Coupling chemiluminescence with capillary electrophoresis to analyze single human red blood cells

In this paper, we describe a new method for determination of hemoglobin of single red blood cells by coupling chemiluminescence with capillary electrophoresis (CL-CE). The chemiluminescent detection is based on the catalytic effects of hemoglobin on the luminol-hydrogen peroxide reaction. The conditions of chemiluminescent reaction and capillary electrophoresis were investigated. Hemoglobin in human blood samples was detected with the present method, the linear range from 1.7 μg mL⁻¹ to 6.8 μg mL⁻¹ was tested, and the correlation coefficient of 0.997 and low detection limit of 0.17 μg mL⁻¹ (approximately 2.2 pg, S/N = 3) were obtained. Cell injection procedure was improved, and the method was successfully used to determine hemoglobin of single red blood cells and the statistical result of the average content of hemoglobin in 26 human red blood cells was 23.6 pg. Compared to other current methods, CE with CL system is simple, sensitive and will become an attractive alternative method for single cell analysis.     

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.     

Separation of nicotine metabolites by capillary zone electrophoresis and capillary zone electrophoresis/mass spectrometry

The use of capillary zone electrophoresis (CZE) and capillary zone electrophoresis/mass spectrometry (CZE/MS) has been demonstrated, in principle, for the separation of nicotine and nicotine metabolites. The buffer system developed for separation and detection by CZE/UV was modified for use in CZE/MS analysis. Several of the metabolites are isobaric and tandem mass spectrometric (MS/MS) techniques have been used to differentiate such analytes.     

High-voltage power supplies to capillary and microchip electrophoresis

Over the past years, the development of capillary electrophoresis (CE) and microchip electrophoresis (ME) systems has grown due to instrumental simplicity and wide application. In both CE and ME, the application of a high voltage (HV) is a crucial step in the electrokinetic (EK) injection and separation processes. Particularly on ME devices, EK injection is often performed with three different modes: gated, pinched, and unpinched. In all these cases, different potential values may be applied to one or multiple channels to control the injection of small sample volumes as well as the separation process. For this reason, the construction of reliable HV power supplies (HVPS) is required. This review covers the advances of the development of commercial and laboratory-built HVPS for CE and ME. Moreover, it intends to be a guide for new developers of electrophoresis instrumentation.     

Rapid determination of globin chains in red blood cells by capillary electrophoresis using INSTCoated fused‐silica capillary

A laboratory‐made INSTCoated fused‐silica capillary has been newly used for CE separation of four mixtures of proteins in sodium phosphate BGEs at pH 3.0 and 2.5, respectively. The obtained separation efficiencies range from 145 000 theoretical plates per meter for myoglobin to 1 216 000 m^?1 for lysozyme. A total of 49–89% of the number of theoretical plates was obtained in a commercial polyvinyl alcohol coated capillary compared to the INSTCoated capillary under the same experimental conditions, 0–86% was obtained in a laboratory polyacrylamide‐coated capillary, and only 0–6% was obtained in an uncoated fused‐silica capillary. The RSD values for the intraday repeatability for an INSTCoated capillary were 0.1–1.0% (migration time) and 0.3–2.4% (peak area); RSD values for the interday repeatability in the same capillary are 0.6–1.4% (migration time) and 2.4–5.5% (peak area); RSD values for interday repeatability between different capillaries equaled 1.7–2.1% (migration time) and 2.8–10.9% (peak area). The INSTCoated capillary has been further used for rapid determination of globin chains isolated from red blood cells. A separation of α and β chains prepared from adult blood has been completed in 3 min with a peak resolution of 1.3, and the separation of α and ^Gγ chains prepared from newborn blood took 3 min with a peak resolution of 3.6.     

Ultrarapid capillary electrophoresis method for the determination of reduced and oxidized glutathione in red blood cells

We describe a very rapid high-performance capillary electrophoresis method for the separation and quantification of reduced (GSH) and oxidized (GSSG) glutathione in red blood cells. Two procedures for sample preparation have been compared, Microcon-10 membrane filtration and acid precipitation. The separation is obtained in an uncoated capillary using a high ionic strength borate buffer at pH 7.8. The intra-assay coefficients of variation (CVs%) are 1.53 and 1.66 for GSH and GSSG, respectively. The run is shorter than 90 s and the migration time is highly reproducible both for GSH (CV% 0.22) and GSSG (CV% 0.17). When the filtration step is used only GSH is found, whereas both GSH and GSSG are detectable after acid precipitation, suggesting that GSSG revealed after acid treatment may be an artefact due to GSH oxidation. Because of its good analytical performance this method could be used for routine red blood cell glutathione measurement in healthy or pathological conditions.     

Polypyrrole-coated capillaries for capillary zone electrophoresis

Fused-silica capillaries are permanently coated by silanization with 3-{[3-(N-pyrrole)-2-hydroxypropyl]amino}propyltriethoxysilane followed by oxidative polymerization of the pyrrole moieties with iron (III) or peroxodisulfate in the presence of chloride, perchlorate, or dextransulfate as anions. This approach allows to modulate the EOF in its magnitude as well as in its direction. With the small anions chloride and perchlorate, the EOF is reversed below pH 5 while with the large dextransulfate polyanions (DS) the EOF is relatively constant over the pH range from 2.5 to 9.4. This can be of advantage at low pH, at which the EOF of uncoated capillaries is close to zero. Application for separation of some herbicides is shown. The lifetime of PP-modified capillaries is satisfactory: the decrease in EOF is less than 3% during 80 analyses (160 min) and less than 5% over three months of storage. The reproducibility of capillary modification is about 5% (RSD of EOF).     

Fraction collector for capillary zone electrophoresis

An instrument is described which is capable of collecting fractions from a capillary zone electrophoresis apparatus. The fraction collector is characterized in terms of discretely collecting the separated components of a multi-component sample. In addition, the fraction collector permits the study of the effect of capillary zone electrophoresis on the biological activity of alpha-chymotrypsin.     

Split-flow injector for capillary zone electrophoresis

A simple construction of a split-flow injector eliminating some common problems connected with the use of such devices is described. It consists of a low-pressure pump, an injection valve and a delivery tube in which the separating capillary inlet is fixed. The sample is injected without moving the separating capillary inlet and without interrupting the applied voltage. The grounded electrophoretic electrode is close to the injection valve so that all metal parts of the injector are kept at a sufficiently low potential. Minimum length and small internal diameter of delivery tube minimizes additional sample zone broadening. The effects of some experimental parameters, such as the position of the separation capillary inlet with respect to the background solution flow direction and background solution flow-rate are experimentally studied. The injector was tested primarily for the electrokinetic injection.     

System zones in capillary zone electrophoresis

When working with capillary zone electrophoresis (CZE), the analyst has to be aware that the separation system is not homogeneous anymore as soon as a sample is brought into the background electrolyte (BGE). Upon injection, the analyte creates a disturbance in the concentration of the BGE, and the system retains a kind of memory for this inhomogeneity, which is propagated with time and leads to so-called system zones (or system eigenzones) migrating in an electric field with a certain eigenmobility. If recordable by the detector, they appear in the electropherogram as system peaks (or system eigenpeaks). However, although their appearance can not be forecasted and explained easily, they are inherent for the separation system. The progress in the theory of electromigration (accompanied by development of computer software) allows to treat the phenomenon of system zones and system peaks now also in very complex BGE systems, consisting of several multivalent weak electrolytes, and at all pH ranges. It also allows to predict the existence of BGEs having no stationary injection zone (or water zone, EO zone, gap, dip). Our paper reviews the theoretical background of the origin of the system zones (system peaks, system eigenpeaks), discusses the validity of the Kohlrausch regulating function, and gives practical hints for preparing BGEs with good separation ability not deteriorated by the occurrence of system peaks and by excessive peak-broadening.     

High-voltage contactless conductivity detection of metal ions in capillary electrophoresis

The detection of alkali, alkaline earth and heavy metal ions with a high-voltage capacitively coupled contactless conductivity detector (HV-C(4)D) was investigated. Eight alkali, alkaline earth metal ions and ammonium could be separated in less than 4 min with detection limits in the order of 5 x 10(-8) M. The heavy metals Mn2+, Pb2+, Cd2+ Fe2+, Zn2+, Co2+, Cu2+ and Ni2+ could also be successfully resolved with a 10 mM 2-(N-morpholino)ethanesulfonic acid/DL-histidine (MES/His)-buffer. Zn2+, Co2+, Cu2+ and Ni2+ showed an indirect response. The detection limits for the heavy metals were determined to range from about 1 to 5 microM.     

Quantitative capillary zone electrophoresis of inorganic anions

Despite the availability of commercial capillary electrophoresis systems for over ten years, where quantitative analysis is required, capillary zone electrophoresis (CZE) has often failed to replace ion chromatography as the method of choice for a large number of analytes, not least inorganic anions. To investigate the reasons for this apparent failing, a review is presented of work that has been carried out to-date involving the quantitative application of CZE to the determination of inorganic anions in industrial and environmental samples. This review summarizes work both investigating and improving the quantitative aspects of the CZE of inorganic anions. A complete survey of how CZE has been applied to the determination of inorganic anions in real samples is given, including what, if any, analytical performance parameters were investigated and quoted, and if quality assurance data and validation methods were briefly considered, thoroughly investigated or simply ignored.     

毛细管区带电泳法测定粉针剂中头孢拉定的含量

用毛细管区带电泳法测定头孢拉定的含量 ,未涂层毛细管柱 (75 μm×48.5cm ,有效长度 40cm) ,电压 2 8kV ,检测波长 2 3 0nm ,温度 2 0℃ ,进样 5×1 0 3Pa× 3s。运行缓冲液为 2 5mmol/L硼砂缓冲液。方法的线性范围 3 1 .2 2μg/mL～ 749.2 8μg/mL ,检测限为 1 .1 7μg/mL。     

Separation of nitrophenols by capillary zone electrophoresis

Separation conditions suitable to a rapid resolution of a group of eight nitrophenols by capillary zone electrophoresis (CZE) were found. Required differences in their effective mobilities were achieved via host-guest complexation of -cyclodextrin combined with intermolecular interactions involved by polyvinylpyrrolidone. When both additives were present in the carrier electrolyte at pH=9.1 nitrophenols could be separated in the column of a, 300 m I.D. and 180 mm in the length within 8–9 minutes. It is shown that the column of such an I.D. providing enhanced sample load capacity, can operate with high separation efficiencies as maintaining zone dispersions due to Joule heating on a tolerable level. CZE on-line coupled with isotachophoretic sample pretreatment is shown to provide the concentration limits of detection at low ppb concentrations by using an on-column photometric detector operating at 254 and 405 nm detection wavelengths.     

Analytical potential of enzyme-coated capillary reactors in capillary zone electrophoresis

Enzymes immobilized on the inner surface of an electrophoretic capillary were used to increase sensitivity and resolution in capillary zone electrophoresis (CZE). Sensitivity is enhanced by inserting a piece of capillary containing the immobilized enzyme into the main capillary, located before the detector, in order to transform the analyte into a product with a higher absorptivity. This approach was used to determine ethanol. In order to improve resolution, capillary pieces containing immobilized enzymes were inserted at various strategic positions along the electrophoretic capillary. On reaching the enzyme, the analyte was converted into a product with a high electrophoretic mobility, the migration time for which was a function of the position of the enzyme reactor. This approach was applied to the separation and determination of acetaldehyde and pyruvate. Finally, the proposed method was validated with the determination of ethanol, acetaldehyde, and pyruvate in beer and wine samples.     

Contemporary chiral simulators for capillary zone electrophoresis

For separation of enantiomers in presence of a chiral selector, data obtained with the 1D dynamic simulators SIMUL5complex and GENTRANS are compared to data predicted by PeakMaster 6, a recently released generalized model of the linear theory of electromigration. Four electrophoretic systems with stereoisomers of weak bases were investigated. They deal with the estimation of input data for complexation together with the elucidation of the origin of observed system peaks, the interference of analyte and system peak migration, the change of enantiomer migration order as function of the selector concentration and the inversion of analyte migration direction in presence of a multiply negatively charged selector. For all systems, data predicted with PeakMaster 6 are in agreement with those of the dynamic simulators and simulation data compare well with experimental data that were monitored with setups featuring conductivity and/or UV absorbance detection along the capillary. SIMUL5complex and GENTRANS provide the full dynamics of any buffer and sample arrangement and require very long execution time intervals. PeakMaster 6 is restricted to conventional CZE, is based on an approximate solution of the transport equations, provides data for realistic experimental conditions within seconds and represents a practical tool for an experimentalist.     

Formamide as solvent for capillary zone electrophoresis

A comprehensive investigation of a number of aspects when using formamide as background electrolyte solvent in capillary zone electrophoresis was presented. It included (i) the change of the ion mobility with ionic strength, (ii) the influence of the ionic strength on diffusion coefficients, and (iii) on the separation efficiency expressed by the maximum reachable plate numbers (when only longitudinal diffusion contributed to zone broadening), (iv) the effect of the solvent on pKa values (taken from the literature) of neutral and cation acids, (v) the establishment of the a pH scale in formamide by dissolving acids with known pKa values and their salts at defined proportion (thus circumventing the problem of calibrating the pH meter), (vi) the agreement between the experimentally derived and the theoretical dependence of the effective mobility on pH, (vii) the uptake of water of this hygroscopic solvent from the humidity of the environment and its consequence to the ion mobilities, pKa values, and the chemical stability of the solvent (e.g., hydrolysis), and finally (viii) the use of conductivity and indirect UV absorption to enable detection of analytes below the optical cutoff of formamide.     

System zones in capillary zone electrophoresis.

This paper brings an overview of system zones (SZs) in capillary zone electrophoresis (CZE) and their effects upon the migration of zones of analytes. It is shown that the formation and migration of SZs is an inherent feature of CZE, and that it depends predominantly on the composition of an actual background electrolyte (BGE). One can distinguish between stationary SZs and migrating SZs. Stationary SZs, which move due to the electroosmotic flow only, are induced in any BGE by sample injection. Migrating SZs may be induced by a sample injection in BGEs which show at least one of the following features: (i) BGE contains two or more co-ions, (ii) BGE has low or high pH whereby H+ or OH- act as the second co-ion, and (iii) BGE contains multivalent weak acids or bases. SZs do not contain any analyte and show always BGE-like composition. They contain components of the BGE only and the concentrations of these components are different from their values in the original BGE. Providing that some of the ionic components of the BGE are visible by the detector, the migrating SZs can be detected and they are present as system peaks/dips in the electropherogram. It is shown that a migrating SZ may be characterized by its mobility, and examples are given how this mobility can depend on the composition of the BGE. Further, the effects of the migrating SZs (either visible or not visible by the detector) upon the zones of analytes are presented and the typical disturbances of the peaks (extra broadening, zig-zag form, schizophrenic behavior) are exemplified and discussed. Finally, some conclusions are presented how to cope with the SZs in practice. The proposed procedure is based on the theoretical predictions and/or measurements of the mobilities of SZs and on the so-called unsafe region. Then, such operational conditions should be selected where the unsafe region is outside of the required analytical window.     

Indirect photometric detection in capillary zone electrophoresis

An indirect photometric detection method is described which is based on the use of an absorbing co-ion as the principal component of the background electrolyte. The zones of non-absorbing ionic species are revealed by changes in light absorption due to charge displacement of the absorbing co-ion. Theoretical considerations are given for selecting a suitable absorbing co-ion to achieve a high sensitivity of detection.The role of electromigration dispersion is illustrated by experiments and the effects of the differences in the effective mobilities of sample ions and that of the absorbing co-ion are discussed. The highest sensitivity can be achieved for sample ions having an effective mobility close to the mobility of the absorbing co-ion. In such a case, the concentration of the sample component in its migrating zone can be high while electromigration dispersion is still negligible. The useful dynamic range of the detection is then limited by the linearity and noise of the detector, the former parameter being given mostly by the shape of the on-column detection cell. The best sensitivities can be obtained in low-concentration background electrolytes containing a co-ion with high absorption at a given detection wavelength.It is shown that indirect photometric detection can be useful for detecting substances that have no optical absorption in the UV and/or visible region, provided that the composition of the background electrolyte is selected correctly.