Non-aqueous capillary electrophoresis

The benefits of non-aqueous capillary electrophoresis have been described in a number of recent publications. The wide selection of organic solvents, with their very different physicochemical properties, broadens our scope to manipulate separation selectivity. The lower currents present in non-aqueous solvents allow the use of high electric field strengths and wide bore capillaries, the latter in turn allowing larger sample load. In many cases detection sensitivity can also be enhanced. The potential of non-aqueous capillary electrophoresis is discussed throughout the paper, and the feasibility of capillary electrophoresis under non-aqueous media is demonstrated with reference to several applications.     

Analysis of neutral surfactants by non-aqueous capillary electrophoresis using an electroosmotic flow reversal

The separation of KM 20, that is in fact a mixture of non-ionic surfactants, was carried out by non-aqueous capillary electrophoresis. This complex mixture resulting from the condensation of ethylene oxide with fatty alcohols does not have chromophoric moieties. So, we analysed it after derivatization by means of 3,5-dinitrobenzoyl chloride. The proposed approach is based both on the formation of complexes with alkaline or ammonium cations in methanol and on the utilisation of a positively charged capillary. From a comparative study on the capillary treatment procedure, we used hexadimethrine bromide as electroosmotic flow reverser in order to obtain both repeatable analyses and good resolutions of the largest KM 20 oligomers. Then, among the five cations used to form complexes with KM 20, we pointed out that ammonium cation led to the best resolutions. Moreover, we evidenced that the counter-ion of this cation had a great influence on resolution because it modified the magnitude of electroosmotic flow. Ion pair formation that is more or less strong between ammonium and its counter-ion was involved in this variation of electroosmotic flow. So, we calculated the association constants for various ammonium salts in methanol. Then, using ammonium chloride as background electrolyte, we optimised the concentration of this salt, in methanol, in order to reach the optimal separation of KM 20 oligomers. Thus, a baseline separation was obtained by using 6 x 10(-2) mol/L NH4Cl as running electrolyte. In these conditions, we separated, in about 30 min, more than 30 oligomers of KM 20. The distribution of these oligomers that was determined from the optimal separation, appeared consistent with that obtained from HPLC analyses. Indeed, we determined that the mean ethoxylation number was equal to 18 while its real value is equal to 20.     

Capillary zone electrophoresis in non-aqueous solutions: pH of the background electrolyte

Although the establishment of a pH scale and the determination of the pH in water is not problematic, it is not a straightforward task in non-aqueous solvents. As capillary zone electrophoresis (CZE) in organic solvents has gained increasing interest, it seems to be valuable to re-discuss the concept of the pH in such media, especially pointing to those aspects, which make pH measurement uncertain in non-aqueous solvents. In this review, the relevant aspects when dealing with primary standard (PS) and secondary standard (SS) as recommended by the International Union of Pure and Applied Chemistry (IUPAC), and the usage of the operational pH are discussed with special emphasis to non-aqueous solvents. Here, different liquid junction potentials, incomplete dissociation of the electrolytes (especially in solvents with low or moderate relative permittivity) and the occurrence of homo- and heteroconjugation must be taken into account. Problems arising in capillary zone electrophoresis practice are addressed, e.g. when the background electrolyte (BGE) consists of organic solvents, but the measuring electrode (normally the glass electrode) is calibrated with aqueous buffers, and the liquid junction potentials between the solvents do not cancel each other. The alternative concept of establishing a certain pH is described, using mixtures of reference acids or bases with known pKa in the organic solvent, and their respective salts, at a certain concentration ratio, relying to the Henderson-Hasselbalch equation. Special discussion is directed to those organic solvents most common in capillary zone electrophoresis, methanol (MeOH) and acetonitrile (ACN), but other solvents are included as well. The potential significance of small amounts of water present in the organic solvent on changes in pKa values, and thus on the pH of the buffering components is pointed out.     

The effect of water on separations in non-aqueous capillary electrophoresis systems

Summary Water, in concentrations up to 10%, has been added to organic solvents (dimethylsulphoxide,N-methylformamide, acetonitrile and methanol) used as the buffer solvents in electrophoresis media for non-aqueous capillary electrophoresis. Anionic and cationic test substances have been used to study the effect on separation selectivity and efficiency. The effect on the electroosmotic flow has also been studied. Water added in concentrations up to 0.5% had only a minor effect on the separation selectivity, efficiency or electroosmotic flow in the systems studied. These results indicate that small variations in the water-content of organic solvents are of only minor importance to the reproducibility of non-aqueous capillary electrophoresis systems. The reproducibility of selectivity might, however, be slightly improved by adding 0.1–0.5% water, because true non-aqueous solvents are likely to cause problems as a result of the variable absorption of water.     

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.     

Chiral separation of amines with N-benzoxycarbonylglycyl-L-proline as selector in non-aqueous capillary electrophoresis using methanol and 1,2-dichloroethane in the background electrolyte

N-Benzoxycarbonylglycyl-L-proline (L-ZGP) has been introduced as a chiral selector for enantioseparation of amines in non-aqueous capillary electrophoresis. Methanol mixed with different proportions of dichloromethane, 1,2-dichloroethane or 2-propanol containing L-ZGP and ammonium acetate was used as the background electrolyte. Enantioseparation of different types of pharmacologically active amines was performed, e.g. the local anaesthetic bupivacaine and the beta-adrenoceptor blocking agent pindolol. Addition of the solvents (dichloromethane, 1,2-dichloroethane or 2-propanol) gave an improved chiral separation partly due to a distinct decrease in the electroosmotic flow. The use of 1,2-dichloroethane in the background electrolyte gave higher precision in migration time (RSD 2.2%) compared to the systems containing dichloromethane. An enantiomeric separation of mepivacaine was performed within 72 s by use of short-end injection with an effective capillary length of 8.5 cm.     

Separation of chlorins and carotenoids in capillary electrophoresis

The present study reports the investigation of capillary electrophoresis (CE) for the separation of the photosynthetic pigments (chlorophyll derivatives as well as carotenoids) together. Various CE methods, such as micellar electrokinetic chromatography, capillary electrokinetic chromatography, and nonaqueous capillary electrophoresis (NACE) are tested, with coated and uncoated capillary columns to evaluate optimal separation conditions using diode array detection. The effect of different type and composition of organic solvents and surfactants on the separation is discussed. Detection limits are found in the range of 1.14-2.45 ppm. According to the system suitability results, the most effective separation is observed using NACE with Aliquat 336 as cationic surfactant in coated capillary and mixture of MeOH-ACN-THF (5:4:1, v/v/v) as solvent. Quantitative evolution is investigated, and recovery percentage values are found to be 96.7-102%.     

Extension of the application range of UV-absorbing organic solvents in capillary electrophoresis by the use of a contactless conductivity detector

A contactless conductivity detection (CCD) system is used for capillary zone electrophoresis (CZE) with non-aqueous solvents of the buffering background electrolyte, which exhibit strong UV absorbance below 230 nm. It is found that the CCD characteristics with such solvents (propylene carbonate, N,N-dimethylformamide and N.N-dimethylacetamide as examples) is the same as with aqueous solutions: the same signal and noise is obtained for a given electric conductance of the background electrolyte, independent of the kind of the solvent. Therefore CCD enables the extension of the application range to solvents with restricted use for common UV detection in CZE due to their unfavourable or even unfitting optical properties. The applicability of CCD is demonstrated by CZE of aliphatic ammonium compounds in these solvents.     

Tentative Comparison of Tube Radial Distribution Chromatography and CZE

Tube radical distribution chromatography (TRDC) uses an untreated open tubular capillary tube and a ternary mixture of solvents (water and hydrophilic/hydrophobic organic solvents) as a carrier solution. A model analyte mixture comprising 1-naphthol, 1-naphthoic acid, 1-naphthalenesulfonic acid, 2,6-naphthalenedisulfonic acid, and 1,3,6-naphthalenetrisulfonic acid was examined by the TRDC and capillary zone electrophoresis (CZE) systems that comprised mainly a capillary tube and a detector. In the TRDC system the elution order of analytes could be changed by altering the component ratios of the solvents, whereas in the CZE system the elution order was changed by altering the electroosmotic flow direction. The experimental data obtained provide clues about the features and utility of TRDC as a new separation method.     

Effects of organic solvents on sample pretreatment and separation performances in capillary electrophoresis

A review of recent developments in theoretical as well as application studies concerning the use of organic solvents, either as purely nonaqueous solvents, hydro-organic mixtures, or a combination of an organic solvent with another organic modifier(s), in the sample matrix and/or separation buffer for effecting sample pretreatment and/or improving separation performances in capillary electrophoresis (CE) is presented. In particular, recent advances made in furthering the basic understanding of selectivity changes that occur in capillary zone electrophoresis due the presence of organic solvents in the separation medium, based on in-depth studies of fundamental processes, such as acid-base chemistry, ion-ion and ion-solvent interactions, were discussed in detail. The utilization of organic solvents for improving the resolution of highly challenging and important separations, i.e., those involving the separation of positional and optical isomers, was also critically reviewed. Furthermore, a comprehensive survey of the use of organic solvents for on-line sample pretreatment, e.g., minimizing aggregation and maximizing solubilization of hydrophobic analytes, improving concentration detection sensitivity for analytes via the use of sample stacking, was presented and discussed. Moreover, recent applications involving the use of organic solvents for improving the CE separations of a variety of molecular species with significance in various disciplines, including biological, environmental and pharmaceutical areas, were summarized and tabulated.     

Non-aqueous capillary electrophoresis of the positional isomers of a sulfated monosaccharide

A non-aqueous capillary electrophoresis (NACE) method coupled to indirect absorbance detection has been developed for the separation of the three positional isomers of monosulfated fucose. The optimized electrolyte was composed of 12 mM ethanolamine, 2 mM trimesic acid buffer in a methanol-ethanol (1:1, v/v) mixture. As the retained electrolyte entails no separating agent other than the pH buffer, the NACE separation of the positional isomers has been ascribed mainly to selective ion-pairing with the electrolyte counter-ion and the possibility of a selective solvation effect in the alcohol mixture. In the absence of pure isomeric standards, peak identification was completed by MS and NMR spectroscopy and selective enzymatic desulfation. This method should be of interest for the structure elucidation of monosulfated fucose-based polysaccharides and for the screening of sulfoesterase of unknown activity.     

Characteristics of the electroosmotic flow of electrolyte systems for nonaqueous capillary electrophoresis

Nonaqueous capillary electrophoresis (NACE) is a powerful tool for the analysis of surface-active substances, which represent a broad class of analytes containing cationic and anionic species, such as surfactants, phosphoric acid esters, and amines. In order to conduct an efficient method development in NACE, the influence of the electrolyte composition on the electroosmotic flow (EOF) of organic separation systems was systematically investigated. Background electrolytes and background chromophores appropriate for direct and indirect UV-detection were considered, as the majority of surface-active substances do not absorb UV-light. It was found that theoretical models developed to describe the EOF in aqueous electrolyte systems are insufficient for organic electrolyte systems. Experimental data on electroosmosis in a variety of organic solvents and mixtures of methanol and acetonitrile applying different background chromophores and basic or acidic additives are given. Differences between them are discussed with relation to the physicochemical properties of the organic solvents.     

Study of the acid hydrolysis of (3-methacryloxypropyl)trimethoxysilane by capillary electrophoresis-ion-trap mass spectrometry

The sol-gel method is a widely used technique for the synthesis of various functional coating films. Alkoxysilanes such as (3-methacryloxypropyl)trimethoxysilane (MEMO) are largely used as precursors for inorganic-organic hybrid sol-gel materials. Indeed, these compounds can form complex network, through hydrolysis and condensation reactions. The latter have to be perfectly controlled to obtain the required properties. In such a context, we have studied the potentialities of capillary electrophoresis-ion-trap mass spectrometry (CE-MS) coupling to resolve both separation and characterization of the synthesized compounds as a function of the hydrolysis time. The study of acid hydrolysis of MEMO was carried out as an example. After optimization of the running electrolyte in capillary zone electrophoresis (CZE) with UV detection, we characterized the synthesized compounds in CE-MS by using positive detection mode. The obtained resolution in CZE-UV was not entirely satisfactory because of the very closed charge/mass ratio of formed solute but also because of the interaction between the solutes and the capillary walls. Nevertheless, several oligomers were characterized in CE-MS. The absence of detection with regard to oligomers that possess higher molecular masses than octamer is discussed in this work.     

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.     

Parameters influencing separation and detection of anions by capillary electrophoresis

Electrolyte composition is critical in optimizing separation and detection of ions by capillary electrophoresis. The parameters which must be considered when designing an electrolyte system for capillary electrophoresis include electrophoretic mobility of electrolyte constituents and analytes, detection mode, and compatibility of electrolyte constituents with one another. An electrolyte system based on pyromellitic acid is well suited for use with indirect photometric detection, and provides excellent separations of anions. The ability to modify the electrophoretic mobility of pyromellitic acid as a function of ph provides flexibility in matching electrophoretic mobilities of analytes. Additionally, the use of alkyl amines as electroosmotic flow modifiers allows the rapid separation of anions by reversing the direction of electroosmotic flow in a fused-silica capillary. The optimization of a capillary electrophoresis electrolyte for anion analysis is also discussed in terms of pH, ionic strength and applied voltage. The effect of organic solvent on separation selectivity is also discussed.     

非水溶液毛细管电泳

评述了非水溶液毛细管电泳的应用及发展，对非水溶液毛细管电泳的原理及应用情况作了简要叙述，有机试剂在毛细管电泳中的加入，扩大了分析物质的范围，以纯有机试剂作电泳介质，具有许多水溶液毛细管电不能比拟的优点。     

Determination of low-molecular-mass organic acids in any type of beer samples by coelectroosmotic capillary electrophoresis

A separation and determination of a mixture of 19 low-molecular-mass organic acids usually present in beer samples was developed using coelectroosmotic capillary zone electrophoresis. A polycation (hexadimetrine bromide, HDB) has been added to the electrolyte, which dynamically coats the inner surface of the capillary and causes a fast anodic electroosmotic flow. The main factors affecting reversal of the EOF such as type of modifier and concentration and influence of organic solvents were studied. Three types of modifiers, two alkylammonium salts (cethyltrimethylammonium bromide and tetradecyltrimethylammonium bromide) and a polycation (HDB) were investigated. The composition of the running buffer results on a 25% 2-propanol, 0.001% HDB and 50 mM sodium phosphate. The different instrumental parameters affecting the capillary electrophoretic separation were also optimized resulting on a -15 kV voltage with a hydrodynamic injection for 7 s with a UV detection at 210 nm. The applicability of the present method has been demonstrated for the determination of organic acids in different beer samples.     

Specific background electrolytes for nonaqueous capillary electrophoresis

The use of organic solvents or mixture of solvents in capillary electrophoresis is gaining wider attention. The electroosmotic flow mobility of eight organic solvents (acetonitrile, acetone, dimethylformamide, dimetylsulphoxide, propylene carbonate, methanol, ethanol, n-propanol) and of mixtures of several solvents (methanol-acetonitrile, methanol-propylene carbonate, acetonitrile-propylene carbonate) has been studied. The influence of 1,3-alkylimidazolium salts in different solvents on the separation of different analytes has been investigated. Some of these salts have shown usefulness for matrix-assisted laser desorption ionization matrices and off-line analysis of electrophoresis fractions. It also appears that nonaqueous capillary electrophoresis with 1,3-alkylimidazolium salts as background electrolytes is suitable for separation small inorganic ions.     

Non-aqueous capillary electrophoresis with red light emitting diode absorbance detection for the analysis of basic dyes

Non-aqueous capillary electrophoresis was evaluated for the separation of five hydrophobic basic blue dyes for application in forensic dye analysis. The use of a red light emitting diode as a high intensity, low-noise light source provided sensitive detection of the blue dyes while also allowing the evaluation of solvents that absorb strongly in the UV region. Excellent peak shapes and separation selectivity were obtained in methanol, ethanol, acetonitrile and dimethylsulfoxide, however water, tetrahydrofuran, dimethylformamide and acetone were unsuitable as solvents due to poor peak shapes and a lack of sensitivity, most likely due to adsorption onto the capillary wall. Due to the known compatibility of methanol with capillary electrophoresis–mass spectrometry, this solvent was examined further with the relative acidity/basicity of the electrolyte being optimised with an artificial neural network. The optimised method was examined for the separation of ink samples from 6 fibre tip and 2 ball point blue or black pens and showed that a unique migration time for the main dye component in seven of the eight pens could be obtained.     

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.