Comparison of different capillary isoelectric focusing methods--use of "narrow pH cuts" of carrier ampholytes as original tools to improve resolution

Two capillary isoelectric focusing (CIEF) systems have first been optimized: one uses a bare silica capillary and 30% (v/v) of glycerol in the separation medium while the other uses a coated capillary and an aqueous background electrolyte. To perform permanent capillary coating, two neutral polymers have been compared: hydroxypropylcellulose (HPC) and polyvinylalcohol (PVA). HPC coating gave best results for electroosmotic flow (EOF) limitation on a wide pH range: as compared to a bare silica capillary, it allowed to decrease EOF by 96% at pH 7.2 after acidic and basic treatments, whereas PVA coating lead only to a 76% decrease. The glycerol CIEF system was more satisfying for the separation of model proteins classically used as pI markers. Finally, the use of "narrow pH cuts" of carrier ampholytes added to commercial ampholyte mixtures allowed increasing resolution up to a factor 2.4 at a chosen pH for the separation of pI markers and milk proteins.     

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.     

Potential of prodendronic polyamines with modulated segmental charge density as novel coating for fast and efficient analysis of peptides and basic proteins by CE and CE‐MS

In this work, the suitability of a new polymer family has been investigated as capillary coatings for the analysis of peptides and basic proteins by CE. This polymer family has been designed to minimize or completely prevent protein–capillary wall interactions and to modify the EOF. These coating materials are linear polymeric chains bearing as side cationizable moiety a dentronic triamine derived from N,N,N’,N’‐tetraethyldiethylenetriamine (TEDETA), which is linked to the backbone through a spacer (unit labeled as TEDETAMA). Four different polymers have been prepared and evaluated: a homopolymer which comprised only of those cationizable repetitive units of TEDETAMA, and three copolymers that randomly incorporate TEDETAMA together with neutral hydrosoluble units of N‐(2‐hydroxypropyl) methacrylamide (HPMA) at different molar percentages (25:75, 50:50 and 75:25). It has been demonstrated that the composition of the copolymers influences the EOF and therefore the separation of the investigated biopolymers. Among the novel polymers studied, poly‐(TEDETAMA‐co‐HPMA) 50:50 copolymer was successfully applied as coating material of the inner capillary surface in CE‐UV and CE‐MS, providing EOF reversing together with fast and efficient baseline separation of peptides and basic proteins. Finally, the feasibility of the polymer‐coated capillary was shown through the analysis of lysozyme in a cheese sample.     

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.     

N‐methyl‐2‐pyrrolidonium methyl sulfonate acidic ionic liquid as a new dynamic coating for separation of basic proteins by capillary electrophoresis

A simple and economical CE method has been developed for the analysis of four model basic proteins by employing N‐methyl‐2‐pyrrolidonium methyl sulfonate ionic liquid (IL) as the dynamic coating material based on the interaction of both between electrostatic attraction and hydrogen bond, and between the organic cations of IL and the inner surface of bare fused‐silica capillary. The N‐methyl‐2‐pyrrolidonium‐based IL modified capillary not only generated a stable suppressed electroosmotic flow, but also effectively eliminated the wall adsorption of proteins. Several important parameters such as the IL concentration, pH values, and concentrations of the background electrolyte were optimized to improve the separation of basic proteins. Consequently, under the optimum separation conditions, a satisfied separation of basic proteins including lysozyme, cytochrome c, ribonuclease A, and α‐chymotrypsinogen A with theoretical plates ranging from 2.09 × 10⁵ to 4.48 × 10⁵ plates/m had been accomplished within 15 min. The proposed method first illustrated the effect of hydrogen bond between coating material and inner capillary surface on the coating, which should be a new strategy to design and select more effective coating materials to form more stable coatings in CE.     

CE‐MS analysis of heroin and its basic impurities using a charged polymer‐protected gold nanoparticle‐coated capillary

The first application of charged polymer‐protected gold nanoparticles (Au NPs) as semi‐permanent capillary coating in CE‐MS was presented. Poly(diallyldimethylammonium chloride) (PDDA) was the only reducing and stabilizing agent for Au NPs preparation. Stable and repeatable coating with good tolerance to 0.1 M HCl, methanol, and ACN was obtained via a simple rinsing procedure. Au NPs enhanced the coating stability toward flushing by methanol, improved the run‐to‐run and capillary‐to‐capillary repeatabilities, and improved the separation efficiency of heroin and its basic impurities for tracing geographical origins of illicit samples. Baseline resolution of eight heroin‐related alkaloids was achieved on the PDDA‐protected Au NPs‐coated capillary under the optimum conditions: 120 mM ammonium acetate (pH 5.2) with addition of 13% methanol, separation temperature 20°C, applied voltage ?20 kV, and capillary effective length 60.0 cm. CE‐MS analysis with run‐to‐run RSDs (n=5) of migration time in the range of 0.43–0.62% and RSDs (n=5) of peak area in the range of 1.49–4.68% was obtained. The established CE‐MS method would offer sensitive detection and confident identification of heroin and related compounds and provide an alternative to LC‐MS and GC‐MS for illicit drug control.     

Adsorptive BSA Coating Method for CE to Separate Basic Proteins

A novel and simple coating method was developed by coating bovine serum albumin (BSA) onto the inner surface of a fused-silica capillary, to avoid the adsorption of analytes during CE. The advantage presented here was that the coating process is more simple, fast, stable, and reproducible. The coated capillary avoided the adsorption of analytes onto the inner surface of a fused-silica capillary and might be a promising candidate for separation of complex biological samples with further development. Meanwhile, the efficiencies of the coated capillary were evaluated by EOF, chromatographic peak shape, and theoretical plate number (N m^?1) of RNase A. The optimal coating conditions were obtained from the results. The pH value of coating buffer PB was 4.2, the standing time was 12 h at 4 °C, and the coating concentration of BSA was 1.5 mg mL^?1. The stability of the coating on the inner wall of the capillary and the reproducibility of the coated capillaries were good. The theoretical plate number values of RNase A were over 1.3 × 10⁵ (N m^?1) in the coated capillary. After successive electrophoresis for 48 h using the coated capillary, the RSD values of EOF and the theoretical plate number were 4.14 % and 9.14 %, respectively. In addition, the RSD values of EOF and the theoretical plate number (N m^?1) in the coated capillaries were 13.19 % and 8.96 %, respectively. Finally, the coated capillary was successfully applied to separate the mixture of four basic proteins (RNase A, lysozyme, trypsin and myoglobin).     

Novel covalently coated diazoresin/polyvinyl alcohol capillary column for the analysis of proteins by capillary electrophoresis

A novel method for the preparation of covalently linked capillary coatings of PVA was demonstrated using photosensitive diazoresin (DR) as coupling agents. Layer‐by‐layer self‐assembly film of DR and PVA based on hydrogen bonding was first fabricated on the inner wall of capillary, then the hydrogen bonding was converted into covalent bonding after treatment with UV light through the unique photochemistry reaction of DR. The covalently bonded coatings suppressed basic protein adsorption on the inner surface of capillary, and thus a baseline separation of lysozyme, cytochrome c and BSA was achieved using CE. Compared with bare capillary or noncovalently bonded DR/PVA coatings, the covalently linked DR/PVA capillary coatings not only improved the CE separation performance for proteins, but also exhibited good stability and repeatability. Due to the replacement of highly toxic and moisture‐sensitive silane coupling agent by DR in the covalent coating preparation, this method may provide a green and easy way to make the covalently coated capillaries for CE.     

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.     

Novel cationic coating agent for protein separation by capillary electrophoresis†

A novel positively charged surfactant N‐dodecyl‐N,N‐dimethyl‐(1,2‐propandiol) ammonium chloride was used for the dynamic coating of the inner wall of a silica capillary. This paper covers the evaluation of dynamic coating and study of the influence of the analysis conditions for the magnitude and direction of electroosmotic flow as well as for the effective and selective separation of chosen proteins (ribonuclease A, cytochrome c, lysozyme, and myoglobin). The concentration of 0.1 mM of N‐dodecyl‐N,N‐dimethyl‐(1,2‐propandiol) ammonium chloride enabled the reversal of the electro‐osmotic flow, however, to separate basic as well as neutral proteins the higher concentration of the studied surfactant was necessary. The final conditions for the separation of studied proteins were set at 100 mM sodium acetate pH 5.5 with 10.0 mM of the studied surfactant. The results were also compared with those of two commercially available cationic surfactants, cetyltrimethylammonium bromide and dodecyltrimethylammonium bromide. Additionally, the developed method for protein separation was applied for the determination of lysozyme in a cheese sample. The limits of detection and quantification of lysozyme were 0.9 and 3.0 mg/L, respectively. The mean concentration of lysozyme found in the cheese sample was 167.3 ± 10.3 mg/kg.     

Separation and determination of flavone and xanthone glycosides in Tibetan folk medicinal species Swertia mussotii and S. franchetiana by capillary electrophoresis

The contents of five pharmacologically active flavone and xanthone glycosides, namely, swertianolin, swertisin, isoorientin, mangiferin, and 7-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-xylopyranosyl]-1,8-dihydroxy-3-methoxyxanthone, extracted from Tibetan folk medicinal species Swertia mussotii and S. franchetiana were determined by capillary electrophoresis with diode-array detection. The separation of five components has been optimized with a capillary column with a total length of 48.5 cm and effective length of 40 cm (50 μm i.d). The influence of the running buffer, the sodium dodecyl sulfonate (SDS) concentration, organic modifier, etc. on the resolution was evaluated. The background electrolyte contained 30 mM borate buffer, 28 mM SDS, 1.0% (v/v) acetonitrile, and was adjusted to pH 9.0 with 0.1 M NaOH. A good baseline resolution was obtained for the separation of five components within 5 min with the working voltage of 24 kV and a column temperature of 25°C. The established method was rapid and reproducible for the separation and determination of five flavone and xanthone glycosides from the extracts of S. mussotii and franchetiana plant samples. The text was submitted by the authors in English.     

Co‐electroosmotic capillary electrophoresis of basic proteins with 1‐alkyl‐3‐methylimidazolium tetrafluoroborate ionic liquids as non‐covalent coating agents of the fused‐silica capillary and additives of the electrolyte solution

The paper reports the results of a study carried out to evaluate the use of three 1‐alkyl‐3‐methylimidazolium‐based ionic liquids as non‐covalent coating agents for bare fused‐silica capillaries and additives of the electrolyte solutions (BGE) for CE of basic proteins in the co‐EOF separation mode. The three ionic liquids are differentiated from each other by the length of the alkyl group on the imidazolium cation, consisting of either an ethyl, butyl or octyl substituent, whereas tetrafluoroborate is the common anionic component of the ionic liquids. Coating the capillary with the ionic liquid resulted in improved peak shape and protein separation, while the EOF was maintained cathodic. This indicates that each ionic liquid is effective at masking the protein interaction sites on the inner surface of the capillary, also when its adsorption onto the capillary wall has not completely neutralized all the negative charges arising from the ionization of the silanol groups and the ionic liquid is not incorporated into the BGE employed for separation. Using the coated capillaries with BGE containing the ionic liquid employed for the coating, at concentration low enough to maintaining the EOF cathodic, both peak shape and protein separation varied to different extents, based on the particular ionic liquid used and its concentration. Fast and efficient separation of the model basic protein mixture in co‐electroosmotic CE is obtained with the 1‐butyl‐3‐methylimidazolium tetrafluoroborate coated capillary and 100 mM acetate buffer (pH 4.0) containing 4.4 mM 1‐butyl‐3‐methylimidazolium tetrafluoroborate as the BGE.     

Sol‐gel technique for the preparation of β‐cyclodextrin gold nanoparticles as chiral stationary phase in open‐tubular capillary electrochromatography

A novel open‐tubular capillary electrochromatography column coated with β‐cyclodextrin was prepared using the sol‐gel technique. In the sol‐gel approach, owing to the three‐dimensional network of sol‐gel and the strong chemical bond between the stationary phase and the surface of capillary columns, good chromatographic characteristics and unique selectivity in separating enantiomers were shown. The influences of capillary inner diameter, coating time, organic modifier, buffer pH, and buffer concentration on separation were investigated. The sol‐gel‐coated β‐cyclodextrin column has shown improved enantioseparation efficiency of chlorphenamine, brompheniramine, pheniramine, zopiclone in comparison with the sol‐gel matrix capillary column. The migration time relative standard deviation of the separation of the enantiomers was less than 0.89% over five runs and 2.9% from column to column. This work confirmed that gold nanoparticles are promising electrochromatographic support to enhance the phase ratio of open‐tubular capillary electrochromatography column in capillary electrochromatography.     

Capillary ion electrophoresis-capacitively coupled contactless conductivity detection of inorganic cations in human saliva on a polyvinyl alcohol-coated capillary

Capillary ion electrophoresis–capacitively coupled contactless conductivity detection (CIE-C4D) with a polyvinyl alcohol chemically coated capillary (PVA capillary) was used to analyze inorganic cations (Na⁺, K⁺, NH₄⁺, Mg²⁺, and Ca²⁺) commonly found in human saliva. The PVA capillary, which was made by our laboratory, minimized electro-osmotic flow in the wide pH range of the background electrolyte (BGE), and the PVA layer adsorbed to capillary wall did not affect the conductimetric background level. In this study, we determined an optimized BGE of 30 mM lactic acid/histidine plus 3 mM 18-crown-6 for the CIE-C4D system using the PVA capillary, which could simultaneously improve the separation of Mg²⁺ and Ca²⁺ from Na⁺ and that of K⁺ from NH₄⁺. This system obtained highly reproducible separation of cations in human saliva samples within 8 min at 20 kV without deprotonation. The quantifiability of cations in human saliva samples on the CIE-C4D system was demonstrated through identification by ion chromatography with satisfactory results.     

Capillary electrophoresis of intact basic proteins using noncovalently triple‐layer coated capillaries

The usefulness of a noncovalent, positively charged capillary coating for the efficient analysis of intact basic proteins with CE was studied. Capillaries were coated by subsequent flushing with solutions of 10% w/v Polybrene (PB), 3% w/v dextran sulfate (DS), and again 10% w/v PB. Coating characterization studies showed that stable coatings could be produced which exhibited a pH‐independent and highly reproducible EOF. The PB–DS–PB coating was evaluated with Tris phosphate BGEs of various pH using the four basic model proteins: α‐chymotrypsinogen A, ribonuclease A, cytochrome c, and lysozyme. Typical migration time RSDs for the proteins were less than 0.85%, and apparent plate numbers were above 125 000 using a capillary length of 40 cm. The high separation efficiency allowed detection of several minor impurities in the model proteins. Using a BGE of medium pH, the CE system with triple‐layer coating appeared to be useful for the repeatable profiling of recombinant humanized mouse monoclonal immunoglobulin G₁ showing a characteristic pattern of glycoforms. The CE system was also applied to the characterization of two llama antibodies, which were produced in Saccharomyces cerevisiae, revealing the presence of a side product in one of the antibodies. The high migration time stability allowed the reliable determination of antibody–antigen binding by monitoring migration time shifts. Finally, the feasibility of using the PB–DS–PB coated capillaries for CE with mass spectrometric detection was shown by the characterization of the impure llama antibody sample.     

Studies on the analytical performance of a non-covalent coating with N,N-didodecyl-N,N-dimethylammonium bromide for separation of basic proteins by capillary electrophoresis in acidic buffers in 25-and 50-μm capillaries

Capillaries (25-and 50-μm inner diameter) coated with a double-alkyl-chain cationic surfactant N,N-didodecyl-N,N-dimethylammonium bromide (DDAB) were used for the separation of four basic standard proteins in buffers of pH 4 at various ionic strengths. The choice of buffer is critical for the analytical performance. Ammonium ions must be avoided in the buffer used in the non-covalent coating procedure owing to competition with the surfactant. Phosphate buffer gave a better separation performance than some volatile buffers; the reason seems to be a complex formation between the proteins and dihydrogenphosphate ions, which decreases tendencies for adsorption to the capillary surface. The DDAB coating was easy to produce and stable enough to permit, without recoating, consecutive separations of the proteins for up to 100 min with good precision in migration times and peak areas. A strong electroosmotic flow gives rapid separations, which is of special importance when commercial instruments are used, since the choice of the length of the capillary is restricted. Figure EOF stability in 25 micrometer i.d. capillaries. Consecutive injections of mesityloxide performed after an initial coating with 1.0     

CE coupled to MALDI with novel covalently coated capillaries

CE offers the advantage of flexibility and method development options. It excels in the area of separation of ions, chiral, polar and biological compounds (especially proteins and peptides). Masking the active sites on the inner surface of a bare fused silica capillary wall is often necessary for CE separations of basic compounds, proteins and peptides. The use of capillary surface coating is one of the approaches to prevent the adsorption phenomena and improve the repeatability of migration times and peak areas of these analytes. In this study, new capillary coatings consisting of (i) derivatized polystyrene nanoparticles and (ii) derivatized fullerenes were investigated for the analysis of peptides and protein digest by CE. The coated capillaries showed excellent run‐to‐run and batch‐to‐batch reproducibility (RSD of migration time ≤0.5% for run‐to‐run and ≤9.5% for batch‐to‐batch experiments). Furthermore, the capillaries offer high stability from pH 2.0 to 10.0. The actual potential of the coated capillaries was tested by combining CE with MALDI‐MS for analysing complex samples, such as peptides, whereas the overall performance of the CE‐MALDI‐MS system was investigated by analysing a five‐protein digest mixture. Subsequently, the peak list (peptide mass fingerprint) generated from the mass spectra of each fraction was entered into the Swiss‐Prot database in order to search for matching tryptic fragments using the MASCOT software. The sequence coverage of analysed proteins was between 36 and 68%. The established technology benefits from the synergism of high separation efficiency and the structure selective identification via MS.     

Separation and Determination of Stereoisomeric Impurity of Folinic Acid Diastereomers by CE with Vancomycin as a Selector

A highly sensitive and rapid method was developed that involves capillary electrophoresis for separation and determination of the stereoisomeric impurity of folinic acid diastereomers. In this method, vancomycin was used as the chiral selector, and a solution of poly(dimethylacrylamide) (PDMA) was prepared for dynamic coating of the capillary wall to minimize the adsorption of vancomycin. This method was optimized for six factors including concentrations of the organic modifier and vancomycin, pH and concentration of the background electrolyte, column temperature, and separation voltage. The following conditions were established: 100 mM Tris-phosphate buffer (pH 6.0) containing 1.0 mM vancomycin and 5 % acetonitrile at 30 °C, and −15 kV applied voltage on the PDMA dynamically coated capillary. Preliminary validation was performed with the determination of limit of quantification and detection, accuracy, precision, and linearity. Under our optimized method, the folinic acid diastereomers were baseline-separated within 7.5 min, and a (6S,2′S)-calcium folinate sample with 0.08 % stereoisomeric impurity was determined.

     

New physically adsorbed polymer coating for reproducible separations of basic and acidic proteins by capillary electrophoresis

In this work, a new physically adsorbed coating for capillary electrophoresis (CE) is presented. The coating is based on a N,N-dimethylacrylamide-ethylpyrrolidine methacrylate (DMA-EPyM) copolymer synthesized in our laboratory. The capillary coating is simple and easy to obtain as only requires flushing the capillary with a polymer aqueous solution for 2 min. It is shown that by using these coated capillaries the electrostatic adsorption of a group of basic proteins onto the capillary wall is significantly reduced allowing their analysis by CE. Moreover, the DMA-EPyM coating provides reproducible separations of the basic proteins with RSD values for migration times lower than 0.75% for the same day (n = 5) and lower than 3.90% for three different days (n = 15). Interestingly, the electrical charge of the coated capillary wall can be modulated by varying the pH of the running buffer which makes possible the analysis of basic and acidic proteins in the same capillary. The usefulness of this coating is further demonstrated via the reproducible separation of whey (i.e. acidic) proteins from raw milk. The coating protocol should be compatible with both CE in microchips and CE-MS of different types of proteins.     

Poly(N,N‐dimethylacrylamide‐co‐4‐(ethyl)‐morpholine methacrylamide) copolymer as coating for CE

In this work, a new physically adsorbed coating for CE is presented. This coating is based on a poly(N,N‐dimethylacrylamide‐co‐4‐(ethyl)‐morpholine methacrylamide) (DMA/MAEM) copolymer synthesized in our laboratory. It is demonstrated that the direction and magnitude of the EOF in CE can be modulated by varying the composition of the DMA/MAEM copolymer and the type and pH of the BGE. Moreover, the DMA/MAEM coating provides %RSD_n = 5 values for migration times lower than 0.9% for the same capillary and day, whereas the %RSD_n = 25 obtained for the interday assay was lower than 2.9%. The stability of the coating procedure is also tested between capillaries obtaining %RSD_n = 15 values lower than 2.9%, demonstrating that this physically adsorbed copolymer gives rise to a stable and reproducible coating in CE. Finally, the usefulness of this new cationic copolymer as CE coating is demonstrated through different applications. Namely, it is demonstrated that the CE separation of basic proteins, nucleotides and organic acids is achieved in a fast and easy way by using the DMA/MAEM coated capillary. The use of fused bare silica capillaries did not allow the separation of these compounds under the same analytical conditions. These results demonstrate that this type of coating in CE provides the option of using BGEs that are useless when utilized together with bare silica capillaries making wider the application and possibilities of this analytical technique.