Capillary electrophoresis using core‐based hyperbranched polyethyleneimine (CHPEI) static‐coated capillaries

With unique 3‐D architecture, the application of core‐based hyperbranched polyethyleneimine (CHPEI), as a capillary coating in capillary electrophoresis, is demonstrated by manipulation of the electroosmotic mobility (EOF). CHPEI coatings (CHPEI5, M_w ≈? 5000 and CHPEI25, M_w ≈? 25 000) were physically adsorbed onto the inner surface of bare fused‐silica capillary (BFS) via electrostatic interaction of the oppositely charged molecules by rinsing the capillaries with different CHPEI aqueous solutions. The EOF values of the coated capillaries were measured over the pH range of 4.0–9.0. At higher pH (pH >6) the coated capillary surface possesses excess negative charges, which causes the reversal of the EOF. The magnitudes of the EOF obtained from the coated capillaries were three‐fold lower than that of BFS capillary. Desirable reproducibility of the EOF with % RSD (n = 5) ? 2 was obtained. Effect of ionic strength, stability of the coating (% RSD = 0.3) and the dependence of the EOF on pH (% RSD = 0.5) were also investigated. The CHPEI‐coated capillaries were successfully utilized to separate phenolic compounds, B vitamins, as well as basic drugs and related compounds with reasonable analysis time (<20 min) and acceptable migration‐time repeatability (<0.7% RSD for intra‐capillary and <2% RSD for inter‐capillary).     

Determination of ammonium and metal ions by capillary electrophoresis-potential gradient detection using ionic liquid as background electrolyte and covalent coating reagent

A capillary zone electrophoresis (CZE)-potential gradient detection (PGD) method coupled with field-amplified sample injection was developed to determine alkali metal, alkaline-earth metal, nickel, lead and ammonium ions. The capillary surface was coated with dialkylimidazolium-based ionic liquid and thus the electroosmotic flow (EOF) of the capillary was reversed. The buffer composed of 7.5 mM lactic acid, 0.6 mM 18-crown-6, 12 mM alpha-cyclodextrin (alpha-CD); it was adjusted to pH 4.0 by 1-hexyl-3-methylimidazolium hydroxide. The 11 cations were baseline separated within 14 min with 5.1-18.9 x 10(4) plates (for 40-cm-long capillary) in separation efficiency, and the detection limits were in the range of 0.27-7.3 ng/ml. The method showed good reproducibility in terms of migration time with RSD < or = 0.90% for run-to-run and < or = 1.65 for day-to-day assessment.     

Capillary zone electrophoresis of proteins applying ionic liquids for dynamic coating and as background electrolyte component

The use of ionic liquids in capillary electrophoresis, either as coating material or as components of the background electrolyte needs systematic standardization to set up optimal conditions. Excellent separation of the proteins was achieved using 1-ethyl-3-methylimidazolium tetrafluoroborate ([emim][BF₄]) or 1-butyl-3-methylimidazolium tetrafluoroborate ([bmim][BF₄]) ionic liquids using the properly made ionic-liquid–water binary mixtures for the experiments. The binary mixture has a distinctly stable and well perceptible low pH, which depends on the concentration of the ionic liquid, and on the preparation time of the mixture. Optimal conditions for the electrophoretic separation were obtained upon a multivariate analysis of the experimental parameters (applied voltage, migration time, concentration, and type of the ionic liquid). The standardized condition provides a low electroendosmotic flow toward the anode, which, however, did not hinder the proteins to migrate toward the cathode. The migration of cytochrome c, lysozyme, myoglobin, trypsin, and apo-transferrin at a pH around 2, far below the isoelectric points of the proteins, showed RSD values of the migration times less than 7.5% and less than 6.5% when using [emim][BF₄] or [bmim][BF₄], respectively, either in run-to-run or day-to-day experiments. The determination of the extent of the EOF is not possible with the commonly used EOF markers, due to interaction with the ionic-liquid constituents. The interaction of the ionic liquids with the proteins influences the migration order in zone electrophoresis. This method has been applied successfully for the analyses of real biological samples such as proteins from egg whites and human tears.     

Triamine‐bonded stationary phase for open tubular capillary electrochromatography

A multi‐functional separation column modified with 3‐[2‐(2‐aminoethylamino)ethylamino] propyl‐trimethoxysilane was developed for open tubular capillary electrochromatography. This functional hydrophilic triamine‐bonded open tubular column could generate both anodic and cathodic EOF. When the pH of the running buffer was below 5.3 (30% 3‐[2‐(2‐aminoethylamino)ethylamino] propyl‐trimethoxysilane, v/v), the anodic EOF was exhibited, which greatly prevented the undesired adsorptions of basic proteins on the capillary inner wall. Favorable separation of four basic proteins (viz. trypsin, ribonuclease A, lysozyme and cytochrome c) was successfully achieved at pH 3.5 of 10 mmol/L phosphate buffer. The column efficiencies of proteins were in the range from 87 000 to 110 000 plates/m, and the RSD values for migration time of four proteins were less than 1.2% (run‐to‐run, n=5). The ionic analytes were also separated efficiently in the co‐electroosmotic mode. The average efficiencies ranged from 81 000 to 190 000 plates/m for seven aromatic acids and 186 000–245 000 plates/m for four nucleoside monophosphates, respectively, and good capillary column repeatability was gained with RSD of the migration time not more than 3.0%. The triamine‐bonded open tubular capillary column is favorable to be an alternative functional medium for the further analysis of basic proteins and anionic analytes.     

Separation of amino acids and amines by capillary electrophoresis using poly(ethylene oxide) solution containing cetyltrimethylammonium bromide

We describe simultaneous analysis of naphthalene-2,3-dicarboxaldehyde (NDA)-amino acid and amine derivatives by capillary electrophoresis in conjunction with light-emitting diode-induced fluorescence (LEDIF) detection using poly(ethylene oxide) (PEO) containing cetyltrimethylammonium bromide (CTAB). In the presence of CTAB and acetonitrile (ACN), adsorption of PEO on the capillary wall is suppressed, leading to generation of a fast and reproducible electroosmotic flow (EOF). In order to optimize separation resolution and speed, 100 mM Tris–borate solution (pH 7.0) containing 20 mM CTAB and 25% ACN was used to fill the capillary and to prepare 1.2% PEO that entered the capillary via EOF. The analysis of 14 NDA-amino acid and -amine derivatives by this approach is rapid (< 4 min), efficient ((0.9–6.4) × 10⁵ theoretical plates), and sensitive (the LODs (S/N = 3) range from 9.5 to 50.5 nM). The RSD values (n = 5) of the migration times and peak heights of the analytes for the intraday analysis are less than 1.5 and 1.2%, respectively. We have validated the practicality of this approach by quantitative determination of 10 amino acids and amines in a beer samples within 4 min.     

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.     

毛细管电泳法快速检测消毒剂中的醋酸氯己定

建立了消毒剂中活性成分醋酸氯己定(又名:醋酸洗必泰)的毛细管电泳快速检测法。采用15 mmol/L磷酸盐-乙腈( 体积比为60∶40)缓冲体系,将醋酸氯己定在50 cm×75 μm i.d.的石英毛细管柱中进行电泳分离,电泳电压为15 kV,检 测波长为254 nm。同时,对毛细管电泳分析醋酸氯己定的条件(如缓冲液的种类、pH值、浓度及电泳电压等)进行了优化 。用该方法对消毒剂样品进行测定,在4 min内可完成分析。醋酸氯己定在质量浓度为0.01～0.10 g/L时线性良好,检测 限为0.004 mg/L,吸光度值的相对标准偏差为3.97%,迁移时间的相对标准偏差为2.99%,样品加标回收率为91.4%～116.6%。将该方法 与高效液相色谱法进行比较,两种方法测定结果的相对误差≤4%。所建立的检测醋酸氯己定含量的毛细管区带电泳法简单 、快速,适用于消毒剂样品的测定。     

Branched polyethyleneimine-bonded tentacle-type polymer stationary phase for peptides and proteins separations by open-tubular capillary electrochromatography

A novel tentacle-type polymer stationary phase covalently modified with branched polyethyleneimine (PEI) was developed for peptides and proteins separations by open-tubular CEC (OT-CEC). The preparation procedure included the silanization of capillary inner wall, in situ graft polymerization and PEI functionalization. A wrinkly polymer surface of multitudinous steric amine groups was evenly formed on the capillary inner wall, and anodic EOF could be gained within a wide pH range of 2.5-7.5. The electroosmotic mobility was examined for its dependence on pH as well as PEI concentrations. Good repeatability was gained with RSD for the migration time of EOF marker within 4.8% and satisfactory chemical stability was validated. Due to the existence of amine groups on the surface of tentacle-type polymer stationary phase, the silanol effect that occurs between the positively charged biomolecules and the silanols of the capillary column was greatly suppressed. Compared with a monolayer-coating capillary, seven enkephalin-related peptides were well resolved on the PEI-bonded column with high efficiencies. Favorable separations of peptides and proteins with high column efficiencies were obtained in 144,000-189,000 and 97,000-170,000 plates/m. Branched PEI-bonded tentacle-type polymer stationary phase has been proven to afford satisfactory retention and resolution of peptides and proteins.     

Enantiomeric separation by capillary electrophoresis with an electroosmotic flow-controlled capillary

Perfect control of electroosmotic flow (EOF) was achieved by dovetailing successive multiple ionic-polymer layer (SMIL) coated capillaries. The direction and magnitude of the EOF was perfectly controllable over the pH range 2-13. Zone diffusion was not observed, even if the inner wall of the dovetailed capillary was discontinuous, or if the sample zone passed through the connected part of the capillary because the RSDs of migration time, theoretical plates, symmetry factor and S/N of the marker were almost the same when seamless capillary and dovetailed capillary were compared. The dovetailed capillary was applied to cyclodextrin modified capillary zone electrophoresis. The control of the EOF enabled us to control both the resolution and the migration order of the enantiomers. The migration time was also controllable and, therefore, the best condition between separation and migration time could be determined by controlling the EOF. Partial filling affinity electrokinetic chromatography with a protein used as a chiral selector was also studied. The migration of the pseudostationary phase was controllable by EOF, and detection of the solute at 214 nm was possible. Therefore, the EOF-controlled dovetailed capillary has great potential to expand the application of the separation technique.     

Suppression of electroosmotic flow and its application to determination of electrophoretic mobilities in a poly(vinylpyrrolidone)-coated capillary

A hydrophilic polymer, poly(vinylpyrrolidone) (PVP), was employed for suppressing the electroosmotic flow (EOF). A capillary was filled with aqueous PVP solution for coating the capillary wall with PVP; the PVP solution was then replaced by a migration buffer solution containing no PVP. Three types of PVP with different molecular weights were examined. The EOF was suppressed more effectively as the molecular weight of PVP increased. The EOF in the coated capillary was approximately 10-fold smaller than that of a bare capillary and was constant in the pH range of 6-8. The suppressed EOF was stable even when no PVP was added to the migration buffer. However, the EOF increased significantly when sodium dodecyl sulfate was added into the migration buffer. The method was applied for determining the electrophoretic mobilities of inorganic anions that have negative electrophoretic mobilities larger than the electroosmotic mobility of the bare capillary. A novel method for determining the electrophoretic mobilities was proposed based on the linear relationship between electric current and electrophoretic mobility. The electrophoretic mobility was proportional to the electric current. Therefore, the intercept of the regression equation represents the electrophoretic mobility at room temperature. The electrophoretic mobilities were in good agreement with the absolute electrophoretic mobilities.     

Efficient and reproducible analysis of peptides by capillary electrophoresis using noncovalently bilayer-coated capillaries

The usefulness of a noncovalent capillary coating consisting of two layers of oppositely charged polymers for the separation of peptides with capillary electrophoresis (CE) was studied. Capillaries were coated simply by subsequently flushing with solutions of 1% m/v Polybrene and 1% v/v poly(vinylsulfonate) (PVS) forming a bilayer, which showed to produce a strong and highly reproducible electroosmotic flow (EOF) at low pH. Using this coating in combination with a background electrolyte (BGE) containing sodium phosphate (pH 2.5) and 0.01% v/v PVS, initially broadened and overlapping peaks were obtained for some test peptides. By omitting the PVS from the BGE, the peak width and shape of the peptides improved resulting in baseline separation. A systematic study of the influence of the BGE composition showed that considerable further enhancement of the separation efficiency was achieved by increasing the ionic strength of the BGE. Using a BGE of 200 mM tris(hydroxymethyl)aminomethane (Tris)-phosphate (pH 2.5) plate numbers for the peptides were in the 300 000-600 000 range and the relative standard deviation of the peptide migration times was less then 0.3% (n = 5). The use of Tris-phosphate instead of sodium phosphate allowed the current to stay within acceptable limits when 30 kV was used as separation voltage. Overall, the bilayer coating showed a remarkable EOF repeatability, as well as long-term stability. Compared to bare fused-silica capillaries the intraday and interday repeatability of migration times was very favorable and coated capillaries could be used for over a month performing analyses with low and high ionic strength BGEs without any performance deterioration. The usefulness of the bilayer-coated capillaries for the analysis of positively charged peptides was demonstrated by the fast and efficient separation of various closely related enkephalins and the baseline separation of an isomeric peptide/peptoid couple exhibiting efficiencies of over 550 000 plates.     

Selectivity control in the separation of aromatic amino acid enantiomers with sulphated beta-cyclodextrin

Control of selectivity in the enantiomeric separation of three aromatic amino acids (phenylalanine, tyrosine and tryptophan) is demonstrated by electrokinetic capillary chromatography utilising temperature variations coupled with the use of sulphated-beta-cyclodextrin (s-beta-CD) as a pseudostationary phase. The concentration of s-beta-CD and temperature were used as experimental variables to control the observed selectivity. A double-coated capillary was used and proved very robust with reproducibility of migration times being <2.0% R.S.D. between runs and <2.6% on using a new capillary. The system was modelled successfully using an artificial neural network (ANN) comprising one input layer, two hidden layers and one output layer. The model accurately described the observed separations with a correlation coefficient of 0.999 being observed between predicted and observed migration times. Selectivity optimisation was achieved using the normalised resolution product and minimum resolution criteria, with both providing optima at different experimental conditions. The selectivity changes observed also allowed the estimation of electrolyte temperatures within the capillary at high operating currents (>100 microA). Using a 50 microm i.d. capillary and an electrolyte comprising 20 mM phosphate and 15 mM s-beta-CD, a temperature of 52 degrees C was calculated within the capillary at an applied voltage of +30 kV.     

A new and selective capillary column coated with cationic diazacrown ether for separation of organic and inorganic anions by capillary electrophoresis

A new coated capillary has been introduced for capillary electrophoretic separation of anions by using a positively charged diazacrown ether with a 12-membered ring. A positive charge spread over the inner capillary surface led to a substantial anodic electroosmotic flow (EOF) over the range of migrating buffer of pH 2-11. Under the optimum conditions of 25 mM phosphate buffer at pH 7, the diazacrown-coated capillary showed a successful simultaneous separation of 7 inorganic anions and 13 aromatic anions (including positional isomers) in less than 15 min. The migration times of the sample anions and EOF marker for consecutive runs on a single column were highly reproducible, giving a relative standard deviation of 1%. Theoretical treatment of the migration behavior clearly demonstrated that ion association between the diazacrown and analyte anions is strongly dependent on the nature of the functional groups of anions (e.g., sulfonate groups > carboxyl groups) and the number of negative charges (e.g., trivalent anions > divalent anions > monovalent anions) on the analyte.     

Comparision of succinate- and phthalate-functionalized etched silica hydride phases for open-tubular capillary electrochromatography

Two open-tubular (OT) capillary electrochromatographic (CEC) columns were prepared by chemically bonding ionizable mono-(2-(methacryloyloxy)ethyl) succinate (MES) and phthalate-functionalized (MEP) ligands onto silica hydride-based phases through surface etching, silanization, and hydrosilation reactions, starting with a bare fused-silica tube. An analysis of the effect of performance of electrophoretic flow (EOF) on the changes in pH values, ionic strength, and the amount of acetonitrile modifiers helped to reveal that some silanol groups remained in the surface composite of the modified capillaries and to prove that MEP capillaries actually exerted greater EOF than MES ones. To explore the potential utilization of these two columns in various fields, three categories of samples, which spanned a wide range of polarities, were prepared and analyzed through many systematic trials of optimizing CEC conditions. For the separation of a mixture of nucleosides and thymine, guanine and adenine with purine uncleobases, which exhibit greater aromaticity than pyrimidine nucleobases, performed a higher retention in the MEP capillary through a π–π interaction than in the MES capillary. While four steroids were used as test samples, their migration order revealed that the MES stationary phase is hydrophilic in comparison with the MEP. An addition of methanol modifier (30%, v/v) into 10 mM borate buffer (pH 9.55 for MEP; pH 10.0 for MES) was necessary to accomplish a baseline separation of nine flavonoids in the MEP and MES capillaries. Studies on the elution order of these solutes revealed the presence of chromatographic activity in addition to electrophoretic migration. Especially in the MEP capillary, hydrophobic characteristics and π–π interactions with aromatic solutes were found and further improved to resolve an enantiomeric pair, catechin and epicatechin. Overall, the hydride-based stationary phases with ionizable ligands were successfully applied to the OT-CEC separations, and these results confidently propose an ideal route to the synthesis of a novel OT-CEC column.     

Advantages and limitations of a new cationic coating inducing a slow electroosmotic flow for CE-MS peptide analysis: a comparative study with commercial coatings

Capillary coatings are crucial for high-quality separation performance in capillary electrophoresis analysis of proteins or peptides as they prevent analyte adsorption at the capillary wall. These coating materials have to fulfill many requirements such as a good separation performance and ensuring a good repeatability. The number of commercially available coating materials is still limited, especially with regard to the charge density on the coating material and the induced electroosmotic flow (EOF) velocity. In this work, we compare the separation performance of the novel self-made cationic capillary coating OHNOON and two commercially available coating materials, the acrylamide based, neutral LN® and the cationic hexadimethrine bromide (Polybrene), using the same coating procedure for all three coating materials. The coatings are investigated regarding the separation efficiency, analyte resolution, coating stability, and migration time stability in tryptic peptide analysis. Good separation performance was confirmed for all three coating materials: all coatings provided high plate numbers of up to 400,000–500,000 and a repeatability of the EOF and the analyte migration times in the range of 1 % relative standard deviation or below. Our results reveal a moderate EOF velocity for the novel OHNOON coating in comparison to the Polybrene coating. We present a detailed discussion of the impact of this reduced EOF velocity and the separation performance. The results presented here will help to define the necessary properties of coating materials to achieve the best compromise between speed of analysis and resolution for the respective application. We show that our novel OHNOON coating is especially valuable for the analysis of low mobility analytes and for samples with a broad range of analyte mobilities.     

Separation of organic and inorganic arsenic species by capillary electrophoresis using direct spectrophotometric detection

Capillary electrophoresis (CE) with UV detection was used for the determination of arsenite, arsenate, monomethylarsonic acid, dimethylarsinic acid, p-aminophenylarsonic acid, 4-hydroxy-3-nitrobenzenearsonic acid, 4-nitrophenylarsonic acid, phenylarsonic acid, and phenylarsine oxide. The electrophoretic mobilities of these anionic species were determined in a 20 mM phosphate buffer in a pH range from 4 to 11, which established pH 10 as the optimum for the separation. The target analytes were then separated in a fused-silica capillary using 20 mM NaHCO(3)-Na(2)CO(3) buffer, pH 10, as electrolyte and detected at 192 nm. Both normal- and reversed electroosmotic flow (EOF) separation modes were investigated and in the latter case, poly(diallydimethylammonium chloride) (PDDAC), was used for dynamic coating of the capillary and to provide a stable and reproducible reversed EOF (relative standard deviation RSD, 0.39%). The influence of electrolyte pH and composition, applied voltage, as well as EOF reversal protocols upon the method performance criteria were investigated. The optimised method provided limits of detection for the target analytes of 1.62, 6.22, 1.45, 1.83, 0.34, 0.40, 0.40, 0.18, and 0.30 mg/L As, respectively. Linearity was obtained in the range of 0.5-40 mg/L As (for aryl compounds) and from 5-100 mg/L As (for the remaining analytes). Reproducibility of peak areas was in the range of 0.8-5.5% RSD. The method was applied to the determination of four aryl arsenic compounds used as additives in animal feed. Analytes were extracted with 40 mM hydrochloric acid - acetonitrile 4:1 v/v, and then cleaned up by passing through a C(18) solid-phase extraction cartridge before analysis by CE with detection at 200 nm. Recoveries for the four analytes were in the range of 78.8-108.3%.     

Novel, selective sample stacking microemulsion electrokinetic capillary chromatography induced by reverse migrating pseudostationary phase for the determination of the new ultra-short acting hypnotic “HIE-124” in mice serum

Microemulsion electrokinetic capillary chromatography (MEEKC) with sample stacking induced by reverse migrating pseudostationary phase (SRMP) technique in a suppressed electro-osmotic flow (EOF) strategy was investigated for analysing the new ultra-short hypnotic HIE-124 in mice serum. The proposed method utilized fused-silica capillary with a total length of 50 cm (effective length 40 cm), applied voltages for stacking and separation were 5.0 kV for 4.30 min and subsequently 25 kV, respectively, with a sample injection of 0.5 psi for 90 s. All the runs were carried out at 25 °C and detected at 213 nm. The optimum microemulsion background electrolyte (BGE) solution consisted of 0.8% (v/v) ethyl acetate, 6.6% (v/v) butan-2-ol, 1.0% (v/v) acetonitrile, 2.0% (w/v) sodium dodecyl sulfate (SDS), and 89.6 mL with 25 mM phosphate buffer pH 8. When this preconcentration technique was used, the sample stacking and the separation processes took place successively with changing the voltage with an intermediate polarity switching step. The proposed method was validated carefully with respect to high specificity of the method, good linearity (r = 0.9994), fair wide linear concentration range (66-1500 ng mL⁻¹), limit of detection and quantitation were 21.6 and 65.5 ng mL⁻¹, respectively. The mean relative standard deviation (RSD) of the results of intra- and inter-day precision and accuracy were less than 6.0%, and overall recovery higher than 95% of HIE-124 in mice serum. The developed method could be used for the trace analyses of HIE-124 in serum and was finally used for the pharmacokinetic study investigation of HIE-124 in mice serum.     

Effect of ionic strength, pH and polymer concentration on the separation of DNA fragments in the presence of electroosmotic flow

DNA separations in the presence of electroosmotic flow (EOF) using poly(ethylene oxide) (PEO) solutions have been demonstrated. During the separations, PEO entered capillaries filled with Tris-borate (TB) free buffers by EOF and acted as sieving matrices. We have found that ionic strength and pH of polymer and free solutions affect the bulk EOF and resolution differently from that in capillary zone electrophoresis. The EOF coefficient increases with increasing ionic strength of the free TB buffers as a result of decreases in the adsorption of PEO molecules. In contrast, the bulk EOF decreases with increasing the ionic strength of polymer solutions using capillaries filled with high concentrations of free TB buffers. Although resolution values are high due to larger differential migration times between any two DNA fragments in a small bulk EOF using 10 mM TB buffers, use of a capillary filled with at least 100 mM TB free buffers is suggested for high-speed separations. On the side of PEO solutions, 1.5% PEO solutions prepared in 100 to 200 mM TB buffers are more proper in terms of resolution and speed. The separation of DNA markers V and VI was accomplished less than 29 min in 1.5% PEO solutions prepared in 100 mM TB buffers, pH 7.0 at 500 V/cm using a capillary filled with 10 mM free TB buffers, pH 7.0.     

Monitoring of the electroosmotic flow of ionic liquid solutions in non-aqueous media using thermal marks

The possibility of applying a new method employing thermal marks to measuring the rate of the electroosmotic flow (EOF) in non-aqueous capillary electrophoresis (NACE) was investigated. The thermal marks were monitored by using a contactless conductivity detection. During one experiment and in between the series of experiments the reproducibility of the method was excellent. The EOF rate was measured 4-7 times during one experiment, the precision of measurement being around 0.5%. In this study, the influence of 1-butyl-3-methyl-imidazolium salts in organic solvents on the rate of the EOF was investigated. Various organic solvents were mixed with an ionic liquid of various concentrations and the EOF rate was measured using thermal marks. The accuracy of the method was compared with that of the neutral marker one. Five benzoic acid derivatives were separated while the EOF was monitored. The relative standard deviations of the corrected effective mobilities of the above analytes were in the range of 1.0-6.1%.     

Capillary modified with covalently attached coating for enhanced CE separation of biopolymers

δ‐Gluconolactone was covalently coupled with aminopropyl‐derivatized capillary, creating hydrophilic brushes on the inner wall of the capillary. The hydrophilic coating provided suppression of EOF and minimized protein adsorption, resulting in the separation of basic proteins and DNA with efficiencies up to 450 000 plates/m. The intra‐ and inter‐day repeatabilities of the coating referring to the migration times of the four tested proteins were satisfactory with RSD of no more than 1.1 and 1.8% (n=5), respectively. Two hundred consecutive runs were performed with negligible change in migration times and efficiency.