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.     

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.     

Success and failure with phthalate buffers in capillary zone electrophoresis

Phthalate buffers are currently used in capillary electrophoresis as robust electrolyte systems for indirect detection. This contribution demonstrates that these buffers show regularly not only successful regions of mobilities of analytes (sample window) but also regions of failure where the migration of analytes is strongly deteriorated due to the presence of a system zone. System zones in phthalate buffers may be easily detected by UV detection and manifest themselves as peaks or dips. Peak shape diagrams are advantageously used for the prediction of the migration behavior of system zones in phthalate background electrolyte (BGE) systems at various pH. It is shown that the mobility of the system zone varies strongly with pH, is practically zero at pH values below 4 and above 7, and shows a maximum at pH 5. Thus, the system peak may coincide either with the peaks of various analytes or with the electroosmotic flow (EOF) peak. Experiments are given showing the effects of such coincidences as, e.g., zigzag detection patterns, double EOF peaks, and/or unusually broad peaks/dips. The message of this contribution is to show how to understand the electrophoretic properties of phthalate BGEs that, regardless of possible failure regions, may be successfully used in the analytical practice of capillary zone electrophoresis (CZE).     

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.     

Conductivity detection in capillary zone electrophoresis: inspection by PeakMaster

A simple rule stating that the signal in conductivity detection in capillary zone electrophoresis is proportional to the difference between the analyte mobility and mobility of the background electrolyte (BGE) co-ion is valid only for systems with fully ionized electrolytes. In zone electrophoresis systems with weak electrolytes both conductivity signal and electromigration dispersion of analyte peaks depend on the conductivity and pH effects. This allows optimization of the composition of BGEs to give a good conductivity signal of analytes while still keeping electromigration dispersion near zero, regardless of the injected amount of sample. The demands to achieve minimum electromigration dispersion and high sensitivity in conductivity detection can be accomplished at the same time. PeakMaster software is used for inspection of BGEs commonly used for separation of sugars (carbohydrates, saccharides) at highly alkaline pH. It is shown that the terms direct and indirect conductivity detection are misleading and should not be used.     

Eigenmobilities in background electrolytes for capillary zone electrophoresis: III. Linear theory of electromigration

A mathematical model of capillary zone electrophoresis (CZE) based on the conception of eigenmobilities, which are the eigenvalues of a matrix M tied to the linearized governing equations is presented. The model considers CZE systems, where constituents, either analytes or components of the background electrolyte (BGE), are weak electrolytes--acids, bases, or ampholytes. There is no restriction on the number of components nor on the valence of the constituents nor on pH of the BGE. An electrophoretic system with N constituents has N eigenmobilities. In most BGEs one or two eigenmobilities are very close to zero so their corresponding eigenzones move very slowly. However, there are BGEs where no eigenmobility is close to zero. The mathematical model further provides: the transfer ratio, the molar conductivity detection response, and the relative velocity slope. This allows the assessment of the indirect detection, conductivity detection and peak broadening (distortion) due to electromigration dispersion. Also, we present a spectral decomposition of the matrix M to matrices allowing the assessment of the amplitudes of system eigenpeaks (system peaks). Our model predicted the existence of BGEs having no stationary injection zone (or water zone, gap, peak, dip). A common practice of using the injection zone as a marker of the electroosmotic flow must fail in such electrolytes.     

System zones in capillary zone electrophoresis.

This paper brings an overview of system zones (SZs) in capillary zone electrophoresis (CZE) and their effects upon the migration of zones of analytes. It is shown that the formation and migration of SZs is an inherent feature of CZE, and that it depends predominantly on the composition of an actual background electrolyte (BGE). One can distinguish between stationary SZs and migrating SZs. Stationary SZs, which move due to the electroosmotic flow only, are induced in any BGE by sample injection. Migrating SZs may be induced by a sample injection in BGEs which show at least one of the following features: (i) BGE contains two or more co-ions, (ii) BGE has low or high pH whereby H+ or OH- act as the second co-ion, and (iii) BGE contains multivalent weak acids or bases. SZs do not contain any analyte and show always BGE-like composition. They contain components of the BGE only and the concentrations of these components are different from their values in the original BGE. Providing that some of the ionic components of the BGE are visible by the detector, the migrating SZs can be detected and they are present as system peaks/dips in the electropherogram. It is shown that a migrating SZ may be characterized by its mobility, and examples are given how this mobility can depend on the composition of the BGE. Further, the effects of the migrating SZs (either visible or not visible by the detector) upon the zones of analytes are presented and the typical disturbances of the peaks (extra broadening, zig-zag form, schizophrenic behavior) are exemplified and discussed. Finally, some conclusions are presented how to cope with the SZs in practice. The proposed procedure is based on the theoretical predictions and/or measurements of the mobilities of SZs and on the so-called unsafe region. Then, such operational conditions should be selected where the unsafe region is outside of the required analytical window.     

Peak deformation in cationic analysis caused by system zones.

In electrophoretic processes, often zones migrate through the separation compartment, with a composition different from that of the background electrolyte (BGE) but which do not contain, however, any component of the sample mixture. These zones migrate with a mobility mainly determined by the composition of the BGE and are called system zones (SZs). If these SZs are visible in electropherograms they are called system peaks (SPs). If sample components have a mobility close to that of a SZ, the separation process can be disturbed and the sample peak shapes are deformed. SZs can appear applying BGEs containing more co-ionic species or if BGEs are used at high or low pH. Recently, the existence of SZs has been described applying BGEs containing weak multivalent anionic species. In this paper, the diverse kinds of system zones, are discussed for cationic systems and the effect of invisible SZs on separations is shown. As an example of a weak multivalent cation, the behavior of the divalent cation histamine is studied, which can be used as co-ion in BGEs for the separation of cations in the indirect UV mode. Applying BGEs containing histamine, SZs are visible in the electropherograms and there existence could also be established theoretically by the use of SystCharts. A mathematical model for the calculation of the mobility of SZs is verified and it has been shown that an unsafe region with a mobility window of msp +/- 10% can be indicated, for the separation of fully ionized sample components.     

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.     

Experimental assessment of electromigration properties of background electrolytes in capillary zone electrophoresis

Electromigration dispersion (EMD) properties of background electrolytes (BGEs) used in capillary zone electrophoresis (CZE) are of key importance for the success of an analysis. The knowledge of these properties may serve well for the prediction of the asymmetry of peaks of analytes, for the prediction of unsafe regions where a strong interference of system zones may be expected, and for the selection of optimum conditions where the analytes of interest may give sharp and practically symmetric peaks. Present theories enable one to calculate and predict EMD properties of many BGEs but there is also a lot of BGEs that are beyond the present theoretical models as far as their composition and equilibria involved are considered. This contribution brings a method for assessment of EMD properties of any BGE from easily accessible experimental data. The method proposed is illustrated by model examples both for cationic and anionic separations. Imidazole acetate, histamine acetate, and histidine acetate served as model BGEs for cationic separations; as the model BGE for anionic separations, Tris-borate and sodium-borate BGEs have been selected since these buffers are frequently used and borate is well-known for its complexing equilibria in aqueous solutions.     

Perphenylcarbamoylated beta-cyclodextrin bonded-silica particles as chiral stationary phase for enantioseparation by pressure-assisted capillary electrochromatography

Perphenylcarbamoylated beta-cyclodextrin bonded-silica particles (5 microm) were packed into 75-mum fused-silica capillaries, and used for the enantiomer separation of neutral and basic solutes by pressure-assisted capillary electrochromatography. Triethylammonium acetate and phosphate buffer were employed as the BGEs. A cathodic EOF was observed with these two BGEs. Seven chiral analytes were successfully resolved into their enantiomers under optimized conditions, and five of them could be baseline-separated within 12 min due to their high electrophoretic mobility. Better results were achieved with phosphate buffer as the BGE. The effects of organic content and pH on the enantioseparation were also investigated.     

Physicochemical characterization of phosphinic pseudopeptides by capillary zone electrophoresis in highly acidic background electrolytes

Phosphinic pseudopeptides (i.e., peptide isosteres with one peptide bond replaced by a phosphinic acid moiety) were analyzed and physicochemically characterized by capillary zone electrophoresis in the pH range of 1.1-3.2, employing phosphoric, phosphinic, oxalic and dichloroacetic acids as background electrolyte (BGE) constituents. The acid dissociation constant (pK(a)) of phosphinate group in phosphinic pseudopeptides and ionic mobilities of these analytes were determined from the pH dependence of their effective electrophoretic mobilities corrected to standard temperature and constant ionic strength of the BGEs. It was shown that these corrections are necessary whenever precise mobility data at very low pH are to be determined. Additionally, it was found that the ionic mobilities of the phosphinic pseudopeptides and pK(a) of their phosphinate group are affected by the BGE constituent used. The variability of migration behavior of the pseudopeptides can be attributed to their ion-pairing formation with the BGE components.     

System zones in capillary zone electrophoresis

When working with capillary zone electrophoresis (CZE), the analyst has to be aware that the separation system is not homogeneous anymore as soon as a sample is brought into the background electrolyte (BGE). Upon injection, the analyte creates a disturbance in the concentration of the BGE, and the system retains a kind of memory for this inhomogeneity, which is propagated with time and leads to so-called system zones (or system eigenzones) migrating in an electric field with a certain eigenmobility. If recordable by the detector, they appear in the electropherogram as system peaks (or system eigenpeaks). However, although their appearance can not be forecasted and explained easily, they are inherent for the separation system. The progress in the theory of electromigration (accompanied by development of computer software) allows to treat the phenomenon of system zones and system peaks now also in very complex BGE systems, consisting of several multivalent weak electrolytes, and at all pH ranges. It also allows to predict the existence of BGEs having no stationary injection zone (or water zone, EO zone, gap, dip). Our paper reviews the theoretical background of the origin of the system zones (system peaks, system eigenpeaks), discusses the validity of the Kohlrausch regulating function, and gives practical hints for preparing BGEs with good separation ability not deteriorated by the occurrence of system peaks and by excessive peak-broadening.     

Theory of system zones in capillary zone electrophoresis

In the last years, it has been shown that the formation and migration of system zones is an inherent feature of capillary zone electrophoresis (CZE) and that it depends predominantly on the composition of an actual background electrolyte (BGE). In most of the currently used BGEs, the SZs are invisible by the UV absorbance detection system, however, the comigration of SZs with the zones of analytes deteriorates the analytical performance of CZE and may be fatal for its utilization. Therefore, the theoretical predictions of the existence and migration of SZs is of key importance for the expediency of CZE. This is a review of the theoretical treatments of SZs which reveals the origin and the properties of SZs and shows how to cope with them. Also, a table of some typical BGEs is presented where the existence and mobilities of SZs are given.     

Separation and investigation of structure-mobility relationships of insect oostatic peptides by capillary zone electrophoresis

Capillary zone electrophoresis (CZE) has been applied to qualitative analysis, separation, and physicochemical characterization of synthetic insect oostatic peptides (IOPs) and their derivatives and fragments. Series of homologous IOPs were separated in three acidic background electrolytes (BGEs; pH 2.25, 2.30, 2.40) and an alkaline BGE (pH 8.1). Best separation was achieved in acid BGE composed of 100 mM H3PO4, 50 mM Tris, pH 2.25. The effective electrophoretic mobilities, mu(ep), of all IOPs in four BGEs were determined and several semiempirical models correlating effective mobility with charge-to-size ratio (mu(ep) versus q/Mr k) were tested to describe the migration behavior of IOP in CZE. None of models was found to be unambiguously applicable for the whole set of 20 IOPs differing in size (dipeptide - decapeptide) and charge (-2 to +0.77 elementary charges). However, a high coefficient of correlation, 0.9993, was found for the subset of homologous series of IOPs with decreasing number of proline residues at C-terminus, H-Tyr-Asp-Pro-Ala-Prox-OH, x = 6 - 0, for the dependence of mu(ep) on q/Mr k with k = 0.5 for IOPs as anions in alkaline BGE and with k = 2/3 for IOPs as cations in optimized acidic Tris-phosphate BGE. From these dependences the probable structure of IOPs in solution could be predicted.     

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.     

Eigenmobilities in background electrolytes for capillary zone electrophoresis: IV. Computer program PeakMaster

We are introducing a computer implementation of the mathematical model of zone electrophoresis (CZE) described in Stedry, M., Jaros, M., Hruska, V., Gas, B., Electrophoresis 2004, 25, 3071-3079 program PeakMaster. The computer model calculates eigenmobilities, which are the eigenvalues of the matrix tied to the linearized continuity equations, and which are responsible for the presence of system eigenzones (system zones, system peaks). The model also calculates other parameters of the background electrolyte (BGE)-pH, conductivity, buffer capacity, ionic strength, etc., and parameters of the separated analytes--effective mobility, transfer ratio, molar conductivity detection response, and relative velocity slope. This allows the assessment of the indirect detection, conductivity detection and peak broadening (peak distortion) due to electromigration dispersion. The computer model requires the input of the BGE composition, the list of analytes to be separated, and the system instrumental configuration. The output parameters of the model are directly comparable with experiments; the model also simulates electropherograms in a user-friendly way. We demonstrate a successful application of PeakMaster for inspection of BGEs having no stationary injection zone.     

Regular properties of simple electrophoretic BGEs with multiprotic weak acids: discovery of complex hybrid system boundaries

In electrophoresis, the introduction of a sample into the BGE creates two new zone boundaries, the front and rear boundary of the sample zone which is sandwiched between the BGE. During an electrophoresis run, these boundaries move and split into other new boundaries demarcating the zones of analytes. Besides the analyte zones migrating out of the original sample location, system zones may be also formed. Such zones are formed by the BGE components and differ from the BGE only in their concentrations. The front and rear boundaries of such system zones evolve during electromigration and may show sharp (S), dispersed (D), and/or hybrid character. This contribution brings the results of theoretical and experimental investigation of system properties of very simple BGEs formed by one weak acid and one weak or strong base provided that the acid is polyprotic. It is shown that system boundaries of the hybrid type occur even in these simple systems represented, e.g., by the very common phosphate buffer. Theory reveals that a phosphate buffer system (formed by phosphoric acid and a strong cation) may exhibit a very unusual complex shape of the electromigration dispersion velocity curve showing three turns. This leads to the formation of complex hybrid boundaries having D-S-D, S-D-S, or even S-D-S-D or D-S-D-S concentration profile types that have not been reported so far. The velocity diagram method allows theoretical analysis of such complicated profiles and the results are in good accordance with both computer simulation and experiments.     

Counterion condensation in short cationic peptides: limiting mobilities beyond the Onsager-Fuoss theory

We investigated the effect of the background electrolyte (BGE) anions on the electrophoretic mobilities of the cationic amino acids arginine and lysine and the polycationic peptides tetraarginine, tetralysine, nonaarginine, and nonalysine. BGEs composed of sodium chloride, sodium propane-1,3-disulfonate, and sodium sulfate were used. For the amino acids, determination of the limiting mobility by extrapolation, using the Onsager-Fuoss (OF) theory expression, yielded consistent estimates. For the peptides, however, the estimates of the limiting mobilities were found to spuriously depend on the BGE salt. This paradox was resolved using molecular modeling. Simulations, on all-atom as well as coarse-grained levels, show that significant counterion condensation, an effect not accounted for in OF theory, occurs for the tetra- and nonapeptides, even for low BGE concentrations. Including this effect in the quantitative estimation of the BGE effect on mobility removed the discrepancy between the estimated limiting mobilities in different salts. The counterion condensation was found to be mainly due to electrostatic interactions, with specific ion effects playing a secondary role. Therefore, the conclusions are likely to be generalizable to other analytes with a similar density of charged groups and OF theory is expected to fail in a predictable way for such analytes.