Capillary electrophoresis and column chromatography in biomedical chiral amino acid analysis

Free amino acids are typically quantified as the sum of their enantiomers, because in terrestrial organisms they mainly exist in the left-handed form. However, with increasing understanding of the biological significance of right-handed amino acids interest in enantioselective quantification of amino acids has steadily increased. Initially, electrophoretic and chromatographic methods using chiral (pseudo)-stationary phases or chiral eluents were applied to the separation of amino acid enantiomers. Later, derivatization of amino acids prior to chromatography with chiral reagents gained in popularity, because the diastereomers formed can be resolved on conventional reversed-phase columns. Novel multi-interaction chiral columns turned attention back to direct chiral chromatographic methods. Hyphenation to mass spectrometry has increasingly replaced optical detection because of superior selectivity, although this has not obviated the need for baseline resolution of amino acid enantiomers. Despite the progress made, enantioselective separation and quantification of amino acids remains an analytical challenge owing to frequently incomplete resolution of all naturally occurring enantiomers and insufficient sensitivity for the determination of the trace amounts of d-amino acids typically found in biological fluids and tissues. Chiral GC-MS analysis of heptafluorobutanol/pentafluoropropionanhydride amino acid derivatives on an Rt-gDEXsa column     

Automated extraction of direct,reactive, and vat dyes from cellulosic fibers for forensic analysis by capillary electrophoresis

Systematic designed experiments were employed to find the optimum conditions for extraction of direct, reactive, and vat dyes from cotton fibers prior to forensic characterization. Automated microextractions were coupled with measurements of extraction efficiencies on a microplate reader UV–visible spectrophotometer to enable rapid screening of extraction efficiency as a function of solvent composition. Solvent extraction conditions were also developed to be compatible with subsequent forensic characterization of extracted dyes by capillary electrophoresis with UV–visible diode array detection. The capillary electrophoresis electrolyte successfully used in this work consists of 5 mM ammonium acetate in 40:60 acetonitrile–water at pH 9.3, with the addition of sodium dithionite reducing agent to facilitate analysis of vat dyes. The ultimate goal of these research efforts is enhanced discrimination of trace fiber evidence by analysis of extracted dyes. Figure Fitted absorbance response surface for extraction of a direct dye, C. I. yellow 58, using a ternary solvent system.     

Optical sensing in microfluidic lab-on-a-chip by femtosecond-laser-written waveguides

We use direct femtosecond laser writing to integrate optical waveguides into a commercial fused silica capillary electrophoresis chip. High-quality waveguides crossing the microfluidic channels are fabricated and used to optically address, with high spatial selectivity, their content. Fluorescence from the optically excited volume is efficiently collected at a 90° angle by a high numerical aperture fiber, resulting in a highly compact and portable device. To test the platform we performed electrophoresis and detection of a 23-mer oligonucleotide plug. Our approach is quite powerful because it allows the integration of photonic functionalities, by simple post-processing, into commercial LOCs fabricated with standard techniques. Figure Femtosecond laser written waveguides can selectively excite fluorescence in a microfluidic channel of a commercial lab-on-a-chip. A compact scheme for on-chip detection by laser induced fluorescence is applied to capillary electrophoresis of a 23-mer Cy3-labeled oligonucleotide     

A viscosity-tunable polymer for DNA separation by microchip electrophoresis

A thermo-responsive separation matrix, consisting of Pluronic F127 tri-block copolymers of poly(ethylene oxide) and poly(propylene oxide), was used to separate DNA fragments by microchip electrophoresis. At low temperature, the polymer matrix was low in viscosity and allowed rapid loading into a microchannel under low pressure. With increasing temperatures above 25°C, the Pluronic F127 solution forms a liquid crystalline phase consisting of spherical micelles with diameters of 17–19 nm. The solution can be used to separate DNA fragments from 100 bp to 1500 bp on poly(methyl methacrylate) (PMMA) chips. This temperature-sensitive and viscosity-tunable polymer provided excellent resolution over a wide range of DNA sizes. Separation is based on a different mechanism compared with conventional matrices such as methylcellulose. To illustrate the separation mechanism of DNA in a Pluronic F127 solution, DNA molecular imaging was performed by fluorescence microscopy with F127 polymer as the separation matrix in microchip electrophoresis. Figure Temperature dependence of the viscosity of 20% w/w Pluronic F127 solution in 1xTBE buffer. Dotted approximates resultant curve.     

Chiral separation of raltitrexed by cyclodextrin-modified micellar electrokinetic chromatography

A rapid and effective method was developed for the chiral separation of raltitrexed (RD) enantiomers by carboxymethyl-beta-cyclodextrin (CM-β-CD)-modified micellar electrokinetic chromatography (MEKC). Optimization of conditions including the type and concentration of the chiral selector, concentration of sodium dodecyl sulfate (SDS), pH and concentration of the background electrolyte (BGE), capillary temperature, and applied voltage was investigated. The enantiomers of raltitrexed could be separated with satisfactory resolution and linear response by using 75 mM Tris-phosphate at pH 8.0 containing 30 mM SDS and 8 mM CM-β-CD as buffer system. Furthermore, the usefulness of this method was demonstrated in a purity test of a real synthetic drug sample. Figure Chiral separation of raltitrexed by CM-β-CD MEKC was optimized and applied to test the purity of a synthetic drug sample     

Forensic analysis of anthraquinone,azo, and metal complex acid dyes from nylon fibers by micro-extraction and capillary electrophoresis

The extraction and separation of dyes present on textile fibers offers the possibility of enhanced discrimination between forensic trace fiber evidence. An automated liquid sample handling workstation was programmed to deliver varying solvent combinations to acid-dyed nylon samples, and the resulting extracts were analyzed by an ultraviolet/visible microplate reader to evaluate extraction efficiencies at different experimental conditions. Combinatorial experiments using three-component mixture designs varied three solvents (water, pyridine, and aqueous ammonia) and were employed at different extraction temperatures for various extraction durations. The extraction efficiency as a function of the three solvents (pyridine/ammonia/water) was modeled and used to define optimum conditions for the extraction of three subclasses of acid dyes (anthraquinone, azo, and metal complex) from nylon fibers. The capillary electrophoresis analysis of acid dye extracts is demonstrated using an electrolyte solution of 15 mM ammonium acetate in acetonitrile/water (40:60, v/v) at pH 9.3. Excellent separations and discriminating diode array spectra are obtained even for dyes of similar color. Figure Capillary electropherogram of three acid dyes extracted from nylon 6,6 thread     

A high-efficiency sample introduction system for capillary electrophoresis analysis of amino acids from dynamic samples and static dialyzed human vitreous samples

A low-volume automated injection system for the analysis of chemically complex, amino acid samples is presented. This system utilizes submicroliter sample volumes stored on a 75-μm inner diameter capillary. A pulse of positive pressure (82 kPa) is used to load nanoliter sample volumes into an in-house fabricated interface and onto a separation capillary. Residual sample solution in the interface is immediately washed away by a continuous transverse flow through the injection interface, yielding a sharp and reproducible sample plug. By performing multiple injections of a static sample, one may average the signals to yield a signal-to-noise ratio improvement of up to 4.07-fold for 20 injections compared with a theoretical maximum of a 4.47-fold improvement. Without interruption of the applied voltage, injections performed every 150 s were used to monitor the progress of the reaction of multiple amino acids with the fluorogenic dye 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde. Analysis of dialyzed clinical vitreous samples demonstrates the resolution and quantitation of arginine, lysine, leucine, glutamine, and glutamate. Observed levels are comparable with those of nonautomated injection methods and reports by others. Figure Multiple injections of fluorescently labeled human vitreous with a detailed view of a single injection (above) and with all injections segmented and averaged for signal-to-noise ratio improvement (right)     

Advances in amino acid analysis

Amino acids are important targets for metabolic profiling. For decades, amino acid analysis has been accomplished by either cation-exchange or reversed-phase liquid chromatography coupled to UV absorbance or fluorescence detection of pre-column or post-column-derivatized amino acids. Recent years have seen great progress in the development of direct-infusion or hyphenated mass spectrometry in the analysis of free amino acids in physiological fluids, because mass spectrometry not only matches optical detection in sensitivity, but also offers superior selectivity. The advent of cryo-probes has also brought NMR spectroscopy within the detection limits required for the analysis of free amino acids. But there is still room for further improvement, including expansion of the analyte spectrum, reduction of sample preparation and analysis time, automation, and synthesis of affordable isotope standards. Figure Fully automated gas chromatography-mass spectrometry analysis of amino acids.     

Phase-changing sacrificial layers in microfluidic devices: adding another dimension to separations

The use of polymers in microchip fabrication affords new opportunities for the development of powerful, miniaturized separation techniques. One method in particular, the use of phase-changing sacrificial layers, allows for simplified designs and many additional features to the now standard fabrication of microchips. With the possibility of adding a third dimension to the design of separation devices, various means of enhancing analysis now become possible. The application of phase-changing sacrificial layers in microchip analysis systems is discussed, both in terms of current uses and future possibilities. Figure Phase-changing sacrificial materials enable multilayer microfluidic device layouts     

Chiral ligand-exchange chromatography of amino acids using porous graphitic carbon coated with a dinaphthyl derivative of neamine

In this paper, we describe the preparation and the evaluation of a porous graphitic carbon (PGC) column coated with a new dinaphthyl derivative of neamine for chiral ligand-exchange (LE) chromatography. It was shown that the graphitic surface/dinaphthyl anchor system efficiently (1.15 μmol/m²) and stably (three months of intensive use) adsorbs the neamine template onto the chromatographic support. The resulting coated PGC stationary phase showed appreciable LE-based enantioselective properties towards several native amino acids. Chromatographic separation of methionine enantiomers using a dinaphtyl neamine-based ligand-exchange chiral stationary phase     

Ice chromatography: current progress and future developments

Ice chromatography, in which water-ice particles are employed as a chromatographic stationary phase, has proven an efficient technique for probing the solution/ice interface. The preparation of fine ice particles has allowed us to not only obtain higher-resolution separation but also investigate the molecular processes occurring on the ice surface in more detail. Chromatographic investigations have revealed that two or more hydrogen bonds are simultaneously formed between a solute and the dangling bonds on the ice surface when the solute gives measurable retention. Several compounds, including estrogens, amino acids, and acyclic polyethers, have been successfully separated by ice chromatography with a hexane-based mobile phase. In addition, this method effectively probes the surface melting of the ice stationary phase and the liquid phase that coexists with water ice at thermodynamic equilibrium. The thickness of the surface liquid layer and the size of the liquid phase that grows inside an ice particle have been evaluated. The perspectives of this method are also discussed.      

Recent advances in capillary electrophoretic analysis of individual cells

Because variability exists within populations of cells, single-cell analysis has become increasingly important for probing complex cellular environments. Capillary electrophoresis (CE) is an excellent technique for identifying and quantifying the contents of single cells owing to its small volume requirements and fast, efficient separations with highly sensitive detection. Recent progress in both whole-cell and subcellular sampling has allowed researchers to study cellular function in the areas of neuroscience, oncology, enzymology, immunology, and gene expression.      

Critical evaluation of sample pretreatment techniques

Sample preparation before chromatographic separation is the most time-consuming and error-prone part of the analytical procedure. Therefore, selecting and optimizing an appropriate sample preparation scheme is a key factor in the final success of the analysis, and the judicious choice of an appropriate procedure greatly influences the reliability and accuracy of a given analysis. The main objective of this review is to critically evaluate the applicability, disadvantages, and advantages of various sample preparation techniques. Particular emphasis is placed on extraction techniques suitable for both liquid and solid samples. Figure Miniaturised extraction techniques allow sensitive analysis of also small sample volumes.     

Sequential preconcentration by coupling of field amplified sample injection with pseudo isotachophoresis–acid stacking for analysis of alkaloids in capillary electrophoresis

A novel on-column sequential preconcentration method based on the combination of field-amplified sample injection induced by acetonitrile and pseudo isotachophoresis (ITP)–acid stacking is developed for simply but efficiently concentrating alkaloid cations in a high-salt sample matrix in capillary electrophoresis. Acetonitrile (70%) added to a sample solution with a high-salt sample matrix not only induces field-amplified sample stacking by decreasing conductivity but also acts as a termination reagent in the succeeding pseudo ITP. After sample injection had been completed, a plug of H⁺ was injected electrokinetically and a neutralization reaction between H⁺ and tartrate from the buffer solution produced a low conductivity zone, in which the injected analyte cations were further concentrated. With the sequential preconcentration method, a 3 orders of magnitude detection sensitivity (1,400-fold) increase could be observed compared with the conventional electrokinetic injection method, without compromising separation efficiency and peak shape, and detection limits of 0.1 ng/mL for myosmine and 0.3 ng/mL for anabasine with the conditions selected were achieved. The calibration curves demonstrated good linearity in the concentration ranges 1.3–600 ng/mL for myosmine and 4.9–900 ng/mL for anabasine, respectively. The proposed method has been used to analyze successfully trace alkaloids in cigarette samples. Figure Sequential preconcentration processes: a sample injection; b introduction of HCl; c capillary zone electrophoresis separation. A ⁻ tartrate, white circles acetonitrile, black circles Na⁺, sample zone, myosmine, anabasine     

Rapid analysis of native neomycin components on a portable capillary electrophoresis system with potential gradient detection

A simple method based on capillary electrophoresis with potential gradient detection was developed to separate and detect neomycin components within 4 min without a derivatization step. Satisfactory separation and good repeatability were obtained using a separation buffer composed of 1 mM ammonium citrate (pH 3.5). The linearity of the method ranged from 10 to 1000 ppm with a limit of detection for neomycin B of about 7 ppm. After a simple dilution and filtering pretreatment step, neomycin components in three real samples were successfully analyzed without any major interference. Due to its simplicity and reliability, this method could provide an excellent alternative to the assays currently listed in U.S. and European Pharmacopoeia. The experiments were performed on a portable capillary electrophoresis system and, hence, the method can be readily applied to field analysis and point-of-care testing. Figure Photo of portable CE-P2-PGD system     

A novel approach for the detection of DNA using immobilized peptide nucleic acid (PNA) probes and signal enhancement by real-time immuno-polymerase chain reaction (RT-iPCR)

A new approach for the detection of DNA target molecules is described, using capture probes and subsequent signal enhancement by a uniform polymerase chain reaction (PCR). Peptide nucleic acid probes were immobilized in real-time PCR-compatible microtiter plates. After hybridization of biotinylated DNA targets, detection was performed by real-time immuno-PCR, a method formerly used for protein detection. We demonstrate the feasibility of this strategy for the qualitative detection of DNA oligonucleotides with a detection limit (LOD) of 6 attomol. Furthermore, the method was applied to PCR-amplified samples from genetically modified maize DNA (Mon810). A 483-bp DNA fragment was detected in mixture with 99.9% of noncomplementary DNA with a sensitivity down to the level of attomole. Figure       

Capillary HPLC–ICP MS mapping of selenocompounds in spots obtained from the 2-D gel electrophoresis of the water-soluble protein fraction of selenized yeast

A method based on ICP collision-cell MS detection in capillary HPLC was developed to gain an insight into the purity and identity of selenium-containing proteins separated by 1-D and 2-D electrophoresis. The bands and spots obtained after the separation of water-soluble proteins in selenized yeast were digested with trypsin prior to chromatography. Selenium could be detected down to the subpicogram level. The method, assisted by information obtained by MALDI TOF MS on the 5000 Da cut-off fraction, permitted the purity of bands and spots to be estimated and the efficiency of tryptic digestion and the quantity of selenium present in individual peptides to be evaluated. Owing to the high sensitivity and the lack of matrix suppression effects, the method provided chromatograms with signal-to-noise ratios of 10–1000 in conditions where the common ES Q–TOF MS detection failed.      

Sensitivity improvement in capillary electrophoresis using organo-aqueous separation buffers and thermal lens detection

It is shown that organo-aqueous separation buffers show much promise when used in capillary electrophoresis separations with photothermal (thermal lens) detection systems. Acetonitrile–water and methanol–water mixtures were selected, as conventionally used in capillary electrophoresis. It is shown that, despite more sophisticated experimental conditions (significant heat outflow from the capillary body) and peak detection, the theoretical ratio of the thermal lens signal for a binary mixture to the thermal lens signal for an aqueous solution (or the corresponding ratio obtained experimentally under bulk batch conditions) can be used to predict the sensitivity of thermal lens detection in capillary electrophoresis. The limits of detection for 2-, 3-, and 4-nitrophenols selected as model compounds in 70% v/v acetonitrile separation buffers are 1×10⁻⁶ M, 1×10⁻⁶ M and 3×10⁻⁷ M, respectively, and are therefore decreased by a factor of six compared to thermal lens detection in aqueous separation buffers. The overall increase in the thermal lens detection sensitivity in a 100% ACN buffer is a factor of 13.      

Micro free-flow electrophoresis: theory and applications

Free-flow electrophoresis (FFE) is a technique that performs an electrophoretic separation on a continuous stream of analyte as it flows through a planar flow channel. The electric field is applied perpendicularly to the flow to deflect analytes laterally according to their mobility as they flow through the separation channel. Miniaturization of FFE (μFFE) over the past 15 years has allowed analytical and preparative separation of small volume samples. Advances in chip design have improved separations by reducing interference from bubbles generated by electrolysis. Mechanisms of band broadening have been examined theoretically and experimentally to improve resolution in μFFE. Separations using various modes such as zone electrophoresis, isoelectric focusing, isotachophoresis, and field-step electrophoresis have been demonstrated.

Capillary electrophoretic separation of humic substances using hydroxyethyl cellulose as a buffer additive and its application to characterization of humic substances in a river water sample

We have developed a concise tool for the investigation of the transition of humic substances in environmental water. The separation of water-soluble humic substances was achieved rapidly and effectively by capillary electrophoresis using a polyacrylamide-coated capillary and a phosphate electrophoretic buffer solution (pH 7.0) containing hydroxyethyl cellulose. The separation mechanism was assessed using the ultrafiltration technique. The effect of the complexation of humic substances with metal ions was studied by using the proposed method. When Fe(III) ions or EDTA was added to the sample solution of fulvic acid, a distinct change in the electropherogram pattern based on the conformational change of fulvic acid was observed. The successful application of the proposed method to the characterization of humic substances in a river water sample was also demonstrated. Figure Addition of Fe(III) ions or EDTA to a solution containing fulvic acid (FA) results in a distinct change in the electropherogram pattern, which reflects the conformational change of FA: this forms the basis for the characterization of humic substances in river water samples