Metabolite profiling of human urine by CE-ESI-MS using separation electrolytes at low pH

We investigated the potential of CE coupled to electrospray MS (CE-ESI-MS) in metabolite profiling of human urine without any sample prefractionation step. A heterogeneous mixture of biologically relevant compounds covering a broad range of physicochemical properties was used to optimize separation conditions in fused-silica capillaries. A running electrolyte containing 50 mM of acetic acid and 50 mM of formic acid at pH 2.5 was used for the CE separations. A sheath-flow electrospray interface was employed for CE-ESI-MS analysis. Sheath liquids containing 80:20 v/v methanol/water with 0.1% v/v of acetic acid or 60:40 v/v isopropanol/water with 0.5% v/v of ammonia were selected for optimum detection in positive and negative ESI modes, respectively. Reproducibility and sensitivity were studied, and strategies for identification of the separated urinary compounds are suggested. We report major advantages and disadvantages of CE-ESI-MS for metabolite profiling of human body fluids. This work may be regarded as a first step in the use of CE-ESI-MS for reliable differential analysis of body fluids from healthy and diseased individuals.     

Ionene-dynamically coated capillary for analysis of urinary and recombinant human erythropoietin by capillary electrophoresis and online electrospray ionization mass spectrometry

In this article, a series of ionene polymers were synthesized and used to coat fused-silica capillaries for the separation of recombinant and urinary human erythropoietin (rhEPO and uEPO) standards by CE. The influence of the charge density of coatings on the separation of rhEPO and uEPO glycoforms was investigated. Then, we further studied the method for fast separation and detection of rhEPO and uEPO standards by CE-ESI-MS. The influence of several CE and MS operating parameters, such as the concentration of CE running buffer, applied external pressure, and the composition and flow rate of sheath liquid on CE-ESI-MS was studied. The results demonstrated that when the capillary was permanently coated with 6,6-ionene and the pH value of acetic acid-ammonium acetate running buffer was 4.80 and 5.50, respectively, a significantly reproducible separation was achieved for rhEPO and uEPO glycoforms. In the online CE-ESI-MS experiments, we not only achieved the online MS signal of uEPO, but also obtained baseline separation of three major rhEPO glycoforms successfully and reproducibly on the 6,6-ionene-coated capillaries. Furthermore, the standard mixture of rhEPO and uEPO was separated, and two incompletely resolved peaks that were identified to be rhEPO and uEPO by the unique MS "fingerprint" were obtained. Additionally, the molecular weight of rhEPO and uEPO were verified and compared to the results by MALDI-TOF-MS. It can be concluded that, in contrast to other indirect methods, the online CE-ESI-MS technique with the combination of the advantages of both CE and MS shows great potential for the separation and detection of rhEPO doping directly in competitive sports.     

Determination of stimulants and narcotics as well as their in vitro metabolites by online CE-ESI-MS

A simple, rapid and sensitive CE-ESI-MS method for the simultaneous analysis of seven stimulants and narcotics (amphetamine, ephedrine, methadone, pethidine, tetracaine, codeine and heroin) was developed. The CE-ESI-MS experimental conditions were optimized as follows: 20?mmol/L ammonium acetate with pH 9.0 as running buffer, the separation voltage of 22?kV and the sheath liquid of isopropanol/water (1:1 v/v) containing 7.5?mmol/L acetic acid with 3.0?μL/min flow rate. Under the optimized conditions, the stimulants and narcotics were well separated within 4.6?min using a 70-cm length fused-silica capillary (50?μm id). The detection limits (S/N=3) of the CE-ESI-MS analysis were in the range of 0.40-1.0?ng/mL. Method repeatability of intra-day and inter-day was satisfactory. The recoveries obtained from the analysis of spiked urine samples were between 84.1 and 108%. The developed method was successfully applied for the simultaneous analysis of methadone, pethidine and codeine and their in vitro metabolites.     

Analysis of benzalkonium chloride by capillary electrophoresis-tandem mass spectrometry

Conditions for the separation and determination of benzalkonium chloride (BAC) homologues by CE with UV-detection and CE coupled to MS (IT) using electrospray as ionization source were established. The separation was performed using fused-silica capillaries of 50 microm id and 100 mM acetic acid-ammonium acetate buffer solution at pH 4.5 with 80% of ACN as carrier electrolyte. CE-MS coupling parameters were optimized and methanol-10 mM acetic acid (90:10 v/v) was selected as sheath liquid. Detection limits, based on an S/N of 3:1, were calculated, and values between 0.8 and 1.3 mg/L with CE-ESI/MS and around 0.5 mg/L with CE-ESI-MS/MS, using hydrodynamic injection (15 s, 3.5 kPa), were obtained. Good run-to-run and day-to-day precisions on concentration were achieved with RSDs lower than 8%. Quantitative analysis was carried out by the internal standard method and the calibration curves showed good linearities (r(2) > 0.98). The CE-ESI-MS/MS method was successfully applied to the analysis of BAC in different ophthalmic solutions, allowing the direct determination, identification and confirmation of the BAC homologues presented in these samples.     

Gold nanoparticle-coated capillaries for protein and peptide analysis on open-tubular capillary electrochromatography

We report a new method of immobilization of gold nanoparticles (AuNPs) on a fused-silica capillary through covalent binding. The resulting modified capillary was applied to electrophoretic systems to improve the efficiency of separation and the selectivity of selected solutes. The immobilization of AuNPs on the capillary wall was performed in a very simple and fast way without requiring heating. The surface features of an AuNP-coated capillary column were determined using the scanning electron microscopy. The chromatographic properties of AuNP-coated capillaries were investigated through variation of the buffer pH and separation voltage. Effective separations of synthetic peptides mixture were obtained on the AuNP-coated capillaries. The method shows a remarkable stability since it was reused about 900 times. The capacity factor was duplicated. Therefore, this modification is stable and can be applied to different separation purposes. A complex mixture of tryptic peptide fragments of HSA was analyzed in both the bare- and the AuNP-coated capillaries. Better electrophoretic peptide profile was observed when using the AuNP-coated capillary.     

Positional and geometrical anionic isomer separations by capillary electrophoresis-electrospray ionization-mass spectrometry

Capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) was applied to the analysis of polar positional and geometrical anionic isomers. Since the investigated positional and geometrical anionic isomers have different pK(a) values, they could be separated by CE-ESI-MS under simple analytical conditions using a bare fused-silica capillary and volatile ammonium acetate buffer after optimizing buffer pH. Ortho-, meta-, para-hydroxybenzoate positional isomers were completely separated on a fused-silica capillary with 20 mM ammonium acetate buffer at pH 10.0, and cis-, trans-cyclohexane dicarboxylate geometrical isomers could be also separated with 20 mM ammonium acetate buffer at pH 4.0. Several analytical parameters affecting ESI-MS sensitivity were also investigated. It was found that both running buffer pH and sheath liquid pH had significant effects on the selectivity and the sensitivity on CE-ESI-MS analysis while sheath flow rate and other parameters had little influence. Under optimized conditions, linearity, detection limit, and repeatability of the analysis of hydroxybenzoate isomers were examined, and good results were obtained. It was found that the method presented in this paper is a simple, robust, and cost-effective method for simultaneous analysis of positional and geometrical anionic isomers as well as of other small anionic compounds.     

Single-run separation of cationic, anionic, and polyanionic compounds by CE-ESI-MS

A method for a single-run separation of cationic, anionic, and polyanionic compounds by CE hyphenated to ESI MS (CE-ESI-MS) is described. One of the main issues for coupling CE to MS with an ESI source consists in maintaining an electric contact for the electrophoretic separation. This condition is only performed if a liquid flow arising from the separation capillary is directed to the needle, making it coupling-compatible. This latter situation is incompatible with the separations of polyanionic compounds of higher electrophoretic mobility (in absolute value) than the electroosmotic mobility, performed in bare fused-silica capillaries under a negative polarity. In this study, several alternative approaches were evaluated to circumvent this difficulty, and applied to the setup of the CE-MS separation of a mixture containing both cationic and polyanionic compounds, which are synthesis intermediates of contrast agents for medical imaging. Eventually, the detection of the cationic and anionic compounds in a single run could be obtained by either using neutrally coated polymethylsiloxane (DB-1) capillaries and simultaneously applying a negative voltage polarity and a pressure allowing to compensate for the residual cathodic EOF or by dynamically modifying the inner wall of a bare fused-silica capillary with a polycationic polymer (hexadimethrine bromide) and using it afterwards under negative voltage polarity.     

Confirmation analysis of ethyl glucuronide and ethyl sulfate in human serum and urine by CZE-ESI-MS(n) after intake of alcoholic beverages

CZE coupled to sheath liquid-based electrospray ionization (ESI) and multiple-stage ion trap mass spectrometry (MS(n) ) was used for the confirmation analysis of ethyl glucuronide (EtG) and ethyl sulfate (EtS) in human serum and urine collected after intake of alcoholic beverages. Electrophoretic separations were performed in uncoated fused-silica capillaries using a pH 9.5 ammonium acetate background electrolyte and normal polarity. MS detection of EtG and EtS occurred after negative ionization using a spray liquid containing 0.5%?v/v ammonia in isopropanol/water (60:40%, v/v). CZE-MS and CZE-MS2 results obtained after injection of solid-phase extracts for EtG and EtS and of diluted urine confirmed the presence of EtG and EtS in samples whose levels were previously determined by CZE with indirect UV detection. Detection limits of each compound were estimated to be around 2.0 (injection of diluted urine) and 0.2?μg/mL (extracts).     

Pressurized liquid extraction-capillary electrophoresis-mass spectrometry for the analysis of polar antioxidants in rosemary extracts

A method based on capillary electrophoresis-electrospray-mass spectrometry (CE-ESI-MS) was developed to qualitatively characterize natural antioxidants from rosemary (Rosmarinus officinalis L.) in different fractions obtained by pressurized liquid extraction (PLE) using subcritical water. The parameters of CE-ESI-MS were adjusted allowing the separation and characterization of different compounds from rosemary in the PLE fractions. These parameters for CE are kind, pH and concentration of the separation buffer, parameters for ESI-MS are dry gas temperature and flow, nebulizing gas pressure, and make-up flow. The following analytical conditions were found most favorable: aqueous CE buffer (40 mM ammonium acetate/ammonium hydroxide, pH 9); sheath liquid containing 2-propanol-water (60:40, v/v) and 0.1% (v/v) triethylamine at a flow rate of 0.24 mL/h; drying gas flow rate equal to 7 L/min at 350 degrees C, nebulizing gas pressure of 13.8 kPa (2 psi), using a compound stability of 50%. Different antioxidant compounds (e.g., rosmarinic acid and carnosic acid) could be detected in the rosemary extracts by CE-ESI-MS without any additional treatment, enabling the determination of variations in the extract composition caused by the different PLE conditions (i.e., 60 and 100 degrees C). The results provide complementary information to HPLC analysis.     

Capillary zone electrophoresis on chemically bonded imidazolium based salts

In the present paper, fused-silica capillaries were chemically modified with an analogue of the imidazole-based ionic liquid and zwitterionic salt. The coated capillaries were examined for the behavior of the electroosmotic flow in both aqueous and non-aqueous electrolytes. The electroosmotic flow in the capillary coated with an ionic liquid analogue was found to be anodic (reversed) and dependent on the pH of the separation buffer. In the case of a zwitterionic capillary, the electroosmotic flow was cathodic and its velocity remained almost constant in the pH range of 4-7. The zeta-potentials of the modified surfaces were also calculated. The effectiveness of coating was investigated by comparing a separation of five inorganic ions and seven alkylphosphonic acids/monoesters in the modified and uncoated capillaries. All separations were successfully carried out in simple buffers and completed during a short analysis time. Finally, the run-to-run and day-to-day reproducibility of the coated capillaries in terms of the migration time of a neutral marker was determined.     

High-efficiency peptide analysis on monolithic multimode capillary columns: Pressure-assisted capillary electrochromatography/capillary electrophoresis coupled to UV and electrospray ionization-mass spectrometry

High-efficiency peptide analysis using multimode pressure-assisted capillary electrochromatography/capillary electrophoresis (pCEC/pCE) monolithic polymeric columns and the separation of model peptide mixtures and protein digests by isocratic and gradient elution under an applied electric field with UV and electrospray ionization-mass spectrometry (ESI-MS) detection is demonstrated. Capillary multipurpose columns were prepared in silanized fused-silica capillaries of 50, 75, and 100 microm inner diameters by thermally induced in situ copolymerization of methacrylic monomers in the presence of n-propanol and formamide as porogens and azobisisobutyronitrile as initiator. N-Ethylbutylamine was used to modify the chromatographic surface of the monolith from neutral to cationic. Monolithic columns were termed as multipurpose or multimode columns because they showed mixed modes of separation mechanisms under different conditions. Anion-exchange separation ability in the liquid chromatography (LC) mode can be determined by the cationic chromatographic surface of the monolith. At acidic pH and high voltage across the column, the monolithic stationary phase provided conditions for predominantly capillary electrophoretic migration of peptides. At basic pH and electric field across the column, enhanced chromatographic retention of peptides on monolithic capillary column made CEC mechanisms of migration responsible for separation. The role of pressure, ionic strength, pH, and organic content of the mobile phase on chromatographic performance was investigated. High efficiencies (exceeding 300 000 plates/m) of the monolithic columns for peptide separations are shown using volatile and nonvolatile, acidic and basic buffers. Good reproducibility and robustness of isocratic and gradient elution pressure-assisted CEC/CE separations were achieved for both UV and ESI-MS detection. Manipulation of the electric field and gradient conditions allowed high-throughput analysis of complex peptide mixtures. A simple design of sheathless electrospray emitter provided effective and robust low dead volume interfacing of monolithic multimode columns with ESI-MS. Gradient elution pressure-assisted mixed-mode separation CE/CEC-ESI-MS mass fingerprinting and data-dependent pCE/pCEC-ESI-MS/MS analysis of a bovine serum albumin (BSA) tryptic digest in less than 5 min yielding high sequence coverage (73%) demonstrated the potential of the method.     

Analysis of selected withanolides in plant extract by capillary electrochromatography and microemulsion electrokinetic chromatography

Microemulsion electrokinetic chromatography (MEEKC) coupled with a diode-array detector was developed for the simultaneous analysis of natural steroidal compounds, withanolides including withaferin A, withacnistin and iochromolide. Optimal resolution was obtained with a microemulsion consisting of 70 mM octane, 800 mM 1-butanol, 100 mM sodium dodecyl sulfate (SDS), and 10 mM phosphate-borate buffer (pH 7) using a fused-silica capillary at 25 kV and 40 degrees C. Since this technique is not compatible with mass spectrometry detection, a capillary electrochromatographic method was developed to separate the investigated withanolides. The effects of mobile phase composition and pH were systematically investigated. Complete separation was obtained with a capillary electrochromatography (CEC) Hypersil C18 bonded silica column (packed length, 25 cmx100 microm ID and 375 microm OD), packed with 3 microm particles. The mobile phase consisted of formic acid-ammonia, pH 8 / acetonitrile (40/60 v/v); the voltage was set at 25 kV and the temperature at 20 degrees C. Under these conditions, resolution of these closely related compounds, including the critical pair withacnistin and iochromolide, was achieved in less than 5 min. The separations by MEEKC and CEC were compared with that obtained by reversed-phase liquid chromatography and showed similar retention order, indicating the analogy of the retention mechanism of these techniques. To further improve specificity and sensitivity, the developed CEC method was interfaced with electrospray ionization mass spectrometry using a Teflon connection between the CEC column and a void fused-silica capillary. Finally, the described methods were applied to the qualitative analysis of withanolides in Iochroma gesnerioides plant extract.     

Evaluation of volatile eluents and electrolytes for high-performance liquid chromatography-electrospray ionization mass spectrometry and capillary electrophoresis-electrospray ionization mass spectrometry of proteins. II. Capillary electrophoresis.

Peptides and proteins were separated by capillary electrophoresis (CE) in fused-silica capillaries coated with an irreversibly adsorbed monolayer of derivatized polystyrene nanoparticles. Whereas phosphate buffer, pH 3.10, enabled the highly efficient separation of basic proteins with plate counts up to 1,400,000 m-1, volatile buffer components such as formic acid or acetic acid titrated with ammonia to the desired pH had to be used for the direct coupling of CE with electrospray ionization mass spectrometry (ESI-MS). Compared to 40 mM phosphoric acid-sodium hydroxide, pH 3.10, a background electrolyte containing 125 mM formic acid-ammonia, pH 4.00, was shown to yield equivalent separation efficiency. Investigation of the influence of buffered electrolytes on the ESI-MS signal of lysozyme at pH 2.70-4.00 showed that the charge state distribution shifted to lower charge states at higher pH with a concomitant five-fold decrease in signal intensity of the most abundant signal. The presence of trifluoroacetic acid in the background electrolyte greatly increased the level of baseline noise and completely inhibited the observation of any mass signals related to proteins. Full scan spectra could be acquired from 50-500 fmol amounts of proteins during coupled CE-ESI-MS utilizing 100-125 mM formic acid-ammonia, pH 3.10. However, compared to UV detection, considerable band broadening is observed with ESI-MS detection which is mainly attributed to column overloading, band spreading in the interface, and scanning data acquisition. Finally, the major whey proteins beta-lactoglobulin A, beta-lactoglobulin B, and alpha-lactalbumin were identified in a whey drink by comparison of molecular masses determined by CE-ESI-MS to molecular masses calculated from the amino acid sequence.     

Separation and quantitation of milk whey proteins of close isoelectric points by on-line capillary isoelectric focusing--electrospray ionization mass spectrometry in glycerol-water media

On-line coupling between CIEF and ESI/MS based on the use of bare fused-silica capillaries and glycerol-water media, recently developed in our laboratory, has been investigated for the separation of milk whey proteins that present close pI values. First, a new rinsing procedure, compatible with MS detection, has been developed to desorb these rather hydrophobic proteins (α-casein (α-CN), bovine serum albumin (BSA), lactoferrin (LF)) from the inner capillary wall and to avoid capillary blockages. Common hydrochloric acid washing solution was replaced by a multi-step sequence based on the use of TFA, ammonia and ethanol. To achieve the separation of major whey proteins (β-lactoglobulin A (β-LG A), β-lactoglobulin B (β-LG B), α-lactalbumin (α-LA) and BSA, which possess close pI values (4.5-5.35), CIEF parameters i.e. carrier ampholyte nature, capillary partial filling length with ampholyte/protein mixture and focusing time, have been optimized with respect to total analysis time, sensitivity and precision on pI determination. After optimization of sheath liquid composition (80:20 (v/v) methanol-water+1% HCOOH), quantitation of β-LG A, β-LG B, α-LA and BSA was performed. The limits of detection obtained from extracted ion current (EIC) and single ion monitoring (SIM) modes were in the 57-136 nM and 11-68 nM range, respectively. Finally, first results obtained from biological samples demonstrated the suitability of CIEF-MS as a potential alternative methodology to 2D-PAGE to diagnose milk protein allergies.     

A new method for screening and determination of diuretics by on-line CE-ESI-MS

A rapid, high-resolution and effective new method for analyzing 12 diuretics by CE-ESI-MS was established in this paper. Ten diuretics (except two neutral compounds) could be fast separated by CE with a DAD at 214 nm with a 20 kV voltage within 6 min, using a 50 microm id and 48.5 cm effective length uncoated fused-silica capillary in a 40 mM ammonium formate buffer (pH 9.40). CE was coupled to the mass spectrometer applying an orthogonal electrospray interface with a triple-tube sheath liquid arrangement. The sheath liquid was composed of isopropanol-water (1:1 v/v) containing 30 mM acetic acid with a flow rate of 4 microL/min. Mass spectrum was employed in the positive mode and both full scan mode and SIM scan mode were utilized. All 12 diuretics could be detected and confirmed by MS in a single analysis. Under optimized conditions, LODs for the 12 diuretics were in the range of 0.13-2.7 micromol/L at an S/N of 3, and the correlation coefficients R(2 )were between 0.9921 and 0.9978. The RDSs (n = 5) of the method was 0.24-0.94 % for migration times and 1.6-8.8 % for peak areas. The recoveries of spiked samples of 12 diuretics were between 72.4% and 118%. The real urine samples were injected directly for analysis, with only simple filtration through a 0.22 microm membrane filter in order to remove solid particles, which may cause capillary blockage. Based on the migration times and characteristic ions, the diuretics in urine samples were detected successfully. This CE-ESI-MS method for analyzing diuretics will hopefully be applied to doping control.     

Open tubular capillary electrochromatography of underivatized amino acids using Rh(III) tetrakis(phenoxyphenyl)porphyrinate as wall modifier

The separation of 17 “common” underivatized amino acids was attempted by open tubular capillary electrochromatography (OT-CEC) in fused-silica capillaries coated with Rh(III) tetrakis(phenoxyphenyl)porphyrinate (Rh(III)TPP(m-OPh)₄OAc) using sodium phosphate and Tris–phosphate buffers as background electrolytes (BGEs). The OT-CEC separation of amino acids was compared with that obtained by capillary zone electrophoresis in bare fused-silica capillaries using the same BGEs. The amino acids were not derivatized and the UV-absorption detection was set at 200 nm. Depending on the experimental conditions at least 15 amino acids were separated. The best separations were obtained in a Rh(III)TPP(m-OPh)₄OAc-coated capillary in 50 mM Tris–100 mM phosphate buffer at pH 2.25. Separation of the critical triplet Val–Ile–Leu was always at least indicated being better at higher BGE concentrations. Regarding the sensitivity of the method, lower concentration limits of detection (LODs) in the coated capillary were obtained for Thr, Gly, Tyr, and Val; the other amino acids exhibited lower LODs in the uncoated capillary. The separation of acidic amino acids was not achieved.     

Separation of lidocaine and its metabolites by capillary electrophoresis using volatile aqueous and nonaqueous electrolyte systems

The separation of the basic drug lidocaine and six of its metabolites has been investigated both by using volatile aqueous electrolyte system, at low pH and by employing non-aqueous electrolyte systems. In aqueous systems, the best separation of the compounds under the investigated conditions was achieved by using the electrolyte 60 mM trifluoroacetic acid (TFA)/triethylamine (TEA) at pH 2.5 containing 15% methanol. With this electrolyte, all seven compounds were well separated with high efficiency and migration time repeatability. The separations with bare fused-silica capillaries and polyacrylamide-coated capillaries were compared with higher separation efficiency with the latter. On the other hand, near baseline separation of all the seven compounds was also obtained by employing the non-aqueous electrolyte, 40 mM ammonium acetate in methanol and TFA (99:1, v/v), with comparable migration time repeatability but lower separation efficiency relative to the aqueous system.     

A sheath-flow nanospray interface for capillary electrophoresis/mass spectrometry

We have developed a novel sheath-flow interface for low-flow electrospray ionization mass spectrometry (ESI-MS) and capillary electrophoresis/electrospray mass spectrometry (CE/ESI-MS). The interface is composed of two capillaries. One is a tapered fused-silica ESI emitter suitable for microliter and nanoliter flow rate electrospray and the other is a tail-end gold-coated CE separation column that is inserted into the emitter. A sheath liquid is supplied between the column and the emitter capillaries. The gold coating and the sheath liquid are used as the conducting media for ESI and the CE circuit. This novel design was initially evaluated by an infusion ESI-MS analysis of the most common antiretroviral dideoxynucleosides, followed by CE/MS coupling analysis of several antidepressant drugs. With infusion studies, the effects of the sheath liquid and the sample flow rates on detection sensitivity and signal stability were investigated. For an emitter with an internal diameter of 30 microm, the optimum flow rates for the sheath and the sample were 200 and 300 nL/min, respectively. The main improvement of this approach in comparison with conventional sheath liquid approaches using an ionspray interface is the gain in sensitivity. Sensitivities were three times better for dideoxynucleosides analyzed by infusion and 12 times higher for antidepressant drugs analyzed by CE/MS with this interface compared with ionspray. The emitter is durable, disposable, and simple to fabricate.     

Use of short-end injection capillary packed with a glycopeptide antibiotic stationary phase in electrochromatography and capillary liquid chromatography for the enantiomeric separation of hydroxy acids

A new chiral stationary phase (CSP) was prepared by reacting MDL 63,246 (Hepta-Tyr), a glycopeptide antibiotic belonging to the teicoplanin family, with 5-μm diol-silica particles. The CSP mixed with 5-μm amino silica particles (3:1) was packed into 75-μm fused-silica capillaries for only 6.6 cm and used for electrochromatographic experiments analyzing several hydroxy acid enantiomers. A reversed electroosmotic flow carried both analytes and mobile phase towards the anode in a short time (1–3 min), being baseline resolved all the studied analytes. In order to achieve the fastest enantiomeric resolution of the studied hydroxy acids, the effect of several experimental parameters such as mobile phase composition (organic modifier type and concentration, pH of the buffer and ionic strength), capillary temperature and applied voltage on enantioresolution factor, retention time, enantioselectivity were evaluated. The packed capillary column allowed the separation of mandelic acid enantiomers in less than 72 s with resolution factor R_s=2.18 applying a voltage of 30 kV and eluting with a mobile phase composed by 50 mM ammonium acetate (pH 6)–water–acetonitrile (1:4:5, v/v). The CSP was also tested in the capillary liquid chromatography mode resolving all the studied enantiomers applying 12 bar pressure to the mobile phase [50 mM ammonium acetate (pH 6)–water–methanol–acetonitrile, 1:4:2:3, v/v)], however, relatively long analysis times were observed (12–20 min).     

Capillary electrochromatography using polyelectrolyte multilayer coatings

This review covers recent progress in polyelectrolyte multilayer (PEM) coatings applied to analytical separations using open-tubular capillary electrochromatography (OT-CEC). The simple preparation procedure involved in the PEM approach has provided some attractive features over other modes of capillary electrophoresis-based separations including packed column capillary electrochromatography (PC-CEC) and micellar electrokinetic chromatography (MEKC). PEM coatings have been used to alleviate the adsorption of basic analytes, to improve separations, and to improve the stability of the electroosmotic flow. Fundamental aspects of PEM coatings on surfaces and analytical separation platforms are briefly outlined in this review. In addition, applications of PEM coatings to fused-silica capillaries or microchip separation devices for the separation of small achiral or chiral analytes, as well as large biomolecules, are discussed.