Marilda Rigobello-Masini

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%.

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.

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

As atividades da Anidrase Carbonica (AC) extra e intracelular foram estudadas na microalga marinha Tetraselmis gracilis (Kylin) Butcher (Chlorophyta, Prasinophyceae) crescendo em cultivos laboratoriais. Durante dez dias de cultivo, determinacoes diarias do pH, numero de celulas, atividades enzimaticas, carbono inorgânico total dissolvido (CID) e suas principais especies CO2 e HCO3- foram feitas. A atividade enzimatica aumentou na medida em que a populacao celular em crescimento retirava carbono inorgânico do meio de cultivo. A concentracao de dioxido de carbono decresceu rapidamente, especialmente no terceiro dia do cultivo, quando um significante aumento na atividade enzimatica intracelular foi observado. A concentracao de bicarbonato teve seu maior decrescimo no meio de cultivo no quarto dia, quando a atividade da enzima extracelular teve seu maior aumento, sugerindo seu uso pela alga atraves da atividade da AC. Apos o quarto dia de cultivo, metade das culturas passou a ser aerada com ar atmosferico sem CO2, o que causou um aumento na atividade total e externa da enzima, fazendo com que esses cultivos entrassem na fase estacionaria do crescimento antes que aqueles aerados com ar atmosferico normal. O pH do meio foi medido diariamente, aumentando desde o primeiro ate o quarto dia e permanecendo quase constante ate o fim do cultivo. Material algal transferido para o escuro perdeu toda a atividade enzimatica.

Application of modified Gran functions and derivative methods to potentiometric acid titration studies of the distribution of inorganic carbon species in cultivation medium of marine microalgae

This paper presents an evaluation of the modified Gran functions (MGFs) and derivative methods for assessing the concentrations of inorganic carbon species, CO2, HCO3 − and CO3 2− , in the cultivation medium of the marine microalgae Tetraselmis gracilis. Both methods were applied to simulated and experimental potentiometric titration data and were able to detect small variations of inorganic carbon species during 10 days of batch cultivation. The MGF were able to determine the concentration of an additional titratable species with p Kb values between 9.5 and 9.9 that appears in the medium after the 4th day of cultivation. This species may be assigned to carboxylate groups of organic carbon compounds excreted by the algae. Treatment of the titration data with the derivative method did not permit the quantitative determination of the concentration of the additional titratable species, but no interference was observed in the results obtained for the inorganic carbon species.

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.

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).

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.

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.

A voltammetric study on the adsorption of Cd(II) and Zn(II) on marine microalgae Tetraselmis gracilis (Kylin) butcher

Proton and metal binding capacities of living cells of the microalgae Tetraselmis gracilis were determined by potentiometric titration and anodic stripping voltammetry, respectively. Binding of Cd(II) and Zn(II) was studied in seawater (pH 8.2) by additions of either single metallic species or binary mixtures. Computation of metal concentrations considered the cell-metal aggregates as slow diffusing. Adsorption capacities and conditional equilibrium constants were 2.9 ± 0.3 µmol g-1 and 6.9 ± 0.3 L g-1 for Cd(II) and 18.5 ± 0.9 µmol g-1 and 8.8 ± 0.2 L g-1 for Zn(II), respectively. For titrations with binary mixtures of Cd(II) and Zn(II), the adsorption capacity of Cd(II) decreased to 0.14 ± 0.01 µmol g-1 and that for Zn(II) to 15.9 ± 1.1 µmol g-1. The results suggest that binding of Zn(II) inhibits that of Cd(II). Under conditions of depleted Zn(II), the alga surface binds Cd(II), a process that can lead to bioaccumulation.