Capillary zone electrophoresis for polymide composition analysis

Capillary zone electrophoresis (CZE) was employed in polyimide composition analysis. Polymide was decomposed to its corresponding aromatic diamine and aromatic acid monomers by an alkali fusion reaction. Sample treatment is much simpler than published methods, and electropherograms show a good separation of decomposed products under the proper conditions.     

Capillary zone electrophoresis for the characterization of latex particles

Capillary zone electrophoresis (CZE) was applied to the separation of acrylic styrene copolymer emulsion particles. Fast separations could be performed on samples containing chemically identical latex particles of different size, as well as on samples with particles of the same size but differing in chemical composition. The developed method was also used for the analysis of water soluble fractions of urethane dispersions. Additionally, the physical interaction between different particles (e.g., acrylic and urethane particles) could be studied using this method. The separation mechanism is based on the zeta potential of the particles and the relaxation effect under the applied analytical conditions.     

Capillary zone electrophoresis for determination of carbohydrate-deficient transferrin in human serum

Carbohydrate-deficient transferrin (CDT) is the most specific marker for diagnosis of chronic excessive alcohol consumption and includes the serum transferrin (Tf) isoforms with two or less sialic acid residues (di-, mono-, and asialo-Tf). To monitor serum CDT, we developed a capillary zone electrophoresis (CZE) method based on the dynamic capillary coating with diethylenetriamine (DETA). The separation was performed in a bare fused-silica capillary (50 microm ID, 57 cm in length), applying a voltage of 25 kV and a temperature of 40 degrees C. Using a 100 mmol/L borate buffer, pH 8.4 with 3 mmol/L DETA, the Tf isoforms (asialo- to pentasialo-Tf) were resolved within 16 min. Enzymatic cleavage of sialic acid residues with neuraminidase and immunosubtraction were used to identify CDT isoforms. The relative amount of CDT expressed as area % of disialo-Tf isoform related to the area of tetrasialo-Tf in 50 healthy donors (24 males and 26 females; aged 25-50 years) was 3.15 +/- 0.76% (mean +/- SD). The comparison between CDT values obtained by this CZE procedure and the "Axis-Shield %CDT" kit gave r = 0.644, p < 0.001 (n = 290). This easy to use and inexpensive CZE procedure could be an ideal tool to investigate CDT proteins for clinical or forensic purposes.     

Capillary zone electrophoresis for the analysis of glycoforms of cellobiohydrolase

Cellobiohydrolase (CBH) is an important enzyme for the conversion of lignocellulosic biomass to ethanol. This work separated the glycoforms of CBH possessing different numbers of neutral mannoses using capillary zone electrophoresis (CZE) in a 50 mM, pH 7.5 phosphate buffer. The method analysed CBH in an intact form using a polyacrylamide coated fused silica capillary without requiring additives or labelling of the enzyme. The migration time of the major peak was found to be 21.6±0.1 min (n=3) and the approach is suitable for testing of batch-to-batch consistency of CBH. Ease-of-use, automation and speed are the other benefits due to which the use of CZE for analysing glycoforms of CBH was concluded to be ideal.     

Capillary electrophoresis for monitoring dityrosine and 3-bromotyrosine synthesis

Protein oxidation affects the structure of many amino acids. Variants of tyrosine are increasingly important in medical and food sciences. The synthesis of standards is essential for monitoring the disease state of patients with various illnesses and the quality of a number of food products. A method for monitoring standard synthesis of dityrosine and 3-bromotyrsoine from tyrosine using capillary electrophoresis (CE) is presented. Optimum separation was achieved using an isoelectric buffer consisting of 100mM iminodiacetic acid (IDA)+75 mM lauryl sulfobetaine (SB 3-12)+0.02% hydroxypropyl methylcellulose (HPMC) in a 27 cm x 75 microm capillary at 22 kV and 45 degrees C. Using these conditions the tyrosine adducts could be easily separated in less than 4 min. The resolution of the CE method was similar to HPLC separations, but analysis time was distinctly shorter.     

Conditions for purification of proteins by free-flow zone electrophoresis

The separation of proteins by free-flow zone electrophoresis is generally impaired by a number of secondary effects which cause spreading of the protein streams with resultant loss in resolution. A strategy is outlined, based on experimental observations and numerical modeling, which allows the operating conditions and separation chamber dimensions to be chosen so as to obtain complete separation of two proteins of known mobility. This approach takes into account some dispersive phenomena such as molecular diffusion, electroosmosis and residence time gradients. In some cases, the right operating conditions cannot be achieved on earth and electrophoresis separations in microgravity may then be justified.     

Capillary zone electrophoresis in laboratory-made fluorinated ethylene propylene capillaries

Capillaries made of fluorinated ethylene propylene (FEP) with an inner diameter of 50 microm have been employed in capillary zone electrophoresis with UV-Vis absorbance detection. The capillaries were made in the laboratory with a recently developed technique using fluoropolymer heat shrink/melt tubing and a tungsten wire as a template for the channel. An electroosmotic flow was obtained in the channels and it is shown that an FEP capillary is more effective for a cationic test solute than a fused-silica capillary. The compatibility of FEP capillaries with optical detection is evaluated briefly.     

Capillary electrophoresis of liposomes functionalized for protein binding

CE enabled assessing the attachment of hexa-histidine-tagged proteins to functionalized phospholipid liposomes. The liposomes were made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, phosphatidyl-ethanolamine, cholesterol and distearoyl-glycero-3-phosphoethanolamine-N-methoxy(polyethylene glycol) in a molar ratio of 29:26:40:5. The unilamellar vesicles, which had an average diameter of 170 nm, were labelled by inclusion of FITC-dextran for fluorescence detection. CE was carried out in poly(vinyl alcohol) (PVA)-coated capillaries at 25 degrees C with a BGE consisting of Tris-HCl (50 mM, pH 8.0). For conjugation of the liposomes with the proteins (soluble synthetic receptor fragments with molecular mass of 60 and 70 kDa, respectively), Ni(2+) was implanted into the vesicle surface by an anchor lipid containing a nitrilotriacetate acid (NTA) group as complexation agent for the metal ions. The difference in surface charge enabled the separation of the different species of interest by CE: plain vesicles, vesicles functionalised with Ni-NTA, vesicle-protein complexes and the species formed upon removal of the Ni-ions by complexation with EDTA. Loss of the Ni-ions resulted in the release of the proteins and the reappearance of the plain Ni-free NTA-liposome species in the electropherograms.     

Capillary zone electrophoresis method for the simultaneous determination of cations in honey

A capillary electrophoresis system for the simultaneous determination of cations in honey samples has been developed. The complete separation and quantification of K+, Ca2+, Na+, Mg2+, Mn2+, Ni2+ and Li+, which represent more than 99% of the total content of cations in honey, can be achieved in 4 min with only a dilution and filtration of the honey sample. Electrolyte solution was composed by 10 mM imidazole as the carrier buffer and background absorbance provider and acetic acid as the complexing agent (pH 3.60). The running voltage was + 25 kV at 25 degree C. Indirect UV detection was achieved at 185 nm. Under the optimum conditions the detection limits ranged from 0.02 to 48.2 mg/kg and the quantification limits have ranged from 0.41 to 48.7 mg/kg. Precision data in honey samples analysed have shown repeatability and reproducibility RSD (%) lower than 2.84 and 6.62%, respectively. Recoveries of cations in honey samples analysed have ranged from 88.5 to 101.8%. These cations were identified by their relative migration times with regard to Ba2+ migration time used as reference standard and they were quantified by using an external standard calibration. Twenty-five honey samples were analysed to test the proposed method. Mean contents of 1.22 x 10(3), 93, 85, 54, 11, 1.9 and 2.3 mg/kg were found, respectively, for K+, Ca2+, Na+, Mg2+, Mn2+, Ni2+ and Li+ cations in analysed honeys. These results were similar than the obtained by other authors.     

Capillary zone electrophoresis of cationic and anionic drugs in methanol

The actual mobilities and dissociation constants of acidic and basic pharmaceuticals were determined in methanol. Actual mobilities were derived from the dependence of the effective mobilities of the analytes on the pH of the methanolic background electrolyte solution (pH(MeOH)). The pKa values of the pharmaceuticals in methanol (pK(a,MeOH)) were calculated by non-linear curve fitting to the measured mobility values. It was found that the shift in pKa value (when compounds were transferred from water to methanol) increased with the acidity of the analyte. The average pKa shift for compounds exhibiting acidic properties in water was ca. 5.5 units, and the shift for basic compounds about 2 units. As was shown for a mixture of beta-blockers, the calculated actual mobilities and pKa values can be utilised in the optimisation of pH conditions for separation. The practical value of the method was illustrated by the analysis of urine samples.     

Capillary electrophoresis monitoring of halide impurities in ionic liquids

Quantification of impurities in ionic liquids is a crucial task in assessing the reliability of physical constants and solvent properties: taking into account the particularities of the ionic matrix, a simple routine method using capillary electrophoresis (CE) is developed to determine the halide content at the ppm level in water-immiscible ionic liquids.     

Capillary zone electrophoresis in wide bore capillary tubes with fiber-coupled diode array detection

This feasibility study deals with the use of a wide bore (320 μm I.D.) capillary tube for the detection and identification of capillary zone electrophoresis (CZE) analytes by optical fiber-coupled diode array detection. A 250-μm mean effective pathlength of the detection cell with an inherently enhanced photon flux through the cell were significant contributors in reaching 0.2–1 μmol/l concentration detectabilities of the CZE analytes by this combination. Experiments with model analytes (p-sulfanilic, sorbic and naphthalene-2-sulfonic acids, tryptophan and asulam) revealed that spectral confirmations of their identities were still possible when their concentrations in the loaded samples (200 nl) were 1–5 μmol/l. Here, chemometry procedures (target transformation factor analysis, fixed size moving window-target transformation factor analysis, fixed size moving window-evolving factor analysis and orthogonal projection approach) employed in the data processing effectively contributed to reliable confirmation of the identities of the analytes also in critical situations (e.g. peak overlaps). The CZE separations were carried out in tandem-coupled columns of identical I.D. This made it possible to use, in the first column of the tandem, carrier electrolyte solutions that provide the desired separative effects, while in the second (detection) column the compositions of the carrier electrolyte solutions employed could reflect favorable conditions for obtaining spectral data. Mixtures containing model constituents at significantly differing concentrations and Maillard’s reaction products spiked with tryptophan enantiomers were employed in experiments aimed at assessing practical applicabilities and limits of the present approach to the analysis of samples characterized by complex ionic matrices.     

On-line nanoliter cycle sequencing reaction with capillary zone electrophoresis purification for DNA sequencing

An integrated system for DNA sequencing based on a nanoreactor for cycle-sequencing reaction coupled with on-line capillary zone electrophoresis (CZE) for purification and capillary gel electrophoresis (CGE) for separation is presented. Less than 100 nl of premixed reagent solution, which includes dye-labeled terminator pre-mix, bovine serum albumin and template, was hydrodynamically injected into a fused-silica capillary (75 microm I.D.) inside a laboratory-made microthermocycler for cycle sequencing reaction. In the same capillary, the reaction products were purified by CZE followed by on-line injection of the DNA fragments into another capillary for CGE. Over 540 base pairs (bp) of DNA can be separated and the bases called for single-standed DNA with 0.9% error rate. The total time was about 3.5 h, or a cycle time of 2 h with staggered operation. For double-stranded DNA, a longer reaction time was required and base calling up to 490 bp with 1.2% error rate was achieved. The whole system is readily adaptable to automated multiplex operation for DNA sequencing or polymerase chain reaction analysis.     

Capillary zone electrophoresis of alpha-helical diastereomeric peptide pairs with anionic ion-pairing reagents

The present study uses an unique capillary electrophoresis (CE) approach, that we have termed ion-interaction capillary zone electrophoresis (II-CZE), for the separation of diastereomeric peptide pairs where a single site in the centre of the non-polar face of an 18-residue amphipathic alpha-helical peptide is substituted by the 19 L- or D-amino acids. Through the addition of perfluorinated acids at very high concentrations (up to 400 mM), such concentration levels not having been used previously in chromatography or CE, to the background electrolyte (pH 2.0), we have been able to achieve baseline resolution of all 19 diastereomeric peptide pairs with an uncoated capillary. Since each diastereomeric peptide pair has the same sequence, identical mass-to-charge ratio and identical intrinsic hydrophobicity, such a separation by CZE has previously been considered theoretically impossible. Excellent resolution was achieved due to maximum advantage being taken of even subtle disruption of peptide structure/conformation (due to the presence of D-amino acids) of the non-polar face of the amphipathic alpha-helix and its interaction with the hydrophobic anionic ion-pairing reagents. In addition, due to the excellent resolution of diastereomeric peptide pairs by this novel CZE approach, we have also been able to separate a mixture of these closely-related alpha-helical peptides.     

Capillary zone electrophoresis for simultaneous determination of seven nonsteroidal anti-inflammatory drugs in pharmaceuticals

A simple capillary zone electrophoresis (CZE) method has been developed for analyzing seven nonsteroidal anti-inflammatory drugs (NSAIDs)—sulindac (SU), ketoprofen (KE), indomethacin (IN), piroxicam (PI), nimesulide (NI), ibuprofen (IB), and naproxen (NA). The separation was run using borate buffer (60 mmol L^–1, pH 8.5) containing 13% (v/v) methanol at 20 kV, and detected at 200 nm. Several conditions were studied, including concentration and pH of borate buffer, methanol percentage, and separation voltage. In method validation, the calibration plots were linear over the range 40.0–500.0 mol L^–1. In intra-day and inter-day analysis, relative standard deviations (RSD) and relative errors (RE) were all less than 5%. The limits of detection were 10 mol L^–1 for SU, IN, PI, and 20 mol L^–1 for KE, NI, IB, NA (S/N = 3, sampling 6 s by pressure). All recoveries were greater than 95%. This method was applied to the quality control of six NSAIDs in pharmaceuticals using NI as internal standard (IS). The assay results were within the labeled amount required by USP 25.     

Capillary zone electrophoresis for the determination of light-absorbing anions in environmental samples.

A rapid and simple method for separation and determination of inorganic anions by capillary zone electrophoresis was described. The detection was carried out directly with a diode array detector. The experimental conditions, such as concentration of carrier electrolyte, capillary length, voltage, and temperature were optimized. In order to improve selectivity, different organic modifiers were also investigated. The baseline separation of 10 light-absorbing anions was accomplished within 3.5 min with a background electrolyte consisting of 50 mM sodium tetraborate containing 5% MeOH. Linear plots were obtained in the concentration range of 0.1-10 microg/ml. With sample stacking injection, the quantitation limits of the anions were found to be in the range of 0.02-0.1 microg/ml. The proposed method was successfully applied to the determination of inorganic anions in environmental samples and in effluents of a power plant.     

Capillary zone electrophoresis of rare earth metals with indirect UV absorbance detection

The separation of 14 lanthanides by capillary zone electrophoresis was studied in the background electrolyte containing hydroxyisobutyric acid as complexing counter-ion and creatinine as a UV absorbing coion for indirect detection of lanthanide zones. A complete separation was achieved in less than 5 min and the applicability of the method for the analysis of real samples was demonstrated.     

Capillary zone electrophoresis in methanol: migration behavior and background electrolytes

Nonaqueous (NA) solutions are often used as background electrolytes (BGEs) and NA solvents are added to aqueous BGEs as organic modifiers in capillary zone electrophoresis (CZE), in order to optimize the separations. This can be tricky, however, because the pH* and pK* concepts may be totally different in NA solvents, whereas often less knowledge is available concerning phenomena, such as system zones, applying NA solvents. In this paper, the concepts of pH* and pK* are considered for methanol as a solvent and pK* values are determined for several components in mixtures of water and methanol. With a mathematical model, adapted for calculations in methanol, parameters are calculated describing the fronting or tailing character of peaks and the question of peaks or dips, and the existence of system zones is discussed for pure methanol as a solvent. These aspects are experimentally verified, applying BGEs useful for the separation of cationic species in the indirect UV mode. It can be concluded that the mathematical model developed for aqueous BGEs is applicable to BGEs in methanol, too, and that the behavior of BGEs in methanol is comparable with that in water concerning the fronting or tailing character of peaks and the question of peaks and dips, although the mobilities and pK values can change significantly.     

Capillary zone electrophoresis with electroosmotic flow controlled by external radial electric field.

A new way of regulation of electroosmotic flow (EOF) in capillary zone electrophoresis (CZE) by external electric field has been developed. A set of three high-voltage power supplies is used to form a radial electric field across the capillary wall. One power supply is applied in the usual way as a driving force of CZE and EOF to the ends of the inner capillary compartment dipped into the electrode vessels and filled with background electrolyte. Two power supplies are connected to the ends of the outer low-conductivity coating of the capillary which is formed by the dispersion of copolymer of aniline and p-phenylenediamine in polystyrene matrix. The difference between electric potentials on the outer capillary surface and inside the capillary determines the voltage of radial electric field across the capillary wall and affects the electrokinetic potential at the solid-liquid interface inside the capillary. The effect of magnitude and polarity of external radial electric field on the flow rate of EOF, on the migration times of charged analytes and on the separation efficiency and resolution of CZE separations of synthetic oligopeptides, diglycine, triglycine and octapeptide fragments of human insulin was evaluated. Through the EOF control by external electric field the dynamic effective length of the capillary was obtained and the speed of analysis and resolution of CZE separations of peptide analytes could be optimized.