Stacking and low-temperature technique in nonaqueous capillary electrophoresis for the analysis of 3,4-methylenedioxymethamphetamine

Low-temperature and ambient-temperature nonaqueous stacking techniques in capillary electrophoresis (CE) are described for the first time. A low-temperature bath was used to control the temperature from ambient to subzero degrees, by which a novel hyphenated method, low-temperature bath-nonaqueous capillary electrophoresis stacking (LTB-NACE stacking) is demonstrated. 3,4-Methylenedioxymethamphetamine (3,4-MDMA) was determined at a concentration of 4.7 x 10(-6) M (at a 92.1% confidence level) by normal nonaqueous capillary zone electrophoresis (NACZE) and this was improved to 2.6 x 10(-8) M and 5.0 x 10(-9) M, respectively, when the NACZE stacking and LTB-NACZE stacking techniques were applied. The content of 3,4-MDMA in an illicit drug and a suspect urine sample was readily detected. Upon application of the LTB to the separation of isomers the resolution (R) for the separation of 2,3-/3,4-MDMA was improved from 0.6 (LTB, 22 degrees C) to 1.6 (LTB, -55 degrees C) and for (+)3,4-MDMA/(-)3,4-MDMA, from 0.4 (LTB, 25 degrees C) to 1.0 (LTB, -10 degrees C).     

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.     

Stacking in capillary zone electrophoresis

Due to the short light path of the capillaries, the CE detection limit based on concentration, is far less than that of HPLC and not sufficient for many practical applications. Several methods, based on different electrophoretic maneuvers, can concentrate the sample (stack) easily on the capillary before the separation step of capillary zone electrophoresis (CZE). These methods incorporate different types of discontinuous buffers as the means for invoking different velocities to the same analyte molecules to produce a sharpening of the band (stacking). In CZE, these buffers can be often very simple such as sample dilution or adding to the sample a high concentration of a fast mobility ion. However, in other applications these buffers can be as complicated as those required for isotachophoresis. Stacking can often yield a concentration factor of 5-30-fold, which can improve greatly in CZE the detection limits bringing them very close to those of HPLC. Different methods of stacking, the importance of discontinuous buffers and the different mechanism for concentration on the capillary are reviewed here. As there is a need for more practical applications, there will be more methods devised for stacking in CZE.     

Recent advances in nonaqueous capillary electrophoresis

Nonaqueous capillary electrophoresis is undergoing rapid development. The scope of the investigations has clearly expanded, covering both basic studies and analytical applications. This review provides a comprehensive overview at the current status of the technique. Special attention is given to theoretical, methodological, and technical challenges, and the achievements of capillary electrophoresis in nonaqueous media are demonstrated with reference to recent applications.     

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.     

Efficiency studies in nonaqueous capillary electrophoresis

Nonaqueous capillary electrophoresis (NACE) is a relatively new area with several advantages that include enhanced efficiency and improved detection sensitivity. The goal of this study was to investigate the influence of NACE compared to aqueous CE on the separation efficiency of oligosaccharides. The applied voltage and buffer concentration were optimized for the aqueous and nonaqueous buffer media to minimize the band broadening effects of Joule heating and electrophoretic dispersion. At the optimized conditions a 1.5-fold enhancement in efficiency was obtained with the nonaqueous buffer medium.     

Ionic liquids as electrolytes for nonaqueous capillary electrophoresis

Acetonitrile is a well-suited medium for nonaqueous capillary electroseparations and enables extending the range of applications of capillary electrophoresis (CE) techniques to more hydrophobic species. In this study, the dialkylimidazolium-based low temperature melting organic salts know as "ionic liquids" (ILs) are used as electrolytes. At room temperature these liquids are miscible with acetonitrile which makes it easy to use them for adjustment of analyte mobility and separation. The anionic part as well as the concentration of an IL influence the general electrophoretic mobility of the buffer system. The separation of different analytes is achieved because they become charged in the presence of ILs in separation media. There is also a possibility for a complex formation between the solute and the electrolyte which alters the mobility of the solute. A selected application of separations of phenols and aromatic acids will be discussed.     

Analysis of steroids by nonaqueous capillary electrophoresis

A simple method for the separation and determination of steroids (estradiol valerate, triamcinolone, levonorgestrel and ethinylestradiol) in single and compound tablets by nonaqueous capillary electrophoresis with ultraviolet (UV) spectrophotometric detection has been developed for the first time. After optimizing the electrophoretic parameters, including the nature of electrolytes and composition of organic solvent, the running buffers of methanol-acetonitrile (95: 5, v/v) containing 20 mM sodium acetate (pH 6.5) and methanol-acetonitrile (90: 10, v/v) containing 25 mM sodium acetate (pH 7.0) were found to be most suitable for determining estradiol valerate and triamcinolone, respectively. Reliable separation and simultaneous determination of levonorgestrel and ethinylestradiol were achieved in methanol containing 20 mM of ammonium acetate and 10 mM of sodium dodecyl sulfate (SDS). Tamoxifen was used as internal standard. Performance of the method, including migration time and peak area reproducibility, linearity, sensitivity and accuracy, were also evaluated. The limits of detection (S/N = 3) for four analytes were in the range of 9.8–19.5 μ g/mL. The relative standard deviations (RSD) of the migration times and peak areas of the analytes were in the range of 0.14–1.0% and 0.7–2.7% (intraday), 0.5–2.8% and 1.5–4.2% (interday), respectively. Within the tested concentration range, linear relationships between peak area ratios and concentrations of the analytes were obtained (correlation coefficients: 0.9987–0.9996). The method has been successfully applied to the determination of ingredients with recoveries over the range of 96.6–100.6%. The text was submitted by the authors in English.     

Interface for coupling nonaqueous wide-bore capillary electrophoresis with mass spectrometry

A liquid-junction-type interface where a thin spraying capillary is inserted inside the separation capillary was constructed for coupling nonaqueous wide-bore capillary electrophoresis (CE) to mass spectrometry (MS). The robust structure of the interface provided fairly easy capillary handling. The study was carried out with uncoated CE capillaries of 200 and 320 microm inner diameter (ID). 1-Propanol-acetonitrile (80:20 v/v) with acetate electrolyte provided a low conducting medium for CE and good spraying conditions for electrospray ionization (ESI) without sheath-flow and drying gas. Methamphetamine, alprenolol, and levorphanol served as model compounds. Approximate detection limits with the 200 microm ID capillary were 35-265 ng/mL.     

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.     

Chiral separations by capillary electromigration techniques in nonaqueous media. I. Enantioselective nonaqueous capillary electrophoresis

Enantiomer separations by CE employing nonaqueous conditions are reviewed. The general focus of this article is directed towards solvent effects on chiral recognition and the separation mechanism. After a general discussion of solvent effects on the individual processes involved in CE enantiomer separation, specifics for various selector classes are discussed together with a few applications of enantioselective nonaqueous CE.     

Stacking and discontinuous buffers in capillary zone electrophoresis

Discontinuous buffers for capillary zone electrophoresis (CZE) can be used under less rigid conditions compared to those for isotachophoresis for stacking. They can be prepared simply by modifying the sample itself, either by addition of small inorganic ions, low conductivity diluents, or both, and also by adjusting its pH, meanwhile injecting a large volume on the capillary. Zwitterionic and organic-based buffers such as triethanolamine and tris(hydroxymethyl)aminomethane (Tris) are well suited for stacking due to their low conductivity, provided the buffer is discontinuous as demonstrated here. A simple mechanism based on discontinuous buffers is described to explain many of the observed stacking types in CZE, pointing out the many similarities to transient isotachophoresis.     

Improved nonaqueous capillary electrophoresis for tetracyclines at subparts per billion level

A NACE method with laser-induced fluorescence detection was modified for sensitive detection of 4 tetracyclines (TCs) in biological samples and feeds. The changes in injection mode, injection times, id of capillary, excitation wavelength, and the use of surfactant and sample stacking technique all contributed to improved LODs of TCs to sub-ng/mL level. With the optimized conditions, the instrumental LODs could reach 1.33 ng/mL for chlorotetracycline (CTC) and 13.3 ng/mL for TC, oxytetracycline (OTC), and doxycycline (DC), an improvement of 10-100-fold over past studies. A simple SPE procedure was further developed for the extraction and concentration of TCs in plasma, urine, feed, and milk. Taken together, the instrumental LOD and feasible SPE concentration factors the overall LODs for CTC could reach 65 pg/mL in feed and milk and 260 pg/mL in plasma and urine. Detection limits for TC, OTC, and DC at sub-ng/mL level were also achieved. The modified CE-LIF method was found to be less complicated and more sensitive than the best current methods using UV or LIF detection, and has been applied successfully to assess oral absorption of DC in swine and chickens and to confirm suspected TC-positive bovine serum samples.     

Alcohols and wide-bore capillaries in nonaqueous capillary electrophoresis.

The feasibility of using C1-C5 alcohols as electrolyte solutions in nonaqueous capillary zone electrophoresis was investigated. The separation of basic narcotic analgesics and acidic diuretics was modified by changing the alcohol in an electrolyte solution containing alcohol-acetonitrile-acetic acid (50:49:1, v/v) and 20 mM ammonium acetate while other experimental conditions were kept constant. The alcohols studied were methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, and 1-pentanol. The results indicate that even longer-chain alcohols can be used in nonaqueous capillary zone electrophoresis and, because of the lower currents they allow, they are especially advantageous in wider capillaries. Basic analytes were separated in 200 microm and 320 microm ID capillaries with 1-butanol-acetonitrile-acetic acid (50:49:1, v/v) containing 20 mM ammonium acetate as electrolyte solution. Problems related to the use of wide-bore capillaries are discussed.     

Special aspects of detection methodology in nonaqueous capillary electrophoresis

Over the recent years considerable efforts have been directed to the design of powerful detector arrangements for capillary electrophoresis (CE). The analytical characteristics of the detector have a great influence on the overall analytical performance of CE investigations. The major detection methods in CE, such as UV-Vis absorbance, fluorescence, mass spectrometry and electrochemical detection, have successfully been adapted also to nonaqueous capillary electrophoresis (NACE). However, the different properties of organic solvent systems require some modification of detector concepts and design compared to aqueous CE. The advances of detector development and application in NACE are reported and discussed with emphasis on methodical aspects.     

Rapid separation of basic drugs by nonaqueous capillary electrophoresis

Summary Nonaqueous capillary electrophoresis (NACE) has been used to achieve rapid separations of basic drugs. A high electric field was obtained by using short capillaries. Baseline separations of basic drugs, including amphetamines, tropane alkaloids and local anesthetics, were achieved in 1 min by selection of the appropriate organic solvent and electrolyte composition. Thus, high-throughput analyses can be performed. Peak efficiency up to 9154 theoretical plates s⁻¹ was achieved in a separation performed at 923V cm⁻¹. No discernible loss in resolution was observed when a conventional capillary (64.5cm) was replaced by a short (32.5 cm) capillary.     

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.     

Noncovalent coatings for the separation of synthetic polypeptides by nonaqueous capillary electrophoresis

Recently, we demonstrated the possibility to extend the range of capillary electrophoresis (CE) applications to the separation of non-water-soluble synthetic polymers. This work focuses on the control of the electro-osmotic flow (EOF) and on the limitation of the solute adsorption in nonaqueous electrolytes. For these purposes, different strategies were investigated. For the initial, a viscous additive (ethylene glycol or glycerol) was used in the electrolyte in order to decrease the EOF magnitude and, possibly, to compete with solute adsorption. A second strategy was to modify, before separation, the fused-silica capillary wall by the adsorption of poly(ethylene oxide) (PEO) via hydrogen bonding. The influence of the molecular mass of the adsorbed PEO on the EOF magnitude and direction was studied in electrolytes based on methanol/acetonitrile mixtures containing ammonium ions. For PEO molecular masses above 1000 g/mol, reversed (anodic) EOF were reported in accordance with previous results obtained with PEO covalently bonded capillaries. The influence of the nature and the concentration of the background electrolyte cation on the EOF magnitude and direction were also investigated. A third strategy consisted in modifying the capillary wall by the adsorption of a cationic polyelectrolyte layer. Advantageously, this polyelectrolyte layer suppressed the adsorption of the polymer solutes onto the capillary wall. The results obtained in this work confirm the high potential and the versatility of CE for the characterization of ionizable organic polymers in nonaqueous media.     

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.     

Evaluation of electroosmotic markers in aqueous and nonaqueous capillary electrophoresis

The most common method to determine the EOF in CE is to measure the migration time for a neutral marker. In this study, 12 compounds (three novel and some previously used) were investigated as EOF markers in aqueous and nonaqueous BGEs. In the aqueous buffer systems (ammonium acetate, sodium phosphate, and sodium borate) the evaluation included a wide pH range (2–12). Two BGEs contained chiral selectors (sulphated‐β‐CD, (?)‐diketogulonic acid) and one that contained a micellar agent (SDS) were included in the study. The majority of the evaluated compounds were found to migrate with the EOF in the water‐based BGEs and are thus useful as EOF markers. However, in the SDS‐based BGE only four of the compounds (acetone, acrylamide, DMSO, and ethanol) were found to be applicable. In the nonaqueous BGEs 11 markers (acetone, acetophenone, acrylamide, anthracene, benzene, 4‐(4‐methoxybenzylamino)‐7‐nitro‐2,1,3‐benzoxadiazole, benzyl alcohol, 2,5‐diphenyloxazole, ethanol, flavone, and mesityl oxide) seemed to be functional as EOF markers. Even though several of the evaluated compounds can be used as EOF markers in the investigated BGEs, the authors would recommend the use of acrylamide as a general marker for UV detection. Furthermore, the four fluorescent markers (of which three were novel) gave RSD values equal to the other markers and can be used for the determination of the EOF in CE or microchip CE with fluorescence detection.