Determination of selected biogenic amines in red wines by automated on-line combination of capillary isotachophoresis-capillary zone electrophoresis.

A screening analytical method based on an automated on-line combination of capillary isotachophoresis-capillary zone electrophoresis (cITP-CZE) in hydrodynamically closed separation system, equipped with photometric detection at 280 nm, was developed for a routine determination of the selected biogenic amines, namely histamine, 2-phenylethylamine and tyramine, in red wines. The evaluated limits of detection (LODs) were 0.35 mg L(-1) for histamine, 0.33 mg L(-1) for 2-phenylethylamine and 0.37 mg L(-1) for tyramine. The repeatability of the migration time and peak area for histamine were 1.1% and 2.6%, respectively, for 2-phenylethylamine 0.7% and 2.0%, respectively, and for tyramine 0.8% and 2.1%, respectively. The method recoveries were 92.1% for histamine, 96.4% for 2-phenylethylamine and 95.5% for tyramine. The developed automated cITP-CZE-UV method was applied for the determination of histamine, 2-phenylethylamine and tyramine in seven red wine samples originating from Czech Republic.

On-line capillary isotachophoresis-capillary zone electrophoresis analysis of bromate in drinking waters in an automated analyzer with coupled columns and photometric detection.

A new, sensitive, and robust analytical method based on capillary zone electrophoresis with on-line capillary isotachophoresis sample pretreatment (ITP-CZE) using a column-coupling (CC) arrangement of automated capillary electrophoretic analyzer was developed for determination of bromate in different type of drinking water samples. Both columns were provided with contact-less conductivity detectors and in CZE step UV detection at 200 nm wavelength was used. Electroosmotic flow of the buffer solutions was suppressed with the addition of 0.1% or 0.05% (m/v) methylhydroxyethylcellulose into the leading and terminating electrolyte, respectively. Hydrodynamic and electroosmotic flows of the buffer solutions were successfully suppressed and therefore, only the electrophoretic transport of ions was significant. Limit of detection for bromate approaching 0.6 µg/L was achieved. Good repeatabilities of migration time (RSD less than 0.3%) and peak area (RSD less than 4.0%) at concentration level 1 µg/L were obtained. Robustness of proposed ITP-CZE method and validation parameters were evaluated. Developed automated ITP-CZE method was applied to the determination of bromate in drinking water samples with different content of inorganic macroconstituents without the need of further sample preparation.

Analysis of therapeutic peptides in human urine by combination of capillary zone electrophoresis-electrospray mass spectrometry with preparative capillary isotachophoresis sample pretreatment.

The presented study deals with the off-line coupling of preparative isotachophoresis (pITP) with on-line combination of capillary zone electrophoresis with electrospray mass spectrometric detection (CZE-ESI-MS) used for the analysis of therapeutic peptides (anserine, carnosine, and buserelin) in complex matrix (urine). Preparative capillary isotachophoresis, operating in a discontinuous fractionation mode in column-coupling configuration, served as a sample pretreatment technique to separation, and fractionation of mixture of therapeutic peptides present in urine at low concentration level. The fractions isolated by pITP procedure were subsequently analyzed by capillary zone electrophoresis with electrospray mass spectrometric detection. Acetic acid at 200 mmol L(-1) concentration served as background electrolyte in CZE stage and it is compatible with MS detection in positive ionization mode. In pITP fractionation procedure, sodium cation (10 mmol L(-1) concentration) as leading ion and beta-alanine as terminating ion (20 mmol L(-1) concentration) were used. While using CZE-ESI-MS, the limits of detection were 0.18 µg mL(-1) for carnosine, 0.17 µg mL(-1) for anserine and 0.64 µg mL(-1) for buserelin in water and 0.19 µg mL(-1) for carnosine, 0.50 µg mL(-1) for anserine and 0.74 µg mL(-1) for buserelin in 10 times diluted urine, respectively. The cleaning power of pITP sample pretreatment was proved as the peptides provided the higher MS signals at lower concentration levels resulting from the minimized matrix effects. The quality of obtained MS/MS spectra was very good so that they can provide information about the structure of analytes, and they were used for verification of the analytes identities. The pITP pretreatment improved the detection limits of the analyzed therapeutic peptides at least 25 times compared to the CZE-ESI-MS itself.

Some possibilities of an analysis of complex samples by a mass spectrometry with a sample pretreatment by an offline coupled preparative capillary isotachophoresis.

This work deals with an analysis of biologically important compounds in complex matrices using preparative isotachophoresis (pITP) in column coupling configuration as a sample pretreatment technique followed by a direct infusion mass spectrometry with nano-electrospray ionization (DI-nESI-MS). Busereline was chosen as a model analyte, and urine was chosen as an example of complex matrix. In pITP experiments, sodium cation (10 mmol/L concentration) was used as a leading ion and ß-alanine as terminating ion (20 mmol/L concentration). The fractions, obtained by pITP pre-separation with the assistance of the mixture of discrete spacers, were finally analyzed by DI-nESI-MS. It was shown that pITP performed before DI-nESI-MS analysis can significantly simplify complex matrix, and, due to its concentration power, pITP can consequently decrease the concentration limit of detection. The concentration of buserelin in the urine samples analyzed by pITP-DI-nESI-MS was 10 µg/L (reflecting at a 8.10⁻9 mol/L concentration) in our work but from the ion intensities obtained in MS as well as MS/MS analyses, it is clear that this concentration level could be several orders of magnitude lower for reliable detection and identification of buserelin in urine analyzed using pITP with DI-nESI-MS detection.

Preparative isotachophoresis with surface enhanced Raman scattering as a promising tool for clinical samples analysis.

A surface enhanced Raman scattering (SERS) spectrometry is an interesting alternative for a rapid molecular recognition of analytes at very low concentration levels. The hyphenation of this technique with advanced separation methods enhances its potential as a detection technique. Until now, it has been hyphenated mainly with common chromatographic and electrophoretic techniques. This work demonstrates for a first time a power of preparative isotachophoresis-surface enhanced Raman scattering spectrometry (pITP-SERS) combination on the analysis of model analyte (buserelin) in a complex biological sample (urine). An off-line identification of target analyte was performed using a comparison of Raman spectra of buserelin standard with spectra obtained by the analyses of the fractions from preparative isotachophoretic runs. SERS determination of buserelin was based on the method of standard addition to minimize the matrix effects. The linearity of developed method was obtained in the concentration range from 0.2 to 1.5 nmol L(-1) with coefficient of determination 0.991. The calculated limit of detection is in tens of pico mols per liter.

Determination of brominated phenols in water samples by on-line coupled isotachophoresis with capillary zone electrophoresis.

A method for determination of nine brominated phenols as environmental risk compounds was developed by on-line coupled capillary isotachophoresis and capillary zone electrophoresis (ITP-CZE). For ITP step, 1x10(-2) mol L(-1) hydrochloric acid with 3x10(-2) mol L(-1) ammediol pH 9.1 was used as the leading electrolyte, and 3x10(-2) mol L(-1) beta-alanine with 2x10(-2) mol L(-1) sodium hydroxide pH 10.05 was used as the terminating electrolyte. As the background electrolyte for CZE separation, 2.5x10(-2) mol L(-1) beta-alanine with 2.5x10(-2) mol L(-1) lysine pH 9.6 was used. All electrolytes contained 0.05% or 0.1% (m/v) hydroxyethylcellulose to suppress the electroosmotic flow. UV detection at wavelength 220 nm was used. Detection limits in order of tens of nmol L(-1) were achieved. Good repeatability of migration times (less than 0.33% RSD) and good repeatability of peak areas (less than 7.19% RSD) at concentration level 5x10(-8) mol L(-1) were observed. Developed ITP-CZE method was applied to determination of brominated phenols in spiked tap and river water samples.

Analysis of buserelin in urine by online combination of capillary zone electrophoresis with electrospray mass spectrometry.

A fast and precise analysis of the synthetic peptide buserelin in urine using CZE-ESI-MS method has been demonstrated. Formic acid at 50 mmol/L concentration served as background electrolyte in CZE stage and it is compatible with MS detection in positive ionization mode. Two injection modes were tested, i.e. pressure (50 mbar for 5 s) and electrokinetic injection (5 kV for 5 s), of which electrokinetic injection provided better calibration parameters. Buserelin LODs were 0.47 microg/mL in water and 0.63 microg/mL in ten times diluted urine samples using pressure injection, while they were 0.32 microg/mL in water and 0.34 microg/mL in ten times diluted urine samples using electrokinetic injection. Repeatability of buserelin migration times was below 6% (pressure injection mode) and 1% (electrokinetic injection mode). Repeatability of buserelin peak area in SIM mode (m/z=620.5+/-0.5) was less than 12% (pressure injection mode) and 5.8% (electrokinetic injection mode). In this work, no interferences were observed during the analyses of spiked urine samples.

Determination of ammonium, calcium, magnesium, potassium and sodium in drinking waters by capillary zone electrophoresis on a column-coupling chip.

This work deals with simultaneous determination of ammonium, calcium, magnesium, sodium and potassium in drinking waters by capillary zone electrophoresis (CZE) on a column-coupling (CC) chip with suppressed hydrodynamic and electroosmotic transports. CZE separations were carried out in a propionate background electrolyte at a low pH (3.2) containing 18-crown-6-ether (18-crown-6) to reach a complete resolution of the cations. In addition, triethylenetetramine (TETA) coated the inner wall surface of the chip channels. The concentration limits of detection (cLOD) for the studied cations ranged from 4.9 to 11.5 microg/l concentrations using a 900 nl volume of the sample injection channel. 93-106% recoveries of the cations in drinking waters indicate a good predisposition of the present method to provide accurate analytical results.

Enhancement of analyte detectability in isotachophoresis exploiting spectrophotometric diode-array detection with chemometric data processing.

The potentialities of capillary ITP combined with diode-array detection (DAD) with subsequent chemometric data processing have been investigated in this work. A series of different migration configurations were created using model analytes, interferents and appropriate spacers. Special attention has been paid not only to constituents migrating in fully developed ITP zones but also to the spike mode of ITP migration. The purity assessment and identity confirmation of model analytes migrating in both modes were performed by means of multivariate curve resolution and target transformation factor analysis (TTFA). Their successful applications have revealed a smart way to increase in the analytical information obtained by ITP separation even in the instance of trace analysis.

Possibilities of column coupling electrophoresis provided with a fiber-based diode array detection in enantioselective analysis of drugs in pharmaceutical and clinical samples.

The present work illustrated possibilities of column coupling electrophoresis combined with ionizable chiral selector and diode array detection (DAD) for the enantioselective analysis of trace drugs (pheniramine and its analogs) in pharmaceutical and clinical samples. Isotachophoresis (ITP), on-line coupled with capillary zone electrophoresis (CZE), served as an ideal injection technique (high sample load capacity, narrow and sharp drugs zones) of on-line pretreated samples (preseparation, purification and preconcentration of drugs) for the CZE stage. Enhanced (enantio)separation selectivity of CZE with ionizable chiral selector (carboxyethyl-beta-cyclodextrin recognized between drugs enantiomers on one hand as well as between drugs and sample matrix constituents on the other hand) enabled to obtain pure zones of the drugs enantiomers, suitable for their detection and quantitation. DAD in comparison with single wavelength UV detection enhanced value of analytical information verifying purity of drugs enantiomers zones (indicating interferents with different spectra to those of drugs). Obtained results indicated pure zones of interest confirming effective ITP-CZE (enantio)separation process. Distinguishing the trace analytes signals superposed on the baseline noise was provided with sufficient reliability (for this purpose the background correction and smoothing procedure had to be applied to the raw DAD spectra). The proposed ITP-CZE-DAD methods were characterized by favorable performance parameters (sensitivity, linearity, precision, recovery, accuracy, robustness, selectivity) and successfully applied for (i) enantiomeric purity testing of dexbrompheniramine in commercial pharmaceutical tablets and (ii) enantioselective metabolic study of pheniramine in human urine.

Potentialities of ITP-CZE method with diode array detection for enantiomeric purity control of dexbrompheniramine in pharmaceuticals.

The present work illustrates potentialities of on-line combined isotachophoresis-capillary zone electrophoresis (ITP-CZE) separation techniques coupled with on-capillary diode array detector (DAD) for enantiomeric purity testing of drugs in pharmaceuticals. The general advantages of the proposed method are its (i) high selectivity, (ii) low concentration limit of detection (LOD) obtainable, (iii) enhanced sample loadability, and (iv) enhanced reliability. For separation of brompheniramine (BP) enantiomers, serving as model analytes, carboxyethyl-beta-cyclodextrin (CE-beta-CD) was appropriate chiral selector providing complete enantioresolution. Given by a high sample load capacity (30 microl sample injection volume) and preconcentration of the analytes in ITP stage, concentration LOD of levobrompheniramine (LBP), serving as model impurity, was 2.5 ng/ml (8 x 10(-9)mol/l). Such separation and detection conditions enabled to easily determine LBP in samples containing a 10(3) excess of dexbrompheniramine (DBP). DAD detection in comparison with single wavelength detection can enhance value of analytical information when analytes and interferents have different spectra (distinguishing impurities in analyte zone, confirmation of migration positions of migrants). In this context purity of BP zones was confirmed with higher reliability in pharmaceutical sample. Moreover, distinguishing the trace analyte signal superposed on the baseline noise was provided with sufficient reliability (for this purpose the background correction and smoothing procedure had to be applied to the raw DAD spectra). Successful validation and application of the proposed ITP-CZE-DAD method suggest its routine use for the enantiomeric purity testing of pharmaceuticals.

Comparative TOF-SIMS and MALDI TOF-MS analysis on different chromatographic planar substrates.

A comparison is made between two high resolution, surface-based, mass spectrometric methods: time-of-flight secondary ion mass spectrometry (TOF-SIMS) and matrix-assisted laser desorption/ionisation mass spectrometry (MALDI TOF-MS) in indication of abietic and gibberellic acids molecular profiles on different chromatographic thin layers. The analytes were applied to silica gel chromatographic thin layers with SIMS on-line interfacing channel, monolithic silica gel ultra-thin layers, and thin layers specifically designed for direct Raman spectroscopic analysis. Two MALDI matrices were used in this research: ferulic acid and 2,5-dihydroxybenzoic acid. The silica gel SIMS-interfacing channel strongly supported formation of numerous different MALDI MS fragments with abietic and gibberellic acids, and ferulic acid matrix. The most intense fragments belonged to [M-OH](+) and [M](+) ions from ferulic acid. Intense conjugates were detected with gibberellic acid. The MALDI MS spectrum from the monolithic silica gel surface showed very low analyte signal intensity and it was not possible to obtain MALDI spectra from a Raman spectroscopy treated chromatographic layer. The MALDI TOF MS gibberellic acid fragmentation profile was shielded by the matrix used and was accompanied by poor analyte identification. The most useful TOF-SIMS analytical signal response was obtained from analytes separated on monolithic silica gel and a SIMS-interfacing modified silica gel surface. New horizons with nanostructured surfaces call for high resolution MS methods (which cannot readily be miniaturised like many optical and electrochemical methods) to be integrated in chip and nanoscale detection systems.

Enantioselective analysis of pheniramine in urine by charged CD-mediated CZE provided with a fiber-based DAD and an on-line sample pretreatment by capillary ITP.

Application potentialities of CZE on-line coupled with capillary ITP and DAD to the identification and determination of trace concentration levels (microg/L) of pheniramine (PHM) enantiomers and their metabolites present in complex ionic matrices of biological origin (urine) are shown. An enhanced (enantio)selectivity of the CZE separation system obtained by the addition of carboxyethyl-beta-CD (CE-beta-CD) to the carrier electrolyte provided CZE conditions for a reliable identification of similar/identical DAD spectra of structurally related compounds (PHM enantiomers and their metabolites) in clinical urine samples differing in qualitative and quantitative composition of sample matrix constituents. A high sample loadability (a 30 microL sample injection volume), partial sample clean-up (removing macroconstituents from the sample), and preconcentration of the analytes in ITP stage resulted in the decrease of concentration LOD for PHM enantiomers in urine to 5.2 and 6.8 microg/L (2.2 x 10(-8) and 2.8 x 10(-8) mol/L), without using any sample pretreatment technique. The background correction and smoothing procedure applied to the raw DAD spectra provided analytically relevant DAD spectra of PHM enantiomers and their metabolites also when they were present in urine sample (30 microL injection volumes of ten-times diluted urine sample) at a 9 x 10(-) (8) mol/L concentration. DAD spectra of PHM enantiomers present in urine samples matched their reference spectra with reasonable certainties. DAD spectra of PHM metabolites were compared with the reference spectra of PHM enantiomers and a good match was found which indicates the similarities in the structures of enantiomers and their metabolites detected in the urine samples. This fact allows performing the quantitative analyses of PHM metabolites in the urine samples by applying the calibration parameters of PHM enantiomers also for PHM metabolites and the results show the possibilities of using the ITP-CZE-DAD combination for the direct analysis of PHM enantiomers and/or their metabolites in urine without any sample pretreatment. ITP-CZE-DAD method with oppositely charged selector is suggested to use in clinical research as it provides favorable performance parameters including sensitivity, linearity, precision, recovery, and robustness with minimal demands on sample preparation.

Combination of large volume sample stacking and dynamic pH junction for on-line preconcentration of weak electrolytes by capillary electrophoresis in comparison with isotachophoretic techniques.

An on-line preconcentration capillary electrophoresis (CE) technique, which combines a large volume sample stacking with a dynamic pH junction technique, is introduced in this paper. This dynamic pH junction with co-electroosmotic migration is formed between sodium borate pH 9.5 and sodium phosphate pH 2.5 with 150 mM sodium dodecylsulfate (SDS). A full capillary based injection allows determination of weak acidic compounds at ppb concentration levels (achieved LOD for benzoic acid was 11 nmol L(-1)). The proposed preconcentration method was compared with ITP/ITP (LOD 120 nmol L(-1)), ITP/CZE (LOD 740 nmol L(-1)) and a simple CZE method (LOD 23,330 nmol L(-1)). The analytical potential of this method was assessed with juice test samples.

Zone electrophoresis separation of perfluorocarboxylic acids on a chip with conductivity detection.

Perfluorinated carboxylic acids (PFCAs), amphiphiles of anthropogenic origin, are spread worldwide throughout the environment. This work deals with their zone electrophoresis (ZE) separation on a chip with coupled columns and integrated conductivity detection. Analogies with the electrophoretic behavior of PFCAs and fatty acids were employed in a search for electrolyte conditions suitable for their separation. ZE separations in the water-ethanol electrolyte systems, based on differences in the ionic mobilities of the anions of PFCAs, provided favorable resolution and detection conditions of the homologues containing up to 10 carbon atoms in the alkyl chain. Concentration limits of detection of 0.3-6.5 micromol/L were attained for PFCAs (loaded by a 900 nL volume sample injection channel of the chip) under these separation conditions. The material of which the chip was made [poly(methylmethacrylate)] restricted its use in investigations of the separations of higher PFCA homologues as it was damaged by ethanolic and/or methanolic background electrolyte solutions required in experiments with these amphiphilic compounds.

Determination of total sulfite in wine. Zone electrophoresis-isotachophoresis quantitation of sulfate on a chip after an in-sample oxidation of total sulfite.

This work deals with the determination of total sulfite in wine. The determination combines an in-sample hydrogen peroxide oxidation of total sulfite in alkalized wine to sulfate with the separation and quantitation of the latter anion by zone electrophoresis (ZE) on-line coupled with isotachophoresis (ITP) on a column-coupling chip. Sample clean up, integrated into the ITP-ZE separation, eliminated wine matrix in an extent comparable to that provided by a highly selective distillation isolation of sulfite. At the same time, conductivity detection, employed to the detection of sulfate in the ZE stage of the ITP-ZE combination, provided for sulfate the concentration limit of detection corresponding to a 90 microg/l concentration of sulfite in the loaded sample (0.9 microl). Such a detectability allowed a reproducible quantitation of total sulfite when its concentration in wine was 15 mg/l. Formaldehyde binding of free sulfite in wine, included into the pre-column sample preparation, prevented an uncontrolled oxidation of this sulfite form. This step contributed to an unbiased determination of sulfate present in the original wine sample (this determination corrected for the concentration of sulfate determined in the sample after the peroxide oxidation of sulfite to the value equivalent to the total sulfite). The 99-101% recoveries of sulfite, determined for appropriately spiked wine samples, indicate a very good accuracy of the present method. Such a statement also supports excellent agreements of the results of quantitation based on the in-sample peroxide oxidation of the total sulfite (bound sulfite released at a high pH) with those in which this analyte was isolated from wine by distillation (bound sulfite released at a very low pH).

Determination of organic acids in wine by zone electrophoresis on a chip with conductivity detection.

An appropriate combination of separation mechanisms (simultaneous use of differences in pK values, host-guest complexations, and the ionic strength dependences of the actual ionic mobilities) provided zone electrophoresis (ZE) resolution of 22 organic and inorganic acids expected in wines on a polymethylmethacrylate (PMMA) chip with integrated conductivity detection. These separating conditions offered a framework for the ZE determination of organic acids responsible for some important organoleptic characteristics of wines (tartrate, malate, succinate, acetate, citrate, and lactate). The ZE procedure developed in this context is simple and rapid (ca. 10 minutes' analysis time), while affording reproducible migration and quantitation data for the acids. For example, 0.8-2.0% RSD values characterized the migration times of the acids for 25 repeated ZE runs with the same sample carried out in 5 days in the background electrolyte solution prepared freshly on a daily basis, while 3-5% RSD values were typical for the accompanying peak area data. The concentration ranges within which the acids of analytical interest could be determined in one ZE run covered all wine samples included in our study (100-400-fold sample dilutions were needed to work under the conditions corresponding to the validities of the calibration data). 90-110% recoveries of the acids as obtained repeatedly for one of the reference wine samples used in our experiments indicate a good predisposition of the present method to provide accurate analytical results. This statement also supports the results from the determination of the acids in reference wine samples with claimed concentrations of malic (five samples), tartaric (one sample), and lactic (one sample) acids.

Fractionation of glycoforms of recombinant human erythropoietin by preparative capillary isotachophoresis.

This feasibility study deals with the use of preparative capillary isotachophoresis (CITP), operating in a discontinuous fractionation mode, to the separations and isolations of glycoforms of recombinant human erythropoietin (rhEPO). The preparative CITP separations were monitored by capillary zone electrophoresis (CZE) with a hydrodynamically closed separation unit. Such a CZE system, suppressing fluctuations of the migration data linked with fluctuations of EOF and hydrodynamic flow, made possible to evaluate and compare the preparative CITP separations performed within a longer time frame. Preparative CITP, carried out in the separation unit with coupled columns of enhanced sample loadability, separating 100 microg of rhEPO in a run lasting ca. 30 min, gave the production rate higher than 55 ng/s for the rhEPO glycoforms. The preparative separations included valve isolations of the glycoforms from the ITP stack into four or six fractions. Such numbers of the fractions corresponded to typical numbers of the major glycoform peaks as resolved in CZE of rhEPO. With respect to close effective mobilities of the glycoforms and a multicomponent nature of rhEPO, the fractions contained mixtures of glycoforms with the dominant glycoforms enriched 10-100-fold, relative to the original rhEPO sample.

Separation of proteins by zone electrophoresis on-line coupled with isotachophoresis on a column-coupling chip with conductivity detection.

This feasibility study deals with the separations of proteins by an on-line combination of zone electrophoresis (ZE) with isotachophoresis (ITP) on a poly(methylmethacrylate) column-coupling (CC) chip with integrated conductivity detection. ITP and ZE provided specific analytical functions while performing the cationic mode of the separation. ITP served, mainly, for concentrations of proteins and its concentrating power was beneficial in reaching a low dispersion transfer (injection) of the proteinous constituents, loaded on the CC chip in a 960 nL volume, into the ZE separation stage. This was complemented by an electrophoretically driven removal of the sample constituents migrating in front of the focused proteins from the separation system before the ZE separation. On the other hand, ZE served as a final separation (destacking) method and it was used under the separating conditions providing the resolutions and sensitive conductivity detections of the test proteins. In this way, ITP and ZE cooperatively contributed to low- or sub-microg/mL concentration detectabilities of proteins and their quantitations at 1-5 microg/mL concentrations. However, a full benefit in concentration detectabilities of proteins, expected from the use of the ITP-ZE combination, was not reached in this work. Small adsorption losses of proteins and detection disturbances in the ZE stage of separation, very likely due to trace constituents concentrated by ITP, appear to set limits in the detection of proteins in our experiments. The ITP-ZE separations were carried out in a hydrodynamically closed separation compartment of the chip with suppressed hydrodynamic and electroosmotic flows of the electrolyte solutions. Such transport conditions, minimizing fluctuations of the migration velocities of the separated constituents, undoubtedly contributed to highly reproducible migrations of the separated proteins (fluctuations of the migration time of a particular protein were typically 0.5% RSD in repeated ITP-ZE runs).

Determination of free sulfite in wine by zone electrophoresis with isotachophoresis sample pretreatment on a column-coupling chip.

This work deals with the determination of free sulfite in wine by zone electrophoresis (ZE) with on-line isotachophoresis (ITP) sample pretreatment on a column-coupling (CC) chip with conductivity detection. A rapid pre-column conversion of sulfite to hydroxymethanesulfonate (HMS), to minimize oxidation losses of the analyte, was included into the developed analytical procedure, while ITP and ZE were responsible for specific analytical tasks in the separations performed on the CC chip. ITP, for example, eliminated the sample matrix from the separation compartment and, at the same time, provided a selective concentration of HMS before its transfer to the ZE stage of the separation. On the other hand, ZE served as a final separation (destacking) method and it was used under the separating conditions favoring a sensitive conductivity detection of HMS. In this way, ITP and ZE cooperatively contributed to a 900 microg/l concentration detectability for sulfite as attained for a 60 nl load of wine (a 15-fold wine dilution and the use of a 0.9 microl sample injection channel of the chip) and, consequently, to the determination of free sulfite when this was present in wine at the concentrations as low as 3 mg/l. The separations were carried out in a closed separation compartment of the chip with suppressed hydrodynamic and electroosmotic flows. Such transport conditions, minimizing fluctuations of the migration velocities of the separated constituents, made a frame for precise migration and quantitation data as achieved for HMS in both the model and wine samples. Ninety percent recoveries, as typically obtained for free sulfite in wine samples, indicate promising potentialities of the present method as far as the accuracies of the provided analytical results are concerned.