Analysis of phenolic acids by non-aqueous capillary electrophoresis after electrokinetic supercharging

Electrokinetic supercharging (EKS), a new and powerful on-line preconcentration method for capillary electrophoresis, was utilized in non-aqueous capillary electrophoresis (NACE) to enhance the sensitivity of phenolic acids. The buffer acidity and concentration, leader and terminator length and electrokinetic injection time were optimised, with the optimum conditions being: a background electrolyte of 40 mM Tris-acetic acid (pH 7.9), hydrodynamic injection of 50 mM ammonium chloride (22 s, 0.5 psi) as leader, electrokinetic injection of the sample (180 s, -10 kV), hydrodynamic injection of 20 mM CHES (32 s, 0.5 psi) as terminator, before application of the separation voltage (-25 kV). Under these conditions the sensitivity was enhanced between 1333 and 3440 times when compared to a normal hydrodynamic injection with the sample volume <3% of the capillary volume. Detection limits for the seven phenolic acids were in the range of 0.22-0.51 ng/mL and EKS was found to be 3.6-7.9 times more sensitive than large-volume sample stacking and anion selective exhaustive injection for the same seven phenolic acids.     

Electrokinetic supercharging in CE for the separation and preconcentration of barbiturate drugs in urine samples

Three barbiturate drugs, barbital, phenobarbital, and secobarbital were separated and analyzed by electrokinetic supercharging. The influence of different parameters on electrokinetic supercharging performance was evaluated using both univariated and multivariated optimization processes. The parameters studied were sample pH, concentration, and length of the leading and terminating electrolytes, electrokinetic injection of the sample and composition and hydrodynamic injection of the solvent plug. The leading electrolyte (50 mM NaCl) was hydrodynamically injected (50 mbar × 120 s) prior to the sample that was adjusted to pH 9.6 and electrokinetically injected at ?8.5 kV for 300 s. The terminating electrolyte (100 mM of 2‐(cyclohexylamino) ethanesulphonic acid) was then hydrodynamically injected (50 mbar × 140 s). The results showed that this strategy enhanced detection sensitivity around 1050‐fold compared with normal hydrodynamic injection, providing detection limits ranging between 1.5 and 2.1 ng/mL for standard samples with good repeatability in terms of peak area (values of relative standard deviation, %RSD < 3). The applicability of the optimized method was demonstrated by the analysis of human urine samples spiked with the studied compounds at different concentration levels and further liquid–liquid extraction step. The estimated detection limits obtained in the urine samples extract ranged between 8 and 15 ng/mL.     

Online preconcentration by electrokinetic supercharging for separation of endocrine disrupting chemical and phenolic pollutants in water samples

Online preconcentration using electrokinetic supercharging (EKS) was proposed to enhance the sensitivity of separation for endocrine disrupting chemical (methylparaben (MP)) and phenolic pollutants (2‐nitrophenol (NP) and 4‐chlorophenol (CP)) in water sample. Important EKS and separation conditions such as the concentration of BGE; the choice of terminating electrolyte (TE); and the injection time of leading electrolyte (LE), sample, and TE were optimized. The optimum EKS‐CE conditions were as follows: BGE comprising of 12 mM sodium tetraborate pH 10.1, 100 mM sodium chloride as LE hydrodynamically injected at 50 mbar for 30 s, electrokinetic injection (EKI) of sample at –3 kV for 200 s, and 100 mM CHES as TE hydrodynamically injected at 50 mbar for 40 s. The separation was conducted at negative polarity mode and UV detection at 214 nm. Under these conditions, the sensitivity of analytes was enhanced from 100‐ to 737‐fold as compared to normal CZE with hydrodynamic injection, giving LOD of 4.89, 5.29, and 53 μg/L for MP, NP and CP, respectively. The LODs were adequate for the analysis of NP and CP in environmental water sample having concentration at or lower than their maximum admissible concentration limit (240 and 2000 μg/L for NP and CP). The LOD of MP can be suitable for the analysis of MP exists at mid‐microgram per liter level, even though the LOD was slightly higher than the concentration usually found in water samples (from ng/L to 1 μg/L). The method repeatabilities (%RSD) were in the range of 1.07–2.39% (migration time) and 8.28–14.0% (peak area).     

Electrokinetic supercharging focusing in capillary zone electrophoresis of weakly ionizable analytes in environmental and biological samples

Five non‐steroidal anti‐inflammatory drugs, naproxen, fenoprofen, ketoprofen, diclofenac and piroxicam, were separated and analyzed by electrokinetic supercharging in CZE. Three different setups of the ITP technique were assayed for the separation and preconcentration of these five non‐steroidal anti‐inflammatory drugs. For the setup that gave the best results, we evaluated the influence of different parameters on separation and preconcentration efficiency such as sample pH, concentration of the leading stacker, BGE composition, electrokinetic injection time, composition and hydrodynamic injection of the solvent plug and of the terminating stacker. In the selected setup, the BGE (10 mM Na₂B₄O₇ + 50 mM NaCl in 10% of MeOH aqueous solution) contained the leading electrolyte while the terminating electrolyte, hydrodynamically injected after the sample (50 mbar×12 s), was 50 mM of CHES. Prior to sample injection at (700 s at −2 kV) a short plug of MeOH (50 mbar ×3 s) was hydrodynamically injected. The results show that this strategy enhanced detection sensitivity 2000‐fold compared with normal hydrodynamic injection, providing detection limits of 0.08 μg/L for standard samples with good repeatability (values of relative standard deviation, %RSD < 1.03%). Method validation with river water samples and human plasma demonstrated good linearity, with detection limits of 0.9 and 2 μg/L for river water samples and human plasma samples, respectively (as well as satisfactory precision in terms of repeatability and reproducibility).     

Analysis of flavonoids by capillary zone electrophoresis with electrokinetic supercharging

On-line concentration via Electrokinetic Supercharging (EKS) was used to enhance the sensitivity of the capillary electrophoretic separation of the four flavonoids naringenin, hesperetin, naringin and hesperidin. Separation conditions, including the background electrolyte pH and concentration, the length and choice of terminator and the electrokinetic injection time were optimized. The optimum conditions were: a background electrolyte of 30 mM sodium tetraborate (pH 9.5) containing 5% (v/v) of methanol, electrokinetic injection of the sample (130 s, -10 kV) followed by hydrodynamic injecting of 100 mM 2-(cyclohexylamino)ethanesulfonic acid (CHES) (17 s, 0.5 psi) as terminator, and separation with -20 kV. Under these conditions the four flavonoids could be separated with a sample-to-sample time of 15 min and detection limits from 2.0 to 6.8 ng mL(-1). When compared to a conventional hydrodynamic injection the sensitivity was enhanced between 824 and 1515 times which is 7.6-16 times higher than other CE methods for the on-line concentration of flavonoids. The applicability of the developed method was demonstrated by the detection of the four flavonoids in an aqueous extract of Clematis hexapetala pall.     

Online preconcentration by electrokinetic supercharging for sensitive determination of berberine and jatrorrhizine in biological samples

Electrokinetic supercharging, a convenient and powerful online preconcentration technique in capillary electrophoresis, was introduced and evaluated for the determination of two alkaloids, berberine and jatrorrhizine, in mice fecal samples for the first time. The method depended on using a bare fused silica capillary (50 cm × 50 μm i.d.) and applying the voltage of 25 kV with UV detection at 205 nm. Parameters that affect the separation and preconcentration efficiency have been optimized. The optimum conditions used were as follows: background electrolyte consisting of 40mM sodium dihydrogenphosphate containing 30% methanol (v/v); hydrodynamic injection of 20mM KCl (50 mbar × 150 s) as the leading electrolyte; electrokinetic injection of the sample (+15 kV, 120 s) followed by the hydrodynamic injection of 30mM dodecyl trimethyl ammonium chloride (50 mbar × 12 s) as the terminating electrolyte. The results showed that the detection sensitivity of berberine and jatrorrhizine was, respectively, improved up 2740- and 2928-fold compared with normal injection, providing limits of detection lower than 3 ng/mL with good repeatability in areas (relative standard deviation < 3%). In summary, the developed method proved its ability in analyzing trace alkaloids in complicated biological samples.     

Highly sensitive analysis of catecholamines by counter‐flow electrokinetic supercharging in the constant voltage mode

Electrokinetic supercharging is one of the most powerful sample‐stacking methods that combines field amplified sample injection and transient ITP. In counter‐flow electrokinetic supercharging, a constant counter pressure is applied during sample injection in order to counterbalance the movement of the injected sample zone. As a result, there will be a pronounced increase in the amount of sample injected and the portion of the capillary available for electrophoresis. In this report, counter‐flow electrokinetic supercharging optimization factors such as the electric field application in the constant voltage and constant current modes, the magnitude of counter pressure, and the terminating electrolyte concentrations were investigated. The enrichments obtained with a 30 min injection of 10 nM catecholamines in 5 mM terminating electrolyte solution in the constant voltage mode applying a counter pressure of 1.3 psi were 41000‐fold for dopamine, 50 000‐fold for norepinephrine, and 32 000‐fold for epinephrine, yielding detection limits of 1.3, 1.4, and 1.2 nM, respectively, with absorbance detection at 200 nm.     

Electrokinetic supercharging for on-line preconcentration of seven non-steroidal anti-inflammatory drugs in water samples

The development of new sensitive methods for the analysis of non-steroidal anti-inflammatory drugs (NSAIDs) in water samples is of great importance. In this work, seven NSAIDs were separated within 9 min using 15 mM sodium tetraborate (pH 9.2) containing 0.1% (w/v) hexadimethrine bromide (HDMB) and 10% (v/v) methanol. Field-amplified sample injection (FASI) was examined and found to improve the detection limits by 200-fold providing detection limits of 0.6-2.0 microg/L, but these are insufficient for the determination of NSAIDs as environmental pollutants in water samples. To improve the sensitivity further, electrokinetic supercharging (EKS) was examined. The optimum EKS method involved hydrodynamic injection leading electrolyte (100 mM NaCl, 30 s, 50 mbar), electrokinetic injection of the sample (200 s, -10 kV) and finally injection of the terminating electrolyte (100 mM 2-(cyclohexylamino) ethanesulphonic acid, CHES, 40s, 50 mbar). With this method, the sensitivity was improved by 2400-fold giving detection limits of 50-180 ng/L. The developed method was validated and then applied to the analysis of wastewater samples from a local sewage treatment plant. The detection limits were found to increase by approximately 10-fold, however, this is still lower than levels previously found in wastewater samples from European and Mediterranean cities. The proposed method has the advantage of simplicity and achieving sensitivity through high-preconcentration power without the use of off-line chromatographic sample cleanup.     

Electrokinetic supercharging with a system-induced terminator and an optimized capillary versus electrode configuration for parts-per-trillion detection of rare-earth elements in CZE

A further improvement of electrokinetic supercharging (EKS) methodology has been proposed, with the objective to enhance the sensitivity of the conventional CZE-UV method down to a single-digit part per trillion (ppt) level. The advanced EKS procedure is based on a novel phenomenon displaying the formation of a zone with an increased concentration of the hydrogen ion, capable to perform the function of a terminator, behind the sample zone upon electrokinetic injection. In combination with a visualizing co-ion of BGE, protonated 4-methylbenzylamine, acting as the leading ion, such system-induced terminator a effected the transient ITP state to efficiently concentrate cationic analytes prior to CZE. Furthermore, to amass more analyte ions within the effective electric field at the injection stage, a standard sample vial was replaced with an elongated vial that allowed the sample volume to be increased from 500 to 900 μL. Alongside, this replacement made the upright distance between the electrode and the capillary tips prolonged to 40.0 mm to achieve high-efficiency electrokinetic injection. The computer simulation was used for profiling analyte concentration, pH, and field strength in order to delineate formation of the terminator during sample injection. The proposed preconcentration strategy afforded an enrichment factor of 80,000 and thereby the LODs of rare-earth metal ions at the ppt level, e.g. 0.04 nM (6.7 ng/L) for erbium(III).     

Electroosmotic flow-balanced isotachophoretic stacking with continuous electrokinetic injection for the concentration of anions in high conductivity samples

An EOF counter-balanced ITP boundary has been used to stack anions from high conductivity samples during continuous electrokinetic injection of the sample. In a polystyrenesulfonate/poly(diallyldimethylammonium chloride) polyelectrolyte coated capillary, the time at which the ITP boundary exited the capillary could be prolonged by balancing the movement of the boundary with the EOF. Using a bis-tris-propane electrolyte, the ITP boundary was removed from the capillary within 7 min, while when using triethanolamine the ITP boundary was still at 30% of the capillary after 2 h of injection. Using these systems, the sensitivity of a mixture of simple organic acids in 100 mM Cl⁻ was improved by 700–800-fold using bis-tris-propane with a whole-capillary injection of the sample and 5 min of electrokinetic injection at +28 kV, and 1100–1300-fold using triethanolamine and 60 min of electrokinetic injection under the same conditions. The potential of the method to be applicable to high conductivity samples was demonstrated by stacking a whole capillary filled with urine spiked with naphthalenedisulfonic acid, with limits of detection 450 times lower than those achievable with a normal hydrodynamic injection.     

Field‐amplified sample injection combined with CE for the enantiodetermination of cathinones in urine samples

This work presents a capillary electrophoresis methodology for the enantiodetermination of cathinones in urine employing a liquid–liquid extraction sample pretreatment. The cathinones were enantioseparated by adding a mixture of 8 mM 2‐hydroxypropyl β‐cyclodextrin and 5 mM β‐cyclodextrin to the background electrolyte, which consists of 70 mM of monosodium phosphate aqueous solution at pH 2.5. Field‐amplified sample injection was used as preconcentration strategy to improve the sensitivity. We studied various parameters that affect this stacking strategy, in particular, the sample solvent and its pH, the presence or absence of a low conductivity solvent plug introduced before the sample injection, the nature and volume of this plug, and the voltage and time of the electrokinetic injection of the sample. The optimum conditions were achieved by injecting a plug of isopropanol:H₂O 50/50 at 50 mbar for 5 s prior to the electrokinetic injection of the sample prepared in an aqueous solution of HCl 10^?6 M. The sensitivity enhancement factors were from 562 to 601 in terms of peak area and from 444 to 472 in terms of peak height. The method was validated by analyzing spiked urine samples, obtaining a linear range of 25 to 1000 ng/mL and limits of detection ranging from 15 to 45 ng/mL.     

Determination of malachite green in fish water samples by cloud‐point extraction coupled to cation‐selective exhaustive injection and sweeping‐MEKC

We have employed a high‐sensitivity off‐line coupled with on‐line preconcentration method, cloud‐point extraction (CPE)/cation‐selective exhaustive injection (CSEI) and sweeping‐MEKC, for the analysis of malachite green. The variables that affect CPE were investigated. The optimal conditions were 250 g/L of Triton X‐100, 10% of Na₂SO₄ (w/v), heat‐assisted at 60°C for 20 min. We monitored the effects of several of the CSEI‐sweeping‐MEKC parameters – including the type of BGE, the concentrations of SDS, the injection length of the high‐conductivity buffer, and the injection time of the sample – to optimize the separation process. The optimal BGE was 50 mM citric acid (pH 2.2) containing 100 mM SDS. In addition, electrokinetic injection of the sample at 15 kV for 800 s provided both high separation efficiency and enhanced sweeping sensitivity. The sensitivity enhancement for malachite green was 1.9×10⁴ relative to CZE; the coefficients of determination exceeded 0.9928. The LOD, based on an S/N of 3:1, of CSEI‐sweeping‐MEKC was 0.87 ng/mL; in contrast, when using off‐line CPE/CSEI‐sweeping‐MEKC the sensitivity increased to 69.6 pg/mL. This proposed method was successfully applied to determine trace amounts of malachite green in fish water samples.     

Far infrared-assisted extraction followed by MEKC for the simultaneous determination of flavones and phenolic acids in the leaves of Rhododendron mucronulatum Turcz

A method based on micellar electrokinetic chromatography with amperometric detection and far infrared‐assisted extraction has been developed for the simultaneous determination of two flavones (rutin and farrerol) and three phenolic acids (syringic acid, vanillic acid, and 4‐hydroxybenzoic acid) in the dried leaves of Rhododendron mucronulatum Turcz., a commonly used traditional Chinese medicine. The effects of some important factors such as the voltage applied on the infrared generator, irradiation time, the concentration of borate and sodium dodecylsulfate (SDS), separation voltage, and detection potential were investigated to acquire the optimum conditions. The detection electrode was a 300‐μm diameter carbon disc electrode. The five analytes could be well separated within 8 min in a 40 cm‐long capillary at a separation voltage of 12 kV in a 50 mM borate buffer (pH 9.2) containing 50 mM SDS. The relationship between peak current and analyte concentration was linear over about three orders of magnitude with the detection limits (S/N=3) ranging from 0.20 to 0.46 μM. The results indicated that far infrared irradiations significantly enhanced the extraction efficiency. The extraction time was substantially reduced to 6 min compared with 3 h for conventional hot solvent extraction.     

Use of dispersive liquid-liquid microextraction for the determination of carbamates in juice samples by sweeping-micellar electrokinetic chromatography

Dispersive liquid?Cliquid microextraction (DLLME) has been proposed for the extraction and preconcentration of 12 carbamate pesticides in juice samples, followed by their determination by micellar electrokinetic chromatography with diode-array detection. To improve sensitivity, an on-capillary sample concentration method based on sweeping has been developed. Also, separations were performed in an extended light path fused-silica capillary; the separation buffer consisted of 100?mM borate and 50?mM SDS (pH?9.0) with 5% acetonitrile. Samples were introduced by hydrodynamic injection, dissolved in the separation buffer, but free of micelles. Several parameters of the DLLME procedure (such as type and volume of extraction and dispersive solvents, pH, salt addition, and extraction time) were optimized. Recoveries obtained for fortified juice samples (banana, pineapple, and tomato) at three different concentration levels, ranged from 78% to 105%, with relative standard deviations lower than 9%. The limits of detection ranged from 1 to 7???g l^?1. Moreover, the method is fast, simple, and environmentally friendly.     

In‐line preconcentration capillary zone electrophoresis for the analysis of haloacetic acids in water

Two in‐line enrichment procedures (large volume sample stacking (LVSS) and field amplified sample injection (FASI)) have been evaluated for the CZE analysis of haloacetic acids (HAAs) in drinking water. For LVSS, separation on normal polarity using 20 mM acetic acid–ammonium acetate (pH 5.5) containing 20% ACN as BGE was required. For FASI, the optimum conditions were 25 s hydrodynamic injection (3.5 kPa) of a water plug followed by 25 s electrokinetic injection (?10 kV) of the sample, and 200 mM formic acid–ammonium formate buffer at pH 3.0 as BGE. For both FASI and LVSS methods, linear calibration curves (r²>0.992), limit of detection on standards prepared in Milli‐Q water (49.1–200 μg/L for LVSS and 4.2–48 μg/L for FASI), and both run‐to‐run and day‐to‐day precisions (RSD values up to 15.8% for concentration) were established. Due to the higher sensitive enhancement (up to 310‐fold) achieved with FASI‐CZE, this method was selected for the analysis of HAAs in drinking water. However, for an optimal FASI application sample salinity was removed by SPE using Oasis WAX cartridges. With SPE‐FASI‐CZE, method detection limits in the range 0.05–0.8 μg/L were obtained, with recoveries, in general, higher than 90% (around 65% for monochloroacetic and monobromoacetic acids). The applicability of the SPE‐FASI‐CZE method was evaluated by analyzing drinking tap water from Barcelona where seven HAAs were found at concentration levels between 3 and 13 μg/L.     

On‐line preconcentration strategy for the simultaneous quantification of three local anesthetics in human urine using CZE

The simultaneous determination of usually employed anesthetics (procaine, lidocaine, and bupivacaine) has been developed and validated using CE with ultraviolet detection at 212 nm. The separation of these three drugs has been achieved in less than 7 min, using a temperature of 25ºC and 25 kV, with a 150 mM citrate buffer (pH 2.5) as BGE. Field‐amplified sample injection (FASI) has been used for on‐line sample preconcentration. Ultrapure water and ACN 50/50 (v/v) mixture gave the greatest enhancement factor when it was employed as an injection solvent. Injection voltage and time were optimized, being 13 kV and 13 s, the optimum values, respectively. To avoid the possible irreproducibility associated with the electrokinetic injection, an internal standard such as tetracaine, was employed. The instrumental detection limits (LOD S/N = 3) for the compounds ranged between 2.6 and 7.0 μg L⁻¹ and the quantitation limits (LOQ S/N = 10) between 37.8 and 55.9 μg L⁻¹. The detection limits obtained in real human urine samples ranged between 55.2 and 83.6 μg L⁻¹ and the quantitation limits between 196.0 and 276.0 μg L⁻¹. The proposed method has demonstrated its applicability to the analysis of these local anesthetics in urine samples without any pretreatment, allowing the rapid determination of these target analytes.     

Determination of organic acids in air by capillary electrophoresis and ion-exclusion chromatography

Summary A capillary electrophoretic (CE) method for the determination of organic acids in the low ppm range is described. The buffer consisted of 5 mM 2,6-pyridinedicarboxylic acid and 0.5 mM cetyltrimethylammonium bromide, pH 5.6. The former served as background electrolyte for indirect UV detection at 200 nm, whereas the latter was used to reverse electroosmotic flow. In <5 min 8 low molecular mass organic acids (oxalic, formic, malonic, glutaric, glycolic, acetic, lactic and propanoic) and two inorganic acids (hydrochloric and sulphuric) were separated. Detection limits for anions tested were 0.04 mg L⁻¹ (lactic acid) to 0.6 mg L⁻¹ (malonic acid). The method was applied to the determination of organic acids in air samples. The CE results when compared with ion-exclusion chromatography (IEC) agreed well. The use of electrokinetic injection in CE proved beneficial for preconcentration of organic acids in real samples. Using electrokinetic injection, preconcentration factors ranging from 14 (hydrochloric acid) to 3 (propanoic acid) were obtained. Presented at Balaton Symposium on High-Performance Separation Methods, Siófok, Hungary, September 1–3, 1999     

Optimization of background electrolyte composition for simultaneous contactless conductivity and fluorescence detection in capillary electrophoresis of biological samples

In this article, optimization of BGE for simultaneous separation of inorganic ions, organic acids, and glutathione using dual C⁴D‐LIF detection in capillary electrophoresis is presented. The optimized BGE consisted of 30 mM 2‐[4‐(2‐hydroxyethyl)piperazin‐1‐yl]ethanesulfonic acid, 15 mM 2‐amino‐2‐hydroxymethyl‐propane‐1,3‐diol, and 2 mM 18‐crown‐6 at pH 7.2 and allowed simultaneous separation of ten inorganic anions and cations, three organic acids and glutathione in 20 min. The samples were injected hydrodynamically from both capillary ends using the double‐opposite end injection principle. Sensitive detection of anions, cations, and organic acids with micromolar LODs using C⁴D and simultaneously glutathione with nanomolar LODs using LIF was achieved in a single run. The developed BGE may be useful in analyses of biological samples containing analytes with differing concentrations of several orders of magnitude that is not possible with single detection mode.     

Pulsed amperometric detection with poly(dimethylsiloxane)-fabricated capillary electrophoresis microchips for the determination of EPA priority pollutants

A miniaturized analytical system for separation and detection of three EPA priority phenolic pollutants, based on a poly(dimethylsiloxane)-fabricated capillary electrophoresis microchip and pulsed amperometric detection is described. The approach offers a rapid (less than 2 min), simultaneous measurement of three phenolic pollutants: phenol, 4,6-dinitro-o-cresol and pentachlorophenol. The highly stable response (RSD = 6.1%) observed for repetitive injections (n > 100) reflects the effectiveness of Au working electrode cleaned by pulsed amperometric detection. The effect of solution conditions, separation potential and detection waveform were optimized for both the separation and detection of phenols. Under the optimum conditions (5.0 mM phosphate buffer pH = 12.4, detection potential: 0.7 V, separation potential: 1200 V, injection time: 10 s) the baseline separation of the three selected compounds was achieved. Limits of detection of 2.2 microM (2.8 fmol), 0.9 microM (1.1 fmol), and 1.3 microM (1.6 fmol) were achieved for phenol, 4,6-dinitro-o-cresol and pentachlorophenol, respectively. A local city water sample and two over-the-counter sore-throat medicines were analyzed in order to demonstrate the capabilities of the proposed technique to face real applications.     

Optimization of the separation of phenolic compounds by micellar electrokinetic capillary chromatography

A group of phenolic compounds including phenolic aldehydes, acids and flavonoids are separated by micellar electrokinetic chromatography (MECC). The influence of buffer (concentration and pH), concentration of sodium dodecylsulphate (SDS) and applied voltage were studied. To increase the selectivity of the separation and the resolution of the solutes organic solvents are added to the separation buffer, the best results were obtained when methanol was used at lower percentages. An optimized buffer (150 mM boric acid (pH 8.5)-50 mM SDS-5% methanol) provides the optimum separation with regard to resolution and migration time. This method was applied to the determination of these compounds in wine samples with good results.