Electroosmotic pump-assisted capillary electrophoresis of proteins

A new method for protein analysis, that is, electroosmotic pump-assisted capillary electrophoresis (EOPACE), is developed and demonstrated to possess several advantages over other CE-based techniques. The column employed in EOPACE consists of two linked sections, poly(vinyl alcohol) (PVA)-coated and uncoated capillaries. The PVA-coated capillary column is the section for protein electrophoresis in EOPACE. Electroosmotic flow (EOF) is almost completely suppressed in this hydrophilic polymer coated section, so protein electrophoresis in the PVA-modified capillary is free of irreversible protein adsorption to the capillary inner wall. The uncoated capillary section serves as an electroosmotic pump, since EOF towards cathode occurs at neutral pH in the naked silica capillary. By the separation of a protein mixture containing cytochrome c (Cyt-c), myoglobin and trypsin inhibitor, we have demonstrated the advantages of EOPACE method over other relevant ones such as pressure assisted CE, capillary zone electrophoresis (CZE) with naked capillary and CZE with PVA-coated capillary. A significant feature of EOPACE is that simultaneous separation of cationic, anionic and uncharged proteins at neutral pH can be readily accomplished by a single run, which is impossible or difficult to realize by the other CE-based methods. The high column efficiency and good reproducibility in protein analysis by EOPACE are verified and discussed. In addition, separation of tryptic digests of Cyt-c with the EOPACE system is demonstrated.     

Free solution capillary electrophoresis of proteins using untreated fused-silica capillaries.

Numerous efforts have been made to separate proteins by capillary zone electrophoresis (CZE). The most common optimization techniques are changing the pH of the running buffer, coating the capillary surface with a hydrophilic polymer, or using additives in the sample solution. Surface coatings and solution additives can reduce the adsorption of the protein onto the capillary surface, but they diminish the separation efficiency and the resolution of CZE. This paper reports the successful separation of proteins in a untreated fused-silica capillary by raising the pH of the running buffer and washing between runs with 1.0 M sodium hydroxide. Under these conditions, model proteins and proteins in human serum have been determined by CZE. It is shown that the results from CZE are compatible with those of sodium dodecyl sulphate-polyacrylamide gel electrophoresis.     

Capillary electrophoretic separation of phenolic diterpenes from rosemary

The major phenolic diterpenes responsible for the antioxidant properties of rosemary extracts, namely carnosol and carnosic acid, were separated by capillary zone electrophoresis (CZE) using a 56 cm long uncoated fused-silica capillary and a 50 mM disodium tetraborate buffer of pH 10.1. The effect of the buffer type, pH and concentration, and the capillary length on the separation, was studied. Carnosol and carnosic acid were identified in the electrophoregrams of rosemary extracts through their migration times and UV spectra obtained by CZE analysis of pure compounds isolated from a rosemary extract by HPLC fractionation. The CZE method had good reproducibility (relative standard deviation less than 5%) and was applied to compare the contents of carnosol and carnosic acid in solid and oil-dispersed commercial extracts of rosemary and in rosemary leaves. The separation of carnosol and carnosic acid was accomplished in less than 11 min.     

Electrochemical Behavior of Cu Electrode and Its Application in CE Determination of Polyols

The electrochemical behavior of a copper (Cu) electrode and its application in capillary electrophoresis determination of polyols was investigated in order to understand the redox property of Cu and achieve better separation efficiency. Electrochemical measurements were performed using a Cu electrode (fresh or oxidized) in buffer solutions having different pH values (7.8–13.0) by cyclic voltammetry. The Cu electrode showed higher electroactivity under stronger alkaline conditions. Further, the Cu electrode was found suitable for detecting weak oxidizing or reducing polyhydroxy compounds because of the redox reactions among Cu, Cu(I), and Cu(II) species. Thus, the Cu electrode was used in capillary zone electrophoresis (CZE) for separation and determination of propanediol and glycerol, weak electroactive polyols, using different separation (pH 8.7) and detection (pH 11.04) buffers. Separation and detection buffers with different pH values in CZE technology could offer efficient separation efficiency and detection limits at the same time.     

Correlation of capillary zone electrophoresis with continuous free-flow zone electrophoresis: application to the analysis and purification of synthetic growth hormone releasing peptide

Two carrier-free electrophoretic separation methods, capillary zone electrophoresis (CZE) and continuous free-flow zone electrophoresis (FFZE), have been applied to both microanalysis at the nanogram level and preparative fractionation, with a throughput of 30 mg/h, of synthetic growth hormone releasing peptide (GHRP). A crude product of GHRP, a hexapeptide with the sequence His-D-Trp-Ala-Trp-D-Phe-Lys-NH2, synthesized by the solid phase methodology, was desalted and analyzed by CZE. Based on the results of analytical CZE the separation was converted into a preparative purification procedure by continuous FFZE, employing the same separation medium (0.5 mol/L acetic acid, pH 2.6). The purifity of peptide fractions obtained by FFZE was reevaluated by CZE. The combination of these two techniques proved to be a valuable tool for both peptide analysis and peptide purification. A close correlation of CZE and FFZE, resulting from the fact that both methods are based on the same separation principle (zone electrophoresis) and that both are performed in a free solution of the same composition, was confirmed. However, when transforming data from CZE to FFZE, the different electroosmotic flow, temperature and electric field intensity in the capillary and in the flow-through cell, respectively, have to be taken into account and corresponding corrections have to be made.     

Immunochemical characterisation of structure and allergenicity of peanut 2S albumins using different formats of immunoassays

Proteins of the 2S albumin family, such as Ara h2 and Ara h6, are most frequently involved in peanut allergy. We have developed a reverse enzyme allergo-sorbent test (EAST) in which total serum IgE antibodies are first captured by immobilised anti-human IgE monoclonal antibodies, and then the binding of the anti-Ara h2 and anti-Ara h6 specific IgE to the corresponding labelled allergens is measured. This reverse immunoassay was used either as a direct EAST or as an EAST inhibition assay to study the interactions of whole peanut protein extract and purified Ara h2 and Ara h6 with IgE antibodies from peanut-allergic patients. Finally, we identified some IgE-binding epitopes on Ara h6 using a format of EAST in which the protein is immobilised in a particular, well defined, manner through interactions with specific monoclonal antibodies (mAbs) coated on the micro-plates. The fine specificity of those mAbs has been characterised at the epitope level, and their binding to the allergen thus masks a known particular epitope and makes it unavailable for recognition by IgE antibodies. The reverse EAST increased the ratio specific signal/background. It avoids interferences with competitors such as anti-peanut protein IgG antibodies and allows the study of the specificity and/or affinity of the interactions between IgE antibodies and Ara h2 or Ara h6 with a higher sensitivity and accuracy than the conventional EAST. The EAST results obtained when the allergens are presented by specific mAbs suggest that the homologous molecular domain(s) in peanut 2S albumins encompass major IgE epitope(s) and are strongly involved in peanut allergenicity.     

Capillary electrophoresis of seed 2S albumins from Lupinus species

Two modes of capillary electrophoresis (CE)--free-solution capillary zone electrophoresis (CZE) and sodium dodecyl sulfate capillary electrophoresis (SDS-CE) using a non-gel sieving matrix--have been developed for comparative analysis of low-molecular-mass 2S albumin isoforms from lupins. The albumin fraction and 2S albumins were separated in uncoated fused-silica capillary by CZE with 0.02 M phosphate buffer, pH 7.3, containing the sodium salt of phytic acid. The use of phytic acid (0.025 M) as buffer modifier and ion-pairing agent improved migration reproducibility, peak shape and separation efficiency. The reduced 2S albumins were separated by SDS-CE using a high concentration (0.3-0.5 M) mixture of tris(hydroxymethyl)aminomethane and borate buffers in uncoated fused-silica capillary. Of the various polymers used as non-gel sieving matrix, SDS-CE with a 10% dextran solution was found to be suitable for separation of 2S albumin polypeptides with molecular masses of 4,000-7,000 and 8,000-11,000. The addition of glycerol or ethylene glycol to the SDS separating buffer improved the resolution of polypeptides. The examined Lupinus species showed species-specific CZE and SDS-CE migration profiles of the 2S albumins.     

Use of acidic and basic pH and calcium ion addition in the capillary zone electrophoretic characterization of recombinant human deoxyribonuclease, a complex phosphoglycoprotein.

This paper describes the analysis of recombinant human deoxyribonuclease (rhDNAse), an acidic and complex phosphoglycoprotein, by capillary zone electrophoresis (CZE). Separation performance was found to be dramatically improved by the addition of calcium ions to the CZE running buffer, due to the influence of calcium binding on the charge and the electrophoretic behavior of rhDNAse. The pH dependent calcium binding effects on the electrophoretic separation were demonstrated at both acidic and basic pH, resulting in a two-dimensional (pH 4.8 and 8.0) calcium aided analysis that achieved multipeak resolution of the complex, glycosylation based, charge microheterogeneity of rhDNAse. Two-dimensional investigation of neuraminidase- and alkaline phosphatase-digested protein further demonstrated that the acidic pH resolved acidic charge heterogeneity and that the basic pH discriminated neutral heterogeneity. This work demonstrates the resolving power of CZE for the analysis of a complex microheterogeneous glycoprotein, and emphasizes the importance of employing multiple separation conditions in accordance with known structural characteristics of the protein.     

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.     

Separation of naturally occurring triterpenoidal saponins by capillary zone electrophoresis.

A high-performance capillary electrophoresis (HPCE) was successfully applied to the separation and quantitation of naturally occurring oleanene triterpenoidal saponins. The HPCE adapted to the separation of two pairs of disteriomeric saponins (1-2) or (3-4), obtained from Trifolium alexandrinum seeds, was based on capillary zone electrophoresis (CZE) in borate buffer with UV detection at 195 nm. An usual technique for isolation and group separation of saponins was developed as an appropriate purification step prior to determination of individual saponins by CZE. The separation parameters such as borate concentration, pH and applied voltage were varied in order to find the best compromise that complied with demands for high separation, short duration and sufficiently high detector response. The optimum running conditions were found to be 60 mM borate buffer, pH 10 and 12 kV. Under the alkaline borate electrolyte, no resolution was achieved for the saponins (1 and 3) or (2 and 4) in a single mixture, except when 20 mM beta-cyclodextrin was added to the running electrolyte. With the combined techniques of group separation, purification and CZE, a rapid and efficient method for the determination of naturally occurring diasteriomeric saponins is now available.     

苯胺及其衍生物的毛细管电泳行为研究

采用紫外吸收检测，毛细管电泳分离，研究了九种苯胺及其衍生物在毛细管区带电泳(CZE)体系和胶束电动毛细管色谱(MECC)体系中的行为特征。讨论了缓冲溶液的浓度与pH、胶束浓度及混合胶束等在不同体系中对分离组分的影响，发现在CZE体系中，控制分离的主要因素是pKb值；在MECC体系中，控制分离的主要因素是溶质分子中碳原子数。建立了一种分离测定九种苯胺及其衍生物的高效毛细管电泳方法。     

Separation of phospholipids by capillary zone electrophoresis with indirect ultraviolet detection

A simple method for separation of different anionic and zwitterionic phospholipid classes by capillary zone electrophoresis (CZE), using indirect UV detection with adenosine monophosphate (AMP) as background electrolyte and the UV-absorbing additive, was successfully developed in this study. The separation conditions including apparent pH (pH*) of running buffer, concentration of AMP, organic solvent, applied voltage and capillary temperature were systematically optimized. The application of this method to human blood sample was also briefly examined.     

Can commercial peanut assay kits detect peanut allergens?

Peanut is the food group mostly associated with severe and fatal allergic reactions. In the United States, more than 90% of peanut-allergic individuals' serum IgE recognized peanut proteins Ara h 1 and Ara h 2, thus establishing these proteins as major peanut allergens. The amount of Ara h 1 and Ara h 2 in 3 varieties of peanut cultivars that are commonly processed in the industrialized countries was determined to be 12-16 and 6-9%, respectively. Current commercial peanut test kits use polyclonal peanut-specific antibodies to detect soluble or buffer extractable peanut proteins. Because the 2 major peanut allergens Ara h 1 and Ara h 2 are isolated from soluble peanut proteins, it is generally assumed that these commercial kits can detect peanut allergens, although none of these kits claims to detect peanut allergen. This study showed for the first time that the peanut test kits could, in fact, detect major peanut allergens Ara h 1 and Ara h 2 in both native or heat-denatured structures; therefore, these kits qualified to be classified as peanut allergen enzyme-linked immunosorbent assays.     

Determination of ciprofloxacin and its impurities by capillary zone electrophoresis

Capillary zone electrophoresis (CZE) has been elaborated for separation, identification and determination of ciprofloxacin and its impurities. The separation, phosphate buffer pH 6.0 was supplemented with 0.075 M pentane-1-sulfonic acid sodium salt. The elaborated method was validated. The selectivity, linearity, limits of detection (LOD) and quantification (LOQ), precision, and accuracy of capillary zone electrophoresis were evaluated. The results obtained by CZE were also compared with those obtained by liquid chromatography. Regarding the validation results the CE method fulfils the current European Pharmacopoeia (Eur. Ph.) requirements. The evaluated CE method could be applicable to the analysis of different medicinal products containing ciprofloxacin.     

Determination of Phenolic Acids in Herba Epilobi by ITP–CE in the Column-Coupling Configuration

The combination of capillary isotachophoresis (ITP) and capillary zone electrophoresis (CZE) in the column-coupling configuration has been optimized in a mode in which the background electrolyte employed in the CZE step was different from the leading and terminating electrolytes of the ITP step. The optimum composition of the electrolyte system was 0.01 M HCl, 0.02 M IMI, 0.2% HEC, pH 7.2 (leading electrolyte), 0.01 M HEPES, pH 8.2 (terminating electrolyte), and 25 mM MES, 50 mM TRIS, 30 mM boric acid, 0.2% HEC, pH 8.3 (background electrolyte). All solutions contained 20% methanol. The timing of the transfer of isotachophoretically stacked analyte zones into the CZE column was also optimized. An ITP–CZE method with UV detection at 270 nm was developed for separation of nine phenolic acids (protocatechuic, syringic, vanillic, cinnamic, ferulic, caffeic, ρ-coumaric, chlorogenic, and gentisic acids) in a model mixture and used for assay of some of these acids in a methanolic extract of herba epilobi. Application of ITP–CZE resulted in 100-fold better sensitivity than conventional CZE; limits of detection ranged between 10 and 60 ng mL⁻¹. When MES–TRIS–borate-based buffer, pH 8.3, was used in the CZE separation step the linearity of the ITP–CZE response was satisfactory (correlation coefficients were from 0.9937 to 0.9777). Repeatability was also satisfactory (RSD values ranged between 0.77% and 1.28% for migration times and between 1.65% and 13.69% for peak area). Revised: 23 March and 27 April 2006     

Use of zwitterionic buffer additives to improve the separation of proteins in capillary zone electrophoresis

In CZE, the adsorption of the proteins on the capillary wall is a common problem. This paper describes the simple method of utilizing zwitterionic buffer additives to improve the separation of proteins in untreated fused silica capillaries at neutral pH. Three kinds of zwitterion are evaluated in the separation of acidic, neutral, and basic proteins, including their effect on protein efficiency, mobility, separation, and resolution; the difference between the effects of the different additives are also highlighted. The method has proved to be a possible means of reducing protein adsorption, especially for basic proteins.     

Bioactive components of the hop strobilus: comparison of different extraction methods by capillary electrophoretic and chromatographic methods

In the present study, the composition of the hop strobilus extract by using different extraction methods under different solvent conditions was analysed and compared. Several separation methods were applied to obtaining detailed information about the hop extract: capillary zone electrophoresis (CZE), high-performance liquid chromatography-mass-spectrometry (HPLC-MS/MS) and gas chromatography-mass-spectrometry (GC-MS). The electropherograms of different extracts varied dramatically. The oxidation reaction of the hop strobilus extract was examined.     

Potential of Capillary Electrophoresis for the Profiling of Propolis

The usefulness of capillary electrophoresis (CE) with diode array detection for the profiling of Propolis, a hive product, is investigated. Water extracts of Propolis were analyzed with both capillary zone electrophoresis (CZE) at pH 7.0 and 9.3, and micellar electrokinetic chromatography (MEKC) with sodium dodecyl sulfate at pH 9.3. Characteristic profiles were obtained and several organic acids and preservatives could be identified by means of library comparison of the recorded UV spectra combined with addition of reference compounds to the extracts. The selectivity of the CZE and MEKC system differed considerably but the information obtained with both methods was similar. The dry residues of the water extraction were extracted with ethanol-water (70 : 30, v/v) and analyzed with the MEKC system to enable the separation of the more hydrophobic constituents of the Propolis samples. Complex profiles containing various well separated peaks were obtained allowing the identification of some interesting flavonoids. On the basis of the recorded CZE and MEKC profiles, the Propolis samples could be divided into two clearly different groups which are probably from a different origin.     

Cross-linked poly(vinyl alcohol) as permanent hydrophilic column coating for capillary electrophoresis.

A fast method for the generation of permanent hydrophilic capillary coatings for capillary electrophoresis (CE) is presented. Such interior coating is effected by treating the surface to be coated with a solution of glutaraldehyde as cross-linking agent followed by a solution of poly(vinyl alcohol) (PVA), which results in an immobilization of the polymer on the capillary surface. Applied for capillary zone electrophoresis (CZE) such capillaries coated with cross-linked PVA exhibit excellent separation performance of adsorptive analytes like basic proteins due to the reduction of analyte-wall interactions. The long-term stability of cross-linked PVA coatings could be proved in very long series of CZE separations. More than 1000 repetitive CE separations of basic proteins were performed with stable absolute migration times relative standard deviation (RSD > 1.2%) and without loss of separation efficiency. Cross-linked PVA coatings exhibit a suppressed electroosmotic flow and excellent stability over a wide pH range.     

Capillary electrophoretic determination of the constituents of Artemisiae Capillaris Herba

Two capillary electrophoretic methods, a micellar electrokinetic electrophoretic (MEKC) one and a capillary zone electrophoretic (CZE) one, were developed for the separation of 12 constituents in Artemisiae Capillaris Herba. Detection at 254 nm with 20 mM sodium dodecyl sulfate and 20 mM sodium borate buffer (pH 9.82) in MEKC or with 25 mM sodium borate and 6.75 mg/ml 2,3,6-tri-O-methyl-beta-cyclodextrin buffer in CZE was found to be the most suitable approach for this analysis. Within 42 min, the MEKC method could successfully separate 12 authentic constituents, whereof chlorogenic acid, however, appeared as a broad and split peak, and capillarisin and chlorogenic acid overlapped partially with other coexisting substances in crude extract of the herb. The CZE method could completely overcome these problems and was used to determine the amounts of capillarisin, chlorogenic acid, scopoletin and caffeic acid in the extract. The effect of buffers on the constituent separation and the validation of the two methods were discussed.