Hilmer Sørensen

Separation of glucosinolates by high-performance liquid chromatography

The separation of a series of glucosinolates by reversed-phase ion-pair liquid chromatography is reported. A rapid and adequate separation was achieved on a column of Nucleosil 5 C18, with 0.01 M phosphate buffer at pH 7.0-methanol (3:7) containing 0.005 M tetraheptylammonium bromide as mobile phase. The modifier concentration, the nature of the counter-ion and the pH greatly influence the separation of the glucosinolates from each other and from non-ionic impurities. The isolation of the total glucosinolate fraction was performed by a newly developed ion-exchange chromatographic method. By combination with the separation method described here, acidic and basic conditions (which would lead to decomposition of the glucosinolates) are completely avoided. The proposed method is briefly discussed in relation to methods previously used for the isolation, identification and quantitative determination of glucosinolates.

Recent advances in the analysis of glucosinolates

Occurrence and biochemistry of glucosinolates are briefly discussed. The chemistry of intact glucosinolates and their degradation products is considered in relation to the methods used for their determination. Different methods have been used, including ion- exchange chromatography, paper chromatography, high- voltage electrophoresis,1H- and13C- NMR spectroscopy. The quantitative analysis of trimethylsilylated desulfoglucosinolates by gas chromatography and of intact glucosinolates by high performance liquid chromatography is discussed in relation to previously used methods based on the determination of glucosinolate degradation products.

Changes in acetaldehyde, ethanol and amino acid concentrations in broccoli florets during air and controlled atmosphere storage

Abstract Acetaldehyde, ethanol and non-protein bound amino acids were determined in broccoli florets ( Brassica oleracea L. var. Italica cv. Marathon) stored for 7 days at 10°C in air or controlled atmospheres (0.125, 0.25, or 0.5% O 2 alone or in combination with 20% CO 2 , or 20% CO 2 in N 2 ) followed by 2 days aeration. Floret yellowing was visible at days 7 and 9 in air. Low O 2 or low O 2 plus high CO 2 atmospheres delayed yellowing. Acetaldehyde and ethanol concentrations increased as O 2 concentrations decreased with or without 20% CO 2 . Aeration for 2 days generally reduced acetaldehyde and ethanol concentrations. The total free amino acid concentration increased during air-storage from 244 μmol g −1 dry weight at harvest to 573 μmol g −1 dry weight at day 9. Due to severe soft rot development in the broccoli treated with 0.125 and 0.25% O 2 free amino acids were only determined in samples treated with 0.5% O 2 , 0.5% O 2 +20% CO 2 and 20% CO 2 . Amino acid change in samples stored under 0.5% O 2 were similar to those of air-stored broccoli. Storage for 7 days in the CO 2 -containing atmospheres resulted in an increase in non-protein amino acids and a decrease in protein amino acids, although total amino acid content remained the same. Alanine accumulated in 0.5% O 2 or 20% CO 2 in N 2 atmospheres. The non-protein amino acid, γ-aminobutyrate accumulated in 20% CO 2 but its concentration decreased upon aeration, and these changes were associated with similar but opposite changes in glutamate concentrations. Aspartate content also decreased in 20% CO 2 and increased upon aeration. This coincided with the formation of an unidentified amino acid. In broccoli treated with high CO 2 atmospheres, α-decarboxylation seemed to be an important path of metabolic interconversion, however, these reaction pathways were reversible upon aeration.

Determination of thiocyanate, iodide, nitrate and nitrite in biological samples by micellar electrokinetic capillary chromatography

Abstract Micellar electrokinetic capillary chromatography (MECC) has been developed as an efficient method for the determination of the thiocyanate ion, iodide, nitrite and nitrate. The use of various alkyltrimethylammonium ions was found advantageous for MECC compared to negatively charged surfactants, as this resulted in low migration times for the anions and thereby fast analysis. Moreover, the MECC system, using positively charged surfactants (dodecyltrimethylammonium bromide, DTAB), was effective in separating the anions of interest from interfering organic anions present in extracts from biological samples such as milk and blood. Detection of the anions was performed by direct UV. The performance of the developed method was satisfying; however, a low number of theoretical plates for the thiocyanate ion indicated the need for some improvements in this respect. The sensitivity of the method increased with a factor two by using a 75 μm I.D. capillary instead of the 50μm I.D. capillary, whereas use of a high-sensitivity optical cell assembly increased the sensitivity an additional nine times. Repeatability with the 50-μm capillary was satisfying with respect to migration time, relative migration time and normalised peak area (except for the thiocynate ion) of the anions, with relative standard deviations varying between 0.24 and 0.29%, 0.07 and 0.16%, and 1.75 and 2.37%, respectively, whereas some improvements are still needed for the normalised and relative normalised peak areas for the thiocyanate ion (13.50 and 13.91%). Apart from the thiocyanate ion ( r 2 = 0.9748), linearity studies gave correlation coefficients between 0.9962 and 0.9975 for the normalised peak areas.

Factors influencing the separation and quantitation of intact glucosinolates and desulphoglucosinolates by micellar electrokinetic capillary chromatography

Abstract Micellar electrokinetic capillary chromatography methods using cetyltrimethylammonium bromide as a surfactant have been developed for the qualitative and quantitative determination of intact glucosinolates and desulphoglucosinolates. The influence of changes in separation conditions has been investigated. A great number of different intact glucosinolates and desulphoglucosinolates have been used for the development of efficient separation conditions for the closely related, but structurally different, compounds. Repeatability and linearity of the quantitative analyses have been evaluated, and critical parameters have been determined. Rapid and efficient separations are possible for glucosinolates in crude extracts and for mixtures of glucosinolates isolated from seeds and the vegetative parts of plants.

Chemical composition of the potential new oilseed crops Barbarea vulgaris, Barbarea verna and Lepidium campestre

Barbarea vulgaris, Barbarea verna and Lepidium campestre were selected as potential new oilseed crops. To evaluate the nutritional and technological quality of the seeds, the chemical composition was studied. The major constituents found were dietary fibre, crude fat and crude protein. Barbarea contained about 350 g kg−1 dietary fibre, 295 g kg −1 crude fat and 170 g kg−1 crude protein, while Lepidium contained about 400 g kg−1 dietary fibre, 200 g kg−1 crude fat and 190 g kg−1 protein. The amino acid composition was found to be suitable for human consumption when comparison with the amino acid pattern for high quality protein was made. Fatty acid composition was dominated by erucic acid in B vulgaris (28%) and B verna (50%) and by linolenic acid in L campestre (34%). Insoluble dietary fibres were dominated by Klason lignin in both Barbarea and Lepidium. Uronic acid and glucose residues were also found in large amounts. Soluble dietary fibres were dominated by uronic acid, arabinose and galactose residues. The major glucosinolates found were glucobarbarin in B vulgaris (108 μmol g−1), gluconasturtiin in B verna (106 μmol g−1) and sinalbin in L campestre (110 μmol g−1). No cyanogens were found in any of the seeds. © 1999 Society of Chemical Industry

Chromatography and capillary electrophoresis in food analysis

General Aspects of Experimental Biochemistry Buffers and Micelles Binding, Association, Dissociation and Kinetic Extraction of Native LMW and HMW Biomolecules Spectroscopy and Detection Methods Liquid Chromatography Ion-exchange Chromatography High Performance Liquid Chromatography and Fast Polymer Liquid Chromatography Electrophoresis High Performance Capillary Electrophoresis Analytical Determination of Low Molecular Weight Compounds Protein Purification and Analysis Immunochemical Techniques Analysis of Dietary Fibre Appendix: Supercritical Fluid Extraction (SFE) and Supercritical Fluid Chromatography (SFC) Subject Index.

Determination of phenolic carboxylic acids by micellar electrokinetic capillary chromatography and evaluation of factors affecting the method

Abstract Micellar electrokinetic capillary chromatography (MECC) based on cetyltrimethylammonium bromide (CTAB) was developed for separation of individual cinnamic and benzoic acid derivatives. This method was adapted to the separation of the phenolic carboxylic acids from other anions, phenolics and glucosinolates in samples prepared from plant materials. The influence of temperature, voltage, pH, electrolyte and detergent concentrations in the buffer on migration times for the compounds considered, peak areas, resolution and number of theoretical plates were investigated. It is shown that rapid and efficient separations (270 000 plates/m) are possible, even for structural closely related phenolics.

Isolation of glucosinolates and the identification of o-(α-l-rhamnopyranosyloxy)benzylglucosinolate from Reseda odorata

Abstract A method has been developed for the quantitative isolation of glucosinolates by ion-exchange chromatography and high voltage electrophoresis avoiding strongly alkaline and acidic conditions. The compounds were identified by 1 H and 13 C NMR spectroscopy and through the products arising from enzymatic, acid and alkaline hydrolysis. The method is well suited for the isolation and identification of glucosinolates containing aglucone parts which produce non-volatile compounds on enzymatic hydrolysis. The method has been used in the isolation and identification of 2-hydroxy-2-methylpropylglucosinolate from Reseda alba , 2-hydroxy-2-phenylethylglucosinolate from R. luteola and a new glucosinolate, o -(α- l -rhamnopyranosyloxy)benzylglucosinolate, occurring in R. odorata . The glucosinolate content in different parts of this plant has been determined and the metabolism of glucosinolates is briefly discussed.

Supercritical fluid chromatography as a method of analysis for the determination of 4-hydroxybenzylglucosinolate degradation products.

In the present study analytical and preparative supercritical fluid chromatography (SFC) were used for investigation of myrosinase catalysed degradation of 4-hydroxybenzylglucosinolate (sinalbin). Sinalbin occurs as a major glucosinolate in seeds of Sinapis alba L., in various mustards and other food products. The degradation products were identified and quantified by analysis based on a developed SFC method using a bare silica column. Determinations comprised transformation products of sinalbin, produced both during degradation of isolated sinalbin, and during autolysis of meal from S. alba seeds. The conditions in the developed SFC method were used as basis for the preparative SFC procedure applied for isolation of the components prior to their identification by nuclear magnetic resonance (NMR) spectroscopy. Myrosinase catalysed sinalbin hydrolysis resulted in the reactive 4-hydroxybenzyl isothiocyanate as an initial product at pH values from 3.5 to 7.5 whereas 4-hydroxybenzyl cyanide was one of the major products at low pH values. 4-Hydroxybenzyl isothiocyanate was found to disappear from the aqueous reaction mixtures in a few hours, as it reacted easily with available nucleophilic reagents. 4-Hydroxybenzyl alcohol was found as the product from reaction with water, and with ascorbic acid, 4-hydroxybenzylascorbigen was produced.