Direct determination of sialic acids in serum by capillary electrophoresis with UV detection

Summary A novel method for the determination of N-acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NGNA) has been developed using high-performance capillary electrophoresis with UV detection at 195 nm, without pre or post-column derivatisation. The acids were separated in a 50-cm, fused-silica capillary (50μ i.d, 45.5-cm effective length) with Na₂B₄O₇−Na₂HPO₄ buffer. The detection limit for NANA is a concentration of 9.6×10⁻⁶ M or, in terms of mass:3.879×10⁻¹⁴ mol (39 fmol). This method is applicable to determination of NANA in normal human serum. The results were also compared with those of the colorimetrie method.     

Using capillary electrophoresis for the determination of organic acids in Port wine

The simultaneous separation and determination of organic acids in several samples of white and red Port wines was performed by capillary zone electrophoresis using indirect UV detection with 2,6-pyridinedicarboxylic acid as a background electrolyte buffer. Operational parameters like migration time, temperature, voltage and capillary length were optimized. Sixteen samples of red wine and four samples of white wine were used to analyze for tartaric, malic, lactic, succinic and acetic acids using glyoxylic acid as the internal standard. The method is rapid, sensitive and quantitative, and time-consuming sample preparation, such as solid-phase extraction or liquid-liquid extraction procedure, is not required.     

Determination of phenolic acids by capillary zone electrophoresis and HPLC

Selected phenolic acids are determined by capillary zone electrophoresis and HPLC, each using UV detection. The optimised CZE background electrolyte contained 50 mM acetic acid, 95 mM 6-aminocaproic acid, 0.1% polyacrylamide, 1% polyvinylpyrrolidone, and 10% methanol. Twelve phenolic acids (gallic, p-hydroxybenzoic, 3,4-dihydroxybenzoic, vanillic, syringic, o-coumaric, p-coumaric, caffeic, sinapic, ferulic, salicylic and chlorogenic) were separated within 10 minutes. Chromatographic separation of these phenolic acids was carried out on an Eclipse XBD C8 column using a mobile phase gradient (acetonitrile / methanol / water / 0.1% phosphoric acid); all were separated within 25 minutes. Electrophoretic and chromatographic determinations of ferulic and chlorogenic acids were compared on barley, malt, and potato samples. The methods’ characteristics were: linearity (1–20 mg ml and 0.2–4 mg ml⁻¹), accuracy (recovery 94 ± 5% and 96 ± 4%), intra-assay repeatability (4.1% and 3.5%), and detection limit (0.2 and 0.02 mg ml⁻¹).      

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.     

Analysis of some anhydrides as their corresponding acids by capillary electrophoresis

Summary The potential of capillary electrophoresis for the indirect determination of anhydrides was demonstrated by the analysis of five closely-related anhydrides as their corresponding acids. Direct analysis of such substances is generally difficult because of their susceptibility to hydrolysis. In this work anhydrides were first hydrolyzed in water, hydrolysis being monitored using micellar electrokinetic chromatography (MEKC) which separated neutral anhydrides and anions of corresponding acids. After hydrolysis, separation of the acids was quickly achieved within two minutes by reversed electroosmotic-flow, capillary electrophoresis (REF-CE), with UV detection at 210 nm.     

Chiral separation of halogenated amino acids by ligand-exchange capillary electrophoresis

The chiral separation of halogenated amino acids by ligand-exchange CE is described. Halogenated amino acids attracted increasing interest in recent years because of their physiological activities. Different chiral selectors, as there are L-4-hydroxyproline, L-histidine, and N-alkyl derivatives of L-4-hydroxyproline in form of their copper(II) complexes, are compared for their chiral recognition ability for halogenated amino acids. The influence of various parameters, such as selector concentration, pH, organic modifier, and field strength, on the resolution was investigated. All halogenated amino acids investigated were baseline-separated under optimized conditions.     

Separation of flavonoids and phenolic acids from propolis by capillary zone electrophoresis

The simultaneous determination of twelve different flavonoids, pinocembrin, acacetin, chrysin, rutin, catechin, naringenin, galangin, luteolin, kaempferol, apigenin, myricetin, and quercetin, two phenolic acids, cinnamic acid and caffeic acid, and one stilbene derivative, resveratrol, in propolis extracts used in medicine has been investigated by capillary zone electrophoresis (CZE). With a buffer constituted by sodium tetraborate 30 mM, pH 9.0, and 15 kV applied voltage, the 15 polyphenols were separated on an uncoated fused-silica capillary within 40 min using normal polarity. Under the experimental conditions used, a linear relationship was calculated between the CZE migration times and the molecular weight of polyphenols' expression of the increasing amount of their hydroxyl groups and polarity. Regression equations revealed a linear relationship (correlation coefficients > 0.97) between the peak area of each polyphenol species and their concentration, from 6 to 120 ng. The levels of analytes in three different propolis extracts, ethanolic, aqueous-ethanolic and aqueous-glycolic, used to prepare various commercial medicinal products, were determined. The aqueous-ethanolic propolis extract showed a great percentage of caffeic acid, galangin, quercetin, and chrysin, whilst the ethanolic preparation was composed of a great amount of resveratrol, chrysin, and caffeic acid. On the contrary, the aqueous-glycolic propolis preparation was composed of approx. 11% of caffeic acid and a low amount of the other identified flavonoids due to the presence of approx. 85% of nonidentified compounds. CZE represents a valuable method for the qualitative and quantitative assay of the most relevant polyphenol components of propolis, representing an alternative to obtain typical fingerprints of propolis and a reliable identification of a large number of propolis polyphenolic species.     

Simultaneous determination of uranium carbide dissolution products by capillary zone electrophoresis

Separation and simultaneous determination of a number of organic acid anions (oxalate, mellitate, trimellitate and benzoate) and U(VI) with direct UV detection is developed for analysis of uranium carbide (UC) dissolution products by capillary zone electrophoresis (CZE). Reverse polarity mode is used. It is found that complex formation of U(VI) with carbonate, used as a carrier electrolyte, allows U(VI) to be determined, as negatively charged species, in a single run with organic acid anions. Some parameters such as pH value, composition of electrolyte and detection wavelength are optimized. Under the chosen conditions (carbonate buffer (ionic strength of 100 mM), pH 9.8, 0.15 mM tetradecyltrimethylammonium bromide (TTAB)) a complete separation is achieved. Calibration plots are linear in two ranges of concentration for U(VI) (∼1 × 10⁻⁵ to 1 × 10⁻³), mellitate and trimellitate (∼5 × 10⁻⁶ to 5 × 10⁻⁴), and about one range (∼1 × 10⁻⁴ to 5 × 10⁻³) for oxalate and benzoate. Accuracy of the procedure is checked by the “added-found” method in standard mixture solutions. Relative standard deviation is within the range of 2–10% and the recovery is in the range of 90–110%. This method is applied for the analysis of real UC dissolution samples.     

Separation of twenty underivatized essential amino acids by capillary zone electrophoresis with contactless conductivity detection

Twenty underivatized essential amino acids were separated using capillary zone electrophoresis and consequently detected with contactless conductivity detection (CCD). A simple acidic background electrolyte (BGE) containing 2.3 M acetic acid and 0.1% w/w hydroxyethylcellulose (HEC) allowed the electrophoretic separation and sensitive detection of all 20 essential amino acids in their underivatized cationic form. The addition of HEC to the BGE suppressed both, electroosmotic flow and analyte adsorption on the capillary surface resulting in an excellent migration time reproducibility and a very good analyte peak symmetry. Additionally, the HEC addition significantly reduced the noise and long-term fluctuations of the CCD baseline. The optimized electrophoretic separation method together with the CCD was proved to be a powerful technique for determination of amino acid profiles in various natural samples, like beer, yeast, urine, saliva, and herb extracts.     

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     

Continuous on-line derivatization and determination of amino acids by a microfluidic capillary electrophoresis system with a continuous sample introduction interface

An automatic, rapid and continuous on-line derivatization system coupled to microfluidic capillary electrophoresis (CE) for the determination of amino acids using o-phthaldialdehyde/N-acetyl-l-cysteine (OPA/NAC) as the derivative agents has been developed. By on-line derivatization, amino acids were automatically and reproducibly converted to the UV-absorbing derivatives, which were separated by capillary zone electrophoresis (CZE). Optimization of derivatization and separation condition was carried out to achieve both good sensitivity and separation efficiency. The separation could be achieved within 4 min and sample throughput rate can reach up to 16 h⁻¹. The repeatability (defined as relative standard deviation, R.S.D.) was 2.56, 2.85, 3.24 and 3.60% with peak area evaluation and 2.93, 3.12, 4.20 and 4.91% with peak height evaluation for arginine (Arg), phenylalanine (Phe), serine (Ser) and glycine (Gly), respectively. The limits of detection (S/N=3) were 10.46, 13.14, 34.39 and 44.79 μmol/l for Arg, Phe, Ser and Gly, respectively. Major advantages of the proposed method include improved precision and efficient automation of the derivatization by the FI system and the enhanced sampling frequencies by the combined FI-CE system.     

Investigation of fulvic acids from peat using capillary electrophoresis (CE)

The aim of this work was to characterize the fulvic acids (FA) extracted from peat by use of capillary electrophoresis (CE). Two FA extracts have been obtained using two different extractants (0.1 M NaOH and 0.1 M NaOH plus 0.1 M Na₄P₂O₇, Na+PP). The CE runs wre performed using a coated silica capillary, and several buffer solutions at different PH values. The results obtained show that the Fa extracts may be well characterized by CE even if the isolelectric point of the fulvic compounds is very low. The best fractionation has been obtained using a buffer borate at PH 8.9 as funning buffer. The charcterization of the two extracts also showed some important differences between the NaOH extract and the Na+PP extract.     

Separation of phenolic acids by capillary electrophoresis with indirect contactless conductometric detection

A new method for the electrophoretic separation of nine phenolic acids (derivatives of benzoic and cinnamic acids) with contactless conductometric detection is presented. Based on theoretical calculations, in which the mobility of the electrolyte co- and counterions and mobility of analytes are taken into consideration, the electrolyte composition and detection mode was selected. This approach was found to be especially valuable for optimization of the electrolyte composition for the separation of analytes having medium mobility. Indirect conductometric detection mode was superior to the direct mode as predicted theoretically. The best performance was achieved with 150 mM 2-amino-2-methylpropanol electrolyte at pH 11.6. The separation was carried out in a counter-electroosmotic mode and completed in less than 6 min. The LODs achieved were about 2.3-3.3 microM and could be further improved to 0.12-0.17 microM by using a sample stacking procedure. The method compares well to the UV-Vis detection.     

Critical evaluation of buffering solutions for pKa determination by capillary electrophoresis

The performance of the most common and also some other less common CE buffers has been tested for the pKa determination of several types of compounds (pyridine, amines, and phenols). The selected buffers cover a pH ranging from 3.7 to 11.8. Whereas some buffers, like acetic acid/acetate, BisTrisH+/BisTris, TrisH+/Tris, CHES/CHES-, and CAPS/CAPS- can be used with all type of analytes, others like ammonium/ammonia, butylammonium/butylammonia, ethylammonium/ethylammonia, diethylammonium/diethylammonia, and hydrogenphosphate/phosphate are not recommended because they interact with a wide range of compounds. The rest of the tested buffers (dihydrogenphosphate/hydrogenphosphate, MES/MES-, HEPES/HEPES-, and boric acid/borate) can show specific interactions depending on the nature of the analytes, and their use in some applications should be restricted.     

Analysis of aromatic acids by nonaqueous capillary electrophoresis with ionic‐liquid electrolytes

The separation of six kinds of aromatic acids by CZE with 1‐ethyl‐3‐methylimidazolium chloride (EMIMCl) and 1‐ethyl‐3‐methylimidazolium hydrogen sulfate (EMIMHSO₄)_, two kinds of ionic liquids (ILs) as background electrolytes, and acetonitrile as solvent were investigated. The six kinds of aromatic acids can be separated under positive voltage with low IL concentration with either of the two ILs and separation with EMIMHSO₄ is better in consideration of peak shapes and separation efficiency. But the migration order is different when the IL is different. Under negative voltage with high IL concentration, the six analytes can be separated with EMIMCl as background electrolytes and the migration order of the analytes is opposite to those with low concentration of EMIMCl as background electrolyte. The separations are based on the combination effects of heteroconjugation between the anions and cations in the ILs and the analytes, of which the heteroconjugation between the anions in the ILs and the analytes plays a dominant role. The heteroconjugation between the anions of the ILs and analytes is proton sensitive and only a very small amount of proticsolvents added into the electrolyte solution can harm the separation. When EMIMCl concentration is high, the heteroconjugation between the IL anions and the proton in the analytes make the effective mobility of the analytes much higher than the EOF and their migration direction reversed. Finally, the six aromatic acids in water samples were analyzed by nonaqueous CE with low concentration of EMIMHSO₄ as background electrolytes with satisfactory results.     

Microchip capillary electrophoresis with electrochemical detector for fast measurements of aromatic amino acids

A method based on microchip capillary electrophoresis with amperometric detection was developed for the rapid separation and direct detection of oxidizable aromatic amino acids (without prior derivatization). The working electrode was a thick-film carbon strip electrode positioned opposite the outlet of the separation channel. Factors influencing the separation and detection processes were examined and optimized. The five aromatic amino acids, tyrosine, 5-hydroxytryptophan, tryptophan, p-aminobenzoic acid, and m-aminobenzoic acid, can be well separated within 5 min using a separation voltage of 2000 V and a 25 mM phosphate buffer (pH 7.0) run buffer containing 50 mM sodium dodecylsulfate. Most favorable amperometric detection was obtained at +0.95 V. Linear calibration plots are observed for micromolar concentrations of the oxidizable amino acids. The new protocol offers good stability and for reproducibility, with relative S.D. of less than 5% for both migration times and peak currents (n=8). It should be useful for the analysis of aromatic amino acids, as desired for life sciences.     

Determination of hydroxy acids in cosmetics by chemometric experimental design and cyclodextrin‐modified capillary electrophoresis

A CD‐modified CE method was established for quantitative determination of seven hydroxy acids in cosmetic products. This method involved chemometric experimental design aspects, including fractional factorial design and central composite design. Chemometric experimental design was used to enhance the method's separation capability and to explore the interactions between parameters. Compared to the traditional investigation that uses multiple parameters, the method that used chemometric experimental design was less time‐consuming and lower in cost. In this study, the influences of three experimental variables (phosphate concentration, surfactant concentration, and methanol percentage) on the experimental response were investigated by applying a chromatographic resolution statistic function. The optimized conditions were as follows: a running buffer of 150 mM phosphate solution (pH 7) containing 0.5 mM CTAB, 3 mM γ‐CD, and 25% methanol; 20 s sample injection at 0.5 psi; a separation voltage of ?15 kV; temperature was set at 25°C; and UV detection at 200 nm. The seven hydroxy acids were well separated in less than 10 min. The LOD (S/N = 3) was 625 nM for both salicylic acid and mandelic acid. The correlation coefficient of the regression curve was greater than 0.998. The RSD and relative error values were all less than 9.21%. After optimization and validation, this simple and rapid analysis method was considered to be established and was successfully applied to several commercial cosmetic products.     

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

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

The determination of lansoprazole in pharmaceutical preparation by capillary electrophoresis

Summary A capillary electrophoretic method for the determination of lansoprazole in pharmaceutical preparations is described. The analysis was performed in a fused silica capillary using 20 mM borate buffer at pH 8.7 as a background electrolyte. The best resolution was obtained by applying a potential of 30 kV and vacuum injection of 1 s. Detection was made at 200 nm. Phenobarbital sodium was a good internal standard and the migration times were 4.1±0.2 min (lansoprazole) and 5.7±0.2 min (phenobarbital sodium). A well-correlated calibration equation was found in the range of 1.12×10⁻⁵ and 2.24×10⁻⁴ M. Limit of detection (LOD) and limit of quantitation (LOQ) were 1.4×10⁻⁶ M (RSD 1.44%) and 4.42×10⁻⁶ M (RSD 1.49%), respectively. The method was validated and applied to the capsules containing enteric coated pellets of lansoprazole. The results of the proposed method were compared those of UV spectrophotometry. Insignificant differences were found between the two methods at the 95% probability level. The described CE method is selective, rapid, sensitive and accurate for the analysis of lansoprazole in quality control laboratories.     

Analysis of carboxylic acids in biological fluids by capillary electrophoresis

This review article addresses the different capillary electrophoretic methods that are being used for the study of both short-chain organic acids (including anionic catecholamine metabolites) and fatty acids in biological samples. This work intends to provide an updated overview (including works published until November 2004) on the recent methodological developments and applications of such procedures together with their main advantages and drawbacks. Moreover, the usefulness of CE analysis of organic acids to study and/or monitor different diseases such as diabetes, new-borns diseases or metabolism disorders is examined. The use of microchip devices and CE-MS couplings for organic acid analysis is also discussed.