Effects of background electrolyte composition and addition of selectors on separation selectivity in nonaqueous capillary electrophoresis

This review gives a survey of the approaches employed to obtain, enhance and tune selectivity in nonaqueous capillary electrophoresis (NACE). Recent developments in NACE are described and the effects of background electrolyte composition and addition of selectors on separation selectivity are discussed. The use of one organic solvent, a mixture of several organic solvents or the use of additives to tune separation selectivity in NACE is presented and a list of relevant applications is included.     

Nonaqueous capillary electrophoresis-mass spectrometry

Nonaqueous background electrolytes broaden the application of capillary electrophoresis displaying altered separation selectivity and interactions between analytes and buffer additives compared to aqueous background electrolytes. In addition, nonaqueous capillary electrophoresis (NACE) appears to be ideally suited for online coupling with mass spectrometry due to the high volatility and low surface tension of many organic solvents. Despite these advantages and an increasing use of nonaqueous background electrolytes in CE, coupling of NACE to mass spectrometry has not yet been applied very often to date. The present review summarizes the applications of online NACE-MS with regard to the analysis of drugs, stereoisomers, peptides, alkaloids, polymers and others. A brief discussion of solvent effects in NACE and pH of nonaqueous background electrolyte systems is also presented.     

Control of electroosmotic flow in nonaqueous capillary electrophoresis by polymer capillary coatings

In aqueous capillary electrophoresis the electroosmotic flow (EOF) can be strongly suppressed or eliminated by coating the capillary surface silanols either by buffer additive adsorption or chemical modification. Hydrophilic coatings, e.g., polyvinyl alcohol (PVA) proved to be most efficient for EOF control in applications like DNA analysis. In nonaqueous capillary electrophoresis (NACE), however, the EOF cannot be totally suppressed with these capillaries and coating efficiency turned out to be solvent-depending. In this paper, fused-silica capillaries with monomeric and polymeric coatings differing in hydrophobicity and chemical properties (vinyl, vinyl acetate, vinyl alcohol and acrylates with different alkyl chain length) were investigated. Besides studying the EOF characteristics with different organic solvents and water, gas chromatography (GC) measurements were carried out to probe the silanol reduction via ether retention and the surface hydrophobicity by retention of nonane. Good correlations between GC results and EOF magnitude could be found. It could be demonstrated that the polymeric coating has to be solvatized by the buffer solvent to reduce the EOF. The PVA coating was optimal for aqueous systems but not effective for some nonaqueous buffers. On the other hand, polyvinyl acetate and polyethyl acrylate as polymeric coatings proved to be optimal to reduce the EOF in NACE.     

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%.     

Method development approaches for capillary ion electrophoresis

Capillary ion electrophoresis (CIE) is a capillary electrophoretic technique optimized for rapid determination of low-molecular-mass inorganic and organic ions. CIE predominantly employs indirect UV detection since the majority of the analytes lack specific chromophores. Described are three methods for detection and electrolyte optimization. The first method discussed approaches for optimizing sensitivity, selectivity and peak confirmation using a chromate electrolyte and selected detection wavelengths. Peak confirmation is aided by using both direct detection of analytes. The second and third methods involve an unattended electrolyte development approach for instruments that only provide fresh electrolyte on the injection side of the capillary. The electrolyte composition is changed in both the injection side vial and in capillary before each sample injection while leaving the receiving side electrolyte vial constant at the initial electrolyte composition. In one mode, the concentration of the electroosmotic flow (EOF) modifier used to induce anodic flow is varied while keeping the background electrolyte composition constant. In a second experiment, the background electrolyte co-ion is sequentially changed from high mobility to low mobility while keeping the EOF modifier concentration constant. The end effect is to achieve a broad range of controlled peak symmetry for analytes in a simple matrix. The results are compared to separations obtained when the injection side and receiving side electrolytes are manually matched.     

Nonaqueous capillary electrophoresis: a versatile completion of electrophoretic separation techniques

Nonaqueous capillary electrophoresis (NACE) is the application of a conductive electrolyte dissolved in either one organic solvent or a mixture of several organic solvents to carry out zone electrophoresis or related techniques in fused-silica capillaries. A complete review on the fundamentals, the optimization of analytical methods, practical considerations, and applications is given. To explain the differences to CE in aqueous media, a brief summary on solvent properties and molecular interactions in solutions introduces the reader into these fields. The use of additives to tune separation selectivity by means beyond a pure zone-electrophoretic mechanism is discussed in detail for organic media. Special detection techniques providing high potential for NACE are presented. Data on the precision of NACE methods and a list of relevant applications are included. More specialized applications like the determination of physicochemical constants in NACE or the setup of a semipreparative mode are described.     

Impact of organic solvents on the resolution of synthetic peptides by capillary electrophoresis

The effect of variations in the concentrations of different organic solvents, including acetonitrile, methanol, ethanol, propanol and isopropanol, with aqueous buffer electrolytes of defined composition and pH on the electroosmotic flow velocity, v(EOF), of uncoated fused silica capillaries and on the electrophoretic mobility, mu(e), of synthetic peptides in high-performance capillary electrophoresis (HPCE) has been systematically investigated. In these experiments, the volume fractions of the organic solvent in the aqueous buffer electrolyte were changed from psi = 0.0 to 0.80. The addition of these organic solvents to the aqueous buffer electrolyte reduced the electroosmotic flow (EOF) of the system, but to significantly different extents. For the protic solvents as the alkyl chain of the alcohol increased, at the same volume fraction the greater was the influence on the electroosmotic flow. However, for the aprotic solvent, acetonitrile, the EOF did not change substantially as the volume fraction was varied. The electrophoretic mobility of synthetic peptides under the different buffer electrolyte conditions showed similar trends, confirming that the content and type of the organic modifier can be rationally employed to subtly manipulate the separation selectivity of synthetic peptides. These results, therefore, provide fundamental insight into the experimental options that can be used to maximise resolution of synthetic peptides in HPCE with aqueous buffer-organic solvent mixtures as well as a basis to select optimal binary or ternary buffer electrolyte compositions for the analysis of peptides when hyphenated techniques, such as HPCE-electrospray ionisation mass spectrometry (ESI-MS), are contemplated for the analysis of peptide samples of low abundance as can often be experienced in proteomic investigations.     

Enantiomeric separations by nonaqueous capillary electrophoresis

This paper reviews the recent advances in enantioseparations by nonaqueous capillary electrophoresis (NACE) and the effect of organic solvents on mobility of enantiomers, separation selectivity and resolution. In general, the enantioseparation systems in NACE are similar to those of aqueous capillary electrophoresis (CE) except pure organic solvents are used. The influence of important parameters such as concentration and type of chiral selectors, apparent pH, ionic strength, temperature, and control of electroosmotic flow is discussed. In addition, the reported applications of NACE separations of racemates are presented.     

Polymeric ionic liquid-coated capillary for capillary electrophoresis

A new application of the polymeric ionic liquid (PIL) in capillary electrophoresis is reported. Poly(1-vinyl-3-butylimidazolium bromide) was physically adsorbed on silica capillary as the simple and effective coating for capillary electrophoresis (CE) analysis, in which the PIL is not present in the background electrolyte. The electroosmotic flow (EOF) of the PIL-coated capillary as compared with that of the bare fused-silica capillary shows a different dependence on electrolyte pH values. The EOF is reversed over a wide pH range from 3.0 to 9.0 and shows good repeatability. It is also found that the coated capillary has a good tolerance to some organic solvents, 0.1 M NaOH and 0.1 M HCl. The PIL-coated capillary has been employed in different areas. Both the basic proteins and anionic analytes can be well separated by PIL-coated capillaries in a fast and easy way. The PIL-coated capillary is also able to separate organic acid additives in a grape juice. The results showed that this type of coating provides an alternative to the CE separation of anions and basic proteins.     

Application of 1-Alkyl-3-methylimidazolium-Based Ionic Liquids as Background Electrolytes in Nonaqueous Capillary Electrophoresis for the Analysis of Coptidis Alkaloids

Interest in ionic liquids (ILs) for their potential in analytical chemistry is increasing because they are environmentally benign and are good separation solvents. The aim of the presented investigation was to verify whether ILs would be a suitable background electrolyte (BGE) in nonaqueous capillary electrophoresis (NACE) for organic cations analysis of the closely related analogues. In this study, a novel and very simple NACE method has been established for analyzing seven quaternary alkaloids in Coptis rhizome using 1-alkyl-3-methylimidazolium tetrafluoroborate-based ionic liquid as BGE. The effects of the alkyl group, imidazolium counterion (anionic part), along with the concentration of IL, are investigated and discussed. Baseline separation, high efficiencies, and symmetrical peaks of the seven alkaloids were obtained. The separation mechanism could be hydrophobic and hydrogen-bonding interactions between the alkaloids and the imidazolium cations. The optimum conditions were 70 mM 1-decyl-3-methylimidazolium tetrafluoroborate (1D-3MI-TFB) methanol solution (apparent pH 2.66) and 30 kV applied voltage. The detection was performed at 254 nm. Seven quaternary alkaloids in Coptis rhizome were separated within 14 min. The proposed NACE separation procedure is highly reproducible and can be applied in the qualitative and quantitative analysis of Coptidis alkaloids.     

非水毛细管电泳应用新进展

非水毛细管电泳(NACE)已经被广泛用于药物、环境和生物等领域。由于有机溶剂种类繁多,它们的物理和化学性质各不相同,因此可以针对被分析物的性质及检测方法的不同,选择不同的有机溶剂用于NACE分离,从而拓宽了毛细管区带电泳(CZE)的应用范围。本综述根据近年来NACE在分析领域的应用,对NACE的优势、检测方法、富集方式以及在实际样品中的应用等方面进行了总结,并对其今后的发展进行了展望。     

Nonaqueous capillary electrophoresis in pharmaceutical analysis

This review presents different solvents and electrolytes commonly used as BGEs in NACE for the analysis of pharmaceutical compounds. Most NACE applications carried out since 1998 for the analysis of compounds of pharmaceutical interest are presented in four tables: (i) analysis of drugs and related substances, (ii) analysis of chiral substances, (iii) analysis of phytochemical extracts and (iv) analysis of drugs in biological fluids. These selected examples are used to illustrate the interest in NACE versus conventional aqueous CE.     

Nonaqueous capillary electrophoretic behavior of 2-aryl propionic acids in the presence of an achiral ionic liquid. A chemometric approach

Ionic liquids (ILs) appear really attractive as electrolyte additives in nonaqueous capillary electrophoresis (NACE). These salts may offer new possibilities of interactions to modulate analyte effective mobilities. The presence of 1-n-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide (BMIM NTf2) in acetonitrile/alcohol background electrolytes (BGEs) was investigated in this work. The aim of this study was to elucidate the influence of the IL concentration on the electrophoretic behavior of four arylpropionic acids and to identify the interactions between the analytes and the IL cation. The influence on mobility of the IL concentration, the nature and the proportion of the organic solvents, and the concentration of the ionic components of the BGE was first studied by a univariate approach. A four-factor D-optimal experimental design was then applied to provide a deeper insight into analyte interaction with IL cation present both free in BGE and adsorbed onto the capillary wall.     

Separation of open-cage fullerenes using nonaqueous capillary electrophoresis

In this study, nonaqueous capillary electrophoresis (NACE) was used to separate three open-cage fullerenes. Trifluoroacetic acid (TFA) was used as the nonaqueous background electrolyte to change the analytes’ mobilities. The selectivity and separation efficiency were critically affected by the nature of the buffer system, the choice of organic solvent, and the concentrations of TFA and sodium acetate (NaOAc) in the background electrolyte. The optimized separation occurred using 200 mM TFA/20 mM NaOAc in MeOH/acetonitrile (10:90, v/v), providing highly efficient baseline separation of the open-cage fullerenes within 5 min. The migration time repeatability for the three analytes was less than 1% (relative standard deviation). Thus, NACE is a rapid, useful alternative to high-performance liquid chromatography for the separation of open-cage fullerenes.     

Influence of solvent properties on separation and detection performance in non-aqueous capillary electrophoresis-mass spectrometry of basic analytes

The versatility of non-aqueous capillary electrophoresis (NACE) results mainly from the variety of physico-chemical properties of the different solvents. They provide solubility for a wide range of analytes, enable to control electrophoretic selectivity, but affect in some cases UV absorbance detection. The coupling of NACE to electrospray mass spectrometry (ESI-MS) allows to cope with the high UV cut-off of some CE relevant solvents (e.g., formamides). In this paper the pure organic solvents methanol, acetonitrile, dimethylsulfoxide, formamide, N-methylformamide and N,N-dimethylformamide are evaluated against water for the preparation of ammonium acetate electrolytes to separate the basic model substances 2-aminobenzimidazole, procaine, propranolol and quinine with NACE-MS. MS coupling is assisted with the sheath liquid water-isopropanol (1:4, v/v) with 0.1% formic acid. The goal of the paper is to assess the influence of the solvent on selectivity, separation speed, and peak efficiency for a given set of model compounds on a simple empirical basis. It should give the user an idea how the separation quality is changed when nothing but the running solvent is altered. The obtained efficiency results were discussed with respect to physico-chemical models described in literature (assuming longitudinal diffusion as the only source of band broadening), but no satisfying correlations with solvent properties could be traced. The feasibility of all six organic solvents for MS coupling was demonstrated and the influence of the separation solvent on the MS detection performance was compared. In the seven different solvents, the shortest run time was obtained with acetonitrile, the best peak resolution with the amphiprotic solvents (especially methanol) best peak efficiency with methanol and formamide, and the most sensitive ESI-MS detection with acetonitrile and methanol, but with only slight advantage to water.     

Enantioseparation of selected N-tert.-butyloxycarbonyl amino acids in high-performance liquid chromatography and capillary electrophoresis with a teicoplanin chiral selector

Enantioseparation of N-tert.-butyloxycarbonyl amino acids (N-t-Boc-Aas) with teicoplanin chiral selector was performed in two different separation systems: A teicoplanin-based chiral stationary phase (CSP-TE) was used in reversed-phase HPLC, and the same chiral selector (CS) was added into a background electrolyte (BGE) in HPCE. The enantioselective interaction with the same CSP/CS can be influenced by several factors, such as mobile phase/background electrolyte composition: the buffer concentration, pH, the CS concentration, the presence of organic modifiers. In addition, the charge of the chiral selector related to the charge of the analyte and to EOF are important variables in CE. The effect of these parameters on enantioselectivity and enantioseparation of selected N-t-Boc-Aas was studied. The presence of a sufficient concentration (1% solution) of a triethylamine acetate buffer in the mobile phase was shown to be essential for enantioseparation of these blocked amino acids in HPLC. A certain concentration of teicoplanin aggregates (along with teicoplanin molecules) in the BGE is required to obtain enantioseparation of N-t-Boc-Aas in HPCE.     

Separation of Organomercury Species Using Nonaqueous Capillary Electrophoresis Coupled with Sample Stacking and Electrokinetic Injection Techniques

A nonaqueous capillary electrophoresis (NACE) method, 30 mM ammonium acetate in methanol as background electrolyte (BGE), was developed for separation of the organomercury species without complexing reagents. The effects of different solutes and solvents in BGE were studied. Three species of organomercury, methylmercury, ethylmercury and phenylmercury, were separated well and all the number of theoretical plates were over 10⁶. The present NACE method was also coupled with sample stacking and electrokinetic injection techniques to enhance the detection sensitivity. Under the optimum conditions, the limit of detection (S/N = 3) is 18 ng mL⁻¹ and the linear relation range from 40 to 750 ng mL⁻¹ were obtained for methylmercury.     

Analysis of Calix[4]arenes Using Nonaqueous Capillary Electrophoresis

In nonaqueous capillary electrophoresis (NACE), an organic solvent is used in place of an aqueous medium as the background solution to improve the solubility and selectivity for hydrophobic analytes. In this study, we employed NACE with UV detection for the analysis of eight calix[4]arenes. We examined the influence of several parameters—the buffer composition, the nonaqueous solvent‘s composition and proportion, and the concentration of the electrolyte of the nonaqueous buffer—on the efficiency of the electrophoretic separation. The separation was achieved through the analyte's different effective mobility via different degrees of deprotonation on the phenolic OH groups of the calix[4]arene. This deprotonation can further affect the analyte's ability to form a complex with the metal ion. The optimized background electrolyte (BGE), comprising a mixture of N‐methylformamide/acetonitrile (30:70, v/v) and 100 mM AcOH/20 mM NH₄OAc, provided rapid (<11 min) separation of the calix[4]arenes with good resolution. The relative standard deviations of the migration times for the eight analytes were all less than 1%. Within the calibration concentration range, the coefficients of determination (R²) were all greater than 0.9914. Thus, the present study demonstrated NACE can provide adequate separation for the analysis of calix[4]arenes.     

Electroosmotic flow changes due to interactions of background electrolyte counter-ions with polyethyleneimine coating in capillary zone electrophoresis of proteins

The properties and behavior of polyethyleneimine (PEI) covalently coated capillaries with respect to different background electrolytes used in capillary zone electrophoresis (CZE) are described. The coating stability and changes of inner surface charge in the capillary were followed by measurement of electroosmotic flow (EOF). Interest was focused mainly on conjugate bases of carboxylic acids as anionic background electrolyte components (acetate, citrate, malate, malonate, tartrate, and succinate). An interesting phenomenon was observed in PEI-coated capillaries: The direction (and the magnitude) of EOF depends on the composition of the background electrolyte and at a certain pH it can undergo reversible change. Ionic complex formation was suggested as a hypothesis to explain this behavior. With this knowledge, the PEI-coated capillary was used for the separation of basic proteins in the above-mentioned background electrolytes. A standard protein mixture of cytochrome c, ribonuclease A, and lysozyme at a concentration of 0.25 mg/mL each was chosen as model sample.