Separation and investigation of structure-mobility relationship of gonadotropin-releasing hormones by capillary zone electrophoresis in conventional and isoelectric acidic background electrolytes

Capillary zone electrophoresis (CZE) has been applied to qualitative and quantitative analysis, separation and physicochemical characterization of synthetic gonadotropin-releasing hormones (GnRHs) and their analogs and fragments. Structurally related peptides were separated in conventional and isoelectric acidic background electrolytes (BGEs), pH 2.18-2.50. Best separation was achieved in isoelectric BGE composed of 200 mM iminodiacetic acid, pH 2.32. The effective electrophoretic mobilities, m(ep), of GnRHs in five BGEs were determined and four semiempirical models correlating effective mobility with charge, q, and relative molecular mass, M(r), (m(ep) versus q/M(r)(k), where k is related to the molecular shape) were tested to describe the migration behavior of GnRHs in CZE. None of the models was found to be quite definitively applicable for the whole set of 10 GnRHs differing in size (tetrapeptide-decapeptide) and positive charge (0.91-3.00 elementary charges). Nevertheless, for the dependence of m(ep) on q/M(r)(k), the highest coefficient of correlation, R=0.995-0.999, was obtained for k close to the value 0.5 in all five acidic BGEs. This indicates that the most probable structure of GnRHs in these BGEs can be predicted as a random coil.     

Analysis and characterization of phosphinic pseudopeptides by capillary zone electrophoresis

Capillary zone electrophoresis (CZE) was applied to analysis and characterization of phosphinic pseudopeptides with the general structure N-Ac-Val-Ala(psi)(PO2(-)-CH(2)) Leu-Xaa-NH(2), where Xaa represents one of 20 proteinogenic amino acid residues. Pseudopeptides containing neutral or acidic amino acid residues in position Xaa were analyzed as anions in weakly alkaline (pH 8.1) Tris-Tricine background electrolyte (BGE), pseudopeptides with basic amino acid residues in position Xaa were analyzed as cations in acid BGEs (Tris-phosphate buffers). Acidity of phosphinic acid moiety in peptides with basic amino acid residues was determined from the dependence of effective mobility of these peptides on pH in the acid pH region (pH 1.4-2.8). Additionally, separation of diastereomers of some peptides was achieved.     

Ampholytes as background electrolytes in capillary zone electrophoresis: sense or nonsense? Histidine as a model ampholyte

A lot of phenomena, occuring in capillary zone electrophoresis (CZE), are linked with the ionic concentration of the background electrolyte (BGE). If weak bases and acids are used as BGEs in CZE, at a pH where they are scarcely ionized, the ionic concentration of the BGE is very low and this brings a strong peak broadening, limited sample stacking and low sample load. Because the electromigration dispersion increases extremely, moreover, the existence of low-conductivity BGEs in CZE is a contradiction in terms. The behavior of ampholytes as BGE in CZE is examined, by means of histidine as a model ampholyte. For BGEs consisting of histidine, important parameters, including the ionic concentrations, buffer capacity, transfer ratio, and the indicator for electromigration dispersion E(1)m(1)/E(2)m(2), are calculated at various pH. Although the transfer ratio is fairly constant over the whole pH traject, the ionic concentration and buffer capacity decrease whereas the electromigration dispersion strongly increases near the pI of histidine. I.e., that ampholytes can be applied as BGEs in CZE, however, just not at pH near their pI value, except as the difference between the pK values of the basic and acidic group, the deltapK value, is very small. For ampholytes with a low deltapK value or at high concentrations, all the before-mentioned effects are less fatal, but in that case we can not speak of a real low-conductivity BGE. If ampholytes are used at pH near their pK values, the use of ampholytes as BGE is not advantageously compared with simple weak bases and acids. This has been confirmed by calculations and experiments.     

Tungstate as complex-forming reagent facilitating separation of selected polyphenols by capillary electrophoresis and its comparison with borate

A novel electrophoretic BGE containing tungstate as complex-forming reagent is suitable for the separation of polyphenols. Similar to molybdate-containing BGE reported earlier (cf. M. Polásek, et al.., Talanta 2006, 69, 192) addition of tungstate to BGE affects significantly migration of compounds/ligands with vicinal -OH groups due to the formation of negatively charged complexes involving W(VI) as central ion. Baseline separation of mixtures of flavonoids (apigenin, luteolin, hyperoside, quercetin, and rutin) and phenolic acids (chlorogenic and p-coumaric acid) was achieved within 15 min with optimized BGE of pH 7.4 containing 50 mM N-(2-hydroxyethyl)piperazine-2'-(2-ethanesulfonic acid) (HEPES), 2.2 mM tungstate, and 25% v/v of methanol. The separation was performed in a 75 cm (effective length 42 cm)x 75 microm id uncoated fused-silica capillary at 30 kV with spectrophotometric detection at 275 nm. The calibration curves were rectilinear for 25-175 microg/mL of all analytes (cinnamic acid as the internal standard). The LODs ranged from 1.8 to 6 microg/mL for all analytes except for chlorogenic acid. Intraday precision (n = 6) of migration times (RSD < or = 1.2%) and peak areas (RSD < or = 5.6%) was evaluated. The tungstate-based BGEs can be alternatively utilized for the analysis of polyphenols at considerably lower pH than with conventional alkaline borate-based BGEs.     

Separation of amino acids in plant tissue extracts by capillary zone electrophoresis with indirect UV detection using aromatic carboxylates as background electrolytes

Summary Amino acids in extracts of plant tissue were separated and detected by capillary zone electrophoresis (CZE) with indirect UV detection. Various aromatic carboxylates such as salicylate, benzoate, phthalate and trimellitate were investigated as background electrolytes (BGEs). A BGE of benzoate gave the best resolution and detector response. Amino acids were separated at a highly alkaline pH to charge amino acids negatively. Separation was achieved by the co-electroosmotic flow (Co-EOF) by the addition of the cationic surfactant myristyltrimethyl-ammonium bromide (MTAB) to the electrolyte. The condtions affecting the separation of amino acids, including electrolyte pH, concentrations of both benzoate and MTAB, were investigated and optimised. Separation of a mixture of 17 amino acids at pH 11.20 with indirect UV detection at 225 nm was achieved with a BGE of 10 mM benzoate containing 1.0 mM MTAB at pH of 11.20. Detection limits ranged between 10 and 50 μM. The proposed method was demonstrated by the determination of amino acids in extracts of Eucalypt leaves with direct injection of samples.     

Determination of pK(a) values of diastereomers of phosphinic pseudopeptides by CZE

A CE method was used for the determination of acidity constants (pK(a)) of a series of ten phosphinic pseudopeptides, which varied in number and type of ionogenic groups. Effective electrophoretic mobilities were measured in the 1.8-12.0 pH range in the BGEs of constant ionic strength of 25 mM. Effective electrophoretic mobilities, corrected to standard temperature of 25 degrees C, were subjected to non-linear regression analysis and the obtained apparent pK(a) values were recalculated to thermodynamic pK(a)'s by extrapolation to zero ionic strength according to the extended Debye-Hückel model. The pK(a) values of the phosphinic acid group fell typically in the 1.5-2.25 interval, C-terminal carboxylic groups in the 2.94-3.50 interval, carboxylic groups of the lateral chain of glutamate and aspartate in the 4.68-4.97 interval, imidazolyl moiety of histidine in the 6.55-8.32 interval, N-terminal amino groups in the 7.65-8.28 interval and epsilon-amino group of the lateral chain of lysine in the 10.46-10.61 interval. Further, separation of diastereomers of the phosphinic pseudopeptides was investigated in achiral BGEs. Evaluation of the resolution of the diastereomers as a function of pH of the BGE revealed that most suitable pH region for separation of the diastereomers is around the pK(a) values of the central phosphinic acid group of the pseudopeptides. Successful separation of some diastereomers was, however, achieved in the neutral and alkaline BGEs as well.     

Electrolysis phenomena in electrophoresis

We present a new theoretical approach for calculating changes in the physico-chemical properties of BGEs for measurements by CZE due to the electrolysis in electrode vials (vessels). Electrolysis is an inevitable phenomenon in any measurement in CZE. Water electrolysis, which occurs in most measurements, can significantly alter the composition of the BGE in electrode vials and in the separation capillary and has a negative influence on the robustness and quality of separations. The ability to predict changes in the composition of the BGE is important for evaluation of the suitability of the BGEs for repeating electrophoretic runs. We compared theoretically calculated changes in the physico-chemical properties (pH, conductivity) with those measured using pH-microelectrode and contactless conductivity detection of the BGE after the electrophoretic run. We confirmed the validity of our theoretical approach with a common BGE composed of acid-base pair, where one constituent is fully dissociated while the second constituent is dissociated by only half, and with Good's buffer. As predicted by theoretical approach, the changes in the physico-chemical properties of the Good's buffer after the electrophoretic run were several times lower than in the case of a common BGE composed of a weak acid – strong base pair.     

The preparation of background electrolytes in capillary zone electrophoresis: golden rules and pitfalls

In this article the methodology of the design of suitable background electrolytes (BGEs) in capillary zone electrophoresis (CZE) is described. The principal aspects of the role of a BGE in CZE are discussed with respect to an appropiate migration behavior of analytes, including the transport of the electric current, the buffering of pH, the Joule heat, the electro-endosmotic flow (EOF) and the principal migration and detection modes. The impact of the composition of the BGE upon migration and detection is discussed. It is shown that the total concentration of the BGE is a principal factor and the adjustment of migrating analyte zones according to the Kohlrausch regulating function (KRF) is the principal effect in most of the sample stacking techniques. The number of co-ions and their properties are of key importance for peak shapes of the analyte peaks and for the existence of system zones. The detection of UV-transparent analytes may advanteously be done in the indirect UV mode, by using UV-absorbing co-ions, however, both peaks and dips may be expected in the UV trace in case of multiple co-ionic BGEs. Properties of BGEs can be predicted applying mathematical models and it is shown that with SystCharts, predictions can be given concerning the existence of system zones, detection modes and the peak shapes of analytes for a given BGE. Practical examples of methodological considerations are given in the design of suitable BGEs for four principal combinations of migration and detection modes. The properties of the BGEs selected are exemplified with experimental results. Golden rules are summarized for the preparation of suitable BGEs in CZE.     

Important electromigration effects of carbon dioxide in capillary electrophoresis at high pH

This paper deals with unwanted effects of carbonate in capillary zone electrophoretic analyses of anions in alkaline BGEs with indirect UV absorption and conductivity detection. Computer simulations and experimental study of selected model systems have shown that carbon dioxide absorbed from air into BGEs and samples induce important electrophoretic effects like formation of new additional zones and/or boundaries that may further induce strong and pronounced temporary changes in the migration of analytes. Examples are reduction of the pH of alkaline BGEs around pH 11 by up to 1 unit or formation of a pronounced detectable carbon dioxide peak comparable with peaks of analytes at 1 mM level. The higher the pH of the BGE, the stronger these effects and the broader their spectrum, involving (i) changes of effective mobilities and selectivity due to changes in pH of the BGE, (ii) occurrence of additional system zones appearing in form of peaks, dips or more complex disturbances in the detection signal, (iii) temporary interactions with the sample components and subsequent modification of the separation process and of its result. This paper reveals all these effects and brings the knowledge necessary to prevent problems with qualitative and quantitative evaluation of the analysis results.     

Use of 1-alkyl-3-methylimidazolium-based ionic liquids as background electrolytes in capillary electrophoresis for the analysis of inorganic anions

Separation of inorganic anions in CE is often a challenging task because the electrophoretic mobilities of inorganic anions are comparable to or even greater than the EOF mobility. In this study, we present the use of ionic liquids (ILs) as background electrolytes (BGEs) in CE of inorganic anions. The 1-alkyl-3-methylimidazolium-based ILs as BGEs dynamically coated the capillary wall and induced a reversed EOF. This allowed the anions to comigrate with the EOF and yielded a rapid separation. Increasing the alkyl chain length of the ILs and BGE concentration can significantly improve the separation resolution. With 40 mM 1-butyl-3-methylimidazolium tetrafluoroborate as BGE, good separations of five model anions (Br-, I-, NO2(-), NO3(-), and SCN-) were achieved in a range of buffer pH values. The separation efficiency was as high as 34 600-155 000, and the RSDs of the migration times were less than 0.8% (n = 5).     

Investigation of the effect of ionic strength of Tris-acetate background electrolyte on electrophoretic mobilities of mono-, di-, and trivalent organic anions by capillary electrophoresis

The effect of ionic strength of the background electrolyte (BGE) composed of tris(hydroxymethyl)aminomethane (Tris) and acetic acid on the electrophoretic mobility of mono-, di- and trivalent anions of aliphatic and aromatic carboxylic and sulfonic acids was investigated by capillary zone electrophoresis (CZE). Actual ionic mobilities of the above anions were determined from their CZE separations in Tris-acetate BGEs of pH 8.1 to 8.2 in the 3 to 100 mM ionic strength interval at constant temperature (25 degrees C). It was found that the ionic strength dependence of experimentally determined actual ionic mobilities does not follow the course supposed by the classical Onsager theory. A steeper decrease of actual ionic mobilities with the increasing ionic strength of BGE and a higher estimated limiting mobility of the anions than that found in the literature could be attributed to the specific behavior of the Tris-acetate BGEs. Presumably, not only a single type of interaction of anionic analytes with BGE constituents but rather the combination of effects, such as ion association or complexation equilibria, seems to be responsible for the observed deviation of the concentration dependence of the actual ionic mobilities from the Onsager theory. Additionally, several methods for the determination of limiting ionic mobilities from CZE measured actual ionic mobilities were evaluated. It turned out that the determined limiting ionic mobilities significantly depend on the calculation procedure used.     

Capillary zone electrophoresis of soil humic acid fractions obtained by coupling size-exclusion chromatography and polyacrylamide gel electrophoresis

Capillary zone electrophoresis (CZE) was used for characterisation of soil humic acid (HA) fractions obtained by coupling size-exclusion chromatography with polyacrylamide gel electrophoresis, on the basis of their molecular size and electrophoretic mobility. CZE was conducted using several low alkaline buffers as background electrolyte (BGE): 50 mM carbonate, pH 9.0; 50 mM phosphate, pH 8.5; 50 mM borate, pH 8.3; 50 mM Tris-borate+1 mM EDTA+7 M urea+0.1% sodium dodecyl sulphate (SDS), pH 8.3. Independently of BGE conditions, the effective electrophoretic mobility of HA fractions were in good agreement with their molecular size. The better resolution of HA were obtained in Tris-borate-EDTA buffer with urea and SDS. This results indicated that CZE, mostly with BGE-contained disaggregating agents, is useful for separating HAs in fractions with different molecular sizes.     

Comparison of atmospheric pressure photoionization and ESI for CZE-MS of drugs

The performance of atmospheric pressure photoionization (APPI) and ESI for CZE was compared using a set of seven drugs (basic amines, quaternary amines and steroids) and four different BGEs. The influence of volatile and nonvolatile BGEs of acidic and neutral pH on the MS responses of test compounds was evaluated by infusion of test solutions into the respective ion sources, and by actual CZE-MS experiments. The infusion experiments indicate that sodium phosphate buffers cause ionization suppression in ESI-MS, although for the amines the suppression was modest (25-60% signal reduction). By contrast, APPI-MS responses were not affected by nonvolatile BGEs. With phosphate buffers, ESI-MS responses for the basic amines were still a factor 3-13 higher than the APPI-MS signals, whereas the steroids yielded similar responses in ESI-MS and APPI-MS. The quaternary amines could readily be detected in ESI-MS, but detection in APPI-MS required specific interface conditions. Using typical CZE-APPI-MS settings, quaternary amines remained undetected. Remarkably, the S/Ns observed in CZE-ESI-MS for the test compounds, were generally similar when using volatile and nonvolatile BGEs. For basic compounds, the S/Ns obtained in CZE-ESI-MS were a factor 2-5 higher than in CZE-APPI-MS, whereas steroids yielded equal S/Ns in both methods. Overall, it is concluded that when using relatively low BGE concentrations, the sensitivity of ESI-MS detection in CZE is more favorable than APPI-MS detection, even when nonvolatile BGEs are employed.     

Capillary electrophoresis-mass spectrometry for impurity profiling of basic pharmaceuticals using non-volatile background electrolytes

A generic approach has been developed for coupling capillary electrophoresis (CE) using non-volatile background electrolytes (BGEs) with mass spectrometry (MS) using a sheath liquid interface. CE-MS has been applied for basic and bi-functional compounds using a BGE consisting of 100 mM of TRIS adjusted to pH 2.5 using phosphoric acid. A liquid sheath effect is observed which may influence the CZE separation and hence may complicate the correlation between CE-UV and CE-MS methods. The influence of the liquid sheath effect on the migration behavior of basic pharmaceuticals has been studied by simulation experiments, in which the BGE outlet vial is replaced by sheath liquid in a CE-UV experiment. As a consequence of the liquid sheath effect, phosphate based BGEs can be used without significant loss of MS sensitivity compared to volatile BGEs. The use of buffer constituents such as TRIS can lead to lower detection limits as loss of MS sensitivity can be compensated by better CE performance. TRIS based BGEs permit relatively high injection amounts of about 100 pmol while maintaining high resolution. The ESI-MS parameters were optimized for a generic method with maximum sensitivity and stable operation, in which the composition of the sheath liquid and the position of the capillary were found to be important. Furthermore, the nebulizing pressure strongly influenced the separation efficiency. The system showed stable performance for several days and a reproducibility of about 15% RSD in peak area has been obtained. Nearly all test compounds used in this study could be analyzed with an MS detection limit of 0.05% measured in scan mode using extracted ion chromatograms. As a result, CE-MS was found to be a valuable analytical tool for pharmaceutical impurity profiling.     

Peaks in capillary zone electrophoresis: fact or fiction

Non-steady-state electrophoretic processes can be estimated by a repeated application of a steady-state model based on the electroneutrality equation, the modified version of Ohm's law, and the mass balances of the co- and counterions. With such a mathematical model, all parameters in sample zones in capillary zone electrophoresis (CZE) can be calculated. The relationships between the calculated parameters for sample zones in CZE, such as the pH, concentrations of co- and counterions, and the ratio E1m1/E2m2 versus the mobilities of both anionic and cationic analytes can be visualized in a SystChart, a set of eight panels, for a given background electrolyte (BGE). All properties of a zone, such as the fronting/tailing character and the question of peaks/dips can be read from such a SystChart. Applying n coions, n-1 discontinuities are present in such a SystChart, indicating the presence of system peaks applying that BGE. For BGEs with one coion, no system peaks (discontinuities) exist at moderate pH values. SystCharts calculated for BGEs with a low pH do show discontinuities, however, which indicates that system peaks are present in electropherograms applying BGEs at low pH. Experimentally, it is shown that system peaks are indeed present in electropherograms applying BGEs with one coion at low pH and the mobilities of the system peaks generally increase with decreasing pH. Hydrogen ions seem to act as a second coionic species. Of course, these system peaks are only visible in the UV signal if the BGE has UV-absorbing properties.     

Separation of naturally occurring triterpenoidal saponins by capillary zone electrophoresis.

A high-performance capillary electrophoresis (HPCE) was successfully applied to the separation and quantitation of naturally occurring oleanene triterpenoidal saponins. The HPCE adapted to the separation of two pairs of disteriomeric saponins (1-2) or (3-4), obtained from Trifolium alexandrinum seeds, was based on capillary zone electrophoresis (CZE) in borate buffer with UV detection at 195 nm. An usual technique for isolation and group separation of saponins was developed as an appropriate purification step prior to determination of individual saponins by CZE. The separation parameters such as borate concentration, pH and applied voltage were varied in order to find the best compromise that complied with demands for high separation, short duration and sufficiently high detector response. The optimum running conditions were found to be 60 mM borate buffer, pH 10 and 12 kV. Under the alkaline borate electrolyte, no resolution was achieved for the saponins (1 and 3) or (2 and 4) in a single mixture, except when 20 mM beta-cyclodextrin was added to the running electrolyte. With the combined techniques of group separation, purification and CZE, a rapid and efficient method for the determination of naturally occurring diasteriomeric saponins is now available.     

Determination of amino acids in tobacco samples by capillary electrophoresis/indirect absorbance detection with isolation of the electrolysis compartment and p-Aminobenzoic acid as a background electrolyte.

A fast, convenient and sensitive method of capillary zone electrophoresis (CZE) and indirect UV detection was proposed for the determination of 16 amino acids. p-Aminobenzoic acid (PAB) was selected as a background electrolyte (BGE). An isolated cell included a BGE buffer part and an electrode buffer one, which were jointed with a glass frit. The isolated cell can prevent PAB from the electrode reaction and improve the stability of the detection baseline. The separation conditions of amino acids were investigated, such as different BGEs, BGE concentration, buffer pH and electroosmotic flow (EOF) modifiers. Under the selected separation conditions, 14 amino acid peaks could be separated in 12 min. The detection limits of the amino acids were in the range of 1.7 - 4.5 micromol/L. The isolated cell is suitable for reagents reacting on the electrodes in capillary electrophoresis. The proposed method has been successfully applied to the determination of the amino acids in tobacco samples.     

Capillary electrophoretic analysis of neutral carbohydrates using ionic liquids as background electrolytes

In this study, ionic liquids (ILs) as BGE additives were applied for the analysis of neutral carbohydrates in CE. The ILs served primarily as chromophores for indirect UV detection. The influence of imidazolium-based ionic liquids on the separation, detection limits and mobility of underivatized neutral carbohydrates was investigated. BGEs consisting of 10-50 mM of ILs at pH 12.4 without other additives provided fast separation of neutral sugars. This method was used to determine sucrose, glucose and fructose in certain vegetable juices.     

Reproducible and efficient separation of aggregatable zein proteins by CZE using a volatile background electrolyte

Zein proteins are a complex mixture of polypetides that belong to the alcohol-soluble storage proteins group (prolamines) in corn. These proteins constitute about 50-60% of the total endosperm protein and are classified in different groups on the basis of differences in their solubility and sequence. Among them, zein proteins are considered the majority group showing a high tendency to aggregate what makes their analysis by any analytical method very difficult. Thus, CZE of these proteins requires the use of very complex BGEs noncompatible with online ESI-MS analysis. The aim of this work was to find a new BGE for the CZE separation of zein protein fully compatible with ESI-MS while providing further light on the complex CZE separation of aggregatable proteins. Thus, it is demonstrated in this work that efficient and reproducible CZE separations of zein proteins can be achieved by using a BGE composed of water, ACN, formic acid and ammonium hydroxide. Besides, it is shown that zein analysis is significantly improved by including the effect of an ammonium gradient during their separation. It is experimentally verified that the ammonium gradient can easily be achieved in CZE by either working with a sample zone with a low concentration of ammonium and a BGE with a high concentration, or conversely, working with a sample zone with high ammonium concentration and a BGE with low concentration of ammonium, giving rise in both cases to a significant improvement in the CZE separation of these proteins. It is demonstrated that this procedure can give rise to efficiency improvements of up to 20-fold in the CZE separation of zein proteins. Under optimized conditions, 20 proteins could be separated with average efficiencies higher than 400 000 theoretical plates/m. Some possible explanations of this effect are discussed including stacking, protein-capillary wall adsorption, protein solubility and protein-salt interactions.     

Cloud point preconcentration prior to capillary zone electrophoresis: simultaneous determination of platinum and palladium at trace levels

The incorporation of a cloud point extraction (CPE) step prior to capillary electrophoresis (CE) for simultaneously determining platinum and palladium at sub-microg/L levels is presented and evaluated. The analytes were extracted as 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol complexes, at pH 2.0, mediated by micelles of the nonionic surfactant polyethyleneglycolmono-p-nonylphenyl ether (PONPE 7.5). The separation-determination step was developed from 150 microL of the extracted surfactant-rich phase diluted with 50 microL of acetonitrile (ACN). An exhaustive study of the variables affecting the cloud point extraction with PONPE 7.5 and the CZE step was done. The type and composition of the background electrolytes (BGEs) were investigated with respect to separation selectivity, reproducibility, and stability. A BGE of 50 mM monobasic sodium phosphate containing 30% ACN, pH 4.53 was found to be optimal for the separation of metal chelates. Detection was performed at 576 nm. An enrichment factor of 250 was obtained for the preconcentration of 50 mL of sample solution. The detection limits for the preconcentration of 50 mL of sample were 0.04 microg/L for Pt and 0.08 microg/L for Pd. As an analytical demonstration, ultratrace concentrations of platinum and palladium were conveniently quantitated in spiked water and urine samples.