Cadmium species in river water were kinetically extracted with dithizone by varying the extraction time. The obtained extraction curve showed a three-stage stepwise profile that reflected the rate of the ligand substitution reaction between the dithizone and cadmium species. Corresponding to each stage, we divided these extracted cadmium species into three groups: “highly labile”, “moderately labile” and “slowly labile” species.
Many researchers have invested considerable efforts toward improving capillary electrophoresis (CE)-mass spectrometry (MS) systems so they can be applied better to standard analyses. This review highlights the developments in CE-MS of proteins and peptides over the last five years. It includes the developments in interfaces, sample-enrichment techniques, microfabricated devices, and some applications, largely in capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF) and capillary isotachophoresis formats.
A capillary electrophoresis/mass spectrometry method for the simultaneous chiral analysis of enantiomers of methamphetamine (MA), amphetamine (AP), dimethylamphetamine (DMA), ephedrine (EP), norephedrine (NE) and methylephedrine (ME) in urine has been developed. The background electrolyte was 1 M formic acid (pH 1.7). Using 0.85 mM heptakis(2,6-diacethyl-6-sulfato)-β-cyclodextrin as the chiral selector, the 12 enantiomers were completely separated within 25 min. The detection limits were 0.01 µg mL-1 for the enantiomers of MA, AP, DMA, EP and ME, and 0.02 µg mL-1 for the enantiomers of NE using selected ion monitoring. The reproducibilities of within-run (n = 4) for the migration times and peak areas of the standard mixture were under 0.58% and 7.83%, respectively. The calibration curves of the peak areas of the 12 enantiomers were linear in the range of 0.05 - 10 µg mL-1. This method was applicable to the analysis of urine samples.
A capillary electrophoresis method was developed for the analysis of oligosaccharides combined with derivatization with 2-aminobenzoic acid. Glycosaminoglycan Δ-disaccharides were effectively resolved on a fused-silica capillary tube using 150 mM borate, pH 8.5, as a running electrolyte solution. This analytical method was applied to the identification of glycosaminoglycan in combination with enzymatic digestion. The separation of N-glycans or glucose-oligomers was performed with a phosphate buffer containing polyethylene glycol or borate as an electrolyte solution. This method is expected to be useful in the determination of oligosaccharide structures in a glycoprotein.
We developed a weak-affinity separation system for single-nucleotide polymorphisms (SNPs) based on capillary electrophoresis. In this approach, single-stranded DNA (ssDNA)-polyacrylamide (polyAAm) conjugate was used as a pseudo-immobilized affinity ligand to separate the target DNA, cytochrome P450 2C9 (CYP2C9), and its point mutant. The ligand DNA was designed to be complementary to the normal DNA, and the target DNA was electrophoretically separated by the difference in the affinity with the pseudo-immobilized ligand in the capillary. We showed that the separation efficiency was closely associated with the Tm value of double-stranded DNA (dsDNA) consisting of the target and ligand DNA, which depends on the measurement conditions, such as the base number of the ligand DNA and the concentration of Mg2+ in the buffer solution.
A novel absolute determination method using chirality without any calibration curves or comparison standards has been proposed for phosphorus-containing amino acid-type herbicides, glufosinate (D,L-GLUF) and bialaphos (BIAL). This method is based on a change in the enantiomeric ratio after the spiking of a known amount of the enantiomers with different enantiomeric ratios to a sample. D,L-GLUF was determined by adding a known amount of L-GLUF to the sample, derivatizing them with dansyl chloride, and measuring the ratio of the peak area of the D-isomer to that of the L-isomer by means of γ-cyclodextrin modified capillary zone electrophoresis. The accuracy and precision of the method were evaluated using a synthetic sample. The mean values obtained for D- and L-GLUF agreed with the values taken within 1.6%; also the reproducibility was as good as less than 2.8%. The determination of BIAL was achieved by determining GLUF quantitatively produced by the acid hydrolysis of BIAL. The proposed methods were applied to the analysis of commercial herbicides and the validity and usefulness were evaluated.
A novel method for the on-column sample stacking of proteins is described. The strategy takes advantage of interactions between protein molecules and sodium dodecyl sulfate (SDS) monomers. A long plug of a protein sample (either acidic or basic) is injected into a capillary filled with a background electrolyte (BGE) containing SDS. When a potential is applied, the proteins interact with SDS monomers in the BGE to form protein-SDS complexes that migrate more slowly than the corresponding uncomplexed protein, resulting in protein stacking. Both acidic and basic proteins migrate at an almost identical electrophoretic velocity after stacking, which indicates that the protein-SDS complexes formed in the BGE zone have a similar charge/mass ratio. The mechanism of stacking was investigated using a sample consisting of a basic protein, lysozyme, and a small molecule, methylene blue. The findings clearly show that two interactions with SDS occur, a stepwise binding interaction between protein molecules and SDS monomers and an interaction in which the small molecules enter into micelles formed by SDS molecules. The method was also applied to the detection of a protein labeled with a fluorescent labeling reagent at trace levels. The labeled protein was detected even under labeling conditions where the labeling efficiency was too low to detect by short-plug injection.
Micellar electrokinetic chromatography (MEKC) using a cationic surfactant as a pseudostationary phase was examined to separate anionic metal cyclohexane-1,2-diaminetetraacetic acid (CDTA) complexes. Cetyltrimethylammonium chloride (CTAC) was employed as the cationic surfactant micelle, its addition leading to EOF reversal. Cu(II), Co(II), Zn(II), Mn(II) and Pb(II) were used as test analytes, and the complete separation was obtained by MEKC. On-line sample preconcentration by sweeping was also examined to improve the detection sensitivity. From 15- to 42-fold increases in the detection sensitivity in terms of the peak heights were obtained by sweeping with a cationic micelle in the presence of high EOF. The limits of detection were in the range (0.6 - 1.8) × 10-6 M with UV detection without any off-line preconcentration step.
This paper presents a capillary-to-microchip connection, which can be used as an interface for coupling capillary electrophoresis (CE) with a thermal lens microscope (TLM). It is difficult to directly apply TLM to samples in a capillary with a curved surface, and such an interface chip at the end of a CE separation column is needed for reliable TLM measurements. The dependence of the TLM signal intensity on the TLM detection point in the interface chip and the dependence of the theoretical plate number of CE separation on the channel dimensions of the interface chip were investigated and optimized with a mixture of 4-dimethylaminoazobenzene-4′-sulfonyl(DABSYL)-derivatized amino acids (glycine, alanine, methionine, and proline) as a model sample. By using an optimized interface chip, theoretical plate numbers of DABSYL-glycine, -methionine, -alanine, and -proline were obtained to be 104000, 95000, 104000, and 95000, respectively.
PCR is an indispensable technique used in DNA analysis. However, with the traditional methods, the time spent on amplification and the subsequent analysis of PCR products is generally long. Therefore, it is essential to improve these two steps so that the whole procedure can be made faster. In the present work, with λ-DNA as the control template, the amplification of 300-bp fragment could be completed within 37 s with capillary reaction chambers of LightCycler, and the following analysis of PCR products could be completed within 120 s with microchip electrophoresis as the detector. Since the high detection sensitivity of microchip electrophoresis, PCR products with template concentration as low as 5 fg/µL could be detected only after 435 s of amplification. In addition, based on additional optimized conditions simulated by CoventorWare, PCR microchips with distinct structure of the reaction chambers have been designed and successfully applied to the amplification of 300-bp fragment. By comparison, those chambers with ellipse and racket shapes were found to offer very high amplification efficiency. All of these results demonstrate the promise of integrating PCR and electrophoresis on microchip for developing easy-carrying instruments for the fast in situ detection of DNA.
We developed and optimized a system coupling microchip capillary electrophoresis (MCE) and laser-induced fluorescence (LIF) detection for the analysis of microorganisms. The MCE-LIF system successfully separated pure cultures of lactic acid bacteria and Saccharomyces cerevisiae within 200 s. The results indicate that the MCE system can be conveniently used for the rapid and highly sensitive detection of microorganisms. Thus, MCE can provide a cheap and simple method for the on-line detection of microbial contamination.
Chiral separations of 1-aminoindan (AI) by cyclodextrin electrokinetic chromatography (CDEKC) were investigated on microfluidic quartz chips. By using a microchip electrophoresis (MCE) instrument equipped with a linear-imaging UV detector, the separation process of the enantiomeric compounds was observed. When sulfated β-cyclodextrin was employed as a chiral selector, the baseline separation of AI could be achieved within 1 min with a high repeatability. The relative standard deviation of the migration time was less than 6%. The fastest separation was achieved in 14 s, utilizing a separation length of only 6.1 mm. These results show that the MCE analysis employing a linear imaging UV detector has a significant potential for fast chiral analysis.
The chiral separation of amino acid derivatives by ligand-exchange electrophoresis in a microchannel chip was performed for the first time. A Cu(II) complex with L-prolinamide was used as a chiral selector. The migration behaviors of eleven NBD-DL-amino acids were investigated by ligand-exchange capillary electrophoresis (LE-CE). The enantiomer of five NBD-amino acids (Ser, Thr, Val, Phe and His) could be separated by LE-CE using a 20 mM ammonium acetate buffer (pH 9.0) containing 10 mM copper acetate, 20 mM L-prolinamide and 1 mM SDS. NBD-His was eluted in the order D-form and L-form, while the elution order of another enantiomers was L-form and D-form. Under this condition, the enantioseparation of these five NBD-amino acids by ligand-exchange microchip electrophoresis (LE-ME) was investigated using a glass microchip. The enantioseparation of NBD-Ser, -Thr and -His could be successfully accomplished by LE-ME. LE-ME was superior to LE-CE in terms of the short migration time and a good enantiomeric separation.