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     

Comparison of aqueous and nonaqueous carrier electrolytes for the separation of penicillin V and related substances by capillary electrophoresis with UV and mass spectrometric detection

A method for the determination of penicillin V together with its impurities and by-products formed during biosynthesis, using capillary electrophoresis (CE) with UV and electrospray-mass spectrometric (ESI-MS) detection is presented. Aqueous and nonaqueous electrolytes containing 20 mM ammonium acetate were investigated to determine their suitability for the separation of these analytes. These carrier electrolytes were optimized with respect to the pH and the solvent/s used (water, methanol, acetonitrile, ethanol and isopropanol) and it was shown that although the nonaqueous electrolytes offered unique separation selectivities, the best results in terms of selectivity and sensitivity were obtained for the aqueous system. Finally, the applicability of this method for the analysis of a mixture representative of a real fermentation broth was demonstrated using an aqueous carrier electrolyte with both UV and ESI-MS detection.     

On-column labeling technique and chiral CE of amino acids with mixed chiral selectors and UV detection

A novel method has been developed for the on-column labeling of amino acid enantiomers with 9-fluoroenylmethyl chloroformate (FMOC), followed by chiral CE with a binary chiral selector system and UV detection. Efficient labeling was achieved by sequential injection of amino acids, borate buffer, and FMOC labeling solution at 0.2 psi for 6 s. After injection, the sandwich sections were electrically mixed at 250 V/cm for 6 s and allowed to react (electric field-free) at room temperature for 2 min. With this procedure, successful online-labeling and chiral CE separation of 19 pairs of amino acids (AA) have been conducted, giving 17 pairs fully enantioresolved (R(s) = 1.73-5.79) and two pairs partially resolved (Ala, R(s) = 0.39 and Arg, R(s) = 1.15) using a running buffer of 150 mM borate containing 30 mM beta-CD, 30 mM sodium taurodeoxycholate (STDC), and 15% isopropanol (IPA) at pH 9.0. Chiral CE of some mixed pairs was also demonstrated, much the same as using precolumn labeling. Surprisingly, Met, Asp, Asn, Gln, and His gained even higher enantioresolution (up to 2.5%) compared with the case of precolumn labeling. As validated by both artificially prepared solutions and serum samples, the method was applicable to the quantitative determination of AA, with LODs down to 4.0 microM. The method allowed the determination of D-AA at the ratio of 1:100 (D:L).     

Analysis of drying oils used as binding media for objects of art by capillary electrophoresis with indirect UV and conductivity detection

Capillary electrophoresis (CE) was applied to analyse the long-chain fatty acid composition of vegetable oils, and their degradation products formed upon ageing when drying oils are used as binding media. The analytes were detected with contactless conductivity detection (CCD) and indirect UV absorption, both detectors positioned on-line at the separation capillary. The long-chain fatty acids were resolved in a background electrolyte (BGE) consisting of phosphate buffer (pH = 6.86, 15 mM) containing 4 mM sodium dodecylbenzensulfonate, 10 mM Brij 35, 2% (v/v) 1-octanol and 45% (v/v) acetonitrile. As in this system dicarboxylic analytes, the products of oxidative degradation of unsaturated fatty acids, cannot be determined, a suitable background electrolyte was developed by the aid of computer simulation program PeakMaster. It makes use of a 10 mM salicylic acid, 20 mM histidine buffer, pH 5.85, which combines buffering ability with the optical properties obligatory for indirect UV detection. This buffer avoids system eigenpeaks, which are often impairing the separation efficiency of the system. Separation of the dicarboxylic analytes was further improved by a counter-directed electroosmotic flow (EOF), obtained by dynamically coating the capillary wall with 0.2 mM cetyltrimethylammonium bromide. Long-chain fatty acids and their decomposition products could be determined in recent and aged samples of drying oils, respectively, and in samples taken from two paintings of the 19th century.     

Development and validation of a capillary electrophoresis method for the measurement of short-chain organic acids in natural rubber latex

Short-chain organic acid contents in serum of natural latex are interesting to measure and capillary electrophoresis (CE) has proved to be a good tool for their study. In the present work a method has been developed to identify the short-chain organic acids present in sera of natural rubber latex (oxalic, formic, fumaric, aconitic, succinic, malic, glutaric, citric, acetic, glycollic, propionic and quinic acids), the separation was optimised and the quantification method validated. The separation was performed on a CE system with UV detection at 200 nm. The separation was carried out with an uncoated fused-silica capillary (57 cm x 50 microm I.D.) and was operated at -10 kV potential. The separation buffers were prepared with 0.5 M H3PO4, 0.5 mM cetyltrimethylammonium bromide and pH adjusted by adding NaOH to 6.25 except for propionic acid which was better measured at pH 7.00. Validation parameters are adequate and limits of detection range from 0.005 mM to 1.6 mM. Short-chain organic acids were measured with this method in sera of three different types of latex.     

Rapid and sensitive determination of phosphoamino acids in phosvitin by N-hydroxysuccinimidyl fluorescein-O-acetate derivatization and capillary zone electrophoresis with laser-induced fluorescence detection

A method was developed for the determination of phosphoamino acids by capillary zone electrophoresis-laser-induced fluorescence detection (argon ion laser, excitation at 488 nm and emission at 520 nm) using derivatization with N-hydroxysuccinimidyl fluorescein-O-acetate (SIFA). Different variables affecting the derivatization (SIFA concentration, derivatization pH, reaction temperature and reaction time) and the separation (type, pH and concentration of buffer, applied voltage and injection mode) were investigated in detail. The optimized separation conditions were 40 mM boric acid buffer (pH 9.2) for background electrolyte, 25 kV for the separation voltage, 25 degrees C for the capillary temperature and 5 s at 0.5 psi for the sample injection. Under the optimal conditions, the SIFA-labeled phosphoamino acids were fully separated within 7 min. The detection limits ranged from 0.1 to 0.3 nM, which are the lowest values reported for capillary electrophoresis (CE) methods. The proposed methodology allowed the rapid, sensitive and selective determination of phosphoamino acids in hen egg yolk phosvitin by the standard addition method. The recovery of these compounds in real sample was 94.0-103.5%. The developed method surpasses previously published CE methods in terms of detection limit, separation time, stability and simplicity of the electrophoretic procedure.     

Simultaneous determination of inorganic anions and organic acids in amine solutions for sour gas treatment by capillary electrophoresis with indirect UV detection

A new CE method has been developed for the simultaneous determination of selected inorganic anions (bromide, chloride, thiosulfate, nitrite, nitrate, sulfate, thiocyanate, fluoride and phosphate) and organic acids (oxalic, malonic, formic, tartric, acetic, glycolic, propionic, butyric and cyclohexanoic) in amine solutions from sour gas treatment units. An electrolyte composed of 10 mM trimellitic acid, 200 mM Tris (pH 9.0), 0.1% polyvinyl alcohol provides a satisfactory separation of all analytes of interest. The electroosmotic flow is reversed by using hexadimethrine bromide as a semi-permanent positively charged coating, making the electrolyte free of additive. Indirect UV detection at 240 nm is used because of the weak absorbing properties of most of analytes. The addition of 1% diethanolamine in standard mixtures permits to better preserve them, inhibiting potential degradation processes, especially for thiosulfate. The quantification is performed using internal standardization, by which molybdate is used as internal standard. Moreover, the use of relative migration times and the excellent repeatabilities obtained allow unambiguous identification of analytes in real samples by comparison with standard mixture. It has been shown that no significant matrix effect came from the presence of 30% diethanolamine in amine solution samples and the developed method was characterized in terms of calibration linearity and accuracy using recovery tests. In short, the developed method allows the simultaneous and rapid determination, in difficult matrices, of numerous inorganic anions and organic acids characterized by a large range of electrophoretic mobilities.     

Enantioseparation of α‐hydroxy acids by chiral ligand exchange CE with a dual central metal ion system

Using two kinds of central metal ions in a background electrolyte, ligand exchange CE was investigated for the simultaneous enantioseparation of dl ‐malic, dl ‐tartaric, and dl ‐isocitric acids. Ligand exchange CE with 100 mM d ‐quinic acid as a chiral selector ligand and 10 mM Cu(II) ion as a central metal ion could enantioseparate dl ‐tartaric acid but not dl ‐malic acid or dl ‐isocitric acid. A dual central metal ion system containing 0.5 mM Al(III) ion in addition to 10 mM Cu(II) ion in the background electrolyte enabled the simultaneous enantioseparation of the three α‐hydroxy acids. These results suggest that the use of a dual central metal ion system can be useful for enantioseparation by ligand exchange CE.     

Direct sample injection for capillary electrophoretic determination of organic acids in cerebrospinal fluid

Organic acids in cerebrospinal fluid (CSF) are potential diagnostic markers for neurological diseases and metabolic disorders. A capillary electrophoretic (CE) method for the direct analysis, i.e., without any sample preparation, of six organic acids in CSF was developed. A capillary coating consisting of a triple layer of charged polymers (polybrene-dextran sulfate-polybrene) was used in combination with a negative separation voltage, providing fast and efficient analysis of acidic compounds. Separation conditions, such as background electrolyte (BGE) concentration and pH were optimized, and the influence of albumin and sodium chloride was systematically studied using a set of test compounds. With injection volumes of ca. 44 nL, plate numbers of up to ca. 150,000 were obtained with a BGE of 200 mM sodium phosphate (pH 6.0). It appeared that high sodium chloride concentrations in the sample hardly affected the peak width and shape of the organic acids, most probably due to transient isotachophoresis effects occurring in the sample zone. Adverse effects of CSF proteins, which frequently compromise the CE performance, could be effectively minimized by the triple layer coating in combination with rinses of 0.1 M hydrochloric acid. Overall, the developed CE system allowed direct injections of CSF samples, yielding good separation efficiencies and stable migration times (RSDs < 2%) for organic acids. Validation of the method with artificial and real CSF samples showed good linear responses (r > 0.99), and LODs for the organic acids were in the range of 2–8 μg/mL when applying UV detection. RSDs for migration times and peak areas were <2% and <7%, respectively. The applicability of the CE system is shown for the determination of organic acids in CSF samples.     

Highly efficient separation of isomeric epoxy fatty acids by micellar electrokinetic chromatography

A capillary electrophoresis (CE) method has been developed for simple and direct separation of cis- and trans-12,13-epoxy-9(Z)-octadecenoic acid and 9,10-epoxy-12(Z)-octadecenoic acid isomers. Separation was performed in micellar electrokinetic capillary chromatography (MEKC) using a buffer consisting of 25 mM borate (pH 9.20), 10 mM sodium dodecyl sulfate (SDS) and 10% v/v acetonitrile. The key variables, concentrations of SDS and organic modifier, were optimized by the application of a factorial experimental design. The use of a low micellar concentration, just above critical micelle concentration (CMC), in a background electrolyte containing an organic modifier not only made it possible to dissolve and separate highly hydrophobic fatty acid isomers, but also resulted in improved separation efficiency and selectivity. Separation efficiency up to 4 x 10(5) theoretical plates/m was achieved under an optimized condition. Also investigated were the influence of temperature on separation and the effect of organic modifier concentration on the dynamic exchange of the analytes between micelles and the bulk of the buffer solution. Direct UV was applied for detection of the fatty acids.     

Separation of perfluorocarboxylic acids using capillary electrophoresis with UV detection

A capillary electrophoretic method with UV detection for separation and quantitation of perfluorocarboxylic acids (PFCAs) from C6-PFCA to C12-PFCA has been developed. The optimization of measurement conditions included the choice of the most appropriate type and concentration of buffer in the background electrolyte (BGE), as well as the type and the content of an organic modifier. The optimal separation of investigated PFCAs was achieved with 50 mM phosphate buffer and 40% isopropanol in the BGE using direct UV detection. The optimum wavelength for direct UV detection was optimized at 190 nm. For indirect detection, several chromophores were studied. Five mM 3,5-Dinitrobenzoic acid (3,5-DNBA) in 20 mM phosphate buffer BGE and indirect UV detection at 280 nm gave the optimal detection and separation performance for the investigated PFCAs. The possibility of on-line preconcentration of solutes by stacking has been examined for indirect detection. The detection limits (LODs) determined for direct UV detection ranged from 2 microg/mL for C6-PFCA to 33 microg/mL for C12-PFCA. The LODs obtained for indirect UV detection were comparable to those obtained for direct UV detection.     

Determination of the spectrum of low molecular mass organic acids in urine by capillary electrophoresis with contactless conductivity and ultraviolet photometric detection--an efficient tool for monitoring of inborn metabolic disorders

A mixture of 29 organic acids (OAs) occurring in urine was analyzed by capillary electrophoresis (CE) with capacitively coupled contactless conductivity detection (C⁴D) and UV photometric detection. The optimized analytical system involved a 100 cm long polyacrylamide-coated capillary (50 μm i.d.) and the background electrolyte of 20 mM 2-morpholinoethanesulfonic acid (MES)/NaOH + 10% (v/v) methanol, pH 6.0 (pH is related to the 20 mM MES/NaOH buffer in water). The LOD values obtained by C⁴D for the OAs which do not absorb UV radiation range from 0.6 μM (oxalic acid) to 6.8 μM (pyruvic acid); those obtained by UV photometry for the remaining OAs range from 2.9 μM (5-hydroxy-3-indoleacetic acid) to 10.2 μM (uric acid). The repeatability of the procedure developed is characterized by the coefficients of variation, which vary between 0.3% (tartaric acid) and 0.6% (5-hydroxy-3-indoleacetic acid) for the migration time and between 1.3% (tartaric acid) and 3.5% (lactic acid) for the peak area. The procedure permitted quantitation of 20 OAs in a real urine sample and was applied to monitoring of the occurrence of the inborn metabolic fault of methylmalonic aciduria.     

Determination of sulfophenyl carboxylic acids in agricultural groundwater samples by CE with ultraviolet absorption detection

An analytical method for the determination of six sulfophenyl carboxylic acids, namely (p-sulfophenyl)acetic, 2-(p-sulfophenyl)propionic, 2-(p-sulfophenyl)butyric, 3-(p-sulfophenyl)butyric, 4-(p-sulfophenyl)butyric, and 5-(p-sulfophenyl)valerianic acid, in agricultural irrigation water samples was developed. It involves an SPE procedure, an on-line preconcentration normal stacking mode and subsequent separation and determination using CE with UV detection (CE-UV). p-Sulfobenzoic acid was used as internal standard. The compounds were separated with an uncoated capillary and a 25 mM ammonium acetate/acetic acid buffer solution (pH 5.5) with 2-propanol (30% v/v) and 0.75 mM CTAB. Analyses were run at -25 kV, 25 degrees C, and 100 s of hydrodynamic injection with UV detection at 225 nm. Quantification limits found ranged between 4 and 6 ng/mL. The proposed method was validated using a recovery assay. It was satisfactorily used for the determination of these compounds in groundwater samples to track down the biodegradation of linear alkylbenzene sulfonates in an agricultural soil from the fertile plain of Granada (Spain).     

Analysis of carboxylic acid metabolites from the tricarboxylic acid cycle in Bacillus subtilis cell extract by capillary electrophoresis using an indirect photometric detection method

With a growing interest in metabolome analysis, there is a need for developing robust methods for analysis of intracellular metabolites profiles in real samples like e.g., bacteria cell. Due to their weak absorbance properties, tri- and dicarboxylic acids from TCA cycle (citric, isocitric, 2-oxoglutaric, succinic, fumaric, malic) as well as carboxylic acid metabolites from glycolysis pathway, urea cycle and metabolism of amino compounds (formic, pyruvic, lactic, acetic, glutamic) were analyzed by capillary electrophoresis (CE) with indirect UV detection. Using 4 mM 2,6-pyridinedicarboxylic acid as a highly UV absorbing carrier electrolyte, 0.2 mM cetyltrimethylammonium bromide, 10% ethylene glycol and 10% acetonitrile, pH 3.5, carboxylic acids metabolites were analyzed in Bacillus subtilis cell extract from two different cultures: glucose and malate. CE with an electrokinetic injection mode achieved limits of detection in the range of 13-54 ppb (1.12-10(-7) - 5.96-10(-7) M). The reproducibility and linearity of method was investigated with RSD for migration time less than 1.3% and acceptable correlation coefficients. The optimized CE method was used to compare metabolome content of cell extract derived from two different culture media containing either glucose or malate as a carbon source. The changes in carboxylic acid metabolites profile were observed depending from used culture medium. Carboxylic acid concentrations ranged: in cell extract from malate culture from 59 to 0.5 microM for lactate and citrate, respectively, and in cell extract from glucose culture from 133 to 0.5 microM for glutamate and citrate, respectively. Appropriate concentrations of carboxylic acid in the single bacterium cell were estimated at mM and sub-mM levels.     

Determination of ethyl sulfate--a marker for recent ethanol consumption--in human urine by CE with indirect UV detection

A CE method for the determination of the ethanol consumption marker ethyl sulfate (EtS) in human urine was developed. Analysis was performed in negative polarity mode with a background electrolyte composed of 15 mM maleic acid, 1 mM phthalic acid, and 0.05 mM cetyltrimethylammonium bromide (CTAB) at pH 2.5 and indirect UV detection at 220 nm (300 nm reference wavelength). This buffer system provided selective separation conditions for EtS and vinylsulfonic acid, employed as internal standard, from urine matrix components. Sample pretreatment of urine was minimized to a 1:5 dilution with water. The optimized CE method was validated in the range of 5-700 mg/L using seven lots of urine. Intra- and inter-day precision and accuracy values, determined at 5, 60, and 700 mg/L with each lot of urine, fulfilled the requirements according to common guidelines for bioanalytical method validation. The application to forensic urine samples collected at autopsies as well as a successful cross-validation with a LC-MS/MS-based method confirmed the overall validity and real-world suitability of the developed expeditious CE assay (sample throughput 130 per day).     

Separation of phenolic acids in soil and plant tissue extracts by co-electroosmotic capillary electrophoresis with direct UV detection

Summary A capillary zone electrophoretic method for the analysis of phenolic acids in soil and plant extracts was developed with direct UV detection using a phosphate electrolyte solution. The electrophoretic separation required the phenolic acids to be charged at a pH above their pKa in order to achieve their migration towards the anode. Electroosmotic flow (EOF) was reversed in direction by adding tetradecyltrimethylammonium bromide (TTAB). Factors affecting the separation selectivity, including the buffer pH and EOF modifiers, were investigated systematically. Eight phenolic acids were separated and detected in 10 min using an electrolyte containing 25 mM phosphate, 0.5 mM TTAB and 15% acetonitrile (v/v) at pH of 7.20. Linear plots for the test phenolic acids were obtained in a concentration range of 0.01–1 mM with detection limits in the range of 1.0–7.0 μM. The recoveries ranged from 92.8 to 102.3% in soil and plant tissues samples spiked at 100 μM and the relative standard deviation based on the peak area were ranged 2.0 to 4.5%. The proposed method was used for the determination of phenolic acids in plant tissue and soil extracts with direct injection.     

Determination of α‐hydroxy acids and their enantiomers in fruit juices by ligand exchange CE with a dual central metal ion system

The content of α‐hydroxy acids and their enantiomers can be used to distinguish authentic and adulterated fruit juices. Here, we investigated the use of ligand exchange CE with two kinds of central metal ion in a BGE for the simultaneous determination of enantiomers of dl ‐malic, dl ‐tartaric and dl ‐isocitric acids, and citric acid. Ligand exchange CE with 100 mM d ‐quinic acid as a chiral selector ligand and 10 mM Cu(II) ion as a central metal ion could enantioseparate dl ‐tartaric acid but not dl ‐malic acid or dl ‐isocitric acid. Addition of 1.8 mM Sc(III) ion to the BGE with 10 mM Cu(II) ion to create a dual central metal ion system permitted the simultaneous determination of these α‐hydroxy acid enantiomers and citric acid. The proposed ligand exchange CE was thus well suited for detecting adulteration of fruit juices.     

Capillary electrophoresis method for the analysis of inorganic anions, organic acids, amino acids, nucleotides, carbohydrates and other anionic compounds.

A previously developed capillary zone electrophoresis (CZE) method with indirect UV detection for the simultaneous determination of inorganic and organic anions, amino acids and carbohydrates using 20 mM 2,6-pyridinedicarboxylic acid (PDC) as the background electrolyte was extended to allow determination of 206 anions including those above--mentioned and physiological amino acids, nucleotides, aromatic acids, haloacetic acids, alcohols, phosphorylated saccharides, oxyhalides, metal oxoacids, metal-ethylenediaminetetraacetic acid (EDTA) complexes, forensic anions, Good's buffers and herbicides. Every compound could be analyzed and their electrophoretic mobility determined simply by selecting detection wavelength. This method is simple and universal for anion analysis, and could be readily applied to the simultaneous determination of anionic compounds. In this work, it was used to identify and quantify important anions in sea urchin and sake.     

Analysis of triazolopyrimidine herbicides in soils using field-enhanced sample injection-coelectroosmotic capillary electrophoresis combined with solid-phase extraction

In this work, a combined methodology using off-line solid-phase extraction (SPE), on-line field-enhanced sample injection (FESI) and coelectroosmotic capillary electrophoresis with UV detection (CE-UV) is developed for the trace analysis of five triazolopyrimidine sulfonanilide pesticides (i.e., flumetsulam, florasulam, cloransulam-methyl, diclosulam and metosulam). An adequate background electrolyte (BGE) was obtained for the separation of these pesticides using hexadimethrine bromide (HDB) as electroosmotic flow (EOF) modifier. This BGE consisted of 0.00042% HDB, 11 mM formic acid, 16 mM ammonium carbonate and 2.5 mM alpha-CD solution at pH 7.6. The use of this running buffer together with the FESI preconcentration method provided limits of detection (LODs) in the low microg/L range (i.e., between 13.0 and 31.5 microg/L). The optimized FESI-CE-UV method was combined with off-line SPE using C(18) cartridges and applied to the determination of the selected group of pesticides in soil samples. Recovery percentages ranged between 50 and 84% in these samples with LODs between 18 and 34 microg/kg. This work shows the great possibilities of the combined use of SPE-FESI-CE-UV to improve CE sensitivity allowing the achievement of LODs similar to other analytical techniques as GC or HPLC.     

Direct chiral resolution of tartaric acid by ion-pair capillary electrophoresis using an aqueous background electrolyte with (1R,2R)-(-)-1,2-diaminocyclohexane as a chiral counterion

Chiral resolution of native DL-tartaric acid was achieved by ion-pair capillary electrophoresis (CE) using an aqueous-ethanol background electrolyte with (1R,2R)-(-)-1,2-diaminocyclohexane (R-DACH) as a chiral counterion. Factors affecting chiral resolution and migration time of tartaric acid were studied. By increasing the viscosity of the background electrolyte and the ion-pair formation, using organic solvents with a lower relative dielectric constant, resulted in a longer migration time. The optimum conditions for both high resolution and short migration time of tartaric acid were found to be a mixture of 65% v/v ethanol and 35% v/v aqueous solution containing 30 mM R-DACH and 75 mM phosphoric acid (pH 5.1) with an applied voltage of -30 kV at 25 degrees C, using direct detection at 200 nm. By using this system, the resolution (Rs) of racemic tartaric acid was approximately 1. The electrophoretic patterns of tartaric and malic acids suggest that two carboxyl groups and two hydroxyl groups of tartaric acid are associated with the enantioseparation of tartaric acid by the proposed CE method.