Determination of inorganic anions, carboxylic acids and amino acids in plant matrices by capillary zone electrophoresis

Summary Physiological investigations of solute transport in plants affords knowledge of solute distribution between different tissues. Capillary electrophoresis using indirect UV and laser induced fluorescence (LIF) detection is demonstrated as a useful technique for the simultaneous determination of inorganic anions, amino acids and carboxylic acids in plant samples. Cell sap obtained from plant tissues as well as simple ethanolic or aqueous plant extracts can be analysed directly without any pretreatment. This matrix stability and the very small volumes required allow fast determinations of various compounds in small plant tissue sections. In the case of carboxylic acids, resolution can be optimized using calcium for selective complexation of some of the compounds. Selective and sensitive determination of amino acids is possible using precolumn derivatisation with orthophthaldialdehyde (OPA) and laser induced fluorescence detection. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996     

Investigation of phenolic acids in yacon (Smallanthus sonchifolius) leaves and tubers

Thin-layer chromatographic (TLC) screening of crude extracts of dried leaves and tubers of yacon (Smallanthus sonchifolius, Asteraceae) and products of acid hydrolysis of tubers on the silica gel HPTLC plates using the developing solvents ethyl acetate-formic acid-water (85:10:15, v/v/v) and n-hexane-ethyl acetate-formic acid (20:19:1, v/v/v) proved the presence of chlorogenic, caffeic and ferulic acid. These phenolic acids were isolated from the crude extract of yacon leaves by preparative TLC, and identified after elution by HPLC/MS, as well as by direct injection of the crude extract into the HPLC/MS system. Acid hydrolysis of tubers released the increased amount of phenolic acids (e.g. caffeic acid and ferulic acid), flavonoid quercetin and an unidentified flavonoid, which was detected by TLC analysis. Ferulic acid, isomers of dicaffeoylquinic acid and still an unidentified derivative of chlorogenic acid (Mr = 562) as constituents of yacon leaves and ferulic acid as constituent of yacon tubers are reported here for the first time. These acids gave significant contribution to the radical scavenging activity detected directly on the TLC plate sprayed with 1,1-diphenyl-2-picrylhydrazyl (DPPH).     

High-performance liquid chromatographic analysis of secoisolariciresinol diglucoside and hydroxycinnamic acid glucosides in flaxseed by alkaline extraction

A HPLC method was developed for the analysis of secoisolariciresinol diglucoside (SDG) and hydroxycinnamic acid glucosides in milled defatted flaxseed flour. Direct extraction by 1 M NaOH for 1 h at 20 degrees C resulted in a higher yield than that obtained by hydrolysis of alcoholic extracts. An internal standard, o-coumaric acid, was used and the method was found to be easy, fast, and with good repeatability. On dry matter basis, different samples of flaxseeds varied considerably in their content of (+)-SDG (11.9-25.9 mg/g), (-)-SDG (2.2-5.0 mg/g), p-coumaric acid glucoside (1.2-8.5 mg/g), and ferulic acid glucoside (1.6-5.0 mg/g).     

Determination of Cu and Ni in plants by microdialysis sampling: Comparison of dialyzable metal fractions with total metal content

Microdialysis sampling is presented as an in situ sampling and sample clean-up technique with the potential to be used for determination of metals in plant suspension. Suspensions prepared from ultra pure water and flowers of a Blepharis aspera plant species obtained from a Cu and Ni mineralized site were sampled for Cu and Ni by microdialysis sampling after incorporating an optimal 0.05% (w/v) composition of humic acid in the perfusion liquid. Acid digestion of the plant samples was employed for quantification of Cu and Ni by flame atomic absorption spectrometry. All microdialysis sampling experiments were carried out at room temperature under quiescent conditions using a concentric type of microdialysis probe and electrothermal atomic absorption spectrometry was employed for metal quantification. The versatility of microdialysis as an in situ sampling and sample clean-up technique was demonstrated by the ability to sample Cu and Ni from the complex matrices of plant suspensions. Linear relations between the concentrations of Cu and Ni determined after microdialysis sampling and acid digestion were established and the constant concentration ratios of the metals were found to be 0.0138 and 0.0440 respectively for Cu and Ni thus demonstrating the potential that microdialysis sampling has in prediction of metal concentrations in plant suspension after direct relation with the acid digestion method.     

Uncertainties of gas chromatographic measurement of troublesome pesticide residues in apples employing conventional and mass spectrometric detectors

In recent years the declaration of estimated uncertainty of measurement has become an integral part of analytical results. This study presents the assessment of results generated within the analysis of selected pesticides represented by carbamates, pyrethroides and azoles, residues of which may be found in treated apples. Multiresidue method used for analysis of spiked samples (residues at levels 0.040–0.163 mg/kg) consisted of (i) ethyl acetate extraction, (ii) GPC clean-up and (iii) identification/quantification of residues by GC. Procedures utilizing either conventional (electron-capture, nitrogen–phosphorus) or mass-selective detectors (quadrupole and ion trap analyzer) were evaluated. The results generated through alternative strategies of uncertainty estimation (“bottom-up”, “top-down”) were compared.

Using the “bottom-up” approach uncertainty of extraction which comprises two components—(i) repeatability of extraction and (ii) uncertainty of extraction recovery was shown to represent the main source of combined standard uncertainty (values of uncertainty of extraction for tested pesticides ranged from 4.6% to 21.6%). On the other hand, uncertainties associated with the GC calibration (uncertainties of weighing and diluting standards, uncertainties of purity of standards) were not so important (most of them did not exceed 2%). Combined standard uncertainties associated with the described analytical method ranged for individual compounds from 9.3% to 24.3%. Similar values of combined standard uncertainties were obtained using the alternative “top-down” approach.     

薄层层析法测定纤维素材料水解液中混合糖的研究

提出了用薄层层析测定纤维素材料水解液混合糖中单糖和各种多糖的含量，以硅胶G为固体吸附剂，以玻璃为支撑物，制成薄层析。以V正丁醇：V37％乙酸：V水＝3：2：2为展开剂，以苯胺－草酸为显色剂，分离效果良好，可完成定性和定量分析任务。     

Characterisation of selenium compounds in rye seedling biomass using <Superscript>75</Superscript>Se-labelling/SDS-PAGE separation/γ-scintillation counting,and HPLC-ICP-MS analysis of a range of enzymatic digests

In the present study, selenium-enriched plant biomass was investigated to evaluate the ability of rye seedlings to take up, and assimilate, inorganic selenium. Two different analytical approaches were used. Electrophoretic separation (SDS-PAGE) of proteins extracted from ⁷⁵Se-labelled biomass was used to investigate the biotransformation of selenite into organic forms of the element. Ion-pair chromatography coupled with ICP-MS detection was chosen for the analysis of selenium species, enzymatically extracted from the plant biomass. The results of three enzymatic hydrolysis procedures and three sequential enzymatic extractions procedures are compared. The most effective single extraction was proteolysis (using protease type XIV), giving an overall extraction efficiency of 48%. However, for combinations of enzymes, the most effective was cellulase (Trichoderma viride) followed by sequential extraction of the solid pellet using protease type XIV, giving an extraction efficiency of 70%. The complementary data from the electrophoretic fractionation of proteins, and the HPLC separation of Se-species in the proteolytic digests, reveal the existence of large number of selenium-containing compounds in the rye seedling plant biomass. The results showed the complete biotransformation of inorganic selenium into organic forms during germination of the rye seedlings. HPLC-ICP-MS analysis of extracts from the plant biomass did not show the presence of selenate or selenite. At the time of this study, the lack of suitable organic-MS facilities meant that it was not possible to characterise them fully. However, the data does show that a combination of different enzymes, rather than just the commonly-used protease, should be considered when developing an extraction strategy for selenium in different food types to those already reported in the literature.     

Rapid identification of volatile compounds in aromatic plants by automatic thermal desorption — GC-MS

Summary Thermal desorption is a valuable method for the fractionation of plant volatile components, which can be carried out on-line with GC analysis. The use of coupled GC-MS affords additional qualitative information, of special interest for plant species whose composition has not been previously studied. Some examples of the application of automatic thermal desorption, coupled to GC-MS to the identification and characterization of volatile components of plants of different families are given.     

Determination of heavy metals in soil, mushroom and plant samples by atomic absorption spectrometry

The concentrations of heavy metals in the soil, mushroom and plant samples collected from Tokat, Turkey have been determined by flame and graphite furnace atomic absorption spectrometry after dry ashing, wet ashing and microwave digestion. The study of sample preparation procedures showed that the microwave digestion method was the best. Good accuracy was assured by the analysis of standard reference materials. The relative standard deviations for all measured metal concentrations were lower than 10%. In all cases, quantitative analytical recoveries ranging from 95 to 103% were obtained. Metal accumulation factors were calculated for mushroom and plant samples. High ratio of plants to soil cadmium, zinc and copper concentrations indicate that these elements are accumulated by mushrooms. Results obtained are in agreement with data reported in the literature.     

Characterisation of natural polysaccharides (plant gums) used as binding media for artistic and historic works by capillary zone electrophoresis

The monosaccharide constituents of plant gums were separated by capillary electrophoresis at pH 12.1 and detected with indirect UV absorbance. The plant gums investigated were gum arabic, gum acacia, gum tragacanth, cherry gum and locust bean gum (carob gum). The monosaccharides obtained after hydrolysis with 2M trifluoroacetic acid and lyophilisation of the hydrolysate were arabinose, galactose, mannose, rhamnose, xylose, fucose, and glucose, and the two sugar acids galacturonic and glucuronic acid, in accordance with the literature. They were separated in a background electrolyte consisting of NaOH to adjust the pH, 20 mM 2,6-pyridinedicarboxylic acid as chromophore for detection and 0.5 mM cetyltrimethylammonium bromide as additive to reverse the electroosmotic flow. Based on their electropherograms, the plant gums could be identified by their typical composition (depicted in a decision scheme) as follows: a peak of glucuronic acid, together with that of rhamnose, is indicative for gum arabic. Peaks of galacturonic acid and fucose point to gum tragacanth. Locust bean gum shows a major peak for mannose (with the concomitant galactose peak in ratio 4-1), whereas a glucuronic acid and a mannose peak together with a prominent arabinose peak indicates cherry gum. The method was applied to identify the plant gums in samples like watercolours and in several paint layers like gum tempera or those with egg white or drying oils as additives. Artificial aging experiments of thin layers of gum arabic on paper or glass carried out with UV-A radiation (366 nm) did not result in changes of the saccharide patterns, in contrast to the simultaneously conducted aging of a drying oil layer.