Electroosmotic flow changes due to interactions of background electrolyte counter-ions with polyethyleneimine coating in capillary zone electrophoresis of proteins

The properties and behavior of polyethyleneimine (PEI) covalently coated capillaries with respect to different background electrolytes used in capillary zone electrophoresis (CZE) are described. The coating stability and changes of inner surface charge in the capillary were followed by measurement of electroosmotic flow (EOF). Interest was focused mainly on conjugate bases of carboxylic acids as anionic background electrolyte components (acetate, citrate, malate, malonate, tartrate, and succinate). An interesting phenomenon was observed in PEI-coated capillaries: The direction (and the magnitude) of EOF depends on the composition of the background electrolyte and at a certain pH it can undergo reversible change. Ionic complex formation was suggested as a hypothesis to explain this behavior. With this knowledge, the PEI-coated capillary was used for the separation of basic proteins in the above-mentioned background electrolytes. A standard protein mixture of cytochrome c, ribonuclease A, and lysozyme at a concentration of 0.25 mg/mL each was chosen as model sample.     

Effects of background electrolyte composition and addition of selectors on separation selectivity in nonaqueous capillary electrophoresis

This review gives a survey of the approaches employed to obtain, enhance and tune selectivity in nonaqueous capillary electrophoresis (NACE). Recent developments in NACE are described and the effects of background electrolyte composition and addition of selectors on separation selectivity are discussed. The use of one organic solvent, a mixture of several organic solvents or the use of additives to tune separation selectivity in NACE is presented and a list of relevant applications is included.     

Use of lignins as components of background electrolyte in capillary electrophoresis

The electrophoretic behavior of two lignins of different compositions, i.e., spruce dioxane lignin and lignosulfonate, is studied. The lignins are shown to affect the electrophoretic behavior of negatively charged analytes, such as carboxylic acids and phenols; their migration time increases. The addition of lignins improves the analytical parameters of phenol quantification by capillary electrophoresis. By means of a simple non-modified capillary, a mixture of six phenols was separated (simple phenol, 2-chlorophenol, 3-chlorophenol, 4-chlorophenol, 2,4-dichlorophenol, and perchlorophenol) with the high resolution (up to 20) and efficiency [(1–5) × 10⁵ TPM]. The separation of six phenols takes 10 min, the lower limit of the analytical range makes 1 μg/mL, the relative standard deviation does not exceed 3%. The potency for the determination of simple phenol and m-cresol is shown on an example of the Verrukatsid medication within 7 min.     

Effects of capillary coating and beta-cyclodextrin additive to the background electrolyte on separation of sulphonated azodyes by capillary zone electrophoresis

Fourteen azodyes containing one to five acidic groups were separated by capillary zone electrophoresis (CZE). The effects of the pH and beta-cyclodextrin additive to the background electrolyte on the separation of sulphonated azodyes were investigated. The effects of the working conditions significantly differ in non-coated fused silica capillaries and in capillaries coated with polyacrylamide. Splitting of the zones of metal-complex dyes was observed in polyacrylamide coated capillaries and the background electrolyte with 10 mmol/L beta-cyclodextrin, due to the separation of stereoisomeric forms of the dyes, which were separated for the first time using CE. Relations between the structure of the sulphonated azodyes and the electrophoretic mobilities are discussed. Naphthalene mono- to tetrasulphonic acids were used as the standards for the calibration of migration scale of the analysed dyes.     

Sample matrix influence on the choice of background electrolyte for the analysis of bases with capillary zone electrophoresis

Low levels of impurities need to be determined in drugs. Consequently, if UV detection is used, a large sample amount must be loaded and as narrow peaks as possible obtained. The sample matrix and the stability of the samples as well as the peak resolution should be considered when the electrolyte is chosen. In this study the influence of the sample matrix composition with varying background electrolytes on the peak appearance of model mixtures loaded in large amounts was investigated. A robust electrolyte for analysis of bases in a sample with varying pH was found to consist of a buffering co-ion and a buffering counter-ion (the pH was approximately 4.2 in the electrolyte). If a minor component has higher mobility than the macrocomponent and the co-ion, better peak shape can be obtained if, for instance, enough sodium chloride is added to the sample, i.e., sample self-stacking is exploited. The effect of addition of organic modifiers, isopropanol or acetonitrile, was examined and good linearity and precision have been shown for impurities in the concentration range tested, approximately 0.03 to 5 mol% of the main component, in model mixtures.     

Capillary zone electrophoresis in non-aqueous solutions: pH of the background electrolyte

Although the establishment of a pH scale and the determination of the pH in water is not problematic, it is not a straightforward task in non-aqueous solvents. As capillary zone electrophoresis (CZE) in organic solvents has gained increasing interest, it seems to be valuable to re-discuss the concept of the pH in such media, especially pointing to those aspects, which make pH measurement uncertain in non-aqueous solvents. In this review, the relevant aspects when dealing with primary standard (PS) and secondary standard (SS) as recommended by the International Union of Pure and Applied Chemistry (IUPAC), and the usage of the operational pH are discussed with special emphasis to non-aqueous solvents. Here, different liquid junction potentials, incomplete dissociation of the electrolytes (especially in solvents with low or moderate relative permittivity) and the occurrence of homo- and heteroconjugation must be taken into account. Problems arising in capillary zone electrophoresis practice are addressed, e.g. when the background electrolyte (BGE) consists of organic solvents, but the measuring electrode (normally the glass electrode) is calibrated with aqueous buffers, and the liquid junction potentials between the solvents do not cancel each other. The alternative concept of establishing a certain pH is described, using mixtures of reference acids or bases with known pKa in the organic solvent, and their respective salts, at a certain concentration ratio, relying to the Henderson-Hasselbalch equation. Special discussion is directed to those organic solvents most common in capillary zone electrophoresis, methanol (MeOH) and acetonitrile (ACN), but other solvents are included as well. The potential significance of small amounts of water present in the organic solvent on changes in pKa values, and thus on the pH of the buffering components is pointed out.     

高效毛细管区带电泳二肽类组分迁移行为的研究

本文考察了影响二肽类组分在高效毛细管区带电泳上迁移时间重复性的因素,提出了采用相对指标来作为高效毛细管区带电泳峰定性的依据,并考察了电流以及柱温对迁移时间和相对迁移时间的影响,发现在恒压lgt~m与T^-1有良好的线性关系;并从迁移时间的基本方程定量地解释了上述的变化规律.本文还发现,采用相对迁移时间指标后可以有效地消除操作电流以及柱温对迁移时间所引起的波动,是毛细管区带电泳的一个特征参数.     

Multivalent weak electrolytes - risky background electrolytes for capillary zone electrophoresis

Multivalent weak acids and bases are useful components of buffers in electrophoresis. The use of such buffers as background electrolytes (BGEs) in capillary zone electrophoresis (CZE) is, however, risky due to the existence of unsafe regions in the analytical window of the separation. This contribution discusses the problems and shows that multivalent weak species in BGEs bring about the same effects as mixtures of two independent co-ions, i.e., the presence of two centers of symmetry in the electropherograms and the existence of a migrating system zone with a mobility in between these two centers of symmetry. The system zone deteriorates the analytical separation and detection of the analytes in its neighborhood. Illustrative experimental examples for both cationic and anionic CZE are shown and related discussion is given. Finally, some basic rules are formulated to avoid the preparation of risky BGEs.     

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.     

Stacking in capillary zone electrophoresis

Due to the short light path of the capillaries, the CE detection limit based on concentration, is far less than that of HPLC and not sufficient for many practical applications. Several methods, based on different electrophoretic maneuvers, can concentrate the sample (stack) easily on the capillary before the separation step of capillary zone electrophoresis (CZE). These methods incorporate different types of discontinuous buffers as the means for invoking different velocities to the same analyte molecules to produce a sharpening of the band (stacking). In CZE, these buffers can be often very simple such as sample dilution or adding to the sample a high concentration of a fast mobility ion. However, in other applications these buffers can be as complicated as those required for isotachophoresis. Stacking can often yield a concentration factor of 5-30-fold, which can improve greatly in CZE the detection limits bringing them very close to those of HPLC. Different methods of stacking, the importance of discontinuous buffers and the different mechanism for concentration on the capillary are reviewed here. As there is a need for more practical applications, there will be more methods devised for stacking in CZE.     

Prediction of electrophoretic mobilities of sulfonamides in capillary zone electrophoresis using artificial neural networks

Artificial neural networks (ANNs) were successfully developed for the modeling and prediction of electrophoretic mobility of a series of sulfonamides in capillary zone electrophoresis. The cross-validation method was used to evaluate the prediction ability of the generated networks. The mobility of sulfonamides as positively charged species at low pH and negatively charged species at high pH was investigated. The results obtained using neural networks were compared with the experimental values as well as with those obtained using the multiple linear regression (MLR) technique. Comparison of the results shows the superiority of the neural network models over the regression models.     

Objective testing for the dependence of electrophoretic mobilities upon size in capillary zone electrophoresis

Summary Eighteen peptides have been modeled. From the volumetric data derived, and published mobilities, the relationship between electrophoretic mobility (μ_ep) and the hydrodynamic radius(r) has been examined. Objective testing with respect to size has been achieved by the log-log version of generalized relationship. (1) From the gradient of the plot versus log r(2.02) there is good support for the inverse square law (μ_ep α 1/r²). Equivalent calculations using molecular weight (M_r) and the number of amino acid residues (n) similarly lead to μ_ep α 1/M _r ^2/3 and μ_ep α 1/n^2/3, respectively. However, the strength of the correlation is diminished as the precision of the representation of size is degraded. (2) An examination of the effect of size at fixed charge and a statistical analysis of the charge distribution on the peptides leads to the conclusion that deviations from the averaged behaviour arise from a charge-induced volumetric effect. Taken together, (1) and (2) indicate that whilst net charge and total size can describe average electrophoretic behaviour well, these parameters are inadequate to describe the specific mobilities of individual analytes. Objective analysis of alkylpyridine data indicates μ_ep α 1/r^x where x=2.6–2.8 (depending upon the nature of the r values utilized), but is certainly ≠1 as may have been presumed. A very small range of values may be responsible for this surprising result.     

Rapid optimized separation of bromide in serum samples with capillary zone electrophoresis by using glycerol as additive to the background electrolyte

After decades of neglect, bromide has recently been re-introduced in therapy as an effective anti-epileptic drug. The present paper describes the methodological optimization and validation of a method based on capillary zone electrophoresis for the rapid determination of bromide in serum using a high-viscosity buffer and a short capillary (10 cm). The optimized running buffer was composed of 90 mM sodium tetraborate, 10 mM sodium chloride, pH 9.24 and 25% glycerol. The separation was carried out at 25 kV at a temperature of 20 °C. Detection was by direct UV absorption at 200 nm wavelength. The limit of detection (signal-to-noise ratio = 5) in serum was 0.017 mM. The precision of the method was verified in blank serum samples spiked with bromide, obtaining intra-day and day-to-day tests, relative standard deviation values ≤0.2% in terms of migration times and values <2% in terms of peaks areas, respectively.     

Piperazine quaternary diammonium salts as additives to background electrolytes in capillary zone electrophoresis

The differential behavior of five different quaternary mono- and diammonium salts, among the 18 investigated, in modulating the electroendoosmotic flow (EOF) and analyte separations in capillary zone electrophoresis is evaluated. It is found that quaternary diammonium salts with positive charges separated by more than four carbon atoms, while exhibiting a very strong affinity for chromatographic silica beads, to the point of exhibiting Rf values close to zero, display, on the contrary, a very poor affinity for the silica wall of capillaries. Compounds separated only by a C2 unit (i.e., 1,4-dialkyl-1,4-diazoniabicyclo[2,2,2,]octane, salts 17 and 18) show high Rf values due to strong ion pair association. The unique behavior of quaternary monoammonium salts possessing an iodinated alkyl (butyl or octyl) tail (i.e., 1, 6, and 7) is attributed to their ability to be covalently affixed to the silica wall via alkylation of ionized silanols at alkaline pH values. They thus strongly modulate and typically invert the EOF, even when not present in the background electrolyte. On the contrary, all diammonium salts, devoid of such alkyl tails, are unable to modulate the EOF and to prevent analyte binding to the silica wall, since they are rapidly removed from the wall by the voltage gradient. However, if added in small amount to the background electrolyte, they offer excellent separations of mixtures of very similar organic acids and prevent any interaction with the capillary wall.     

Determination of monosaccharide composition in plant fiber materials by capillary zone electrophoresis

The neutral sugar composition of acid hydrolyzed extracts of cellulose fiber samples, i.e. oat spelt, wheat straw, thermomechanica pulp (TMP) made of spruce, aspen stemwood, and bleached birch kraft pulp, was determined by a new capillary zone electrophoresis (CZE) method employing an alkaline background electrolyte. The method relies on in-capillary reaction and direct UV detection at wavelength 270 nm. Neutral carbohydrates D-(+)-galactose, D-(+)-glucose, L-rhamnose, D-(+)-mannose, D-(-)-arabinose, and D-(+)-xylose were simultaneously separated. The calibration plots were linear over a range from 10 to 150 mg/L for D-(+)-galactose, L-rhamnose, D-(+)-mannose, and D-(-)-arabinose and from 50 to 400mg/L for D-(+)-glucose and D-(+)-xylose. Relative standard deviations (RSDs) of peak areas during a 5-day analysis period varied from 3.3% for galactose to 11.8% for rhamnose. RSDs of migration times varied between 0.3 and 0.7%. The detection limit (at S/N 3) was 5mg/L for each monosaccharide. The results obtained by CZE agreed well with results obtained by high-performance anion-exchange chromatography. Glucose and xylose were the two predominant monosaccharides in the plants, except in the spruce TMP sample where glucose and mannose dominated.     

三角形法和四面体法优化选择毛细管区带电泳背景电解质

建立了两种高效、快速的毛细管区带电泳背景电解质(BGE)的优化方法三角形优化法和四面体优化法。以色谱指纹图谱指数F和色谱指纹图谱相对指数Fr作为评价毛细管电泳分析系统的目标函数,以雪莲药材水提取液为样品,考察一定浓度的硼砂、硼酸、磷酸氢二钠和磷酸二氢钠溶液按三角形优化法和四面体优化法构成背景电解质时对样品的分离情况,通过添加有机改性剂和调节pH进行再优化。用三角形法优化出以50 mmol/L硼砂-含3%乙腈的150 mmol/L磷酸二氢钠(体积比为1∶1)作为BGE时分离效果最佳,用四面体法优化出以50 mmol/L硼砂-150 mmol/L磷酸二氢钠-200 mmol/L硼酸(体积比为1∶1∶2,用0.1 mol/L氢氧化钠调pH 8.55)作为BGE时分离效果最佳,分别获得28个和25个电泳峰。所建立的方法操作简捷,适用于中药材水提取液或醇提取液的毛细管区带电泳BGE的选择。     

Determination of the electrophoretic mobility of bacteria and their separation by capillary zone electrophoresis

Conditions for the determination of electrophoretic mobilities of bacteria by capillary electrophoresis (CE) were explored. Most precise values are obtained using fused silica capillaries of 1–3 m length (0.25 mm inner diameter), a background buffer with an ionic strength of 0.0015 mol/L and a pH value of 7–10 at a field strength of 120 V/cm. Capillary electrophoretic separation of three different bacteria populations on the basis of their mobility differences could be realized. Electrophoretic band widths of all bacteria populations investigated are relatively large compared to molecule bands. It finds its explanation in the different distribution of surface charge density to cross-sectional area of each single cell of a population.     

1,3-dialkylimidazolium-based room-temperature ionic liquids as background electrolyte and coating material in aqueous capillary electrophoresis

The 1-ethyl-3-methylimidazolium (EMIM) cation was found to have constant mobility of 4.5 x 10(-4) cm2 V(-1) s(-1) over the pH range of 3 to 11. The electroosmotic flow of bare silica capillary was reversed by the covalently bonded room-temperature ionic liquid (RTIL) coating. With run buffer of 5 mM EMIM (pH 8.5), NH4+ in human urine was separated from the K+ matrix and was detected to be 0.37 +/- 0.012%. K+, Na+, Li+, Ca2+, Mg2+ and Ba2+ were baseline separated in RTIL-coated capillary with run buffer of 10 mM EMIMOH-acetic acid at pH 5, and the concentration of the above ions in a red wine were detected to be 907, 27.9, 0, 71.0, 83.4 and 31.1 microg/ml, respectively. The RTIL-coated capillary showed stable electroosmotic flow for at least 80 h in the run buffer.     

Determination of the electrophoretic mobility of bacteria and their separation by capillary zone electrophoresis

Conditions for the determination of electrophoretic mobilities of bacteria by capillary electrophoresis (CE) were explored. Most precise values are obtained using fused silica capillaries of 1–3 m length (0.25 mm inner diameter), a background buffer with an ionic strength of 0.0015 mol/L and a pH value of 7–10 at a field strength of 120 V/cm. Capillary electrophoretic separation of three different bacteria populations on the basis of their mobility differences could be realized. Electrophoretic band widths of all bacteria populations investigated are relatively large compared to molecule bands. It finds its explanation in the different distribution of surface charge density to cross-sectional area of each single cell of a population. Received: 30 January 1997 / Revised: 15 May 1997 / Accepted: 22 May 1997     

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.