Influence of ignored and well-known zone distortions on the separation performance of proteins in capillary free zone electrophoresis with special reference to analysis in polyacrylamide-coated fused silica capillaries in various buffers. II. Experimental studies at acidic pH with on-line enrichment

The separation of acidic and basic model proteins was studied in capillary free zone electrophoresis in a polyacrylamide-coated, electroosmosis-free capillary at pH below their isoelectric points (pI) using various buffers at pH 2.7-4.8 with UV detection at 200 nm. The separation performance was significantly dependent on the coating quality, which may even differ within the same batch of capillaries. In addition, a washing step with 2 M HCl and the storage of the capillary in distilled water was essential for the performance. For high efficiency and resolution the choice of buffer constituents was extremely important which is discussed in quantitative terms in Part I. The most promising buffers were ammonium acetate and ammonium hydroxyacetate at pH 4 (ionic strengths: 0.12 and 0.15 M, respectively) with plate numbers up to 1,700,000 plates/m, corresponding to a zone width (2sigma) of only 1 mm in a capillary with 40 cm effective length, when the injected samples were dissolved in a 10-fold diluted background electrolyte (BGE), a zone even narrower than those obtained in polyacrylamide gel electrophoresis, the characteristic feature of which is remarkably thin zones. In the experiment giving this plate number, the calculated variance for longitudinal diffusion was larger than all the other calculated variances (those for the width of the starting zone, Joule heating, sedimentation and the curvature of the capillary). Interestingly, the effect of capillary curvature was significant. In addition, the sum of all other imaginable variances (corresponding to various types of slow on/off kinetics and hyper-sharp peaks) was in the most successful experiments only 28-50% of the variance for longitudinal diffusion. One hundred- to two hundred-fold dilution of the BGE improved the detection limits and provided high precision in both migration times and peak areas with ammonium hydroxyacetate and ammonium acetate as background electrolytes. However, that high dilution increased the variance 140-400% for these buffers, respectively, at least partly due to conductivity or pH differences between the sample and buffer zones (hyper-sharp peaks). Sedimentation of the enriched sample, a factor that has not previously been treated theoretically or experimentally, was probably another reason for our finding that peak heights did not increase when the sample was dissolved in a buffer diluted more than 200-fold, although pH changes and in some cases thermal expansion in the capillary also may contribute. Loss of protein may occur at the ionic strength 0.01 and lower due to precipitation. Limits of detection were in the range 4-17 pmol of proteins with ammonium acetate as BGE. No indication of denaturation of proteins at pH 4 was observed. However, the separation performance at pH 3 was not satisfactory and loss of proteins was observed, possibly indicating such problems. The protein mobilities decreased unexpectedly from pH 4 to 3--a further indication of conformation changes.     

Zone broadening in electrophoresis with special reference to high-performance electrophoresis in capillaries: an interplay between theory and practice

Approximate equations have been derived for the total (final) zone width (plate height, plate number and resolution) as a function of the width of the starting zone and of the zone broadening caused by diffusion. Joule heat, adsorption and the difference in conductivity (delta kappa) between a solute zone and the surrounding buffer. Two cases are treated: (A) the conductivity differences eliminate entirely or (B) partially, the diffusional broadening at one boundary of a zone. When adsorption is negligible one can derive from these equations the field strength - and for case A also the electrical conductivity - that gives the minimum zone broadening (plate height). Interestingly, at this minimum, contributions to the zone broadening from diffusion. Joule heat and conductivity differences have the ratio 4:1:1 in case A. In case B the ratio between the broadening caused by diffusion (including that caused by conductivity and pH differences) and broadening due to Joule heat is 4:1. The total zone width, plate height and optimal field strength calculated from the derived equations agree satisfactorily with experimental values. A simple method to estimate the variance of the zone broadening caused by the Joule heat led to a formula similar to that calculated mathematically. An appropriate width of the starting zone can be calculated rapidly by means of a simple formula. Following a run the true width can be estimated graphically from measurements of plate heights or zone widths at low field strengths. For high resolution the width of the starting zone usually should not exceed 0.5 mm. A new principle for the design of multi-buffer systems which generate sufficiently narrow starting zones has been developed for carrier-free zone electrophoresis. This zone sharpening method permits application of wide zones of concentrations below the detection limit of the monitor. The diffusion coefficient (D) and the universal parameter D/mu (mu = mobility) appear in many of the equations derived and are often the only variables which are not easily accessible. Simple methods have therefore been developed by which they can be determined with sufficient accuracy. Fortunately, they are raised to the power of 1/5 in many formulas and therefore only a rough estimation is required. True plate numbers (calculated in the absence of electroendosmosis) often differ considerably from apparent plate numbers (calculated in the presence of electroendosmosis). A mathematical relationship between the true and apparent plate numbers has been derived.(ABSTRACT TRUNCATED AT 400 WORDS)     

毛细管区带电泳中场增强进样柱内富集的非线性特征

直接柱头场效应进样是一种毛细管区带电泳柱内富集,其进样过程中样品在柱内的分布可分为两部分,即在运行缓冲溶液中的堆积区段和由电渗流引入的样品溶液区段.通过对溶质输运行为的研究表明:两区段长度与进样时间之间并非简单的线性关系,因此进样量与进样时间的关系也非线性,且与溶质淌度有关;进样量的增加并不能导致富集倍数的同步增加,由于层流的作用使得场效应进样柱内富集效果降低.为了在保持柱效基本不变情况下得到好的富集效果,除需使溶质在运行缓冲溶液和样品溶液中的电导率比极大外,进样时间也应与之匹配.     

Formamide as solvent for capillary zone electrophoresis

A comprehensive investigation of a number of aspects when using formamide as background electrolyte solvent in capillary zone electrophoresis was presented. It included (i) the change of the ion mobility with ionic strength, (ii) the influence of the ionic strength on diffusion coefficients, and (iii) on the separation efficiency expressed by the maximum reachable plate numbers (when only longitudinal diffusion contributed to zone broadening), (iv) the effect of the solvent on pKa values (taken from the literature) of neutral and cation acids, (v) the establishment of the a pH scale in formamide by dissolving acids with known pKa values and their salts at defined proportion (thus circumventing the problem of calibrating the pH meter), (vi) the agreement between the experimentally derived and the theoretical dependence of the effective mobility on pH, (vii) the uptake of water of this hygroscopic solvent from the humidity of the environment and its consequence to the ion mobilities, pKa values, and the chemical stability of the solvent (e.g., hydrolysis), and finally (viii) the use of conductivity and indirect UV absorption to enable detection of analytes below the optical cutoff of formamide.     

Carbon nanotubes as separation carrier in capillary electrophoresis

The utility and versatility of carboxylic single-walled carbon nanotubes (c-SWNT) in capillary electrophoresis (CE) is demonstrated, using as model solutes homologues and structural isomers. In the case of homologues of caffeine and theobromine, distinct changes in the electrophoretic parameters occur at a critical concentration of c-SWNT in the run buffer. It is suggested that the c-SWNT of a definite concentration could form a network in the run buffer as a pseudostationary phase on the basis of the unique tubule structure, providing a different separation from sodium dodecyl sulfate (SDS) micelles. In the case of structural isomers of catechol and hydroquinone, differing from the homologues, it is mainly attributable to the functional groups on the c-SWNT that have an effect on the electrophoretic behaviors by forming intermolecular hydrogen bonding with analytes. Furthermore, aggregated c-SWNT serve as anticonvective media and minimize solute diffusion contributing to zone broadening. The presence of charged c-SWNT suppressed the electrodiffusion and decreased the adsorption between capillary wall and solutes, which led to better peak shapes of isomers.     

Capillary zone electrophoresis: Effect of physical parameters on separation efficiency and quantitation

Separation efficiency in capillary zone electrophoresis was examined as a function of capillary length and diameter as well as solute concentration for a borosilicate glass capillary/phosphate buffer system. Electroosmotic flow coefficients in borosilicate, fused silica, and teflon capillaries were measured over the useful operating pH range of 3–8 at constant applied power (0.333 W). Sample introduction by a simple, low-dispersion electromigration method was found to be a reliable procedure for solute quantitation.     

Retention of ionic and non-ionic catechols in capillary zone electrophoresis with micellar solutions

The use of micellar solutions in capillary zone electrophoresis has been primarily relegated to separations of non-ionic solutes, while its applicability to cationic species has been unexplored. We have found that the use of sodium dodecyl sulfate micelles in phosphate buffer allows for tremendous gains in selectivity for several cationic and non-ionic catechols over what can be obtained with normal capillary zone electrophoresis. Complexation of catechols with boric acid alters the net charge on the solutes and changes the partitioning behavior to produce adequate selectivity with improved analysis times. Although the mechanisms of solute interaction with the micellar phase for the cationic species are not decisively known, evidence is presented supporting the existence of ion-pairing equilibria simultaneously accompanied by micellar solubilization.     

The effect of electric field strength,buffer type and concentration on separation parameters in capillary zone electrophoresis

Summary Probe solutes were used to investigate the effect of buffer type, concentration and applied voltage on solute mobility, column efficiency and resolution in capillary zone electrophoresis. With low conductivity buffers higher concentrations and/or higher voltages could be used to improve column efficiency and resolution. Doubling the concentration of the buffer doubles the amount of heat generated inside the column while doubling the applied voltage cause a 4-fold increase. Solute migration time is approximately an inverse function of the charge density of the buffer's cation. Analysis time is increased by about 30% if the buffer concentration is doubled while it is cut in half if the applied voltage is doubled. Column efficiency is improved (higher theoretical plate count) with increasing buffer concentration and/or applied voltage as long as the heat generated is efficiently dissipated. The separation factor is directly related to analysis time and, therefore, selectivity improves with increasing buffer concentration but decreases with increasing applied voltage. Hence, resolution is optimized by increasing buffer concentration at a moderate applied voltage.     

A review of the zone broadening contributions in free-flow electrophoresis

The broadening of analyte streams, as they migrate through a free-flow electrophoresis (FFE) channel, often limits the resolving power of FFE separations. Under laminar flow conditions, such zonal spreading occurs due to analyte diffusion perpendicular to the direction of streamflow and variations in the lateral distance electrokinetically migrated by the analyte molecules. Although some of the factors that give rise to these contributions are inherent to the FFE method, others originate from non-idealities in the system, such as Joule heating, pressure-driven crossflows, and a difference between the electrical conductivities of the sample stream and background electrolyte. The injection process can further increase the stream width in FFE separations but normally influencing all analyte zones to an equal extent. Recently, several experimental and theoretical works have been reported that thoroughly investigate the various contributions to stream variance in an FFE device for better understanding, and potentially minimizing their magnitudes. In this review article, we carefully examine the findings from these studies and discuss areas in which more work is needed to advance our comprehension of the zone broadening contributions in FFE assays.     

Understanding mechanisms of pressure-assisted electrokinetic injection: application to analysis of bromate, arsenic and selenium species in drinking water by capillary electrophoresis-mass spectrometry

The mechanism underlying the enrichment power by pressure-assisted electrokinetic injection (PAEKI) in capillary electrophoresis (CE) was investigated for on-line pre-concentration of arsenic [As(III) and As(V)], selenium [Se(IV) and Se(VI)] and bromate (BrO(3)(-)). Analyte diffusion behaviour from PAEKI sample plugs were evaluated by monitoring peak broadening as a function of stagnant time and position in the capillary. During PAEKI, anionic analytes accumulate at the sample-separation buffer boundary. We proposed that a counter-ion layer formed in PAEKI, where a cation layer was formed at the separation buffer side of boundary. The cation layer served as a soft boundary which impeded zone broadening via electrostatic attraction between layers. This effect likely played an important role in maintaining focused analyte bands by suppressing diffusion. Comparison of analyte behaviour in PAEKI injected sample plugs to behaviour in hydrodynamically injected ones proved the existence of a counter-ion layer. The dependence of analyte diffusion in PAEKI plugs on electrochemical properties (viscosity, conductivity, electrophoretic mobility) further supported the hypothesis. Additionally, it was noted that analytes with low electrophoretic mobility were more efficiently pre-concentrated by PAEKI and were less subject to forces of dispersion than analytes with greater electrophoretic mobility. PAEKI-CE coupled to electrospray tandem mass spectroscopy (ESI-MS/MS) was then optimized and validated for detection of arsenic, selenium and bromate in water samples. On-line enrichment of the target analytes was achieved with 1-3 ng mL(-1) detection limits, which was below the maximum contaminant levels in drinking water for all five anions studied. Noteworthy, the potential of the method for unbiased detection of molecular species in untreated water was demonstrated. No contamination was detected in the water samples tested; however, recovery was 90-118% for spiked samples. The method was demonstrated be comparable to current methods for detection of inorganic contaminants in drinking water and is a good alternative method to ion chromatography/liquid chromatography-MS.     

Preparative capillary zone electrophoresis using a dynamic coated wide-bore capillary

Preparative capillary zone electrophoresis separations of cytochrome c from bovine and horse heart are performed efficiently in a surfactant-coated capillary. The surfactant, dimethylditetradecylammonium bromide (2C(14)DAB), effectively eliminated protein adsorption from the capillary surface, such that symmetrical peaks with efficiencies of 0.7 million plates/m were observed in 50-microm id capillaries when low concentrations of protein were injected. At protein concentrations greater than 1 g/L, electromigration dispersion became the dominant source of band broadening and the peak shape distorted to triangular fronting. Matching of the mobility of the buffer co-ion to that of the cytochrome c resulted in dramatic improvements in the efficiency and peak shape. Using 100 mM bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane phosphate buffer at pH 7.0 with a 100-microm id capillary, the maximum sample loading capacity in a single run was 160 pmol (2.0 microg) of each protein.     

Limitations of the optimization of the resolution by the buffer pH in capillary zone electrophoresis

The model which enables the prediction of the resolution as a function of the buffer pH for capillary zone electrophoresis described elsewhere has two main limitations. One limitation is linked to the accuracy of the data necessary for the calculation, namely actual mobilities, pK values and effective charge numbers of the separands. The other is given by the fact that only longitudinal diffusion is taken as the source of peak dispersion in the model. Examples for deviations from the predicted resolution are discussed, with wall adsorption of small ions contributing significantly to migration and distorsion.This work is dedicated to S. Hjertén on the occasion of his 65th birthday     

New peak broadening parameter for the characterization of separation capability in capillary electrophoresis

The influence of separation conditions on peak broadening is usually estimated by the number of theoretical plates. Using the data available in literature and experimental data, it is shown that in pressure‐assisted capillary electrophoresis the plate number is not directly related to the separation capability of conditions used. The experiments at different electrolyte flow velocities demonstrate that a higher plate number (the best separation efficiency) can be obtained with a lower peak resolution. Since ions are separated by electrophoresis due to the difference in electrophoretic mobilities, the peak width in terms of electrophoretic mobility is suggested as a new peak broadening parameter describing the separation capability of the conditions used. The parameter can be calculated using the tailing factor and the temporal peak width at 5% of the peak height. A simple equation for the resolution calculation is derived using the parameter. The advantage of the peak width in terms of mobility over other parameters is shown. The new parameter is recommended to be used not only in pressure‐assisted capillary electrophoresis but also in general capillary electrophoresis when in a number of runs the virtual separative migration distance and separation capability of the conditions used change widely.     

Separation of nicotine metabolites by capillary zone electrophoresis and capillary zone electrophoresis/mass spectrometry

The use of capillary zone electrophoresis (CZE) and capillary zone electrophoresis/mass spectrometry (CZE/MS) has been demonstrated, in principle, for the separation of nicotine and nicotine metabolites. The buffer system developed for separation and detection by CZE/UV was modified for use in CZE/MS analysis. Several of the metabolites are isobaric and tandem mass spectrometric (MS/MS) techniques have been used to differentiate such analytes.     

Reaction of fluorogenic reagents with proteins II: capillary electrophoresis and laser-induced fluorescence properties of proteins labeled with Chromeo P465

The fluorogenic reagent Chromeo P465 is considered for the analysis of proteins by capillary electrophoresis with laser-induced fluorescence detection. The reagent was first used to label alpha-lactalbumin; the product was analyzed by capillary zone electrophoresis in a sub-micellar sodium dodecyl sulfate (SDS) buffer. The product generated a set of equally spaced but poorly resolved peaks that formed a broad envelope with a net mobility of 4 x 10(-4)cm(2) V(-1) s(-1). The components of the envelope were presumably protein that had reacted with different numbers of labels. The mobility of these components decreased by roughly 1% with the addition of each label. The signal increased linearly from 1.0 nM to 100 nM alpha-lactalbumin (r(2)=0.99), with a 3sigma detection limit of 70 pM. We then considered the separation of a mixture of ovalbumin, alpha-chymotrypsinogen A, and alpha-lactalbumin labeled with Chromeo P465; unfortunately, baseline resolution was not achieved with a borax/SDS buffer. Better resolution was achieved with N-cyclohexyl-2-aminoethanesulfonic acid/Tris/SDS/dextran capillary sieving electrophoresis; however, dye interactions with this buffer system produced a less than ideal blank.     

Separation of alkylbenzyl quaternary ammonium compounds by capillary zone electrophoresis with sample stacking injection

Summary A systematic investigation of operational buffer systems, sample preparation and instrument parameters for achieving the best possible performance for determinating an homologous series of N-benzyl-N-alkyl-N,N-dimethylammonium chloride compounds by capillary zone electrophoresis with direct UV detection. The most effective separation was achieved within 3.5 min with the addition of acetonitrile (40%) in a phosphate buffer (20 mM pH 5.2) using a 40 cm fused-silica capillary operating at 25 KV and 20°C. Degassing of all electrolyte solutions and samples was very important. The linearity and repeatability for each compounds were satisfactory. To improve detection limits, on-column sample preconcentration, sample stacking, was investigated achieving a tenfold enrichment factor and quantitation limits about 10⁻⁷M.     

Two-step stacking in capillary zone electrophoresis featuring sweeping and micelle to solvent stacking: I. Organic cations

Two-step stacking of organic cations by sweeping and micelle to solvent stacking (MSS) in capillary zone electrophoresis (CZE) is presented. The simple procedure involves hydrodynamic injection of a micellar sodium dodecyl sulfate solution before the sample that is prepared without the micelles. The micelles sweep and transport the cations to the boundary zone between the sample and CZE buffer. The presence of organic solvent in the CZE buffer induces the second stacking step of MSS. The LODs obtained for the four beta blocker and two tricyclic antidepressant test drugs were 20-50 times better compared to typical injection.     

Capillary zone electrophoresis for the analysis of glycoforms of cellobiohydrolase

Cellobiohydrolase (CBH) is an important enzyme for the conversion of lignocellulosic biomass to ethanol. This work separated the glycoforms of CBH possessing different numbers of neutral mannoses using capillary zone electrophoresis (CZE) in a 50 mM, pH 7.5 phosphate buffer. The method analysed CBH in an intact form using a polyacrylamide coated fused silica capillary without requiring additives or labelling of the enzyme. The migration time of the major peak was found to be 21.6±0.1 min (n=3) and the approach is suitable for testing of batch-to-batch consistency of CBH. Ease-of-use, automation and speed are the other benefits due to which the use of CZE for analysing glycoforms of CBH was concluded to be ideal.     

Determination of the cis-trans isomerization barrier of several L-peptidyl-L-proline dipeptides by dynamic capillary electrophoresis and computer simulation

Dynamic capillary electrophoresis (DCE) and computer simulation of the elution profiles with the theoretical plate and the stochastic model has been applied to determine the isomerization barriers of the three dipeptides L-alanyl-L-proline, L-leucyl-L-proline, and L-phenylalanyl-L-proline. The separation of the rotational cis-trans isomers has been performed in an aqueous 70 mM borate buffer at pH 9.5. Interconversion profiles featuring plateau formation and peak broadening were observed. To determine the rate constants k1 and k(-1) of the cis-trans isomerization in dynamic capillary electrophoresis, equations have been derived for the theoretical plate model and stochastic model. The electropherograms were simulated with the ChromWin software which uses the experimental data plateau height h(plateau), peak width at half height Wh, the total migration times of the cis-trans isomers tR and the electroosmotic break-through time t0 as well as the peak ratio [cis]/[trans]. From temperature-dependent measurements, the rate constants k1 and k(-1) and the kinetic activation parameters deltaG#, deltaH# and deltaS# of the cis-trans isomerization of the three dipeptides were obtained.     

样品溶液基质对毛细管电泳分离的影响

用样品苯甲醇，苯甲醛和苯甲酸研究了样品溶液基质十二烷基硫酸钠，磷酸二氢钾－四硼酸钠和乙醇对毛细管电泳分离的影响。实验结果显示，随着样品溶液基质种类和浓度的变化，非离子型的苯甲醇和苯甲醛的毛细管电泳分离有相同的变化规律，离子型的苯甲酸有相反的变化规律。乙醇的添加导致了三个组份的区带扩张。