Bare Silica Capillary Research Articles

Simple and Fast Determination of Carbaryl Pesticide in Commercial Topical Formulations by Capillary Electrophoresis

A novel analytical method using capillary zone electrophoresis (CZE) for simple and fast determination of the pesticide carbaryl in commercial topical formulations was developed and validated. The carbaryl was previously hydrolyzed quantitatively under an alkaline medium (NaOH solution) to form 1-naphthol, which was separated and quantified by CZE with spectrophotometric detection at 214 nm. Optimization of the hydrolysis reaction regarding time and NaOH concentration was conducted. The CZE separation was achieved in less than 3 min using a bare silica capillary (60 cm total length) and a 10 mmol L-1 sodium borate buffer (pH 9.3) as background electrolyte. The proposed CZE method was linear in the 0.25 to 70 mg L-1 concentration range, as attested by a coefficient of determination (R2) higher than 0.999 and confirmed by the lack of fit test. Recovery tests at three concentration levels provided recovery percentages ranging from 80 to 115%, indicating acceptable accuracy. The limits of detection (LOD) and quantification (LOQ) were 0.08 and 0.25 mg L-1, respectively. The proposed CZE method was successfully applied for carbaryl determination in commercial samples of topical formulations such as powders and dry bath gel.

Analysis of intact proteins with capillary zone electrophoresis coupled to mass spectromery using uncoated and coated capillaries

Although, in general, the application of coated capillaries is recommended for the separation of intact proteins, bare silica capillary is still the most often used capillary due to its simplicity and cheapness. In this work, the performance of bare fused silica capillary for intact protein analysis was compared to that of different (dynamically coated polybrene (PB) and permanently coated linear polyacrylamide (LPA)) coated capillaries using capillary zone electrophoresis - mass spectrometry (CZE-MS). In cases where low pH (pH=1.8) was used in bare silica capillaries, good precision (0.56-0.78 RSD% and 1.7-6.5 RSD% for migration times and peak areas, respectively), minimal adsorption and separation efficiency (N= 27 000/m - 322 000/m) similar to or even better than those obtained with the coated capillaries (created by an intricate multi-step process) was achieved. The PB and the LPA capillaries demonstrated their slightly better resolving power in terms of separating the different forms/variants of the same protein (e.g., hemoglobin subunits). Among the studied capillaries the one with LPA coating showed the most stable separations in the long term (n=25: 0.18-0.49 RSD% and 3.1-4.9 RSD% for migration times and peak areas, respectively). For the separation of a few proteins or even a larger number of proteins in biological samples (e.g., snake venom) the application of the simple and cheap bare fused silica capillary can be considered as an efficient choice.

Establishing repeatability and ruggedness of chiral separations in micellar electrokinetic chromatography mass spectrometry: Combined use of covalently bonded capillary column and molecular micelles

Although micellar electrokinetic chromatography-mass spectrometry (MEKC-MS) using bare silica capillary filled with molecular micelles is an advantageous hyphenated technique for chiral analysis, it is still in the developmental stage. This is mainly because of the lower repeatability of retention time and peak area associated with the difficulty in controlling electroosmotic flow on bare silica capillaries. Besides the lower robustness and electrospray erosion of the fused-silica capillary tip, the lifetime is limited for 10–15 runs per capillary column. We have tested a new MEKC-MS method to eradicate this problem using a covalently bonded 2-acrylamido-2-methyl-1-propane-sulfonic acid (AMPS) column filled with free floating molecular micelles, polysodium N-undecenoxy carbonyl-L-leucinate (poly-L-SUCL). Simultaneous enantiomeric separations and MS/MS detection of three β-blockers [atenolol (ATEN), metoprolol (METO) and, pindolol (PINDO)] was achieved within 25 min with improved column lifetime of at least 45–50 runs. Excellent repeatability of retention time was observed for β-blockers, as evidenced by the relative standard deviation of less than 2% and 3% for intra-capillary and inter-capillary column, respectively. The linear calibration range of both β-blockers was simultaneously established using enantiomers of PINDO as an internal standard. The limit of detection and the lower limit of quantitation were 0.2 μg/mL and 0.5 μg/mL, respectively, for both ATEN and METO. Acceptable recovery of the enantiomeric content of the commercial METO tablet (98-99.5%) and ATEN tablet (89-92.5%) were obtained with adequate system suitability for the precision of peak area (≤10% RSD) under optimum conditions. The developed MEKC-MS approach was extended, which provided broader repeatibility of chiral separation to a panel of primary, secondary and tertiary amines as well as one anionic chiral compound.

Improving repeatability of capillary electrophoresis\u2014a critical comparison of ten different capillary inner surfaces and three criteria of peak identification

A poor repeatability of migration times caused by the fluctuations of electroosmotic flow (EOF) is an inherent weakness of capillary electrophoresis. Most researchers endeavor to prevent this problem using relative migration times or various capillary coatings which are expensive and not easy in comparison. Herein, we present an original approach to this problem: we apply a model sample designed to induce significant EOF instability, in order to critically compare ten capillary types with different physicochemical characteristics. Moreover, we accompany capillary modification with the evaluation of various criteria of peak identification: migration time, migration times ratio, and electrophoretic mobility. Our results show a great effectiveness of a dynamic coating in the stabilization of migration times, with the average RSD(%) value reduced from 3.5% (bare silica capillary) down to 0.5%. The good outcomes were also obtained for the surfactant-modified silica and amine capillaries. For the capillaries exhibiting significant instability of EOF, electrophoretic mobility turned out to be a more universal and reliable criterion of peak identification than relative migration time. It can be explained by an intrinsic dependency of migration times ratio on EOF change, which should always be considered during the selection of an internal standard.

A comparative study of various physicochemically modified capillaries used in CE technique for the three distinct analytical purposes.

The five different commercially offered capillaries, bare silica, one dynamically and three permanently coated, have been tested with respect to the three distinct bioanalytical issues: (i) achiral and chiral separation of small mass molecules, warfarin and its six isomeric hydroxy-derivatives; (ii) ultraselective separation of transferrin and albumin including differentiation between the diferric, two monoferric and iron-free forms of transferrin; and (iii) pioneering identification of albumin-induced shifts of acid dissociation constant by using CE instrumentation, revealed for warfarin and six hydroxywarfarins. As a result all of these pharmacologically and biochemically-relevant purposes have been reached, but using different capillaries. The dynamically coated silica capillary has allowed for the first time to separate warfarin from its six main hydroxy-metabolites by CE with high resolution. The bare silica capillary has performed well in cyclodextrin-assisted enantioseparation of these compounds. The neutral capillary has provided excellent resolution and performance in separation of proteins. The amine capillary, in turn, has occurred to be the best choice for identification of albumin-induced pKa shifts occurring in vivo, omitting capillary clogging and providing better sensitivity than the neutral capillary. This work reveals specific predispositions and shows that there is no universal capillary which may be applied to all issues.

Coated capillaries with highly charged polyelectrolytes and carbon nanotubes co-aggregated with sodium dodecyl sulphate for the analysis of sulfonylureas by capillary electrophoresis

Sulfonylureas (SUs) are one of the most widely used herbicides to control weeds in crops. Herein, capillary electrophoresis (CE) was used to determine four sulfonylureas in natural waters, namely chlorsulfuron (CS), iodosulfuron methyl (IM), metsulfuron methyl (MSM) and mesosulfuron methyl (MSS). First of all, a bare silica capillary was chosen with 10mM of 1-butyl-3-methylimidazolium tetrafluoroborate (bminBF4) as electrophoretic buffer (pH 9.6) containing 2mgL−1 of surfactant-coated single-wall carbon nanotubes (SC-SWCNTs). A dramatic deviation in migration times was observed. Therefore, a poly(diallyldimethylammonium) chloride (PDADMAC) statically coated cationic capillary was used to improve repeatability and to alter the selectivity of the separation. The electroosmotic flow (EOF) measurement revealed that the SC-SWCNTs were strongly adsorbed at the surface of the PDADMAC coating even in the absence of the surfactant-coated nanotubes in the electrolyte buffer. Consequently, a stable strong cathodic EOF and excellent repeatabilities were obtained with relative standard deviations (RSDs) on migration times and on corrected peak areas below 0.9 and 1.5%, respectively. The separation of the SUs was conducted in only 6min. No regeneration of the coating between analyses was necessary, and high peak efficiencies up to 173,000 theoretical plates were obtained. The bi-layer coating was subsequently used to analyze sulfonylureas in tap water, in several mineral waters as well as in underground waters spiked with SUs and directly injected into the CE capillary.

'Click' chemistry synthesis and capillary electrophoresis study of 1,4-linked 1,2,3-triazole AZT-systemin conjugate.

The Cu(I) catalyzed Huisgen 1,3-dipolar azide-alkyne cycloaddition (CuAAC) was applied for a nucleoside-peptide bioconjugation. Systemin (Sys), an 18-aa plant signaling peptide naturally produced in response to wounding or pathogen attack, was chemically synthesized as its N-propynoic acid functionalized analog (Prp-Sys) using the SPPS. Next, CuAAC was applied to conjugate Prp-Sys with 3'-azido-2',3'-dideoxythymidine (AZT), a model cargo molecule. 1,4-Linked 1,2,3-triazole AZT-Sys conjugate was designed to characterize the spreading properties and ability to translocate of cargo molecules of systemin. CuAAC allowed the synthesis of the conjugate in a chemoselective and regioselective manner, with high purity and yield. The presence of Cu(I) ions generated in situ drove the CuAAC reaction to completion within a few minutes without any by-products. Under typical separation conditions of phosphate 'buffer' at low pH and uncoated fused bare-silica capillary, an increasing peak intensity assigned to triazole-linked AZT-Sys conjugate was observed using capillary electrophoresis (CE) during CuAAC. CE analysis showed that systemin peptides are stable in tomato leaf extract for up to a few hours. CE-ESI-MS revealed that the native Sys and its conjugate with AZT are translocated through the tomato stem and can be directly detected in stem exudates. The results show potential application of systemin as a transporter of low molecular weight cargo molecules in tomato plant and of CE method to characterize a behavior of plant peptides and its analogs.

Hybrid phospholipid bilayer coatings for separations of cationic proteins in capillary zone electrophoresis

Protein separations in CZE suffer from nonspecific adsorption of analytes to the capillary surface. Semipermanent phospholipid bilayers have been used to minimize adsorption, but must be regenerated regularly to ensure reproducibility. We investigated the formation, characterization, and use of hybrid phospholipid bilayers (HPBs) as more stable biosurfactant capillary coatings for CZE protein separations. HPBs are formed by covalently modifying a support with a hydrophobic monolayer onto which a self-assembled lipid monolayer is deposited. Monolayers prepared in capillaries using 3-cyanopropyldimethylchlorosilane (CPDCS) or n-octyldimethylchlorosilane (ODCS) yielded hydrophobic surfaces with lowered surface free energies of 6.0 ± 0.3 or 0.2 ± 0.1 mJ m(-2) , respectively, compared to 17 ± 1 mJ m(-2) for bare silica capillaries. HPBs were formed by subsequently fusing vesicles comprised of 1,2-dilauroyl-sn-glycero-3-phosphocholine or 1,2-dioleoyl-sn-glycero-3-phosphocholine to CPDCS- or ODCS-modified capillaries. The resultant HPB coatings shielded the capillary surface and yielded reduced electroosmotic mobility (1.3-1.9 × 10(-4) cm(2) V(-1) s(-1) ) compared to CPDCS- and ODCS-modified or bare capillaries (3.6 ± 0.2 × 10(-4) cm(2) V(-1) s(-1) , 4.8 ± 0.4 × 10(-4) cm(2) V(-1) s(-1) , and 6.0 ± 0.2 × 10(-4) cm(2) V(-1) s(-1) , respectively), with increased stability compared to phospholipid bilayer coatings. HPB-coated capillaries yielded reproducible protein migration times (RSD ≤ 3.6%, n ≥ 6) with separation efficiencies as high as 200 000 plates/m.

Bis(salicylaldehyde) orthophenylenediamine as complexing reagent in simultaneous determination of gold, chromium, iron, uranyl, and nickel using capillary zone electrophoresis

Summary Bis(salicylaldehyde) orthophenylenediamine (BSOPD) is reported here as a complexing reagent for simultaneous determination of six metal ions, gold, chromium, iron(II)/(III), uranyl, and nickel, by capillary zone electrophoresis. The pre-derivatized complexes were injected onto 50 cm bare silica capillary (i.d. 75 µm) with background electrolyte 50 mM citrate:oxalate buffer in 1:1 at pH 4. All metal ions were separated within 15 min including coexisted ions at applied voltage of −15 kV at detection wavelength of 250 nm. Under the abovementioned conditions, limits of quantification and detection were found to be 0.5, 1.0, 1.0, 10.0, 1.0, and 10.0 µg mL−1; and 0.16, 0.33, 0.33, 3.33, 0.33, and 3.33 µg mL−1 for Au(III), Cr(VI), Fe(III), Fe(II), UO2(II), and Ni(II), respectively. Linear calibration graphs were obtained in the ranges of 0.5–100, 1–200, 10–100, 1–100, 1–100, and 10–200 µg mL−1 with the correlation coefficient values 0.9964, 0.9948, 0.9900, 0.9999, 0.9992, and 0.9918 for Au(III); and Cr(VI), Fe(III), Fe(II), UO2(II), and Ni(II), respectively. Applicability of the method has been evaluated by determining gold from wastewater samples of goldsmith factories and uranyl from ore samples. The developed method was also used for the determination of chromium in selected environmental waters (drinking and polluted). The data obtained with developed method were compared with those obtained from atomic absorption spectroscopy, and the results of the analysis were found to be in good agreement.

Preparation and evaluation of C10-cationic latex particle coated open-tubular column for capillary electrochromatography

A simple way was proposed to prepare reverse phase open tubular column (OTC) for capillary electrochromatography (CEC) by dynamically coating cationic latex particles with C10 functional groups onto the fused silica capillary with surface sulphonation. The latex particles are bounded into the capillary wall via electrostatic attraction force and demonstrate good stability under test conditions. Relative bare silica capillary, reverse electroosmotic flow (EOF) is generated for the OTC due to the existence of cationic latex particles onto the capillary. Preliminary separation of aromatic compounds on an OTC with 50 i.d. has been demonstrated.

Preparation and evaluation of anion exchange open tubular column

An anion open tubular column (OTC) by coating cationic latex particle onto the silica capillary with surface sulfonate functionalized has been prepared and evaluated. The performance of the OTC was observed to be superior to that of those prepared by the bare silica capillary without sulfonation in term of separation efficiency. To increase the surface area of the capillary, acid and base-based etching method for treating silica capillary wall was carried out and compared. An OTC with multiple layers was prepared by alternatively coating cationic and anionic latex particles aiming to further increase the column capacity. The anion OTC with size of 50μm i.d.×1m long demonstrated good separation of common inorganic anions with high efficiency (e.g. for NO2−, its theory plate number is 11655plate/m).

A semipermanent coating for preventing protein adsorption at physiological pH in kinetic capillary electrophoresis

Protein adsorption to the inner capillary wall hinders the use of kinetic capillary electrophoresis (KCE) when studying noncovalent protein-ligand interactions. Permanent and dynamic capillary coatings have been previously reported to alleviate much of the problems associated with protein adsorption. The characteristic limitations associated with permanent and dynamic coatings motivated us to look at a third type of coating - semipermanent. Here, we demonstrate that a semipermanent capillary coating, designed by Lucy and co-workers, comprised of dioctadecyldimethylammonium bromide (DODAB) and polyoxyethylene (POE) stearate, greatly reduces protein adsorption at physiological pH - a necessary requirement for KCE. The coating (i) does not inhibit protein-DNA complex formation, (ii) prevents the adsorption of the analytes, and (iii) supports an electoosmotic flow required for many applications of KCE. The coating was tested in three physiological buffers using a well-known DNA aptamer and four proteins that severely bind to bare silica capillaries as standards. For every protein, a condition was found under which the semipermanent coating effectively suppresses protein adhesion. While no coating can completely prevent the adsorption of all proteins, our findings suggest that the DODAB/POE stearate coating can have a broad impact on CE at large, as it prevents the absorption of several well studied, highly adhesive proteins at physiological pH.

CHARACTERIZING EFFECTS OF CATIONIC SURFACTANT ON ELECTRO-OSMOTIC MOBILITY AND ANION MIGRATION IN CAPILLARY ELECTROPHORESIS

The analysis of anions by capillary electrophoresis (CE) with bare silica capillary often uses cationic surfactant to modify the electro-osmotic mobility (EOM) to ensure anions migrating to anode and being detected. The effect of cetyltrimethylammonium bromide (CTAB) concentration in phosphate background electrolyte (BGE) with various ionic strength and pH on the migration of analytes was investigated and reported. A concentration of 0.08 mM CTAB in the BGE starts to reverse cathodic EOM into anodic EOM, and a concentration of 0.30 mM CTAB in the BGE maximizes anodic EOM. Lower EOM is obtained in BGE of higher phosphate concentration while the CTAB concentration and pH are kept constant. Under conditions of constant CTAB and phosphate concentration, higher cathodic EOM was obtained in the BGE of higher pH and lower anodic EOM was obtained in the BGE of higher pH. Although the effect of CTAB concentration, pH, and phosphate concentration on the migration of aromatic anions is complex, the result indicates that the most rapid and effective separation for the anions can be achieved by using a BGE with 0.20 mM CTAB. Furthermore, the relationship between the electrophoretic mobility of anions and the CTAB concentration can be used to determine the critical micelle concentration of cationic surfactant.

Influence of polyelectrolyte capillary coating conditions on protein analysis in CE

CE of biomolecules is limited by analyte adsorption on the capillary wall. To prevent this, monolayer or successive multiple ionic-polymer layers (SMILs) of highly charged polyelectrolytes can be physically adsorbed on the inner capillary surface. Although these coatings have become commonly used in CE, no systematic investigation of their performance under different coating conditions has been carried out so far. In a previous study (Nehmé, R., Perrin, C., Cottet, H., Blanchin, M. D., Fabre, H., Electrophoresis 2008, 29, 3013-3023), we investigated the influence of different experimental parameters on coating stability, repeatability and peptide peak efficiency. Optimal coating conditions for monolayer and multilayer (SMILs) poly(diallyldimethylammonium) chloride/ poly(sodium 4-styrenesulfonate) coated capillaries were determined. In this study, the influence of polyelectrolyte concentration and ionic strength of the coating solutions, and the number of coating layers on coating stability and performance in limiting protein adsorption was carried out. EOF magnitude and repeatability were used to monitor coating stability. Coating ability to limit protein adsorption was investigated by monitoring variations of migration times, time-corrected peak areas and separation efficiency of test proteins. The separation performance of polyelectrolyte coatings were compared with those obtained with bare silica capillaries.

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 Phix174/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.

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.

Determination of pharmaceuticals in drinking water by CD‐modified MEKC: Separation optimization using experimental design

A suite of 12 widely used pharmaceuticals (ibuprofen, diclofenac, naproxen, bezafibrate, gemfibrozil, ofloxacin, norfloxacin, carbamazepine, primidone, sulphamethazine, sulphadimethoxine and sulphamethoxazole) commonly found in environmental waters were separated by highly sulphated CD-modified MEKC (CD-MEKC) with UV detection. An experimental design method, face-centred composite design, was employed to minimize run time without sacrificing resolution. Using an optimized BGE composed of 10 mM ammonium hydrogen phosphate, pH 11.5, 69 mM SDS, 6 mg/mL sulphated beta-CD and 8.5% v/v isopropanol, a separation voltage of 30 kV and a 48.5 cm x 50 microm id bare silica capillary at 30 degrees C allowed baseline separation of the 12 analytes in a total analysis time of 6.7 min. Instrument LODs in the low milligram per litre range were obtained, and when combined with offline preconcentration by SPE, LODs were between 4 and 30 microg/L.

Use of quasi‐isoelectric buffers to limit protein adsorption in capillary zone electrophoresis

The use of quasi-isoelectric buffers consisting of narrow pH cuts of carrier ampholytes (NC) has been investigated to limit protein adsorption on capillary walls during capillary zone electrophoresis experiments. To quantify protein adsorption on the silica surface, a method derived from that of Towns and Regnier has been developed. alpha-Lactalbumin (14 kDa, pI 4.8) and alpha-chymotrypsinogen A (25 kDa, pI 9.2) have been used as model proteins. Acidic narrow pH cuts of carrier ampholytes (NC, pH 3.0) obtained from fractionation of Serva 4-9 carrier ampholytes were used as BGE in bare-silica capillaries, and allowed to decrease significantly protein adsorption, as compared to experiments performed with classical formate buffer. The use of NC as BGE appeared to be as efficient as the use of polydimethylacrylamide coating to prevent protein adsorption. This increase of protein recovery when using NC was attributed to the interaction of carrier ampholytes with the silica surface, leading to a shielding of the capillary wall.

Influence of polyelectrolyte coating conditions on capillary coating stability and separation efficiency in capillary electrophoresis

Polyelectrolytes are widely used in capillary electrophoresis as coating agents of silica capillaries to prevent adsorption phenomena and improve the repeatability of peptide and protein analysis. A systematic study of the coating experimental conditions has been carried out to optimize coating stability and performance. The main experimental parameters studied were the type and concentration of polyelectrolytes used in several monolayer and multilayer coatings, the ionic strength of coating and stabilizing solutions, and the procedures used for coating and capillary storage. Electroosmotic flow magnitude, direction and repeatability were used to monitor coating stability. Coating ability to limit adsorption was investigated by monitoring variations of migration times, time-corrected peak areas and separation efficiency of test peptides. Capillary-to-capillary and batch-to-batch reproducibility was also studied. In addition, the separation performance of polyelectrolyte coatings were compared to those obtained with bare silica capillaries.

Use of quasi‐isoelectric buffers as anolyte and catholyte to improve capillary isoelectric focusing performances

The use of quasi-isoelectric anolytes and catholytes has been investigated to improve CIEF performances. Narrow pH cuts of carrier ampholytes (NC) have been compared to more conventional couples of anolytes/catholytes (phosphoric acid/sodium hydroxide and glutamic acid/lysine). First, a CIEF setup that consists in a bare silica capillary and 70:30 water/glycerol separation medium has been used. The experiments have shown that when using NC instead of more classical anolytes and catholytes, an increase in the protein detection time was observed and the resolutions obtained for neutral and acidic proteins were doubled. Moreover, according to the NC fraction used, the resolution was modified. In order to investigate further the mechanisms involved, a second setup using a capillary coated with hydroxypropylcellulose was used. With this setup no difference has been observed when changing anolyte and catholyte nature. A simple methodology has then been developed to evaluate EOF during focusing and mobilization steps of CIEF experiments. It highlighted the crucial role played by EOF when using a bare silica capillary. EOF indeed decreased by 33% during mobilization step when using NC instead of classical anolytes and catholytes.