em crescimento retirava carbono inorgânico do meio de cultivo. A concentracão de dióxido de carbono decresceu rapidamente, especialmente no terceiro dia do cultivo, quando um significante aumento na atividade enzimática intracelular foi observado. A concentracão de bicarbonato teve seu maior decréscimo no meio de cultivo no quarto dia, quando a atividade da enzima extracelular teve seu maior aumento, sugerindo seu uso pela alga através da atividade da AC. Após o quarto dia de cultivo, metade das culturas passou a ser aerada com ar atmosférico sem CO2, o que causou um aumento na atividade total e externa da enzima, fazendo com que esses cultivos entrassem na fase estacionária do crescimento antes que aqueles aerados com ar atmosférico normal. O pH do meio foi medido diariamente, aumentando desde o primeiro até o quarto dia e permanecendo quase constante até o fim do cultivo. Material algal transferido para o escuro perdeu toda a atividade enzimática.