Synergism of capillary isotachophoresis and capillary zone electrophoresis

The combination of capillary isotachophoresis and capillary zone electrophoresis may enhance greatly the performance of analytical capillary electrophoresis with respect to both separation power and the concentration sensitivity. The concentrating effects and the separation power of isotachophoresis allow the analysis of diluted samples and the elimination of interferences due to bulk components. The separation process of zone electrophoresis enables one to resolve the stack of trace analytes and detect the resulting individual zones with high sensitivity. The transition of isotachophoresis into zone electrophoresis plays the key role in the overall performance of this hyphenated technique. This article describes the dynamics of the conversion of isotachophoresis into zone electrophoretic mode and shows that the key role is played by the segments of the leading and terminating zones from the isotachophoretic stage. The magnitude of these segments directly effects the detection time as well as the separation width of the peaks of analytes. It is shown that these effects are also important in the analyses by capillary zone electrophoresis where isotachophoresis is induced by the sample itself. Finally, the paper presents a list of recommended, user-friendly, electrolyte systems which enable one to simply predict the performance of the combination isotachophoresis-zone electrophoresis.     

Fast separation of 16 seizure drug substances using non-aqueous capillary electrophoresis

A fast and simple method for separation of 16 seizure drug substances using capillary electrophoresis in a non-aqueous separation medium is described. The separation medium consists of a mixture of acetonitrile, methanol and glycerol with ammonium acetate/acetic acid as the electrolyte. The analytes are detected by UV detection at 214 nm. Injection from the detection end (8.5 cm to detector) combined with the usage of a short capillary (32.5 cm total length) makes it possible to separate all 16 amines within 2 min. The choice of solvents, electrolytes and viscosity increasing additives are discussed with special emphasis to their influence on the separation selectivity.     

Separation and detection of closely related peptides by micellar electrokinetic chromatography coupled with electrospray ionization mass spectrometry using the partial filling technique

Closely related peptides such as neurotensin and angiotensin analogues were separated by capillary zone electrophoresis using a nonionic surfactant, sucrose monododecanoate, as a micelle forming reagent. These peptides were detected by an on-line coupled mass spectrometer using an electrospray ionization interface. However, the presence of the micelles in the separation solution drastically reduced the sensitivity of the mass spectrometer. Therefore, a partial filling technique was employed to prevent the micelles from entering the mass spectrometric interface. A part of the capillary from the injection end was filled with the micellar solution in this technique. Analytes passed through the micellar zone during the electrophoresis and when the separated analytes reached the detection end of the capillary, the micellar zone was still behind the analyte zones, because the nonionic surfactant moved very slowly in acidic conditions. Thus the technique was very useful for mass spectrometric detection for CE when the micellar solution was employed for separation. The optimization of separation and detection conditions was investigated.     

Development of a microfabricated disposable microchip with a capillary electrophoresis and integrated three-electrode electrochemical detection

We have developed microsystems with a capillary electrophoresis and an electrochemical detector. The microfabricated CE-ECD systems are adequate for a disposable type and the characteristics are optimized for application in electrochemical detection. The system was realized by means of a polydimethylsiloxane (PDMS)-glass chip and an indium tin oxide electrode. The injection and separation channels were produced by relatively simple and inexpensive methods. A capillary electrophoresis and a three-electrode electrochemical detector were fabricated on the same substrate with the same fabrication procedure. We measured electropherograms for the testing analytes consisting of catechol and dopamine with different concentrations of 1mM and 0.1mM, respectively. The results showed an efficient and rapid separation and detection of all compounds within a very short time of around 80s using a separate electric field 60 V/cm. We could also successfully achieve an electropherogram of the separation of the 1 kb DNA ladder (8.4 ng/mul) from the 500 bp to 10 kb DNA fragments within just 150 s.     

Evaluation of quantitative structure property relationships necessary for enantioresolution with lambda- and sulfobutylether lambda-carrageenan in capillary electrophoresis

Lambda-carrageenan, a linear, high molecular weight sulfated polysaccharide, was successfully employed in both its native and sulfobutyl derivatized form as a chiral selector in capillary electrophoresis for the separation of enantiomers of basic pharmaceutical compounds. In order to characterize the chiral selectivity properties of this chiral selector, various structurally related racemic compounds were analyzed for enantiomeric interactions using capillary electrophoresis. The results of these studies were then rationalized and analyzed utilizing a general quantitative structure-property relationship (QSPR) evaluation in order to predict critical analyte structural requirements for successful enantiomeric separation. Important structural components of the analytes were found to include the aromatic content, the type of substitution on the aromatic ring, presence of a primary or secondary protonated amine, and an overall positive charge to the molecule.     

Multiwavelength fluorescence detection for DNA sequencing using capillary electrophoresis.

Multiwavelength detection of laser induced fluorescence for dideoxynucleotide DNA sequencing with four different fluorophores and separation by capillary gel electrophoresis is described. A cryogenically cooled, low readout noise, 2-dimensional charge-coupled device is used as a detector for the on-line, on-column recording of emission spectra. The detection system has no moving parts and provides wavelength selectivity on a single detector device. The detection limit of fluorescently labeled oligonucleotides meets the high sensitivity requirements for capillary DNA sequencing largely due to the efficient operation of the CCD detector with a 94% duty cycle. Using the condition number as a selectivity criterion, multiwavelength detection provides better analytical selectivity than detection with four bandpass filters. Monte Carlo studies and analytical estimates show that base assignment errors are reduced with peak identification based on entire emission spectra. High-speed separation of sequencing samples and the treatment of the 2-dimensional electropherogram data is presented. Comparing the DNA sequence of a sample separated by slab gel electrophoresis with sequence from capillary gel electrophoresis and multiwavelength detection we find no significant difference in the amount of error attributable to the instrumentation.     

On-chip isotachophoresis for separation of ions and purification of nucleic acids

Electrokinetic techniques are a staple of microscale applications because of their unique ability to perform a variety of fluidic and electrophoretic processes in simple, compact systems with no moving parts. Isotachophoresis (ITP) is a simple and very robust electrokinetic technique that can achieve million-fold preconcentration and efficient separation and extraction based on ionic mobility. For example, we have demonstrated the application of ITP to separation and sensitive detection of unlabeled ionic molecules (e.g. toxins, DNA, rRNA, miRNA) with little or no sample preparation and to extraction and purification of nucleic acids from complex matrices including cell culture, urine, and blood. ITP achieves focusing and separation using an applied electric field and two buffers within a fluidic channel system. For anionic analytes, the leading electrolyte (LE) buffer is chosen such that its anions have higher effective electrophoretic mobility than the anions of the trailing electrolyte (TE) buffer (Effective mobility describes the observable drift velocity of an ion and takes into account the ionization state of the ion, as described in detail by Persat et al.). After establishing an interface between the TE and LE, an electric field is applied such that LE ions move away from the region occupied by TE ions. Sample ions of intermediate effective mobility race ahead of TE ions but cannot overtake LE ions, and so they focus at the LE-TE interface (hereafter called the "ITP interface"). Further, the TE and LE form regions of respectively low and high conductivity, which establish a steep electric field gradient at the ITP interface. This field gradient preconcentrates sample species as they focus. Proper choice of TE and LE results in focusing and purification of target species from other non-focused species and, eventually, separation and segregation of sample species. We here review the physical principles underlying ITP and discuss two standard modes of operation: "peak" and "plateau" modes. In peak mode, relatively dilute sample ions focus together within overlapping narrow peaks at the ITP interface. In plateau mode, more abundant sample ions reach a steady-state concentration and segregate into adjoining plateau-like zones ordered by their effective mobility. Peak and plateau modes arise out of the same underlying physics, but represent distinct regimes differentiated by the initial analyte concentration and/or the amount of time allotted for sample accumulation. We first describe in detail a model peak mode experiment and then demonstrate a peak mode assay for the extraction of nucleic acids from E. coli cell culture. We conclude by presenting a plateau mode assay, where we use a non-focusing tracer (NFT) species to visualize the separation and perform quantitation of amino acids.     

Determination of uric acid and p-aminohippuric acid in human saliva and urine using capillary electrophoresis with electrochemical detection: potential application in fast diagnosis of renal disease

The monitoring of uric acid (UA) and p-aminohippuric acid (PAH) levels in biological samples is routinely carried out in clinical laboratories as an indication of renal disease. With the aim of investigation of the correlation between the trace amounts of UA and PAH in human saliva or urine and renal diseases, we carried out the determination of UA and PAH in human saliva and urine by using capillary electrophoresis with electrochemical detection (CE-ED) in this work. Under the optimum conditions, UA, PAH and three coexisting analytes could be well separated within 21 min at the separation voltage of 14 kV in 80 mmol/L borax running buffer (pH 9.2). Good linear relationship was established between peak current and concentration of analytes over two orders of magnitude with detection limits (S/N = 3) ranged from 5.01 x 10(-7) to 2.00 x 10(-6) mol/L for all analytes. The result shows that this proposed method could be successfully applied for the study on the correlation between the levels of UA and PAH in human saliva and urine and renal diseases, and provide an alternative and convenient method for the fast diagnosis of renal disease.     

Chemiluminescence determination of pharmacologically active compounds by capillary electrophoresis.

A simple and rapid capillary electrophoresis with direct chemiluminescence method has been developed for the determination of five natural pharmacologically active compounds including rutin, protocatechuic aldehyde, chlorogenic acid, luteolin and protocatechuic acid. The luminol as a component of the separation electrolyte buffer was introduced at the head of the separation capillary. The separation of five compounds was carried out in a fused-silica capillary with 15.0 mmol/L tetraborate, 1.0 mmol/L SDS and 0.42 mmol/L luminol (pH 8.5). The analytes was determined by enhancing the chemiluminescence of luminol with 0.13 mmol/L K3Fe(CN)6 in 0.05 mol/L NaOH, which was introduced at the post-column stage. The voltage applied was 16 kV. Under the optimum conditions, the analytes were separated within 10 min. The excellent linearity was obtained over two to three orders of magnitude with a detection limit (signal:noise = 3) of 0.012-0.055 micromol/L for all five analytes. The method was successfully used in the analysis of pharmaceutical and biological samples, and the assay results were satisfactory.     

Capillary electrochromatographic separation of aromatic amino acids possessing peptides using porphyrin derivatives as the inner wall modifiers

Two different porphyrin derivatives (H2TPP(m-OPh)4 and Rh(III)TPP(m-OPh)4) were investigated with respect to their capability to help resolution of five model aromatic peptides in capillary electrophoresis/open tubular capillary electrochromatography. Though the main separation mechanism was preferentially based on the ionic properties of the separated analytes, involvement of particularly H2TTPP(m-OPh)4-peptide interactions at alkaline pH (8.0) was clearly demonstrated. In combination with Tris-phosphate buffer, a speed up of the separation was observed at pH 2.25 (particularly if Rh(III)TPP(m-OPh)4 was used as capillary coating); in spite of the speed up of the separation the selectivity of the system was sufficient and resulted in a complete separation of the five model peptides. It can be expected that Rh(III)TPP(m-OPh)4 capillary coating in combination with Tris-phosphate buffer can be generally used for a considerable speeding up of lengthy separations of peptides in acidic media with some decrease in the separation power of the system.     

Enantioselective separations using capillary electrophoresis

The preconditions are outlined for enantioselective separations in capillary electrophoresis (CE) with chiral selectors as additives to the background electrolyte. Free solution capillary electrophoresis conditions are characterised by a single solution phase. Chiral separations are reviewed by selector type (chiral ligand exchange, cyclodextrins, crown ethers, glycoproteins) with the extensive studies on cyclodextrins grouped into sections on amino acids, pharmaceuticals, and speciality chemicals, optimisation, biological fluids, and quantitative aspects. In micellar electrokinetic capillary chromatography, enantioselective discrimination occurs by partition in a two-phase system, with a chiral micellar phase as selector. Optimum separation conditions can be readily predicted for a given selector–selectand combination, and absolute values of binding constants determined by CE. Advantages of CE in comparison with HPLC using a chiral stationary phase include robust, rapid assays and the use of small volumes of aqueous solutions; disadvantages include less favourable detection limits. © 1994 Wiley-Liss, Inc.     

Development of Laser-Excited Indirect Fluorescence Detection for High-Molecular-Weight Polysaccharides in Capillary Electrophoresis

The separation and detection of native polysaccharides in capillary zone electrophoresis is presented. To provide differences in the electrophoretic mobility, a pH of 11.5 is used to partially ionize the analytes. Detection is made possible by indirect fluorescence, where the charged analytes displace the anionic fluorescing buffer ion to create negative peaks. Detection limits in the picogram range are possible.     

Analysis of optical purity and impurity of synthetic D-phenylalanine products using sulfated beta-cyclodextrin as chiral selector by reversed-polarity capillary electrophoresis

A new capillary electrophoresis (CE) method has been achieved for simultaneous separation and quantification of phenylalanine, N-acetylphenylalanine enantiomers, and prochiral N-acetylaminocinnamic acid, possibly co-existent in reaction systems or synthesized products of D-phenylalanine. The separation was carried out in an uncoated capillary under reversed-electrophoretic mode. Among the diverse charged cyclodextrins (CDs) examined, highly sulfated (HS)-beta-CD as the chiral selector exhibited the best enantioselectivity. The complete separation of the analytes was obtained under the optimum conditions of pH 2.5, 35 mM Tris buffer containing 4% HS-beta-CD, applied voltage -15 kV, and capillary temperature 25 degrees C. Furthermore, the proposed method was applied to the determination of optical purity and trace impurities in three batches of the asymmetric synthetic samples of D-phenylalanine, and satisfactory results were obtained. The determination recoveries of the samples were in the range of 97.8-103.8%, and precisions fell within 2.3-5.0% (RSD). The results demonstrate that this CE method is a useful, simple technique and is applicable to purity assays of D-phenylalanine.     

Simultaneous determination of flavones and phenolic acids in the leaves of Ricinus communis Linn. by capillary electrophoresis with amperometric detection

Capillary electrophoresis (CE) with amperometric detection (AD) has been developed for the separation and determination of disaccharide glycoside rutin, gentistic acid, quercetin, and gallic acid in the leaves of Ricinus communis Linn. for the first time. The effects of the acidity and the concentration of the running buffer, separation voltage, injection time, and detection potential were investigated to acquire the optimum conditions for the determination of the four analytes. The detection electrode was a 300microm diameter carbon disc electrode at a detection potential of +0.90V (versus saturated calomel electrode (SCE)). The four analytes could be well separated within 10min in a 40cm length fused silica capillary at a separation voltage of 15kV in a 50mM borate buffer (pH 9.0). The relation between peak current and analyte concentration was linear over about 3 orders of magnitude with detection limits (S/N=3) ranging from 0.8 to 2.9microM for all the analytes. The proposed method has been successfully applied to monitor flavones and phenolic acids in the real plant samples with satisfactory assay results.     

Innovative developments in isotachophoresis (displacement electrophoresis)

This Mini Review is aimed at characterizing the innovative developments in isotachophoresis (ITP) during the past few years, discussing in turn new theoretical, analytical, preparative and applicative aspects of this unique separation method. Examples given from our laboratory include the study of the detailed dynamics of the ITP separation of four components by computer simulation and experimental validation in a capillary-type instrument with multiple sensors along the separation trough; the anionic ITP analysis in presence of a strong cathodic electroosmotic flow using an open-tubular fused-silica capillary with on-column multiwavelength detection, and the fractionation of proteins in a screen-segmented, rotating column as well as by recycling ITP.     

Stereoselective determination of ofloxacin and its metabolites in human urine by capillary electrophoresis using laser-induced fluorescence detection

A capillary electrophoresis method for the simultaneous separation and enantioseparation of the antibacterial drug ofloxacin and its metabolites desmethyl ofloxacin and ofloxacin N-oxide in human urine has been developed and validated. Enantioseparation was achieved by adding sulfobutyl β-cyclodextrin to the running buffer. The detection of the analytes was performed by laser-induced fluorescence (LIF) detection using a HeCd-laser with an excitation wavelength of 325 nm. In comparison with conventional UV detection, LIF detection provides higher sensitivity and selectivity. The separation can be performed after direct injection of urine into the capillary without any sample preparation, because no matrix compounds interfere with the assay. Additionally, the high sensitivity of this method allows the quantification of the very low concentrations of enantiomers of both metabolites. The limit of quantification was 250 ng/ml for ofloxacin enantiomers and 100 ng/ml for each metabolites’ enantiomers. This method was applied to the analysis of human urine samples collected from a volunteer after oral administration of 200 mg of (±)-ofloxacin to elucidate stereoselective differences in the formation and excretion of the metabolites. It could be demonstrated that the renal excretion of the S-configured metabolites, especially S-desmethyl ofloxacin, within the first 20 h after dosage, is significantly lower than that of the R-enantiomers.     

Development of a gas chromatography silicon-based microsystem in clinical diagnostics

The accurate determination of biological parameters by means of rapid, on-line measurements at low-concentrations is an important task within the fields of pharmaceutical screening and medical diagnostic. Nevertheless, in biological samples, the analytes of interest are present as minor components in complex mixtures and with interfering species. Biosensors are the best candidates for these applications providing a direct solution to this need of accuracy, but their intrinsic selectivity often excludes all the other components in the sample. A separation step introduced prior to the sensing component could allow both the increase of selectivity with respect the interfering species and the identification of a large spectrum of molecular components in the sample. This work reports the development of a silicon-based integrated separation microsystem for gas chromatography aimed to biomedical applications, with particular emphasis to monitor the homovanillic acid (HVA) and vanillylmandelic acid (VMA) ratios in mass population screening for neuroblastoma diagnosis and prognosis. The miniaturised system consists of two main modules: (i) a metal oxide semiconductor detector and (ii) a micromachined separation capillary column. As first step, the metal oxide semiconductor capability to detect HVA and VMA has been demonstrated. Then, a technology for a silicon separation capillary microcolumn including the on-chip gas sensor housing has been proposed and a first prototype has been developed. The proposed microsystem is an analytical device with biosensing capabilities for diagnostic and biomedical applications, which yield an electronic signal proportional to the concentration of a specific analyte or group of analytes.     

Capillary zone electrophoresis of collagen type I CNBr peptides in acid buffers

Collagen type-I CNBr peptides were separated under acidic conditions by capillary electrophoresis. Separation conditions were: 100 mM phosphate buffer pH 2.5, 50 cm × 50 μm capillary (placed in a cartridge), 8 kV, running time 30–45 min, detection by UV at 200 nm. The peptides were separated strictly by their molecular mass and the overall pattern was well comparable to RP-HPLC separations of these analytes. It is proposed that the separation mechanism may involve hydrophobic sorptions to the capillary wall.     

Hyphenated thermal field flow fractionation--capillary electrophoresis

The possibility was considered to use the transverse thermophoresis of analytes in the capillary for capillary electrophoresis (CE) to control the separation process, decrease the peak width due to thermal effects and provide new separation parameters in CE. As the examination has shown, in non-aqueous buffers the Joule heating in the capillary for CE can provide transverse temperature gradients comparable with the temperature gradients in conventional devices for thermal field flow Fractionation (ThFFF). It was proposed to use the non-uniform velocity profile of analytes caused by the transverse temperature gradient and the temperature dependence of the buffer viscosity for the FFF-like separation of analytes besides CE separation. The expressions for the peak parameters have been derived, where the non-uniform transverse analyte concentration distribution due to the thermophoresis is taken into account, and the possibilities based on FFF-CE principles are discussed. As possible objects of this hyphenated technique, macromolecules and particles are considered.     

Following the lipase catalyzed enantioselective hydrolysis of amino acid esters with capillary electrophoresis using contactless conductivity detection

The progress of the enzymatic hydrolysis of racemic mixtures of the enantiomers of the methyl esters of serine and threonine was monitored. This was possible in a reaction vessel of 1.5 mL by direct sampling of volumes in the nanoliter‐range directly into an electrophoresis capillary. Contactless conductivity detection was used for quantification as the analytes are not accessible by UV‐detection in capillary electrophoresis. Porcine pancreatic lipase and wheat germ lipase both showed a preference for the L‐enantiomers of both amino acid esters. The selectivity of the porcine lipase between the two L‐esters of the two amino acids was also studied and it was found that the production of L ‐threonine had priority over L ‐serine. Chirality, 2010. © 2009 Wiley‐Liss, Inc.