Impact of organic solvents on the resolution of synthetic peptides by capillary electrophoresis

The effect of variations in the concentrations of different organic solvents, including acetonitrile, methanol, ethanol, propanol and isopropanol, with aqueous buffer electrolytes of defined composition and pH on the electroosmotic flow velocity, v(EOF), of uncoated fused silica capillaries and on the electrophoretic mobility, mu(e), of synthetic peptides in high-performance capillary electrophoresis (HPCE) has been systematically investigated. In these experiments, the volume fractions of the organic solvent in the aqueous buffer electrolyte were changed from psi = 0.0 to 0.80. The addition of these organic solvents to the aqueous buffer electrolyte reduced the electroosmotic flow (EOF) of the system, but to significantly different extents. For the protic solvents as the alkyl chain of the alcohol increased, at the same volume fraction the greater was the influence on the electroosmotic flow. However, for the aprotic solvent, acetonitrile, the EOF did not change substantially as the volume fraction was varied. The electrophoretic mobility of synthetic peptides under the different buffer electrolyte conditions showed similar trends, confirming that the content and type of the organic modifier can be rationally employed to subtly manipulate the separation selectivity of synthetic peptides. These results, therefore, provide fundamental insight into the experimental options that can be used to maximise resolution of synthetic peptides in HPCE with aqueous buffer-organic solvent mixtures as well as a basis to select optimal binary or ternary buffer electrolyte compositions for the analysis of peptides when hyphenated techniques, such as HPCE-electrospray ionisation mass spectrometry (ESI-MS), are contemplated for the analysis of peptide samples of low abundance as can often be experienced in proteomic investigations.     

Liposome Capillary Electrophoresis of Peptides and Proteins

Liposome capillary electrophoresis (LCE) using unilamellar liposomes composed of the zwitterionic phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) as a suspended pseudo stationary phase has been investigated for its capability at separating peptides and proteins in bare fused-silica capillaries. The study has explored different strategies for allowing the liposome suspension to act as a disperse pseudo stationary phase with the ability of modulating selectivity, resolution and separation performance of peptides and proteins in bare-fused silica capillaries. Such strategies comprise the use of capillaries either partially or totally filled with the liposome suspension, whereas the electrolyte solution is liposome-free, or the incorporation of the liposomes into the buffer solution employed for rinsing the capillary and as the background electrolyte. Three synthetic peptides of similar amino acid sequence and four basic standard proteins have been employed as test analytes. Varying the volume of the liposome suspension introduced in the capillary promoted differentiated variations in the migration velocity of the three peptides reflecting their selective interactions with the liposomes. Efficient separation of basic proteins was obtained at pH 7.4 in a bare fused-silica capillary with the electrolyte solution containing 60 μM POPC.     

Analysis of synthetic peptides by capillary electrophoresis: effect of organic solvent modifiers and variable electrical potentials on separation efficiencies

In this study, procedures based on volatile ammonium acetate buffer electrolytes of high pH value containing different organic solvent modifiers have been developed to achieve very high efficiency separations of histidine-containing synthetic peptides by high-performance capillary electrophoresis (HPCE) employing untreated fused silica capillaries. Different organic solvents, including acetonitrile, methanol and ethanol, at high volume fractions were used to modify the composition of the background buffer electrolyte. With the peptides investigated, it was found that methanol had the greatest effect in terms of enhancement of separation efficiency, as determined from the evaluation of theoretical plate numbers, N, of these HPCE systems. On the other hand, separation selectivities, e.g. the alpha(ij) values, did not change significantly as the volume fraction, psi, of the organic solvents was increased up to psi = 60% (v/v). Under these conditions, very rapid, e.g. 1-2 min, separation times could be still achieved. Compared to the effect of carrying out the separation of these peptides at constant voltage, a dramatic increase in the separation efficiency was also achieved by applying a linear voltage gradient during the HPCE experiment. Under optimal conditions of organic solvent composition and linear voltage gradient ramps, very high peak efficiencies for the studied set of synthetic peptides with N values of approximately 2-3 million theoretical plates per meter could be routinely obtained with fast analysis times. Moreover, these buffer electrolyte conditions are compatible with direct interfacing of the HPCE effluent to electrospray ionisation and ion trap mass spectrometers, thus expanding the analytical capabilities of these HPCE systems.     

Capillary zone electrophoresis of histidine-containing compounds

Capillary zone electrophoresis has been tested for the separation of angiotensins, cationic heptapeptides and model histidine derivatives. Good separation efficiencies are seen for peptides and model compounds with negative to small positive net charges. For net charge greater than +2, addition of putrescine to pH 6 buffer greatly suppresses ion exchange at anionic sites on fused silica. When operating at pH values where histidine groups are neutral, addition of Zn2+ allows separations based on metal, rather than proton, binding. Separation efficiencies and relative migration times are dependent on capillary length when ion-exchange behavior occurs.     

Comparison of three modifications of fused‐silica capillaries and untreated capillaries for protein profiling of maize extracts by capillary electrophoresis

In this work, capillary electrophoresis was applied to protein profiling of fractionated extracts of maize. A comparative study on the application of uncoated fused‐silica capillaries and capillaries modified with hydroxypropylmethylcellulose, ω‐iodoalkylammonium salt and a commercially available neutral capillary covalently coated with polyacrylamide is presented. The coating stability, background electrolyte composition, and separation efficiency were investigated. It was found that for zeins separation, the most stable and efficient was the capillary coated with polyacrylamide. Finally, the usefulness of these methods was studied for the differentiation of zein fraction in transgenic and nontransgenic maize. Zeins extracted from maize standards containing 0 and 5% m/m genetic modification were successfully separated, but slight differences were observed in terms of the zein content. Albumin and globulin fractions were analyzed with the use of unmodified fused‐silica capillary with borate buffer pH 9 and the capillary coated with polyacrylamide with phosphate buffer pH 3. In the albumin fraction, additional peaks were found in genetically modified samples.     

毛细管电泳中影响径向电场控制电渗的主要因素

利用自制的二维电场毛细管电泳系统研究了不同因素对径向电场控制电渗能力的影响,发现缓冲液的ｐＨ值、浓度、种类以及管壁表面状态、管径等对电渗的电场调控有关键性的影响。有趣的是,添加剂不影响电场的调控能力,而杯芳烃涂层毛细管却能提高电渗对径向电场的响应能力。利用这种涂层效应有可能实现较高ｐＨ值下电渗的电场调控。     

CE Enantioseparation of Betti Bases with Cyclodextrins and Crown Ether as Chiral Selectors

Direct capillary zone electrophoretic methods were developed for the separation of the enantiomers of 1-(α-aminoarylmethyl)-2-naphthol and 2-(α-aminoarylmethyl)-1-naphthol analogues. The effects of selector and buffer concentrations, electrolyte pH and applied voltage on the separation efficiency were studied. Variation of the electrophoretic conditions with the application of negative polarity, hydrodynamic injection, an unmodified silica capillary, different buffers and sulfated cyclodextrins and (S,S)-dimethylpyridino-18-crown-6 ether as chiral selectors led to the baseline resolution of all the compounds investigated.     

A fresh look into background electrolyte selection for capillary electrophoresis‐laser induced fluorescence of peptides and proteins

This study reports a reinvestigation of background electrolyte selection strategy for performance improvement in CE‐LIF of peptides and proteins. This strategy is based on the employment of high concentrations of organic species in BGE possessing high buffer capacity and low specific conductivity in order to ensure excellent stacking preconcentration and separation resolution of fluorescently tagged peptides and proteins. Unlike universal UV detection, the use of such BGEs at high concentrations does not lead to degradation of LIF detection signals at the working excitation and emission wavelengths. At the same buffer ionic strength, pH and electric field, an “inorganic‐species‐free” BGE (or ISF BGE) for CE‐LIF of fluorescently labeled beta amyloid peptide Aβ 1–42 (a model analyte) offered a signal intensity and peak efficiency at least three‐times higher than those obtained with a conventional BGE normally used for CE‐LIF, while producing an electric current twice lower. Good peak performance (in terms of height and shape) was maintained when using ISF BGEs even with samples prepared in high‐conductivity phosphate buffer saline matrix. The advantageous features of such BGEs used at high concentrations over conventional ones in terms of high separation resolution, improved signal intensities, tuning of EOF magnitudes and minimization of protein adsorption on an uncoated fused silica capillary are demonstrated using Alexa‐488‐labelled trypsin inhibitor. Such BGE selection approach was applied for investigation of separation performance for CE‐LIF of ovalbumin labelled with different fluorophores.     

Enantioseparation of aspartyl dipeptides by CE: Comparison between 18-crown-6-tetracarboxylic acid and carboxymethyl-β-cyclodextrin as chiral selector

Summary The simultaneous separation of isomeric α-and β-aspartyl peptides as well as their enantiomers by capillary electrophoresis was investigated. Resolution of the enantiomers of the peptides α/β-AspPhe-NH₂ and α/β-AspPhe-OMe was achieved with 18-crown-6-tetracarboxylic acid (18C6H₄) in untreated fused silica capillaries in the acidic pH range. Baseline resolution for the dipeptides was obtained using polyacrylamide (PAA)-coated capillaries. The selectivity of the crown ether is compared with the selectivity of another chiral selector, namely the negatively charged CD derivative carboxymethyl-β-CD (CM-β-CD).     

Investigation of the separability of thaumatin by capillary electrophoresis

The electrophoretic behaviour of the highly basic protein thaumatin was explored in strongly acid (pH 2) and mildly acid (pH 4.5) separation systems using both bare and coated fused silica capillaries. The separation selectivity for thaumatin I, thaumatin II, and for other sample constituents was insufficient for their baseline separation at pH 2 in an uncoated capillary because the separation efficiency was markedly lower than is common in the electrophoretic separations of proteins. A separation selectivity higher by up to one order of magnitude has been reached at pH 4.5. A pronounced asymmetry of zones, which impaired resolution at this pH, was effectively suppressed by coating of the capillary wall with a polymer. In fact, adsorption on the capillary coating always plays a contributory role whenever a good separation of thaumatin constituents is attained. This indicates that electrochromatographic separation systems based on capillaries coated with the layer of either cationic or hydrophilic uncharged polymer hold promise for the development of methods for thaumatin analysis.     

Electrophoretic separation of aniline derivatives using fused silica capillaries coated with acid treated single-walled carbon nanotubes

This paper reports on a new strategy for coating fused silica capillaries based on the ionic adsorption of acid treated single-walled carbon nanotubes (SWCNTs) on a poly(diallydimethylammonium chloride)-modified fused silica surface. The coated capillaries were used to demonstrate their performance for baseline separation of a mixture of seven nitrogen-containing aromatic compounds compared to capillary zone electrophoresis. This combined layer formed a coating material that could be useful for improvement of the selectivity of the solutes in an electrical field. We reasoned that the interaction of the solutes and the modified capillary wall occurred mainly via ionic interactions with the charged moieties of CNTs. The single-walled CNT modified capillaries were very stable and could be used for over 200 repeated analyses without compromising its analytical performance.     

New isoelectric buffers for capillary electrophoresis: N-carboxymethylated polyethyleneimine as a macromolecular isoelectric buffer

Isoelectric buffers are attractive for electrophoresis because of their low conductivity, and their compatibility with indirect photometric detection in capillary electrophoresis (CE) where they do not interfere with the detection by exhibiting competitive displacement of the UV-absorbing probe ion. N-carboxymethylated polyethyleneimine (CMPEI) was prepared by introducing a half molar equivalent of carboxylate groups onto a polyethyleneimine backbone. Its isoelectric point determined by conductometric titration and from the pH of its dilute aqueous solution is approx. 6.8, which allows isoelectric buffering at a lower pH compared to histidine (pI7.7). Although the isoelectric point is somewhat diffuse, as expected for a polymeric compound, it exhibits a buffering capacity at a pI point of about twice that of histidine. Studies of electroosmotic flow (EOF) profile at various pH values in fused silica capillaries showed that CMPEI adsorbs onto the fused silica wall and reverses the EOF at pH < 6.5. CMPEI was applied as a buffer in an electrolyte containing 0.5 mM of the anionic dye tartrazine used as the probe for indirect detection of anions. The separation system exhibited a stable baseline, no system peaks, separation efficiencies of up to 195,000 theoretical plates, and detection limits down to 0.2 microM or 2 amol of injected analyte.     

Separation of aromatic sulphonic acids by CZE in coated and non‐coated capillaries

Capillary zone electrophoresis (CZE), besides ion‐pairing mode HPLC and salting‐out HPLC, is well‐suited for the analysis of aromatic sulphonic acids widely used as intermediates in the production of synthetic dyes and optical brighteners. The separation selectivity in CZE of many aromatic acids, including positional isomers, can be controlled by the type and concentration of a cyclodextrin additive. The influence of the concentration of β‐cyclodextrin in the working electrolyte on the separation of the positional isomers of naphthalene (poly‐)sulphonic acids, and their amino‐ and hydroxy derivatives, by CZE was studied, both in non‐coated fused silica capillaries and in capillaries coated with polyacrylamide. The migration time scale was calibrated using 4‐alkylbenzenesulphonic acids as the calibration standards. Limiting mobilities of the free acid anions and of their complexes with β‐cyclodextrin were calculated and the effect of the inclusion guest‐host complex formation on the CZE separation was quantitatively characterized. The migration order in coated capillaries is reversed with respect to CZE in non‐coated fused silica capillaries, the separation selectivity is different and the separation of polysulphonic acids such as naphthalene tri‐ and tetrasulphonic acids is significantly accelerated.     

CE separation of various analytes of biological origin using polyether ether ketone capillaries and contactless conductivity detection

The development of efficient and sensitive analytical methods for the separation, identification and quantification of complex biological samples is continuously a topic of high interest in biological science. In the present study, the possibility of using a polyether ether ketone (PEEK) capillary for the CE separation of peptides, proteins and other biological samples was examined. The performance of the tubing was compared with that of traditional silica capillaries. The CE analysis was performed using contactless conductivity detection (C⁴D), which eliminated any need for the detection window and was suitable for the detection of optically inactive compounds. In the PEEK capillary the cathodic EOF was low and of excellent stability even at extremes pH. In view of this fast biological anions were analyzed using an opposite end injection technique without compromising separation. A comparison of the performances of fused‐silica and polymer capillaries during the separation of model sample mixtures demonstrated the efficiency and separation resolution of the latter to be higher and the reproducibility of the migration times and peak areas is better. Furthermore, PEEK capillaries allowed using simple experimental conditions without any complicated modification of the capillary surface or use of an intricate buffer composition. The PEEK capillaries are considered as an attractive alternative to the traditional fused‐silica capillaries and may be used for the analysis of complex biological mixtures as well as for developing portable devices.     

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.     

Coupled fused silica capillaries for rapid capillary zone electrophoresis of proteins

A novel two-dimensional electrophoretic system for the control of electroosmosis in capillary zone electrophoresis has been developed and evaluated for rapid separations of proteins. The system comprises uncoated and polyether-coated fused silica capillaries coupled in series. An equation relating the average electroosmotic flow velocity in the coupled capillaries to the intrinsic electroosmotic velocities of the connected segments and their corresponding lengths has been derived and verified experimentally. This approach has the advantage of enabling the electroosmotic flow to be tuned independently of the applied voltage. As a consequence, rapid protein analysis at relatively low field strength was achieved without sacrificing the high separation efficiencies obtained with surface-modified capillaries.     

卤代乙酸及其结构相近化合物的高效毛细管电泳分离

氟、氯、溴等卤代乙酸是结构非常相近的离子型化合物,对它们的分离测定比较困难。用高效毛细管电泳法在碱性或酸性缓冲液条件下可将它们分离。在酸性缓冲液条件下,可提高有机酸分离的选择性。较低的操作电压有利于提高阴离子的分离度,而改变温度对分离度的影响不大。     

Effect of quasi‐interpenetrating network of polyacrylamide and poly(N,N‐dimethylacrylamide) on separation of dsDNA fragments and basic protein using CE

Quasi‐interpenetrating network (quasi‐IPN) of linear polyacrylamide (LPA) with low molecular mass and poly(N,N‐dimethylacrylamide) (PDMA), which is shown to uniquely combine the superior sieving ability of LPA with the coating ability of PDMA, has been synthesized for application in dsDNA and basic protein separation by CE. The performance of quasi‐IPN on dsDNA separation was determined by polymer concentration, electric field strength, LPA molecular masses and different acrylamide (AM) to N,N‐dimethylacrylamide (DMA) ratio. The results showed that all fragments in Φ×174/HaeIII digest were achieved with a 30 cm effective capillary length at –6 kV at an appropriate polymer solution concentration in bare silica capillaries. Furthermore, EOF measurement results showed that quasi‐IPN exhibited good capillary coating ability, via adsorption from aqueous solution, efficiently suppressing EOF. The effect of the buffer pH values on the separation of basic proteins was investigated in detail. The separation efficiencies and analysis reproducibility demonstrated the good potentiality of quasi‐IPN matrix for suppressing the adsorption of basic proteins onto the silica capillary wall. In addition, when quasi‐IPN was used both as sieving matrix and dynamic coating in bare silica capillaries, higher peak separation efficiencies, and better migration time reproducibility were obtained.     

Rapid separation of peptides and proteins by isocratic capillary electrochromatography at elevated temperature

The use of capillary electrochromatography (CEC) for the separation by isocratic elution of synthetic peptides, proteins as well as the tryptic digest of cytochrome c has been demonstrated. The monolithic porous stationary phase was prepared from silanized fused-silica capillaries of 75 microm I.D. by in situ copolymerization of vinylbenzyl chloride and ethylene glycol dimethacrylate in the presence of propanol and formamide as the porogens. The chloromethyl groups at the surface of the porous monolith were reacted with N,N-dimethylbutylamine to form a positively charged chromatographic surface with fixed n-butyl chains. Results of studies on the influence of temperature and mobile phase composition on the retention and selectivity of separation by CEC demonstrated the feasibility of rapid polypeptide analysis and tryptic mapping at elevated temperature with high resolution and efficiency. Typically the chromatography of a tryptic digest of cytochrome c took about 5 min at 55 degrees C and 75 kV/m with hydro-organic mobile phases containing acetonitrile in 50 mM phosphate buffer, pH 2.5. For peptides and proteins plots of logarithmic k'cec against acetonitrile concentration were nonlinear, whereas Arrhenius plots for the mobilities were nearly linear. Comparison of the separation of such samples under conditions of CEC and capillary zone electrophoresis (CZE) indicates that the mechanism of separation in CEC is unique and leads to a chromatographic profile different from that obtained by CZE.     

Capillary zone electrophoresis with electroosmotic flow controlled by external radial electric field.

A new way of regulation of electroosmotic flow (EOF) in capillary zone electrophoresis (CZE) by external electric field has been developed. A set of three high-voltage power supplies is used to form a radial electric field across the capillary wall. One power supply is applied in the usual way as a driving force of CZE and EOF to the ends of the inner capillary compartment dipped into the electrode vessels and filled with background electrolyte. Two power supplies are connected to the ends of the outer low-conductivity coating of the capillary which is formed by the dispersion of copolymer of aniline and p-phenylenediamine in polystyrene matrix. The difference between electric potentials on the outer capillary surface and inside the capillary determines the voltage of radial electric field across the capillary wall and affects the electrokinetic potential at the solid-liquid interface inside the capillary. The effect of magnitude and polarity of external radial electric field on the flow rate of EOF, on the migration times of charged analytes and on the separation efficiency and resolution of CZE separations of synthetic oligopeptides, diglycine, triglycine and octapeptide fragments of human insulin was evaluated. Through the EOF control by external electric field the dynamic effective length of the capillary was obtained and the speed of analysis and resolution of CZE separations of peptide analytes could be optimized.