Coaxial flow‐gating interface for capillary electrophoresis

A coaxial flow‐gating interface is described in which the separation capillary passes through the sampling capillary. Continuous flow of the sample solution flowing out of the sampling capillary is directed away from the injection end of the separation capillary by counter‐current flow of the gating solution. During the injection, the flow of the gating solution is interrupted, so that a plug of solution is formed at the inlet into the separation capillary, from which the sample is hydrodynamically injected. Flow‐gating interfaces are originally designed for on‐line connection of capillary electrophoresis with analytical flow‐through methods. The basic properties of the described coaxial flow‐gating interface were obtained in a simplified arrangement in which a syringe pump with sample solution has substituted analytical flow‐through method. Under the optimized conditions, the properties of the tested interface were determined by separation of K⁺, Ba²⁺, Na⁺, Mg²⁺ and Li⁺ ions in aqueous solution at equimolar concentrations of 50 μM. The repeatability of the migration times and peak areas evaluated for K⁺, Ba²⁺ and Li⁺ ions and expressed as relative standard deviation did not exceed 1.4%. The interface was used to determine lithium in mineral water and taurine in an energy drink.     

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.     

Therapeutic deferoxamine and deferiprone monitoring in β‐thalassemia patients’ plasma by field‐amplified sample injection and sweeping in capillary electrophoresis

One CE method was established for detecting deferoxamine (DFO) and deferiprone (DFR) in plasma. For β‐thalassemia patients, DFO and DFR are major medicines to treat the iron overload caused by blood transfusion. Field‐amplified sample injection combined with sweeping was used for sensitivity enhancement in CE. This method was performed on an uncoated fused‐silica capillary. After liquid–liquid extraction, the plasma samples were electrokinetically injected into capillary at +10 kV for 180 s. The phosphate buffer (100 mM) containing 50 mM triethanolamine was used as the BGE (pH 6.6). Separation buffer was phosphate buffer (100 mM, pH 3.0) containing 150 mM SDS. This method showed good linearity (r ≥ 0.9960). Precision and accuracy were evaluated by the results of RSD and relative error of intrabatch and interbatch analyses, and all of the absolute values were less than 6.12%. The LODs (S/N = 3) were 200 ng/mL for DFO, and 25 ng/mL for DFR. The LOQ (S/N = 10) of DFO and DFR were 600 and 75 ng/mL, respectively. This method was applied for clinical applications of five β‐thalassemia patients.     

Highly sensitive detection of five typical fluoroquinolones in low‐fat milk by field‐enhanced sample injection‐based CE in bubble cell capillary

Fluoroquinolones are a group of synthetic antibiotics with a broad activity spectrum against mycoplasma, Gram‐positive, and Gram‐negative bacteria. Due to the extensive use of fluoroquinolones in farming and veterinary science, there is a constant need in the analytical methods able to efficiently monitor their residues in food products of animal origin, regulated by Commission Regulation (European Union) no. 37/2010. Herein, field‐enhanced sample injection for sample stacking prior the CZE separation was developed inside a bubble cell capillary for highly sensitive detection of five typical fluoroquinolones in bovine milk. Ethylenediamine was proposed as the main component of BGE for the antibiotics separation. The effect of BGE composition, injection parameters, and water plug length on the field‐enhanced sample injection‐based CE with UV detection was investigated. Under the optimized conditions, described field‐enhanced sample injection‐based CE‐UV analysis of fluoroquinolones provides LODs varying from 0.4 to 1.3 ng/mL. These LOD values are much lower (from 460 to 1500 times) than those obtained by a conventional CE in a standard capillary without bubble cell. The developed method was finally applied for the analysis of fluoroquinolones in low‐fat milk from a Swiss supermarket. Sample recovery values from 93.6 to 106.0% for different fluoroquinolones, and LODs from 0.7 to 2.5 μg/kg, were achieved. Moreover, the proposed ethylenediamine‐based BGE as volatile and compatible with MS system, enabled the coupling of the field‐enhanced sample injection‐based CE with a recently introduced electrostatic spray ionization MS via an iontophoretic fraction collection interface for qualitative fluoroquinolones identification.     

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.     

CE‐ESI‐MS coupled with dynamic pH junction online concentration for analysis of peptides in human urine samples

In this article, an approach has been developed for the analysis of some small peptides with similar pI values by CE‐ESI‐MS based on the online concentration strategy of dynamic pH junction. The factors affected on the separation, detection and online enrichment, such as BGE, injection pressure, sheath flow liquid and separation voltage have been investigated in detail. Under the optimum conditions, i.e. using 0.5 mol/L formic acid (pH 2.15) as the BGE, preparing the sample in 50 mM ammonium acetate solution (pH 7.5), 50 mbar of injection pressure for 300 s, using 7.5 mM of acetic acid in methanol–water (80% v/v) solution as the sheath flow liquid and 20 kV as the separation voltage, four peptides with similar pI values, such as L ‐Ala‐L ‐Ala (pI=5.57), L ‐Leu‐D ‐Leu (pI=5.52), Gly‐D ‐Phe (pI=5.52) and Gly‐Gly‐L ‐Leu (pI=5.52) achieved baseline separation within 18.3 min with detection limits in the range of 0.2–2.0 nmol/L. RSDs of peak migration time and peak area were in the range of 1.45–3.57 and 4.93–6.32%, respectively. This method has been applied to the analysis of the four peptides in the spiked urine sample with satisfactory results.     

Quantification of 5‐aminolevulinic acid by CE using dynamic pH junction technique

An online dynamic pH junction preconcentration method was developed for quantification of 5‐aminolevulinic acid (ALA) by CE with the separation time less than 6 min. The optimal dynamic pH junction of ALA was carried out between pH 9.3 borate buffer (BGE, 40 mM) and pH 2.5 phosphate buffer (sample matrix, 40 mM) when 4.1 cm of sample plug was hydrodynamically injected into an uncoated fused‐silica capillary (48.5 cm in length, id of 50 μm). If a 24 kV separation voltage was applied, the calibration curve of ALA peak area (200 nm) showed good linearity (R² = 0.9991) ranging from 0.01 to 6.5 mg/mL. The reproducibility of this system was excellent with RSDs (n = 10) of 2.5% for peak area response and 0.6% for migration time at ALA concentration of 0.5 mg/mL. The LOD was evaluated as 1.0 μg/mL (S/N > 3). Compared to conventional CE procedure, the sensitivity was successfully improved over 50‐fold. The analytical results of pharmaceutical formulations show a good agreement with those by HPLC (r = 0.94).     

Electrokinetic sample injection for high‐sensitivity CZE (part 2): Improving the quantitative repeatability and application of electrokinetic supercharging‐CZE to the detection of atmospheric electrolytes

Electrokinetic supercharging (EKS) is defined as a technique that combines electrokinetic sample injection with transient ITP. Quantitative repeatability of EKS‐CZE and the other CE methods using electrokinetic sample injection process is usually inferior in comparison with the CE methods using hydrodynamic or hydrostatic injection. This is due to some effects, such as the temperature change and the convection of the sample solution in the reservoir, as well as the change of the distance between an electrode and a capillary end (D_ec). In particular, we have found that the D_ec change might most seriously affect the repeatability, especially when the electrode is a thin Pt wire that could be unintentionally bent during sampling. By using a Teflon spacer to fix D_ec to 1.1 mm, the RSD of peak area (n=5) was decreased from 20 to 3.4% in EKS‐CZE for several metal cations. This D_ec dependence of the sample amount injected was supported by computer simulation using CFD‐ACE+ software. The improved repeatability (down to 5.1% at n=5, averaged RSD for Co²⁺, Li⁺, Ni²⁺, Zn²⁺ and Pb²⁺) was also experimentally attained by increasing the D_ec to ca. 20 mm, which was also effective to obtain high sensitivity. Since the temperature and the convection effects on the repeatability are comparatively small in a proper laboratory environment, these effects were estimated from the EKS‐CZE experiments using conditions such as warming and agitating the sample solution during EKS process. Finally, EKS‐CZE was applied to the detection of ions from atmospheric electrolytes in high‐purity water exposed to ambient air for 2 h. The microgram per liter levels of anions (chloride, sulfate, nitrate, formate, acetate and lactate) and cations (ammonium, calcium, sodium and magnesium) could be detected using conventional UV detector.     

Characterization of various geometric arrangements of “air-assisted” flow gating interfaces for capillary electrophoresis

For connecting flow-through analytical methods with capillary electrophoresis, a chip working in the air-assisted flow gating interface regime is cast from poly(dimethylsiloxane). In the injection space, the exit from the delivery capillary is placed close to the entrance to the separation capillary. Prior to injecting the sample into the separation capillary, the background electrolyte is forced out of the injection space by a stream of air. In the empty space, a drop of the sample with a volume of <100 nL is formed between the exit from the delivery capillary and the entrance into the separation capillary, from which the sample is injected hydrodynamically into the separation capillary. After injection, the injection space is filled with BGE, and the separation can be begun. Three geometric variants for the mutual geometric arrangement of the delivery and separation capillaries were tested: the delivery capillary is placed perpendicular to the separation capillary, from either above or below, or the capillaries are placed axially, that is, directly opposite one another. All of the variants are equivalent from the analytical and separation efficiency viewpoints. The repeatability expressed by RSD is up to 5%. The tested flow gating interface variants are also suitable for continuous and discontinuous sampling at flow rates of the order of units of μL/min. The developed instrument for sequential electrophoretic analysis operates fully automatically and is suitable for rapid sequential monitoring of dynamic processes.     

Determination of chlorine species by capillary electrophoresis – mass spectrometry

The applicability of CZE with mass spectrometric detection for the determination of four chlorine species, namely chloride and three stable chlorine oxyanions, was studied. The main aspects of the proper selection of BGE and sheath liquid for the CE‐MS determinations of anions with high mobility were demonstrated, pointing out the importance of pH and the mobility of the anion in the BGE. The possibility of using uncoated fused silica capillary and common electrolytes for the separation was shown and the advantage of using extra pressure at the inlet capillary end was also presented. The linear range was found to be 1–100 µg/mL for ClO₃^? and ClO₄^?, 5–500 µg/mL for ClO₂^?, and 25–500 µg/mL for Cl^?, but the sensitivity can be greatly improved if larger sample volume is injected and electrostacking effect is utilized. The LOD for ClO₃^? in drinking water was 6 ng/mL, when very large sample volume was injected (10 000 mbar·s was applied).     

Online concentration by field-amplified sample injection in acidic buffer for analysis of fangchinoline and tetrandrine in herbal medicine by flow injection-micellar electrokinetic capillary chromatography

A novel, rapid, and continuous online concentration approach based on field-amplified sample injection for the analysis of fangchinoline and tetrandrine was developed in this paper by combination of flow injection-MEKC. The BGE used was a solution composed of 75 mM H3PO4-triethylamine-2.5% v/v polyoxyethylene sorbitan monolaurate-20% v/v methanol buffer (pH* 5.0). The analytes prepared in 50% v/v aqueous ethanol were used as the test analytes. Sample was injected electrokinetically between plugs of water. When the cations reached the boundary between the water plug and BGE, they slowed down and became concentrated. Thereafter, MEKC was initiated for the separation. This results in 6.8-8.9-fold improvement in concentration sensitivity relative to conventional CE methods. The separation could be achieved within 10 min and sample throughput rate can reach up to 50/h. The repeatability (defined as RSD) was 4.8, 4.4% with peak height evaluation and 3.6, 0.94% with peak area evaluation for TET and FAN, respectively.     

Simple in‐house flow‐injection capillary electrophoresis with capacitively coupled contactless conductivity method for the determination of colistin

An in‐house flow‐injection capillary electrophoresis with capacitively coupled contactless conductivity detection method was developed for the direct measurement of colistin in pharmaceutical samples. The flow injection and capillary electrophoresis systems are connected by an acrylic interface. Capillary electrophoresis separation is achieved within 2 min using a background electrolyte solution of 5 mM 2‐morpholinoethanesulfonic acid and 5 mM histidine (pH 6). The flow‐injection section allows for convenient filling of the capillary and sample introduction without the use of a pressure/vacuum manifold. Capacitively coupled contactless conductivity detection is employed since colistin has no chromophore but is cationic at pH 6. Calibration curve is linear from 20 to 150 mg/L, with a correlation coefficient (r²) of 0.997. The limit of quantitation is 20 mg/L. The developed method provides precision, simplicity, and short analysis time.     

Determination of Ca, K, Mg, Na, sulfate, phosphate, formate, acetate, propionate, and glycerol in biodiesel by capillary electrophoresis with capacitively coupled contactless conductivity detection

In this work, a new method employing capillary electrophoresis (CE) for the determination of several species in biodiesel is introduced. The concentrations of inorganic species (Na⁺, K⁺, Ca²⁺, Mg²⁺, SO₄²⁻, and PO₄³⁻) and glycerol are of interest to the regulatory authorities due to their ability to form undesirable compounds in engines. Additionally, other species of low molecular weight (e.g., acetate, formate, and propionate) are of interest because they contribute towards increasing the acidity. These species are formed by the degradation of biodiesel and cause damage to engines and the environment. The cation separation was performed in background electrolyte (BGE) composed of 30 mmol L⁻¹ of 2-(n-morpholino)ethanesulfonic acid (MES)/L-histidine (His), pH 6. The separation of anionic species was carried out in similar BGE with 0.2 mmol L⁻¹ cetyltrimethylammonium bromide (CTAB) added. For glycerol, a neutral species, its oxidation with periodate was employed. This well-known reaction is specific to polyols and generates iodate. The amount of iodate produced by the reaction was determined by CE. The separation was carried out in approximately 1 min using BGE composed of 30 mmol L⁻¹ acetic acid, pH 3. The analytical parameters evaluated were: linearity (r > 0.99), the RSD values for area and migration time were < 3.4% and 0.9%, respectively, and recovery was in the range of 89 to 107%.     

On-line preconcentration of perfluorooctanoic acid and perfluorooctanesulfonic acid by nonaqueous capillary electrophoresis

Separation of major environmental pollutants as perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) by capillary electrophoresis is reported for the first time. It is not possible to resolve the solutes in an aqueous media. However, the use of methanol and acetonitrile as the background electrolyte (BGE) solvents allowed their rapid separation in an uncoated capillary. A major effort was put into BGE optimization in respect to both separation efficiency and detection for further on‐line preconcentration. 5 mmol.L^?1 naphthalene‐1‐sulfonic acid and 10 mmol.L^?1 triethylamine dissolved in ACN/MeOH (50:50 v/v) provided best separation and detection conditions. Next, the large‐volume sample stacking and the field‐amplified sample injection were applied and compared. Large‐volume sample stacking improved limits of detection (LODs) with regard to the standard injection by 69 times for PFOA and 143 times for PFOS with LODs of 280 and 230 nmol.L^?1, respectively. Field‐amplified sample injection improved LODs 624 times for PFOAand 806 times for PFOS with LODs 31 and 40 nmol.L^?1, respectively. Both preconcentration methods showed repeatabilities of migration times less than 1.2% RSD intraday and 6.6% RSD interday. The method was applied on PFOA and PFOS analysis in a sample of river water treated with solid‐phase extraction, which further improved LOD toward 5.6 × 10^?10 mol.L^?1 for PFOS and 6.4 × 10^?10 mol.L^?1 for PFOA and allows the method to be used for river water contamination screening or decomposition studies.     

Use of high‐conductivity sample solution with sweeping‐micellar electrokinetic capillary chromatography for trace‐level quantification of paliperidone in human plasma

Paliperidone is a new antipsychotic drug with a relatively low therapeutic concentration of 20–60 ng/mL. We established an accurate and sensitive CE method for the determination of paliperidone concentrations in human plasma in this study. To minimize matrix effect caused by quantification errors, paliperidone was extracted from human plasma using Oasis HLB SPE cartridges with three‐step washing procedure. To achieve sensitive quantification of paliperidone in human plasma, a high‐conductivity sample solution with sweeping‐MEKC method was applied for analysis. The separation is performed in a BGE composed of 75 mM phosphoric acid, 100 mM SDS, 12% acetonitrile, and 15% tetrahydrofuran. Sample solution consisted of 10% methanol in 250 mM phosphoric acid and the conductivity ratio between sample matrix and BGE was 2.0 (γ, sample/BGE). The results showed it able to detect paliperidone in plasma samples at concentration as low as 10 ng/mL (S/N = 3) with a linear range between 20 and 200 ng/mL. Compared to the conventional MEKC method, the sensitivity enhancement factor of the developed sweeping‐MEKC method was 100. Intra‐ and interday precision of peak area ratios were less than 6.03%; the method accuracy was between 93.4 and 97.9%. This method was successfully applied to the analysis of plasma samples of patients undergoing paliperidone treatment.     

Dispersive liquid–liquid microextraction based on solidification of floating organic drop combined with field‐amplified sample injection in capillary electrophoresis for the determination of beta(2)‐agonists in bovine urine

Dispersive liquid–liquid microextraction based on solidification of floating organic drop (DLLME–SFO) was for the first time combined with field‐amplified sample injection (FASI) in CE to determine four β₂‐agonists (cimbuterol, clenbuterol, mabuterol, and mapenterol) in bovine urine. Optimum BGE consisted of 20 mM borate buffer and 0.1 mM SDS. Using salting‐out extraction, β₂‐agonists were extracted into ACN that was then used as the disperser solvent in DLLME–SFO. Optimum DLLME–SFO conditions were: 1.0 mL ACN, 50 μL 1‐undecanol (extraction solvent), total extraction time 1.5 min, no salt addition. Back extraction into an aqueous solution (pH 2.0) facilitated direct injection of β₂‐agonists into CE. Compared to conventional CZE, DLLME–SFO–FASI–CE achieved sensitivity enhancement factors of 41–1046 resulting in LODs in the range of 1.80–37.0 μg L^?1. Linear dynamic ranges of 0.15–10.0 mg L^?1 for cimbuterol and 15–1000 μg L^?1 for the other analytes were obtained with coefficients of determination (R²) ≥ 0.9901 and RSD% ≤5.5 (n = 5). Finally, the applicability of the proposed method was successfully confirmed by determination of the four β₂‐agonists in spiked bovine urine samples and accuracy higher than 96.0% was obtained.     

Gold nanomaterials based pseudostationary phases in capillary electrophoresis: A brand‐new attempt at chondroitin sulfate isomers separation

In this work, a CE method with bare gold nanorods (GNRs) based pseudostationary phase was developed and applied for the separation of chondroitin sulfate (CS) isomers, CS, and dermatan sulfate (DS). The separation efficiency was investigated by varying the experimental parameters such as concentration and pH of the BGE, separation voltage, internal diameter of capillary, different size, and morphology of gold nanomaterials. Results showed that different size and morphology of gold nanomaterials had different effects on the separation of CS and DS. The best separation of CS and DS was achieved in the BGE composed of aqueous 150 mmol/L (mM) ethylenediamine + 20 mM sodium dihydrogen phosphate + 30% v/v GNRs, pH 4.5, at the separation voltage of ?10 kV. Capillary was 59.2 cm in length (effective length 49 cm), 50 μm id capillary thermostated at 25°C. CE with bare GNRs used as pseudostationary phase was shown to be a suitable technique for the separation of CS and DS mixtures with wider peaks. RSD of migration time and peak area of CS and DS were 0.13, 0.14 and 0.86, 1.07%, respectively.     

Capillary electrophoresis procedure for the simultaneous analysis and stoichiometry determination of a drug and its counter-ion by using dual-opposite end injection and contactless conductivity detection: Application to labetalol hydrochloride

In this work, a capillary electrophoresis (CE) procedure was developed for the simultaneous determination of a pharmaceutical drug and its counter-ion, namely labetalol hydrochloride. For this purpose, an uncoated fused-silica capillary, a low conductivity background electrolyte (BGE) and a capacitively coupled contactless conductivity detector (C⁴D) were employed. This detection system is highly sensitive and enables detection of inorganic as well as organic ions unlike with direct UV detection. Moreover, to be able to simultaneously analyze the cationic drug (labetalol⁺) and its anionic counter-ion (Cl⁻) in the same electrophoretic run without the need of a coated capillary, a dual-opposite end injection was performed. In this technique, the sample is hydrodynamically injected into both ends of the capillary. This method is simple and easy to perform since the different injection steps are automated by the CE software.This novel CE-C⁴D procedure with dual-opposite end injection has been successfully validated and applied for the analysis of chloride content in an adrenergic antagonist (labetalol hydrochloride). Thus, the hereby developed method has been shown to enable fast (analysis time < 10 min), precise (repeatability of migration times < 0.7% and of corrected-peak areas < 3.3%; n = 6) and rugged analyses for the simultaneous determination of a pharmaceutical drug and its counter-ion.     

Electrokinetic injection of samples into a short electrophoretic capillary controlled by piezoelectric micropumps

Electrokinetic sample injection using two piezoelectric micropumps has been proposed for electrophoresis in short capillaries. The sample is brought to the injection end of the capillary using one of them. Then, the high‐voltage source is turned on and the sample is injected electrokinetically for a defined time. The injection is terminated by removal of the sample zone by the flowing separation electrolyte pumped by the second piezoelectric micropump. The RSD value, expressing the repeatability of the injection, does not exceed 4%. The injection apparatus does not contain any mobile mechanical components, there is no movement of the capillary and both its ends remain constantly in the solution during both the sample injection and separation. Thus, the micropumps replace the six‐way injection valve and linear pump in similar types of injection apparatuses. The injection was tested in the separation and determination of ammonium and potassium ions in two samples of mineral fertilizers. The separation was performed in background electrolyte containing 500 mM of acetic acid + 20 mM Tris + 2 mM 18‐crown‐6 (pH 3.3) in a capillary with id 50 μm and total length/length to the contactless conductivity detector of 10.5/8 cm. The injection and separation took place at a voltage of 5 kV and the separation time equaled 20 s. The measured values of the analyte contents corresponded to the value declared by the manufacturer within the reliability interval, where RSD equaled between 3.5 and 4.7%.     

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.