Milan Hutta

Novel stepwise gradient reversed-phase liquid chromatography separations of humic substances, air particulate humic-like substances and lignins.

RP-HPLC using stepwise gradients of dimethylformamide (DMF) in buffered aqueous mobile phase and a wide-pore (30 nm) octadecylsilica column has been applied to the analysis of soil, peat and air particulate humic and humic-like substances (HSs), as well as lignin, in order to demonstrate the usefulness of the approach for their characterization even at trace concentration level. Tandem combination of spectrophotometric (DAD) and fluorimetric detection was used to get more detailed information on chromatographic behaviour of HSs. The results showed that ten-step gradient can induce distinct features of HSs and lignins. Combination of very good DMF solvating and disaggregating properties for HSs and lignins together with wide pore RP sorbent improves surface interactions of the analytes and suppresses influence of size exclusion effects. Thus it provides reproducibility of characterisation profiles and robustness of the method. Very good reproducibility of retention times (from +/-0.12 to +/-0.36% RSD), of peaks enforced by the step gradient shape supports well defined characterization and/or fractionation of HSs. Evaluated limit of quantitation (S/N = 10) of selected soil humic acid working standard using fluorimetric detection (ex.470 nm/em.530 nm) was 9.7 microg/ml. This corresponds to 0.5 microg of the humic acid per injection. Calculated limit of detection (S/N = 3) was 3.3 microg/ml, what corresponds to 0.15 microg of the humic acid per injection and enabled determination of 0.1% (m/m) humic-like substances in 20 mg of air particulates under defined rules and conditions.

Analysis of asulam in soil by isotachophoresis and liquid chromatography

Abstract Capillary isotachophoresis, preparative capillary isotachophoresis and high-performance liquid chromatography were tested for the determination of the herbicide asulam in soil. Different configurations of analytical systems based on these techniques were evaluated with respect to detection limits, recovery and reliability of quantitative analysis. While capillary isotachophoresis used alone after a simple sample pre-treatment permitted reliable quantitation at concentrations down to 0.5 ppm, the use of preparative capillary isotachophoresis for the sample cleanup before the final isotachophoretic or chromatographic analyses gave, in general, much better results. A combination of preparative and analytical isotachophoresis permitted the reliable quantitation of asulam at fortification levels of 0.1–0.2 ppm with reliable detection at a concentration of 0.02 ppm. Similar results were achieved for the combination of preparative isotachophoresis and liquid chromatography. The recoveries of the complete analytical procedures were in the ranges 81–91% (0.2 ppm) and 95–120% (0.02 ppm).

Liquid chromatographic method development for determination of fungicide epoxiconazole enantiomers by achiral and chiral column switching technique in water and soil

High-performance liquid chromatography (HPLC) in both chiral isocratic and achiral-chiral column switching mode was employed for optimization of separation conditions, separation and determination of fungicide epoxiconazole in real samples. Two enantiomers of commercially available triazole fungicide epoxiconazole (BAS 480 F), first registered in 1993, were resolved for the first time on a microcrystalline cellulose triacetate (MCTA). A low-cost home-packed chiral column (150x3 mm, 15-25 microm, MCTA, Merck) enabled baseline enantiomeric resolution of two enantiomers of the fungicide epoxiconazole produced commercially. The effects of concentration of organic modifiers (methanol, ethanol) in mobile phase, flow-rate and temperature were studied. The isocratic chiral HPLC method allows determination of the enantiomers in tap and surface water within the range 1-1000 mg/l by direct injection (20 microl) of the sample. Using the achiral (C18)-chiral (MCTA) column-switching technique and 1-ml sample volume, injection of 0.050 mg/l of epoxiconazole enantiomers can be conveniently determined by UV detection at 230 nm. The same method applied to methanolic soil extracts allows determination of 0.2 mg/kg of epoxiconazole enantiomers in addition to the other 10 commonly used pesticides in fortified soils.

Identification of Natural Dyestuff in Archeological Coptic Textiles by HPLC with Fluorescence Detection

Abstract High performance liquid chromatography with UV–Vis detection and post-column derivatization with fluorimetric detection was used for analysis of dyes used in Coptic textiles. To obtain the best separation of analyzed compounds composition of mobile phase was optimized. The best results were obtained for 25 mM phosphoric buffer at pH 2.5 with gradient change of methanol concentration (Table 1). As post-column reagents used to enhance fluorescence signal of analyzed compounds, solutions of Al(III), Ga(III), In(III), and Zn(II) were used. The highest increase of fluorescence signal was observed for Ga(III) and 10 mM solution of this ion was used for investigation of plant extracts and extracts from Coptic textiles.

Preparative capillary isotachophoresis as a sample pretreatment technique for complex ionic matrices in high-performance liquid chromatography

Abstract Preparative capillary isotachophoresis (ITP) was studied for sample pretreatment in the high-performance liquid chromatography (HPLC) of ionogenic analytes present in complex ionic matrices (urine and humic substances). Sulphanilate, methyl (4-aminobenzenesulphonyl)carbamate (asulam), 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-methyl-4-chlorophenoxyacetic acid (MCPA) served as model analytes. A high sample load of the ITP pretreatment was achieved by performing the preparative separations in tubes of 2.0 and 1.0 mm I.D. in the column-coupling configuration of the separation unit. The ITP separation according to ionic mobilities was combined with gradient elution HPLC in the ion-suppression mode to achieve highly dissimilar (orthogonal) separation systems in both techniques. The pretreatment provided the sample fraction for the HPLC analysis containing in addition to the analyte (sulphanilate) only ca. 2–3% of the urine matrix (spread along the complete elution profile) when a pair of discrete spacers defined the trapped constituents. Under these conditions the limit of detection for sulphanilate in urine could be reduced by more than two orders of magnitude. A high recovery of the pretreatment procedure [99 ± 1.5% for a 1.7 ppm (w/w) concentration of sulphanilate] was typical. For asulam, 2,4-D and MCPA present in a humic matrix it was shown that the ITP pretreatment may also be effective for multi-residue procedures while favourable analytical characteristics of the pretreatment such as recovery and efficient sample clean-up are maintained.

Analysis of saturated normal fatty acids in hydrocarbon matrices by capillary isotachophoresis

Abstract Separation conditions suitable for the analysis of C l -C 18 saturated normal fatty acids by capillary isotachophoresis were investigated. Operational systems using water-methanol solvent mixtures were suitable only for the separation of C 1 -C 10 acids, whereas a complete resolution of the studied constituents is possible in methanol. Analyses of the acids present in reaction mixtures after the oxidation of alkanoic and/or alkenoic substrates (the substrate or n -butyric acid served as the reaction environment) were carried out without any sample pre-treatment using the operational system proposed in experiments with model mixtures. The time for the analysis ranged from 12 to 25 min, depending on the complexity of the reaction mixture and on the configuration of the separation unit employed.

Some theoretical and practical aspects in the separation of humic substances by combined liquid chromatography methods

Permanent need to understand nature, structure and properties of humic substances influences also separation methods that are in a wide scope used for fractionation, characterization and analysis of humic substances (HS). At the first glance techniques based on size-exclusion phenomena are the most useful and utilized for relating elution data to the molecular mass distribution of HS, however, with some limitations and exceptions, respectively, in the structural investigation of HS. The second most abundant separation mechanism is reversed-phase based on weak hydrophobic interactions beneficially combined with the step gradients inducing distinct features in rather featureless analytical signal of HS. Relatively great effort is invested to the developments of immobilized-metal affinity chromatography mimicking chelate-forming properties of HS as ligands in the environment. Surprisingly, relatively less attention is given to the ion-ion interactions based ion-exchange chromatography of HS. Chromatographic separation methods play also an important role in the examination of interactions of HS with pesticides. They allow us to determine binding constants and the other data necessary to predict the mobility of chemical pollutants in the environment. HS is frequently adversely acting in analytical procedures as interfering substance, so more detailed information is desired on manifestation of its numerous properties in analytical procedures. The article topic is covered by the review emphasizing advances in the field done in the period of last 10 years from 2000 till 2010.

Influence of various biological matrices (plasma, blood microdialysate) on chromatographic performance in the determination of β-blockers using an alkyl-diol silica precolumn for sample clean-up

A HPLC column-switching system with LiChrospher RP-8 ADS precolumn was applied for the determination of beta-blockers (atenolol, pindolol, propranolol) in human plasma. The influence of biological matrices on the changes of the chromatographic parameters such as retention time, peak symmetry, area and selectivity were investigated. After injection of 5 ml plasma a decrease of retention times of the analytes was observed of up to 25% and an increase of asymmetry factors of up to 5%. Peak areas and selectivities were not changed. The observed effect could indicate changes of chromatographic performance caused by contributions of the analytical column or the ADS precolumn. The experiments with microdialysis excluded the contribution of the analytical column. A detailed investigation of experiments have been discussed in this paper.

Isotachophoretic determination of short-chain fatty acids in drinking water after solid-phase extraction with a carbonaceous sorbent

A macroporous carbon sorbent, packed into disposable columns (Separcol-Carb), was investigated for the off-line preconcentration of short-chain fatty acids from drinking water in conjunction with their determination by capillary isotachophoresis (ITP). Of the acids investigated (C1-C9), butyric acid and higher homologues could be enriched into a high degree from samples of drinking water. Their detection limits from the ITP conductivity detector were in the low parts per 10(9) range when an amount equivalent to 8 ml of the sample was taken for analysis. The lowest homologues (C1-C3) were not adsorbed sufficiently to achieve their reasonable enrichment by the sorbent under the working conditions employed (acidification of the sample to pH 2.0). Acetone and diethyl ether were employed for the elution of the adsorbed analytes. The latter was more convenient in the analysis of practical samples as it co-eluted a considerably smaller number of the adsorbed anionic constituents. Octadecyl-bonded silica, evaluated in parallel, was found to be of only very limited utility for the same purpose.

Reversed phase liquid chromatography trace analysis of pesticides in soil by on‐column sample pumping large volume injection and UV detection

The idea of utilization of one hydraulic line of a common commercial HPLC pump for direct on-column sample pumping injection of large sample volumes, 20 mL, was further investigated with the aim to develop multicomponent pesticides trace residues HPLC method in gram soil samples. Target pesticides group involve asulam, atrazine, 2,4-D, PCA, propazine, simazine, 4-chloro-2-methylphenoxyacetic acid, 2-(4-chloro-2-tolyloxy) propionic acid, chlortoluron, metoxuron, epoxiconazole. The results proved the applicability of this approach in experiments with mixtures of analytes at low ng/mL levels. Analysis of 20 mL of soil leachates and extracts of fortified soil samples containing these pesticides at the 10-50 ng/g level (in dry soil) revealed good figures of merit, also in the presence of large excess of humics. LODs achieved by detection at 220 nm evaluated from calibration runs of spiked soil extracts by Hubaux et al. method ranged from 5-12 ng per injected volume. For 20 mL large volume injection it represents 0.25-0.6 ng/mL of diluted soil extract, or 2.5-6 ng/mL of crude extract, or 6-5 ng/g dry soil. Recoveries of pesticides at concentration levels approaching half of maximum allowable concentration of pesticides in soil (100 ng/g) ranged from 85 to 98% with acceptable reproducibility, except asulam and metoxuron.