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.     

Salt effects in capillary zone electrophoresis. V. Adsorption and retention of electrically neutral analytes

Two electrically neutral analytes previously observed to be separated from the neutral marker in capillary zone electrophoresis (CZE) experiments [sulphanilamide (SAA) and sulphaguanidine (SGW)] have been examined to determine the basis for separation. The degree of separation increases markedly with buffer concentration and improves with increasing field strength. On the basis of the apparent electrophoretic mobilities in conventional CZE, migration times in a zero EOF environment were calculated for SAA, SGW and six other sulphonamides that were known to be ionized. These six markers were used to test the legitimacy of our predictions and to correct for small discrepancies between the predicted and observed migration times. It was concluded that SAA and SGW have negligible electrophoretic mobilities and that they are retained in the electrical double layer close to the capillary wall. A mechanism for adsorption is proposed and the generality of the phenomenon is discussed.     

Change of migration time and separation window accompanied by field-enhanced sample stacking in capillary zone electrophoresis.

When field-enhanced sample stacking was used in capillary zone electrophoresis (CZE) analysis of cations, the decrease of migration time and the reduction of separation window was observed with increase of sample plug length. A simple equation expressing the migration velocity in the stacking process was derived to explain the above phenomenon. From experiments and theoretical consideration, we confirmed that this effect was caused by the higher potential gradient and larger eletroosmotic flow (EOF) mobility at the sample plug than those at the supporting electrolyte. A mathematical model appropriate for the computer simulation of such a system was studied considering the experimental results, and it was concluded that electroosmotic velocity (v(eof)) should be introduced to the equation of continuity as a constant.     

Application of 1-alkyl-3-methylimidazolium-based ionic liquids in separation of bioactive flavonoids by capillary zone electrophoresis

Room-temperature ionic liquids (ILs) are liquids that are constituted entirely of ions and can provide a solvent environment quite unlike any other available at room temperature. They continue to attract considerable interest in the chemistry research community as they are good solvents for a wide range of both inorganic and organic materials. In this study, a CZE method has been established for resolving natural flavonoids, quercetin, kaempferol and isorhamnetin in the Chinese herbal extract from Hippophae rhamnoides and its medicinal preparation (Sindacon Tablet). In this method, 1-alkyl-3-methyl-imidazolium-based ILs are used as the additive, and the effects of the alkyl group, imidazolium counterion (anionic part), along with the concentration of IL are investigated and discussed. Baseline separation, high efficiencies and symmetrical peaks of the three flavonoids were obtained. The separation mechanism seems to be the hydrogen-bonding interaction between the imidazolium cations of IL and the flavonoids.     

Th(IV) adsorption on alumina: effects of contact time, pH, ionic strength and phosphate

Adsorption of Th(IV) (total concentration, 10(-5)-10(-4) mol/L) was studied by a batch technique. The effects of pH, ionic strength, contact time, and phosphate on the adsorption of Th(IV) onto alumina were investigated. Adsorption isotherms of Th(IV) on alumina at approximately constant pH and three ionic strengths (0.05, 0.1, 0.5 mol/L KNO3) were determined. It was found that the pH values of aqueous solutions of both the Th(IV)-alumina and phosphate-alumina adsorption systems increase with increasing contact time, respectively. Adsorption of Th(IV) on alumina steeply increases with increasing pH from 1 to 4.5 and the adsorption edge consists of three regions. The phosphate added clearly enhances Th(IV) adsorption in the pH range 1-4. From the adsorption isotherms at approximately constant pH and three different ionic strengths, a reduced ionic strength effect was observed and is contradictory to the insensitive effect obtained from the adsorption edges on three oxides of Fe, Al, and Si at different ionic strengths. Compared with the adsorption edges at different ionic strengths, the adsorption isotherms at approximately constant pH and different ionic strengths are more advantageous in the investigation of ionic strength effect. The significantly positive effect of phosphate on Th(IV) adsorption onto alumina was attributed to strong surface binding of phosphate on alumina and the subsequent formation of ternary surface complexes involving Th(IV) and phosphate.     

Optimization of separation and migration behavior of cephalosporins in capillary zone electrophoresis

The influences of buffer pH, buffer concentration and buffer electrolyte on the migration behavior and separation of 12 cephalosporin antibiotics in capillary zone electrophoresis using three different types of buffer electrolyte, including phosphate, citrate, and 2-(N-morpholino)ethanesulfonate (MES), were investigated. The results indicate that, although buffer pH is a crucial parameter, buffer concentration also plays an important role in the separation of cephalosporins, particularly when cefuroxime and cefazolin, cephalexin and cefaclor, or cefotaxime and cephapirin are present as analytes at the same time. The electrophoretic mobility of cephalosporins and electroosmotic mobility measured in citrate and MES buffers are remarkably different from those measured in phosphate buffer. With citrate buffer, optimum buffer concentration is confined to a small range (35-40 mM), whereas buffer concentrations up to 300 mM can be used with MES buffer. Complete separations of 12 cephalosporins could be satisfactorily achieved with these three buffers under various optimum conditions. However, the separability of 12 cephalosporins with citrate or MES buffer is better than that with phosphate buffer. As a consequence of a greater electrophoretic mobility of cephalosporins than the electroosmotic mobility with citrate buffer at pH below about 5, some cephalosporins are not detectable. The cloudiness of the peak identification and of the magnitudes of the electrophoretic mobility of cefotaxime and cefuroxime reported previously are clarified. In addition, the pKa values of cephradine, cephalexin, cefaclor, and cephapirin attributed to the deprotonation of either an amino group or a pyridinium group are reported, and the migration behavior of these cephalosporins in the pH range studied is quantitatively described.     

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.     

Sorption of Th(IV) ions onto TiO2: Effects of contact time, ionic strength, thorium concentration and phosphate

Summary The sorption of Th(IV) onto TiO₂ was studied by the batch technique as a function of pH and ionic strength at moderate concentration (10^-4-10^-5 mol/l) and in the presence and absence of phosphate. It was found that the sorption rate of Th(IV) was relatively slow, the sorption percent was abruptly increased from pH 2 to 4, and the sorption was decreased with increasing ionic strength as a whole. In the concentration range of Th(IV) from trace concentration to 1.4 ^. 10^-4 mol/l and in the absence of phosphate, the sorption isotherms were roughly fitted the Freundlich equation at different ionic strengths and approximately constant pH. These sorption characteristics of Th(IV) onto TiO₂ were compared with those of uranyl on the same sorbent. In addition, the positive effect of phosphate on the sorption of Th(IV) onto TiO₂ was demonstrated obviously and can be attributed to strong surface binding of phosphate, and the subsequent formation of ternary surface complexes of Th(IV). The difference between the sorption characteristics of Th(IV) ions and uranyl ions onto TiO₂ is discussed.     

Study of ethidium bromide effect on dsDNA separation by capillary zone electrophoresis and laser light scattering

The effect of ethidium bromide (EB) on dsDNA separation was studied by using capillary zone electrophoresis (CZE) and laser light scattering (LLS). By increasing the concentration from 1 to 15 microg/mL, EB showed significant influence on the separation of pBR322 HaeIII in terms of resolution, speed, and signal-over-noise ratio. The physicochemical properties of DNA, such as mobility under electric field, charge-over-size ratio, and hydrodynamic radius, showed two declines, a sharp drop followed by a relaxed decrease, with increasing EB concentration. Such observations could be explained by using two modes of the dye binding process: intercalation and outside binding. The combination of results obtained from CZE and LLS also confirmed the existence of outside binding. Compared with intercalation, outside binding showed a weaker dye concentration dependence in neutralizing the negative charges on the DNA fragments. Static light scattering showed that and of DNA in 1xTris-borate-EDTA (TBE) were increased with increasing EB concentration. / of DNA with dye had a value larger than 1.5, suggesting a random coil conformation.     

Use of cyclodextrins for the enantioselective separation of ergot alkaloids by capillary zone electrophoresis.

Ergot alkaloid enantiomer derivatives were resolved using capillary zone electrophoresis. The effect of cyclodextrins, added to the background electrolyte, on the migration time and the resolution was studied. Good separation for epimeric ergot alkaloid derivatives was also obtained using phosphate buffer at pH 2.5. Separation was improved by supplementing the background electrolyte with 30 mM of gamma-cyclodextrin. Good resolution of racemic ergot alkaloid derivatives in their enantiomers was achieved in a background electrolyte containing either beta-cyclodextrin or its derivative, or gamma-cyclodextrin.     

Polymeric ionic liquid as additive for the high speed and efficient separation of aromatic acids by co-electroosmotic capillary electrophoresis

A simple and reliable co-electroosmotic capillary electrophoresis system for the fast determination of aromatic acids has been developed by employing poly (1-vinyl-3-butylimidazolium bromide) as the background electrolyte modifier. The polymeric ionic liquid was synthesized by the conventional radical polymerization. The reversed electroosmotic flow was obtained by adding a small amount of the polymeric ionic liquid (0.0006%, w/v) to the electrolyte. To further improve the resolution of aromatic acids, conditions including the concentration of polymeric ionic liquid and pH of background electrolytes were optimized. All eight aromatic acids were baseline resolved in one measurement in a short time (less than 3.5 min) under optimized conditions, 100 mM NaH₂PO₄ buffer containing 0.006% (w/v) polymeric ionic liquid, pH 6.0. Separation efficiencies were in the range from 355,000 to 943,000 (plates/m). Satisfactory reproducibility on the basis of the migration time of analytes was achieved. RSDs (n = 3) were less than 0.33% except the p-aminobenzoic acid (0.9%). The applicability of the present method has been demonstrated for the determination of water-soluble aromatic acids in a common drug for external use.     

Enantiomeric separation of D- and L-carnitine by integrating on-line derivatization with capillary zone electrophoresis.

A new capillary zone electrophoretic method has been developed for the enantiomeric separation and quantification of enantiomers of carnitine, D- and L-carnitine were derivatized with 9-fluorenylmethyl chloroformate in a flow system, working on-line with the capillary electrophoretic equipment. The separation was performed using a selective chiral buffer containing 2,6-dimethyl-beta-cyclodextrin (heptakis). Triethanolamine was used as electroosmotic modifier and the separation was carried out in a uncoated capillary. Under the optimal conditions the resolution between D- and L-carnitine was 1.2 and the limits of detection for both isomers were about 5.0 microM. The proposed method was applied to the determination of D-carnitine in excess of L-carnitine in synthetic samples, and the results demonstrated that the maximal D-:L-carnitine ratio determined was approximately 1:100.     

Comparative study of capillary zone electrophoresis and micellar electrokinetic capillary chromatography for the separation of naphthalenedisulfonate isomers

We investigated the separation of a test mixture of nine substituted and unsubstituted naphthalenedisulfonate isomers by capillary electrophoresis with a UV diode array detector. In particular, we focused on how the composition of the running buffer affected the separation selectivity. When capillary zone electrophoresis was carried out, the best results were obtained when organic solvents such as ethanol or propan-2-ol were added. Eight peaks were baseline separated but in no case were all the unsubstituted isomers separated. Therefore, capillary electrophoretic separation of the compounds was examined in the presence of micellar agents, such as sodium dodecyl sulfate, polyethylene glycol dodecyl ether (Brij 35) and cetyltrimethylammonium bromide. All the substituted isomers and two of the unsubstituted isomers were well resolved within 20 min by micellar electrokinetic capillary chromatography when Brij 35 was used as micellar agent. Separations were reproducible, in terms of peak area and migration time, under these conditions.     

Electrophoretic behavior of peptides in capillary electrophoresis influence of ionic strength and pH in aqueous-organic media.

Through correct pH, pKa and activity coefficients values, a model describing the effect of pH on electrophoretic mobility of substances has been applied to a series of peptides in water and in acetonitrile-water mixtures. The derived equations permit prediction of the optimum pH for the electrophoretic separation from only a few experimental values and they also permit determination of pKa values of analytes in the aqueous-organic media employed. Furthermore, the electrophoretic resolution between pairs of substances can be predicted, in order to evaluate electrophoretic separations of the studied peptides.     

Analytical and micropreparative separation of peptides by capillary zone electrophoresis using discontinuous buffer systems.

The tiny injection volumes that are usually necessary to maintain the high efficiency of capillary zone electrophoresis present a major problem if only limited sample amounts are available. To increase the sample load, discontinuous buffer systems were developed that allow the on-column concentration of dilute samples. Injection volumes can be increased in this way by at least a factor of 30. These stacking systems were applied to the analysis of tryptic peptides, to the purity checking of high-performance liquid chromatographic fractions and for the micropreparative separation of peptides with subsequent amino acid sequence analysis.     

Improved separation of metallothionein isoforms by the presence of cyclodextrin in capillary zone electrophoresis

Capillary zone electrophoresis (CZE) with cyclodextrin (CD) in the polyacrylamide-coated capillary was used to study metallothionein (MT) forms in the horse kidney preparation produced commercially by Sigma. It is known that CDs form complexes with hydrophobic amino acids. The results show that the presence of CD improves the separability of the various MT forms, including the MT-IA and the MT-IB forms, metallothionein aggregates, as well as the so far unidentified a and b forms. This was true both below and above the isoelectric points (pIs), although the migration times were somewhat longer at increasing CD concentrations for runs at constant voltage than with constant current.     

Rapid separation of tetracycline derivatives and their main degradation products by capillary zone electrophoresis.

A mixture of five tetracycline (TC) derivatives: minocycline (MC), demeclocycline (DMCTC), doxycycline (DC), and sancycline (SC), as well as each TC derivative from its main degradation product were separated by capillary zone electrophoresis (CZE). The influence of the pH and the concentration and nature of the background electrolyte (BGE) on the separations was investigated. Ethylenediaminetetraacetic acid (EDTA; 1 mM) was used as additive in a 25 mM phosphate buffer (pH 2.3) because this BGE enabled the rapid separation of the TC derivatives and of each TC derivative from its respective degradation product in less than 6 min. After optimization of the separation conditions, the analytical characteristics of the method were investigated. The parameters involved were linearity, precision (repeatability and reproducibility), and limits of detection (LODs). LODs obtained for the five TC derivatives studied were about 3 microg/mL. Finally, the CZE method developed was applied to study the stability of TC derivatives and to analyze the TC derivative content in three different pharmaceutical preparations.     

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     

Recent advances in on-line concentration and separation of amino acids using capillary electrophoresis

This article highlights recent methodological developments in the on-line concentration and separation of amino acids and their enantiomers using capillary electrophoresis. Sections are dedicated to recent contributions to on-line concentration strategies such as field-amplified sample stacking, large-volume sample stacking, dynamic pH junction, transient isotachophoresis, sweeping, and the combination of two methods. The main applications, advantages, and limitations of these procedures in the biological, food, and pharmaceutical fields are addressed. Comprehensive tables listing on-line techniques for the concentration and separation of amino acids and their enantiomers, categorized by the stacking strategies used, background electrolytes, sample matrix, limit of detection, and enhancement factor, are provided.