The effect of electroosmotic and hydrodynamic flow profile superposition on band broadening in capillary electrophoresis

The effect of the superposition of electroosmotic flow and pressureinduced hydrodynamic counterflow on efficiency has been investigated for different capillary electrophoretic systems. Results are shown for 50 and 75 μm internal diameter capillaries at several voltage and counterpressure levels. Hydrodynamic counterflows were successfully applied in electrokinetic chromatography in order to delay the entry of a UV-active pseudostationary phase, tetraphenyl porphyrintetrasulfonate, into the detection zone allowing the separation of neutral nitroaromatics. The separations are based on the weak charge-transfer interactions between the porphyrin and the analytes.     

Dynamic capillary coatings for electroosmotic flow control in capillary electrophoresis

Control of the electroosmotic flow (EOF) is critical for achieving optimal separations by capillary electrophoresis. For instance, manipulation of the EOF can yield either high resolution separations or rapid analyses. Dynamic capillary coatings are a simple and cost-effective approach to altering the EOF. The normal EOF can be slowed using buffer additives such as Mg²⁺ and hexamethonium which ion exchange onto the surface silanols to lower the effective wall charge. Alternatively, cationic polyelectrolytes or cationic surfactants can be used to establish a cationic coating on the capillary wall, which results in a reversed EOF. Practical considerations such as pH stability and reproducibility obtainable with an EOF modifier will be discussed.     

Peak broadening in capillary zone electrophoresis with electroosmotic flow: Dependence of plate number and resolution on charge number

Summary Based on the Einstein-Nernst equation, describing the relation between diffusion coefficient and ionic mobility, and expression for the plate height and plate number can be obtained which is independent of the diffusion coefficient. This approach was supported by experimental data, obtained from anionic and cationic solutes. An expression for the electrophoretic resolution is introduced that is also independent on diffusion coefficients. The effect of electro-osmotic flow on the separation of anions and cations based on the expressions derived for the plate number and the resolution is discussed.     

Investigation of the electroosmotic flow effect on the efficiency of capillary electrophoresis.

The influence of the electroosmotic flow profile on the efficiency and resolution of capillary electrophoresis is studied. The mathematical model is formulated and the set of equations is solved numerically. The results of the analysis are applicable to a wide range of buffer concentrations and capillary diameters. The temperature dependence of electrophoretic mobility, viscosity and thermal conductivity and the dependence of electrical conductivity on temperature and ion concentration in the double layer are taken into account. It is shown that there exists a region of buffer concentrations and capillary diameters where the influence of the electroosmotic flow profile on the efficiency and resolution is much greater than that of the temperature dependence of the electrophoretic mobility. The results are especially essential for small buffer concentrations or capillary diameters comparable with the double electrical layer thickness.     

Mobilization of electroosmotic flow markers in capillary zone electrophoresis

UV-absorbing neutral substances are commonly used as markers of mean electroosmotic flow in capillary electrophoresis for their zero electrophoretic mobility in an electric field. However, some of these markers can interact with background electrolyte components and migrate at a different velocity than the electroosmotic flow. Thus, we tested 11 markers primarily varying in their degree of methylation and type of central atom in combination with five background electrolyte cations differing in their ionic radii and surface charge density, measuring the relative electrophoretic mobility using thiourea as a reference marker. Our results from this set of experiments showed some general trends in the mobilization of the markers based on the effects of marker structure and type of background electrolyte cation on the relative electrophoretic mobility. As an example, the effects of an inadequate choice of marker on analyte identification were illustrated in the electrophoretic separation of glucosinolates. Therefore, our findings may help electrophoretists appropriately select electroosmotic flow markers for various electrophoretic systems.     

Theory of band broadening during cycling temperature capillary electrophoresis

A two-state transport model is presented for cycling temperature CE. In this high-throughput method of mutation detection, the temperature oscillates in time, causing the DNA to periodically transit between an annealed state and a partially melted (denatured) state. The change in state alters the electrophoretic mobility, and the presence of a mutation changes the temperature dependence of the denaturing/annealing kinetics. Asymptotic formulae for the mean velocity and effective diffusivity (dispersivity) of the DNA are computed by a multiple-time scales scheme in the limit where the DNA have experienced many temperature cycles before reaching the detector. Explicit analytical results are presented for the case where the temperature cycle consists of one interval with irreversible annealing, followed by a second interval with an infinitely fast, irreversible denaturation. The lag in the annealing leads to a reduction in the mean velocity and an enhanced dispersion compared to the idealized case where the DNA respond instantaneously to the changes in temperature, with the dispersion scaling quadratically with the electric field. The predicted plate height scales linearly with the electric field, and the optimal separation resolution is predicted for moderate values of the cycle frequency that allow the DNA to relax during each temperature oscillation.     

Control of electroosmotic flow in nonaqueous capillary electrophoresis by polymer capillary coatings

In aqueous capillary electrophoresis the electroosmotic flow (EOF) can be strongly suppressed or eliminated by coating the capillary surface silanols either by buffer additive adsorption or chemical modification. Hydrophilic coatings, e.g., polyvinyl alcohol (PVA) proved to be most efficient for EOF control in applications like DNA analysis. In nonaqueous capillary electrophoresis (NACE), however, the EOF cannot be totally suppressed with these capillaries and coating efficiency turned out to be solvent-depending. In this paper, fused-silica capillaries with monomeric and polymeric coatings differing in hydrophobicity and chemical properties (vinyl, vinyl acetate, vinyl alcohol and acrylates with different alkyl chain length) were investigated. Besides studying the EOF characteristics with different organic solvents and water, gas chromatography (GC) measurements were carried out to probe the silanol reduction via ether retention and the surface hydrophobicity by retention of nonane. Good correlations between GC results and EOF magnitude could be found. It could be demonstrated that the polymeric coating has to be solvatized by the buffer solvent to reduce the EOF. The PVA coating was optimal for aqueous systems but not effective for some nonaqueous buffers. On the other hand, polyvinyl acetate and polyethyl acrylate as polymeric coatings proved to be optimal to reduce the EOF in NACE.     

Indirect amperometric measurement of electroosmotic flow rates and effective mobilities in microchip capillary electrophoresis

End-channel indirect amperometry is based on the principle of Kohlrausch regulating function (KRF). A dilute electroactive ionic species is added to the background electrolyte as a continuously eluting electrophore, which is used as probe. The probe concentration variation with the omega value of KRF in the sampling zone was described schematically in this report. Either cathodic or anodic electroosmotic flow (EOF) rates were monitored in microchip. There was no significant difference between the values of EOF rates measured by present method and current-monitoring method. Detection of electroactive and nonelectroactive analytes can also be accomplished by indirect amperometric method. Hence, the effective mobility of analytes can be accurately calculated. And the response mechanism of nonelectroactive analytes K(+), Na(+) and Li(+) in the indirect method was speculated.     

Characteristics of the electroosmotic flow of electrolyte systems for nonaqueous capillary electrophoresis

Nonaqueous capillary electrophoresis (NACE) is a powerful tool for the analysis of surface-active substances, which represent a broad class of analytes containing cationic and anionic species, such as surfactants, phosphoric acid esters, and amines. In order to conduct an efficient method development in NACE, the influence of the electrolyte composition on the electroosmotic flow (EOF) of organic separation systems was systematically investigated. Background electrolytes and background chromophores appropriate for direct and indirect UV-detection were considered, as the majority of surface-active substances do not absorb UV-light. It was found that theoretical models developed to describe the EOF in aqueous electrolyte systems are insufficient for organic electrolyte systems. Experimental data on electroosmosis in a variety of organic solvents and mixtures of methanol and acetonitrile applying different background chromophores and basic or acidic additives are given. Differences between them are discussed with relation to the physicochemical properties of the organic solvents.     

Sample stacking of fast-moving anions in capillary zone electrophoresis with pH-suppressed electroosmotic flow

On-line sample concentration of fast moving inorganic anions by large volume sample stacking (LVSS) and field enhanced sample injection (FESI) with a water plug under acidic conditions is presented. Detection sensitivity enhancements were around 100 and 1000-fold for LVSS and FESI, respectively. However, reproducibility and linearity of response in the LVSS approach is superior compared to the FESI approach.     

Mobility-based selective on-line preconcentration of proteins in capillary electrophoresis by controlling electroosmotic flow

A simple method to perform selective on-line preconcentration of protein samples in capillary electrophoresis (CE) is described. The selectivity, based on protein electrophoretic mobility, was achieved by controlling electroosmotic flow (EOF). A short section of dialysis hollow fiber, serving as a porous joint, was connected between two lengths of fused silica capillary. High voltage was applied separately to each capillary, and the EOF in the system was controlled independently of the local electric field intensity by controlling the total voltage drop. An equation relating the EOF with the total voltage drop was derived and evaluated experimentally. On-line preconcentration of both positively charged and negatively charged model proteins was demonstrated without using discontinuous background electrolytes, and protein analytes were concentrated by approximately 60-200-fold under various conditions. For positively charged proteins, positive voltages of the same magnitude were applied at the free ends of the connected capillaries while the porous joint was grounded. This provided a zero EOF in the system and a non-zero local electric field in each capillary to drive the positively charged analytes to the porous joint. CE separation was then initiated by switching the polarity of the high voltage over the second capillary. For negatively charged proteins, the procedure was the same except negative voltages were applied at the free ends of the capillaries. Mobility-based selective on-line preconcentration was also demonstrated with two negatively charged proteins, i.e. beta-lactoglobulin B and myoglobin. In this case, negative voltages of different values were applied at the free ends of the capillaries with different values, which provided a non-zero EOF in the system. The direction of EOF was the same as that of the electrophoretic migration velocities of the protein analytes in the first capillary and opposite in the second capillary. By controlling the EOF, beta-lactoglobulin B, which has a higher mobility, could be concentrated over 150-fold with a 15 min injection while myoglobin, which has a lower mobility, was eliminated from the system.     

System peaks and optimization of anion separation in capillary electrophoresis with non-reversed electroosmotic flow

We studied system peaks present in the electropherograms obtained in the separation of anions by capillary electrophoresis with indirect spectrophotometric detection and cathode electroosmotic flow (EOF) with a chromate background electrolyte. The system peaks correspond to the zones with changed concentration of the background electrolyte; they formed when the zones of each analyte passed through the outlet of the capillary and then moved towards the EOF detector. It has been revealed that the height and area of the system peaks linearly depends on the concentration of the corresponding anion and the areas of the system peaks can achieve 10% of the anion peak area. An algorithm has been proposed for the determination of the optimal conditions for anion separation using hydrodynamic pressure for the regulation of the EOF flow rate. This algorithm prevents the overlapping of the anion and system peaks.     

Hydrodynamic suppression of the electroosmotic flow in capillary electrophoresis with indirect spectrophotometric detection

A version of capillary electrophoresis with indirect spectrophotometric detection and the hydrodynamic suppression of electroosmotic flow is studied. It is shown that, to improve the reliability of ion identification, one should calculate electrophoretic mobilities of ions or migration times corrected with regard to the electroosmotic flow rate. Correlations between electrophoretic peak areas of ions and their electrophoretic mobilities are derived. In the studied version of capillary electrophoresis, the accuracy of measuring anion concentrations can be improved using the internal standard method.     

Large-volume stacking in capillary electrophoresis using pH hysteresis of the electroosmotic flow in a bare fused-silica capillary

For large-volume stacking with the electroosmotic flow pump (LVSEP) in capillary electrophoresis of anionic analytes it is required that the electroosmotic mobility (EOM) should be smaller than the magnitudes of the effective mobilities of the analytes. When a fused-silica capillary is treated with an acidic solution, the silanoate group on the silica surface is neutralized to silanol and the EOM is suppressed. Due to the slow deprotonation equilibrium of the silanol group at an intermediate pH, this reduced EOM can be retained during a number of electrophoresis runs. Using a bare fused-silica capillary preconditioned with 0.01 M HCl, successful LVSEP at pH 6.0 was achieved for weakly acidic compounds with two orders of magnitude enhancements in the concentration sensitivity. The repeatability in migration times of ten analytes stacked by LVSEP in a single day was excellent with the relative standard deviation (RSD) less than 1% (n = 6). The day-to-day repeatability was also excellent with RSD less than 3% (n = 3 x 6) when the capillary was preconditioned each day.     

Extracolumn band broadening in capillary liquid chromatography

A commercially available capillary LC instrument was modified to investigate and control the contribution of different instrument components on extracolumn band broadening. Quantitative estimations of dispersion induced by several equipmental parts were carried out. Injection parameters could be optimized to achieve the theoretical value of 12 for a profile factor describing a rectangular sample profile. Additionally, an additive injector flow channel dependent dispersion effect was found. A practical approach for minimizing instrumental effects in capillary LC is suggested. The results were compared with those obtained with an HPLC instrument designed for conventional size columns.     

Analysis of neutral surfactants by non-aqueous capillary electrophoresis using an electroosmotic flow reversal

The separation of KM 20, that is in fact a mixture of non-ionic surfactants, was carried out by non-aqueous capillary electrophoresis. This complex mixture resulting from the condensation of ethylene oxide with fatty alcohols does not have chromophoric moieties. So, we analysed it after derivatization by means of 3,5-dinitrobenzoyl chloride. The proposed approach is based both on the formation of complexes with alkaline or ammonium cations in methanol and on the utilisation of a positively charged capillary. From a comparative study on the capillary treatment procedure, we used hexadimethrine bromide as electroosmotic flow reverser in order to obtain both repeatable analyses and good resolutions of the largest KM 20 oligomers. Then, among the five cations used to form complexes with KM 20, we pointed out that ammonium cation led to the best resolutions. Moreover, we evidenced that the counter-ion of this cation had a great influence on resolution because it modified the magnitude of electroosmotic flow. Ion pair formation that is more or less strong between ammonium and its counter-ion was involved in this variation of electroosmotic flow. So, we calculated the association constants for various ammonium salts in methanol. Then, using ammonium chloride as background electrolyte, we optimised the concentration of this salt, in methanol, in order to reach the optimal separation of KM 20 oligomers. Thus, a baseline separation was obtained by using 6 x 10(-2) mol/L NH4Cl as running electrolyte. In these conditions, we separated, in about 30 min, more than 30 oligomers of KM 20. The distribution of these oligomers that was determined from the optimal separation, appeared consistent with that obtained from HPLC analyses. Indeed, we determined that the mean ethoxylation number was equal to 18 while its real value is equal to 20.     

反向电渗流非水毛细管电泳法快速测定微量赤霉素

赤霉素是一类重要的植物激素,是结构相似的弱酸性二萜类化合物。针对毛细管电泳法分离、测定赤霉素速度慢、效率不佳等问题,该文发展了一种可快速测定微量活性赤霉素的非水毛细管电泳-紫外检测法。以聚环氧乙烷动态涂布毛细管,利用正离子使电渗流反向,将含10 mmol/L醋酸铵的甲醇-水(95∶5,v/v)作为缓冲体系,在0.08%(v/v)醋酸含量下,分离8种内源性赤霉素。结果表明,赤霉素出峰时间的相对标准偏差(RSD)≤2.1%(日内)或≤4.3%(日间),峰面积的RSD≤4.5%(日内)或≤6.9%(日间),检出限(S/N=3)为1.04~2.20 mg/L,相关系数为0.998 2~0.999 3,回收率为87.2%~93.5%。该方法简单、快速、稳定,以含醋酸铵的甲醇水溶液为缓冲体系,预先考虑了质谱测定的需要,可用于实际样品如麦芽中赤霉素的分析,具有一定的应用价值。     

Diffusion as major source of band broadening in field-amplified sample stacking under negligible electroosmotic flow velocity conditions

Theory of field-amplified sample stacking (FASS) also called field-enhanced sample stacking is reevaluated considering the early work of Chien, Burgi and Helmer. The classical theory presented by Chien, Helmer and Burgi predicts the existence of maxima, which are ascribed to the counteracting principles of zone focusing and hydrodynamic dispersion. In contrast to their work, we here focus on cationic analytes separated in an acidic background electrolyte providing a very low electroosmotic flow velocity. Therefore, peak broadening due to differences in the local electroosmotic flow velocities in different compartments of the capillary can be regarded to be negligible. Consequently, peak broadening resulting from hydrodynamic dispersion will not be the dominant limitation of the accessible enrichment efficiency. In our experimental studies we, however, obtain an optimum value for the field enhancement factor (maximum of the enrichment efficiency, when varying the electric conductivity of the sample and the size of the sample injection plug) corresponding to a 10-fold dilution of the BGE in the sample solution. Comparing these experimental data with data modeled according to the revised theory, we show that this limitation of the loadability is caused by the unavoidable decrease of the analyte migration velocity in the BGE compartment of the capillary when injecting of a sample plug of lower electric conductivity (decrease in the local electric field strength). The additional diffusional band broadening limits the obtainable enrichment efficiency.