High-sensitivity analyses of metabolites in biological samples by capillary electrophoresis using dynamic pH junction-sweeping

Emerging fields of biochemical research, such as metabolomics, present challenges to current separation technologies because of the large number of metabolites present in a cell and their often low (submicromolar) concentration. Although capillary electrophoresis (CE) holds great promise as the method of choice for high-resolution separations of biological samples, it suffers from poor concentration sensitivity, especially with the use of UV detection. In CE, sweeping and dynamic pH junction represent two complementary on-line focusing techniques that have been used for sensitivity enhancement of hydrophobic and weakly acidic analytes, respectively. However, the application of either the sweeping or dynamic pH junction technique alone might, in some cases, be less effective for the analysis of certain sample mixtures. Recent work in the development of a hyphenated dynamic pH junction-sweeping technique is presented as an effective on-line method of preconcentration suitable for both hydrophilic (anionic) and hydrophobic (neutral) analytes. Sensitive analyses of flavin metabolites by CE with laser-induced fluorescence (LIF) detection is demonstrated in various biological matrixes, including cell extracts of Bacillus subtilis, pooled human plasma, as well as heat-deproteinized flavoenzymes. Enhanced analyte band narrowing and improved sensitivity is achieved for flavins using dynamic pH junction-sweeping compared to either sweeping or dynamic pH junction alone. This results in over a 1200-fold improvement in sensitivity relative to conventional injection methods, giving a limit of detection (LOD, defined as S/N = 3) of about 4.0 x 10(-12) M. Strategies for sensitive and more comprehensive analyses of other cell metabolites, including nucleotides, coenzymes, and steroids, are also discussed when using on-line focusing techniques in conjunction with multiplexed CE and UV detection.     

Dicholorbenzyl alkyl ether homologs as retention index markers and internal standards for the analysis of environmental samples using capillary gas chromatography

A homologous series of 2,4-dichlorobenzyl alkyl ethers (DCBEs) have been synthesized and purified for use as retention index calibrants and internal standards. They are stable, sensitive to ECD and FID and ideal for GC-MS, with base peaks at m/z 159/161. The retention index calibration against the n-alkanes for a linear temperature program (LTP) series is given and recommendations made for the most suitable members of the DCBE series for use as internal standards in organochlorine (OC) residue analysis.     

Splitless on-line coupling of capillary high-performance liquid chromatography,capillary electrochromatography and pressurized capillary electrochromatography with nuclear magnetic resonance spectroscopy

A mixture of unsaturated fatty acid methyl esters was separated with a new splitless capillary set-up. With the employed apparatus configuration different capillary separation techniques such as capillary high-performance liquid chromatography (cHPLC), capillary electrochromatography (CEC) and pressurized capillary electrochromatography (pCEC) could be applied. The detection and identification of the sample compounds were accomplished by hyphenating these capillary separation techniques with nuclear magnetic resonance (NMR) spectroscopy using a novel configuration of the detection capillary set-up. Using modified electrokinetically driven separation techniques, the electric field was applied solely across the separation column. With this improved interface for capillary liquid chromatography-NMR on-line coupling, the stereochemical assignment of the cis and trans configuration of unsaturated fatty acids could be easily accomplished. Finally, the results of cHPLC-NMR, CEC-NMR and pCEC-NMR coupling experiments were compared.Dedicated to Professor Günter Häfelinger on the occasion of his 65th birthday     

A Novel Method for Quantification of Circulating DNA in Serum by Capillary Zone Electrophoresis

In this paper, we first presented a novel method for quantification of circulating DNA in human serum based on capillary zone electrophoresis with laser-induced fluorescence detection (CZE-LIF). The serum was digested by proteinase to release free DNA, and then CZE-LIF system was used for the quantification of total circulating DNA. This method was successfully used to quantify the circulating DNA levels in sera from healthy individuals and certain cancer patients.We found the significantly elevated circulating DNA levels in certain prostate cancer patients. Our results demonstrated that CZE-LIF system has good linearity, excellent sensitivity (0.5 ng/mL DNA),satisfactory reproducibility (RSDs in one day and between days were both less than 5%) and reliability, and is well suitable to the quantification of the circulating DNA in human serum or plasma.     

Partial-filling affinity capillary electrophoresis

Partial-filling affinity capillary electrophoresis (PFACE) is used to examine the binding interactions between two model biological systems: D-Ala-D-Ala terminus peptides to the glycopeptide antibiotic vancomycin (Van) from Streptomyces orientalis, and arylsulfonamides to carbonic anhydrase B (CAB, EC 4.2.1.1, bovine erythrocytes). Using these two systems, modifications in the PFACE technique are demonstrated including flow-through PFACE (FTPFACE), competitive flow-through PFACE (CFTPFACE), on-column ligand synthesis PFACE (OCLSPFACE), and multiple-step ligand injection PFACE (MSLIPFACE). In PFACE small plugs of sample are injected into the capillary column and an equilibrium is established between receptor and ligand during electrophoresis. Binding constants are then obtained by Scatchard analysis using changes in the migration time of the receptor/ligand on changing the concentration of the ligand/receptor. Data demonstrating the quantitative potential of these methods are presented. This review focuses on the unique capabilities of the different PFACE techniques as applied to two model biological systems.     

Reproducibility of retention indices measurement of alkylbenzenes on crosslinked methyl silicone phase fused silica capillaries

Summary Reproducibility of the Kovats retention indices of alkylbenzenes was studied on conventional “PONA” fused-silica capillary columns coated with crosslinked dimethyl silicone phase, with a film thickness of 0.5 μm. The data were compared with those obtained on conventional OV-101 columns. The tested PONA columns showed reproducible chromatographic properties, capacity ratios and high efficiencies. Having a standard deviation of s=0.03 i.u. of the retention indices on a single column, the column-to-columns differences were found to be up to 0.3 i.u. Higher indices were determined for crosslinked columns, the difference being in most cases about one i.u.     

Comparison of Pulsed Electrochemical Detection Modes Coupled with Microchip Capillary Electrophoresis

There is a need to develop analytical methods that are capable of rapidly measuring small biological markers in the field of metabolomics. Among others, carbohydrates play an important role biologically yet are traditionally hard to detect since they have no chromophore or fluorophore. In the present report, the first application of integrated pulsed amperometric detection (iPAD) coupled with microchip electrophoresis to the analysis of glucose, mannose, sucrose, maltose, glucosamine, lactose, maltotriose and galactose is demonstrated. iPAD is an electrochemical detection mode that can be used for direct detection of carbohydrates, amines and sulfur containing compounds. The effect of different solution parameters, including the buffer concentration, pH and the concentration of SDS on both separation and detection response was analyzed. In addition, a comparison study between PAD and iPAD was performed using glucose, glucosamine, sucrose and maltose as model carbohydrates.     

Binary and Ternary Complexes Between Lauryl Hexaoxyethylene, Benzoate and Cyclodextrin. Part II. β-CD

The characterization of binary and ternary complexes of benzoate, lauryl hexaoxyethylene (C₁₂E₆) and -CD is presented. The complexation equilibrium was characterized by UV-Vis spectrophotometry, titration microcalorimetry, capillary electrophoresis, and 2D ROESY ¹H-NMR. Results suggested that -CD forms one complex with C₁₂E₆in the stoichiometric ratio of -CD : C₁₂E₆1.5 : 1, with a stability constant 1.3 × 10⁵ M^-1.5. The 2-D ROESY ¹H-NMR spectrum indicated that C₁₂E₆is included inside the -CD cavity. The primary binding site of C₁₂E₆ is on the lauryl subunit of this molecule. Analogous to a previously reported study of -CD, the combination of -CD and C₁₂E₆precipitated from the solution. Addition of benzoate seemed to dissolve the precipitate and nearly doubled the apparent stability constant of the complex. Results from the various techniques supported formation of ternary complexes between -CD, C₁₂E₆, and benzoate.     

Characterization of a convection-based support microstructure through a flow resistance parameter

Modern convection-based supports differ substantially in pore size, porosity, and microstructure topology. Due to such variability, it is challenging to evaluate the contribution of a particular microstructure topology on flow resistance. We demonstrated that the flow resistance parameter ($\varphi$) introduced decades ago can be used as a criterion to evaluate the effect of microstructure topology on a pressure drop when the pore size is used as a characteristic support dimension. Furthermore, the $\varphi$ value of simple cubic packing was calculated over the entire range of open porosity and compared to the $\varphi$ values determined for pressure drop models derived for particular convection-based supports and experimental values of various convection-based supports from the literature. It was shown that different convection-based supports become clustered into distinct groups when plotted according to their $\varphi$ and open porosity values, allowing their discrimination.     

Separation of Dyes from Aqueous Solutions by Paper Adsorption

Separation of the dyes methyl violet, methylene blue, and congo red from aqueous solutions by paper capillary permeation adsorption method was studied using paper. Nearly 100% of the investigated dyes could be separated under the optimum conditions. The effect of pH on the separation efficiency was studied in particular. At pH 5–9, 1.3–11, and 7–11, the maximum separation was achieved for methyl violet, methylene blue and congo red, respectively. The effects of dye concentration and some foreign ions on the separatability were examined. Moreover, the selective separation of some dyes was attempted by elution with chemical reagents.