Synthesis of poly(N,N‐dimethylacrylamide)‐block‐poly(ethylene oxide)‐block‐poly(N,N‐dimethylacrylamide) and its application for separation of proteins by capillary zone electrophoresis

A series of well‐defined triblock copolymers, poly(N, N‐dimethylacrylamide)‐block‐poly(ethylene oxide)‐block‐poly(N, N‐dimethylacrylamide) (PDMA‐b‐PEO‐b‐PDMA) synthesized by atom transfer radical polymerization, were used as physical coatings for protein separation. A comparative study of EOF showed that the triblock copolymer presented good capillary coating ability and EOF efficient suppression. The effects of the M_r of PDMA block in PDMA‐b‐PEO‐b‐PDMA triblock copolymer and buffer pH on the separation of basic protein for CE were investigated. Moreover, the influence of the copolymer structure on separation of basic protein was studied by comparing the performance of PDMA‐b‐PEO‐b‐PDMA triblock copolymer with PEO‐b‐PDMA diblock copolymer. Furthermore, the triblock copolymer coating showed higher separation efficiency and better migration time repeatability than fused‐silica capillary when used in protein mixture separation and milk powder samples separation, respectively. The results demonstrated that the triblock copolymer coatings would have a wide application in the field of protein separation.     

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.     

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.     

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.     

Capillary electrophoresis of seed albumins fromVicia species using uncoated and surface-modified fused silica capillaries

Summary Capillary zone electrophoresis has been developed for the separation of seed albumins fromVicia faba using both uncoated and polyoxyethylene ether (Brij-35) coated octadecysilane derivatized capillaries. Optimal separation conditions were found by studying the effect of pH, buffer composition and applied voltage. The nonionic surfactant/C₁₈ coated capillary significantly reduced albumin adsorption and electroosmotic flow (EOF). A gradual washing out of the surfactant from the coated capillary during use altered not only the magnitude of the EOF, but also its reproducibility. The introduction of hydrophilic polymer solutions between analyses for dynamic modification of the Brij/C₁₈ coated capillary surface prevented desorption of coating material, allowed optimization of resolution and ensured stability of the EOF. CE with surface-modified capillaries was then used to compare seed albumin profiles of severalVicia species. This technique appears to provide a powerful tool for use in taxonomic investigations.     

Capillary electrophoresis of seed albumins fromVicia species using uncoated and surface-modified fused silica capillaries

Summary Capillary zone electrophoresis has been developed for the separation of seed albumins fromVicia faba using both uncoated and polyoxyethylene ether (Brij-35) coated octadecylsilane derivatized capillaries. Optimal separation conditions were found by studying the effect of pH, buffer composition and applied voltage. The nonionic surfactant/C₁₈ coated capillary significantly reduced albumin adsorption and electroosmotic flow (EOF). A gradual washing out of the surfactant from the coated capillary during use altered not only the magnitude of the EOF, but also its reproducibility. The introduction of hydrophilic polymer solutions between analyses for dynamic modification of the Brij/C₁₈ coated capillary surface prevented desorption of coating material, allowed optimization of resolution and ensured stability of the EOF. CE with surface-modified capillaries was then used to compare seed albumin profiles of severalVicia species. This technique appears to provide a powerful tool for use in taxonomic investigations.     

Facile preparation of polysaccharide‐coated capillaries using a room temperature ionic liquid for chiral separations

In this study, the dissolution of polysaccharides into an ionic liquid was investigated and applied as a coating onto the capillary walls of a fused‐silica capillary in open‐tubular CEC. The coating was evaluated by examining the chiral separation of two analytes (thiopental, sotalol) with three cellulose derivatives (cellulose acetate, cellulose acetate phthalate, and cellulose acetate butyrate). Baseline separation of thiopental enantiomers was achieved by use of each polysaccharide coating (Rs: 7.0, 8.1, 7.1), while sotalol provided partial resolution (Rs: 0.7, 1.0, 0.9). In addition, reproducibility of the cellulose‐coated capillaries was evaluated by estimating the run‐to‐run and capillary‐to‐capillary RSD values of the EOF. Both stability and reproducibility were very good with RSD values of less than 7%.     

Open tubular columns with mixed‐mode reversed‐phase and weak anion‐exchange stationary phase for capillary electrochromatography

Columns for open tubular capillary electrochromatography, coated with a mixed‐mode (RP/ion‐exchange) stationary phase, were prepared by using the sol–gel method. The synthetic procedure was optimized by changing the ratios of tetraethoxysilane, octyltriethoxysilane, and 3‐aminopropyltriethoxysilane in the initial sol. SEM studies reveal that a coating with about 400 nm thickness can be obtained. The inner surface properties of these capillaries were probed by measuring the EOF as a function of pH. The surface of this stationary phase contains octyl, amine, and residual silanol moieties; the amine and silanol groups determine the net charge on the inner surface of the capillary and can produce a switchable EOF (anodal/cathodal). The performances of the columns were evaluated by open tubular capillary electrochromatography using a wide range of compounds (polycyclic aromatic hydrocarbons, aromatic acids, and aromatic amines).     

Evanescent‐wave particle velocimetry measurements of zeta‐potentials in fused‐silica microchannels

The wall ζ‐potential ζ_w, the potential at the shear plane of the electric double layer, depends on the properties of the BGE solution such as the valence and type of electrolyte, the pH and the ionic strength. Most of the methods estimate ζ_w from measurements of the EOF velocity magnitude u_eo, usually spatially averaged over the entire capillary. In these initial studies, evanescent‐wave particle velocimetry was used to measure u_eo in steady EOF for a variety of monovalent aqueous solutions to evaluate the effect of small amounts of divalent cations, as well as the pH and ionic strength of BGE solutions. In brief, the magnitude of the EOF velocity of NaCl‐NaOH and borate buffer‐NaOH solutions was estimated from the measured velocities of radius α = 104 nm fluorescent polystyrene particles in 33 μm fused‐silica microchannels. The particle ζ‐potentials were measured separately using laser‐Doppler micro‐electrophoresis; ζ_w was then determined from u_eo. The results suggest that evanescent‐wave particle velocimetry can be used to estimate ζ_w for a variety of BGE solutions, and that it can be used in the future to estimate local wall ζ‐potential, and hence spatial variations in ζ_w.     

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).     

Capillary Electrophoretic Separation of Tricyclic Antidepressants Using 1‐Alkyl‐3‐Methylimidazolium‐Based Ionic Liquids as Background Electrolyte

A capillary electrophoretic (CE) method coupled with the use of 1‐ethyl‐3‐methylimidazolium tetrafluoroborate (1E‐3MI‐TFB) ionic liquid as background electrolyte (BGE) has been developed for the simultaneous separation of nine tricyclic antidepressants, viz. amitriptyline (Ami), clomipramine (Clo), desipramine (Des), fluphenazine (Flu), imipramine (Imi), nortriptyline (Nor), promazine (Pro), thioridazine (Thi) and trimipramine (Tri). Resolution of TCAs with similar molecular structures and pK_a values was accomplished by minute manipulation of the electrophoretic velocities of TCAs via reversed electroosmotic flow (EOF) generated by adsorption of 1E‐3MI cations onto the capillary wall. The optimal separation was obtained with a 50 mM 1E‐3MI‐TFB as the sole BGE at pH 3. Symmetric peaks with efficiencies up to 2.4 × 10⁵ plates/m were achieved. RSD values on migration times and peak areas were in the ranges of 0.63–0.95% and 3.41–6.34% (n = 4), respectively. The role of different alkyl groups on the imidazolium cations was also investigated.     

A two‐step method for rapid characterization of electroosmotic flows in capillary electrophoresis

The measurement of electroosmotic flow (EOF) is important in a capillary electrophoresis (CE) experiment in terms of performance optimization and stability improvement. Although several methods exist, there are demanding needs to accurately characterize ultra‐low electroosmotic flow rates (EOF rates), such as in coated capillaries used in protein separations. In this work, a new method, called the two‐step method, was developed to accurately and rapidly measure EOF rates in a capillary, especially for measuring the ultra‐low EOF rates in coated capillaries. In this two‐step method, the EOF rates were calculated by measuring the migration time difference of a neutral marker in two consecutive experiments, in which a pressure driven was introduced to accelerate the migration and the DC voltage was reversed to switch the EOF direction. Uncoated capillaries were first characterized by both this two‐step method and a conventional method to confirm the validity of this new method. Then this new method was applied in the study of coated capillaries. Results show that this new method is not only fast in speed, but also better in accuracy.     

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.     

Novel cationic polyelectrolyte coatings for capillary electrophoresis

The use of bare fused silica capillary in CE can sometimes be inconvenient due to undesirable effects including adsorption of sample or instability of the EOF. This can often be avoided by coating the inner surface of the capillary. In this work, we present and characterize two novel polyelectrolyte coatings (PECs) poly(2‐(methacryloyloxy)ethyl trimethylammonium iodide) (PMOTAI) and poly(3‐methyl‐1‐(4‐vinylbenzyl)‐imidazolium chloride) (PIL‐1) for CE. The coated capillaries were studied using a series of aqueous buffers of varying pH, ionic strength, and composition. Our results show that the investigated polyelectrolytes are usable as semi‐permanent (physically adsorbed) coatings with at least five runs stability before a short coating regeneration is necessary. Both PECs showed a considerably decreased stability at pH 11.0. The EOF was higher using Good's buffers than with sodium phosphate buffer at the same pH and ionic strength. The thickness of the PEC layers studied by quartz crystal microbalance was 0.83 and 0.52 nm for PMOTAI and PIL‐1, respectively. The hydrophobicity of the PEC layers was determined by analysis of a homologous series of alkyl benzoates and expressed as the distribution constants. Our result demonstrates that both PECs had comparable hydrophobicity, which enabled separation of compounds with log P_o/w > 2. The ability to separate cationic drugs was shown with β‐blockers, compounds often misused in doping. Both coatings were also able to separate hydrolysis products of the ionic liquid 1,5‐diazabicyclo[4.3.0]non‐5‐ene acetate at highly acidic conditions, where bare fused silica capillaries failed to accomplish the separation.     

Rapid determination of globin chains in red blood cells by capillary electrophoresis using INSTCoated fused‐silica capillary

A laboratory‐made INSTCoated fused‐silica capillary has been newly used for CE separation of four mixtures of proteins in sodium phosphate BGEs at pH 3.0 and 2.5, respectively. The obtained separation efficiencies range from 145 000 theoretical plates per meter for myoglobin to 1 216 000 m^?1 for lysozyme. A total of 49–89% of the number of theoretical plates was obtained in a commercial polyvinyl alcohol coated capillary compared to the INSTCoated capillary under the same experimental conditions, 0–86% was obtained in a laboratory polyacrylamide‐coated capillary, and only 0–6% was obtained in an uncoated fused‐silica capillary. The RSD values for the intraday repeatability for an INSTCoated capillary were 0.1–1.0% (migration time) and 0.3–2.4% (peak area); RSD values for the interday repeatability in the same capillary are 0.6–1.4% (migration time) and 2.4–5.5% (peak area); RSD values for interday repeatability between different capillaries equaled 1.7–2.1% (migration time) and 2.8–10.9% (peak area). The INSTCoated capillary has been further used for rapid determination of globin chains isolated from red blood cells. A separation of α and β chains prepared from adult blood has been completed in 3 min with a peak resolution of 1.3, and the separation of α and ^Gγ chains prepared from newborn blood took 3 min with a peak resolution of 3.6.     

Preparation and characterization of a polystyrene/bovine serum albumin nanoparticle‐coated capillary for chiral separation using open‐tubular capillary electrochromatography

Polystyrene (PS) nanoparticles coated by BSA, hereafter denoted as PS/BSA, were prepared and chemically immobilized for the first time onto a capillary inner wall for open‐tubular CEC (OTCEC). EOF and scanning electron micrography were used to characterize the prepared nanoparticle‐coated capillaries. To investigate the performance of the prepared columns in OTCEC, chiral separation of d ,l ‐tryptophan (dl ‐Trp) was performed in monolayer BSA‐modified capillary and PS/BSA nanoparticle‐coated columns. The results indicated that the nanoparticle‐modified column afforded a higher resolution compared with the monolayer type. Rapid enantioseparation of dl ‐Trp (within 3 min) was achieved with the PS/BSA‐immobilized column using an electroosmotic pump‐assisted CEC. Enantiomer separations of other compounds like dl ‐tyrosine and warfarin were also achieved with the column. Besides, run‐to‐run and column‐to‐column repeatabilities of the PS/BSA‐coated column in the chiral separation were systematically introduced.     

New pressure‐assisted sweeping on‐line preconcentration for highly polar environmentally relevant nitrosamines: Part 2. Cationic and anionic surfactants with zero‐flow capillaries

We recently introduced a pressure‐assisted sweeping‐reversed migration‐EKC (RM‐EKC) method for preconcentration of neutral polar N‐nitrosamines with low affinity for the micellar phase. The type of surfactant and phase ratio are dominant factors in dictating the magnitude of interactions between analyte and micellar phase, thus four surfactants (anionic and cationic) with a range of functionalities (SDS, ammonium perfluorooctanoate (APFO), bile salts, and cetyltrimethylammonium chloride (CTAC)) were evaluated for sweeping‐RM‐EKC of highly polar N‐nitrosamines. All gave acceptable results for sweeping‐RM‐EKC when used in high concentrations (≥200 mM) with low EOF. While no single surfactant was superior by all measures, all but the bile salts had useful performance characteristics. APFO showed the narrowest peak widths and highest number of theoretical plates, though two species co‐migrated at all concentrations (25–300 mM); SDS and the cationic surfactant CTAC also showed good separation characteristics and could resolve all peaks, but CTAC had wider separation window. Various types of capillaries coated for EOF control were compared for use with anionic and cationic surfactants. A commercial zero‐EOF capillary coated with a polymer bearing sulfonic acid functional groups showed superior EOF suppression and reproducibility of migration time with all surfactants.     

Preparation and evaluation of stable coating for capillary electrophoresis using coupled chitosan as coated modifier

A coated capillary modified with a coupled chitosan (COCH) was developed by using a simple and fast (60 min) process that could be easily automated in capillary electrophoresis instrument. The COCH coating was achieved by first attaching chitosan to the capillary inner wall, and then coupling with glutaraldehyde, and rinsing chitosan again to react with glutaraldehyde. The COCH coating was stable and showed amphoteric character over the pH range of 1.8-12.0. When the pH value was lower than 4.5, the capillary surface possessed positive charges, which caused a reversal in the direction of the electroosmotic flow (EOF). The normal EOF direction could be obtained when the pH value was higher than 4.5. The COCH coating showed strong stability against 0.1 mol/L HCl, 0.1 mol/L NaOH and other solvents compared with conventional chitosan coating. The relative standard deviation of the run-to-run, day-to-day and capillary-to-capillary coating was all below 2% for the determination of EOF. The COCH-modified capillary was applied to acidic and basic proteins analyses and high efficiency could be attained. The comparison between unmodified capillary, chitosan-modified and COCH-modified capillary for the separation of real sample, extract from Elaphglossum yoshinagae with water, was also studied. Better results could be obtained on COCH-modified capillary than the other two capillaries.     

New pressure‐assisted sweeping on‐line preconcentration for polar environmentally relevant nitrosamines: Part 1. Sweeping for polar compounds and application of auxiliary pressure

Typically sweeping reversed migration EKC (RM‐EKC) is used for online enrichment and separation of neutral compounds in CE, however sweeping is not usually suitable for highly polar neutral compounds due to the lack of strong interaction with micellar phase. Since acidic BGE or coated capillaries (BGE pH 2–8) are used to virtually eliminate the EOF, migration of neutral analytes is only through association with the micelles with relatively slow electrophoretic mobility. To decrease the long analysis times that result, an auxiliary pressure can be applied, which also serves to avoid the associated band broadening. In this study, we have modified a commercially available CE instrument to perform pressure‐assisted sweeping. The apparatus described can be used to precisely control the application of pressure, and therefore direction and magnitude of bulk flow in the capillary. This modification allows us to employ longer capillaries and capillaries with larger internal diameter to increase the sensitivity. An optimized method was used for the analysis of a group of seven N‐nitrosamines that have been widely reported in environmental samples and good concentration factors of up to 34 were achieved. When a coated capillary is employed, this method is effective even at neutral pH, making it broadly applicable.     

Preparation and evaluation of amphiphilic silica‐based monolithic column having surface‐bound octanoyl‐aminopropyl moieties for capillary electrochromatography

A novel amphiphilic silica‐based monolithic column having surface‐bound octanoyl‐aminopropyl moieties was successfully prepared by a one‐step in situ derivatization process. As expected, the amphiphilic monolithic column exhibited RP chromatographic behavior toward non‐polar solutes (e.g., alkyl benzenes) with high column performance. As the pH of the buffer inside the column increases, the EOF changed from −2.65×10⁻⁸m² V⁻¹s⁻¹ at pH 3.0 to 1.20×10⁻⁸ m² V⁻¹s⁻¹ at pH 8.0 with the reversion of EOF at about pH 6.4. Using acidic mobile phase, five aromatic acids can be efficiently separated in less than 6 min under co‐EOF conditions. For basic compounds, symmetrical peaks were obtained due to the existence of hydrophilic acyl amide group, which can effectively minimize the adsorption of the positively charged basic analyte to the silica‐based surface of the capillary column.