K. Bächmann

Determination of inorganic cations and anions in single plant cells by capillary zone electrophoresis

Investigation of the solute transport in plants on a molecular level requires the knowledge of the solute distribution between different cells within a tissue. Capillary electrophoresis is demonstrated as a useful technique for the determination of a number of solutes in single plant cell vacuoles. The procedures of sampling vacuolar sap, dilution of the sample, internal standardization and subsequent injection into the separation capillary are described. Extracted vacuolar sap volumes have been in the range of 20 to 60 pl. For obtained sap volumes >20 pl division of the sample droplet into subsamples is possible. This allows repeated injections into the CE and thus high reliability of the obtained data and, using different CE systems, the possibility of determination of many different species in one vacuolar sample. This is demonstrated with the determination of inorganic cation and anion concentrations in the same vacuole of individual wheat epidermal cells using indirect UV detection. The applicability of CE for vacuolar analysis is illustrated with the identification of intercellular solute patterns between different wheat epidermal cell types.

Separation and direct UV detection of sugars by capillary electrophoresis using chelation of copper(II)

Abstract A new method for the capillary electrophoretic separation of neutral sugars with direct UV detection based on the chelation reaction with copper (II) is demonstrated. Using an electrolyte consisting of copper(II) sulphate and ammonia, UV absorption at 245 nm results from the chelation of Cu(II) by the sugars under alkaline conditions. In addition, the sugar mobility increases with increasing Cu(II) concentration in the electrolyte. Thus, the chelation with Cu(II) provides both separation and direct UV detection of neutral sugars. Detection limits for sugars in the range of 50–100 μ M are achieved. Linearity over two orders of magnitude is observed. Furthermore, using the same electrolyte, the simultaneous detection of amino acids due to complexation with Cu(II) and the indirect detection of inorganic cations is possible.

Determination of tobacco alkaloids in single plant cells by capillary electrophoresis

A new capillary electrophoresis system with direct UV detection for the analysis of the tobacco alkaloids nicotine, nornicotine and anabasine in plant microsamples was developed. An electrolyte containing a high concentration of citric acid to provide good buffer capacity at pH 3.6 was found to be most suitable in terms of sensitivity and separation efficiency. At this low pH the tobacco alkaloids are present in cationic form, showing high mobility and increased UV absorption. This system was used for the analysis of nicotine in single epidermal leaf cells of tobacco plants. Only vacuolar concentrations of nicotine were determined, as the vacuole occupies >95% of the entire volume in epidermal cells. The procedure of sample acquisition and preparation for nicotine analysis of vacuolar samples in the pl range is shown. The results indicate a gradient of nicotine from the leaf base to the tip with higher concentrations present in the cells at the tip. Compared to simultaneously measured bulk leaf samples containing all types of cells, tissues and compartments, the concentrations in epidermal cells are much higher. As nicotine is the major defence substance against insects in tobacco and the epidermis is the most exposed leaf tissue this result is physiologically plausible.

New derivatization method for carboxylic acids in aqueous solution for analysis by capillary electrophoresis and laser-induced fluorescence detection

Abstract A new derivatization method for carboxylic acids in aqueous solution for analysis with capillary electrophoresis and laser-induced fluorescence detection was developed. The derivatization is based on a phase-transfer reaction with 4-aminofluorescein as fluorophore and dicyclohexylcarbodiimide as activating agent. This reaction procedure includes an enrichment factor of the analyte up to 10-fold. The limits of detection of carboxylic acids are in the range of 3–150 nmol/l. An advantage of this system is the indifference against inorganic matrices like carbonate, chloride, etc., up to the mmol/l range. Using this technique for the first time, the measurement of diurnal profiles of carboxylic acids (C5–C9) in ambient air with a time resolution of 1 h is possible.

Coelectroosmotic separations of anions in non-aqueous capillary electrophoresis

Abstract The suitability of hexadimethrine bromide (HDB) as electroosmotic flow (EOF) modifier in non-aqueous capillary electrophoresis was investigated. Using HDB as EOF modifier a reversed EOF resulted, which allows very fast separations of anionic compounds in the coelectroosmotic mode. This is demonstrated for the separation of hydroxy- and dihydroxybenzoic acids in various acetonitrile–methanol mixtures. According to the decreasing ratio of dielectric constant and viscosity the EOF mobility showed a linear decrease with increasing methanol concentration in the electrophoresis medium, whereas the analytes showed non-uniform behaviour on addition of methanol. The EOF reproducibility of EOF migration time was excellent, if the concentration of HDB in the running electrolyte was 0.001% (w/v). With this concentration a reproducibility of

Analysis of solutes and metabolites in single plant cell vacuoles by capillary electrophoresis

Abstract Capillary electrophoresis (CE) is demonstrated as a useful technique for the determination of a large number of solutes and metabolites such as inorganic anions, carboxylic acids, sugars and amino acids in single plant cell vacuoles. Sample volumes in the picoliter range can be extracted from single vacuoles and divided into subsamples for subsequent analyses. The challenges involved with such low volume samples are the low analyte amounts present, which require CE systems with sensitive detection approaches for the different compounds, and the danger of contamination and evaporation, which makes minimized sample handling under an inert protective medium essential. Three different separation and detection approaches were used in this investigation. For the determination of inorganic anions and carboxylic acids in vacuolar samples, indirect UV detection using salicylic acid as background electrolyte provides sufficient sensitivity. Amino acids were derivatised on-column with o -phthaldialdehyde-2-mercaptoethanol, separated by micellar electrokinetic chromatography and detected with fluorescence. For sugars a separation and direct UV detection based on chelation with Cu(II) was employed. The determination of these compounds in single vacuoles of epidermal or mesophyll cells of wheat leaves is shown.

Capillary preconditioning for analysis of anions using indirect UV detection in capillary zone electrophoresis: Systematic investigation of alkaline and acid prerinsing techniques by designed experiments

It is widely accepted in capillary zone electrophoresis, that the use of alkaline prerinse procedures can improve the reproducibility of migration times and of corrected peak areas. In this study we present a systematic investigation of alkaline and acid preconditioning procedures for anion analysis using indirect UV detection by designed experiments according to the methodology of Taguchi. Four frequently used electroosmotic flow modifiers (diethylenetriamine, hexamethonium hydroxide, tetradecyltrimethylammonium hydroxide, and hexadimethrine hydroxide) were examined. The optimized procedures were evaluated with regard to the necessary preconditioning time compared to the analysis time. Furthermore, it was demonstrated that the optimized preconditioning technique could be applied to other indirect detection based CE systems. For all examined electrolyte systems, relative standard deviations below 0.5% for migration times and below 5% for corrected peak areas (n=20) were achieved using automated peak integration without further manual reprocessing.

Discontinuous electrokinetic chromatography of parabens using different substituted resonances as pseudostationary phases.

Resorcarene derivatives, negatively charged even at moderate pH, were synthesized and employed as pseudostationary phases to achieve mobilities exceeding that of the electroosmotic flow. Under these conditions, a discontinuous electrolyte system was developed which allows the separation of four uncharged homologous 4‐hydroxybenzoic esters (parabens) within a zone of resorcarene electrolyte, and the detection of these UV active compounds in a resorcarene‐free zone, free from the high UV background absorbance of the resorcarenes. Resorcarenes, with differently charged functionalities (carboxylate and phosphate groups) to provide the electrophoretic mobility and with alkyl residues of different chain lengths responsible for the chromatographic interactions with the analytes, were tested and compared in terms of mobility and selectivity. Only the resorcarene phosphates exhibited sufficient mobilities at low pH exceeding the mobility of the electroosmotic flow (EOF). Retention factors of the parabens were found to increase with increasing chain length of the alkyl residues attached to the resorcarene. However, maximum selectivity was observed for an intermediate chain length (C8). An equation for the calculation of retention factors in discontinuous electrokinetic chromatography (EKC) is presented.