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PubMed2005

The use of etched, chemically modified, rectangular capillaries as a separation medium for open tubular capillary electrochromatography.

The use of etched, chemically modified, capillaries with a rectangular inner channel for open tubular electrochromatography is investigated. Comparisons of separation capabilities are made between circular and rectangular capillaries undergoing the same etching and chemical modification processes. With the long dimension of the rectangular column aligned in the direction of the optical light path, the relative sensitivities of the two capillary geometries are evaluated. The electrochromatographic properties of two catechins found in tea are investigated on the rectangular etched octadecyl-modified capillary.

Carbon↗

PubMed2001

Capillary zone electrophoresis of proteins with poly(2-hydroxyethyl methacrylate)-coated capillaries: fundamental and applications.

Fused silica capillaries have been modified by atom-transfer radical polymerization (ATRP) to generate covalently bonded polymer films of 2-hydroxyethyl methacrylate. Because the kinetics of ATRP have mainly been investigated in bulk solutions, a GC experiment was set up to examine monomer conversion inside narrow-bore capillaries. It was shown that after 1 to 4 h the reaction was nearly complete. The coating process was further optimized by monitoring EOF, because low EOF indicates high surface coverage. To deal with the very low EOF values, a new approach was used to dramatically reduce the measurement time by overlaying hydrodynamic flow on the electroosmotic flow. The corresponding equations are derived separately in detail. Capillaries were then coated under optimum conditions with linear or cross-linked polymer films. The EOF was reduced over a wide range of pH values. A long-term reproducibility test with both types of functionalization showed that the efficiency of the linear polymer coating decreased significantly over time. With cross-linked films, however, the efficiency even increased. Relative standard deviations for protein migration times were also much lower in cross-linked coated capillaries. Highly efficient separations could be performed for basic and acidic proteins in acidic media, and for the latter even in basic media.

Electrophoresis, Capillary↗

PubMed1993

Capillary zone electrophoresis and packed capillary column liquid chromatographic analysis of recombinant human interleukin-4.

Capillary zone electrophoresis (CZE) and packed capillary column liquid chromatography (micro-LC) have been applied to the analysis of the recombinant human protein interleukin-4 (rhIL-4). Separations for both the parent protein and its enzymatic digest were developed for the purpose of characterizing protein purity and identity. CZE separations of the intact protein were investigated over the pH range of 4.5 to 8.0 using uncoated fused silica capillaries. Gradient reversed-phase micro-LC was performed using 0.32 mm packed capillary columns at flow-rates of 5-6 microliters/min. Emphasis was placed on the ability of these methods to separate close structural variants and degradation products of the protein. Peptide mapping of the tryptic digest of rhIL-4 using a combination of CZE and micro-LC provided complimentary high resolution methods for establishing protein identity. Reproducible separations were achieved using sub-picomol amounts of sample. The advantages and problems encountered with these two techniques for characterizing rhIL-4 were assessed.

Capillary Action↗

PubMed1996

Separation of tetracyclines by high-performance capillary electrophoresis and capillary electrochromatography.

Separations of various tetracycline mixtures by high-performance capillary electrophoresis (HPCE) and a new form of electrochromatography (CEC) are compared. The new CEC method involves etching the inner wall of the capillary surface with an appropriate reagent (ammonium dihydrogen fluoride) in order to produce a significant increase in surface area. The etched surface is then modified by a silation/hydrosilation reaction sequence to first produce a hydride intermediate which is then further reacted to attach a C18 moiety. The bare and hydride capillaries are tested under HPCE conditions while the C18 capillary functions in the CEC mode. The effects of pH and the presence of an organic modifier (methanol) are also studied. Detection limits ( < 10 micrograms/ml) are comparable to previous HPLC and HPCE results. Resolutions for mixtures which simulate real analytical problems are equal to or better than those reported for separations on polymeric and diol columns by HPLC and in earlier studies by HPCE and MECC.

Chromatography, High Pressure Liquid↗

PubMed2004

Use of dynamically coated capillaries for the determination of heroin, basic impurities and adulterants with capillary electrophoresis.

Rapid, precise, accurate, and reproducible methodology using capillary electrophoresis (CE) with dynamically coated capillaries for the analysis of heroin and its basic impurities and adulterants is presented. Highly selective determination of the above solutes is obtained by analyzing the same sample preparation by two CE methods. For the determination of heroin, its basic impurities and basic adulterants, dynamic coating of the capillary surface is accomplished using a commercially available reagent kit with an added cyclodextrin ((CD) polycation coating followed by polyanion coating with dimethyl-beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin). The addition of a cyclodextrin to the run buffer significantly improves the separation of these solutes. Neutral, acidic, and weakly basic adulterants which migrate near or after t0 do not interfere with the more mobile basic solutes. The determination of neutral, acidic, and weakly basic adulterants in heroin is accomplished using a modification of the above commercially available reagent kit. After first coating with a polycation, a negative coating is obtained using a surfactant sodium dodecyl sulfate. Micellar electrokinetic chromatography (MEKC) with dynamically coated capillaries gives an excellent separation of the neutral, acidic, and weakly basic solutes, with considerably shorter run times compared to conventional MEKC. In addition for this system, most basic solutes in heroin have longer migration times than the uncharged and acidic compounds.

Electrophoresis, Capillary↗

PubMed2005

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).

Electrophoresis, Capillary↗

PubMed2006

Construction and analytical application of an on-column photo reactor for improved detection of iron-species as plant metabolites in capillary flow injection and capillary electrophoresis.

An on-column photo reactor for CE, which is constructed from UV-transparent capillaries and a small Pen-Ray UV lamp, is applied to the analysis of small, non-covalent iron-species. These iron-species, e.g. phytosiderophores (PS) in grasses and the non-protein amino acid nicotianamine (NA), play an important role in plant metabolism. The photo reactor is placed directly in front of the on-column absorbance detector, illuminating only some centimeters of the capillary. The photo reactor is used for capillary electrophoresis (CE) and also for capillary flow injection analysis (CFIA). Photoinduced sensitivity changes for model iron-species and for plant extracts are investigated, using UV detection and capacitively coupled contactless conductivity detection (C(4)D). The detection sensitivity for iron-species is enhanced in CFIA; the enhancement factor depends on the type of iron-species. In CE, the sensitivity of iron-species is kinetically dependent on the type of iron-species, decreasing with both detectors, but a photo reaction product is detectable. The relationship between irradiation window length and sensitivity is investigated quantitatively using EDTA-Fe(III). Pure ligands without iron are little affected by the photo reaction in both CFIA and CE. In CE, the detector signals of iron-species in real plant samples are selectively decreased with the proposed photo reactor, thus enabling a simple screening method for such photoactive iron-species.

Electrophoresis, Capillary↗

PubMed2002

On-line drug-metabolism system using microsomes encapsulated in a capillary by the sol-gel method and integrated into capillary electrophoresis.

A novel microsome-encapsulation technique using the sol-gel method was developed for the on-line drug-metabolism analytical system integrated into capillary electrophoresis. This analytical system allows both the metabolism of drugs and the determination of the metabolites in a single capillary simultaneously. Microsomes isolated from rat liver were encapsulated in tetramethoxysilane-based silica matrices within a capillary in a single step under mild conditions. The availability of this system was evaluated using UDP-glucuronyltransferase, which is one of the most important microsomal enzymes. 4-Nitrophenol and testosterone, which were metabolized by the different isoforms of UDP-glucuronyltransferase, were used as substrates. The resultant monolithic reactor showed enzymatic activity at the same level as that of the soluble form. The following separation of the unreacted substrates and metabolites in the same capillary also showed high selectivity. Furthermore, the sample amount required for one analysis decreased more than 3 orders of magnitude from conventional reaction schemes in free solution. This on-line system could largely simplify the laborious procedures which were needed in conventional analytical schemes.

Animals↗

PubMed2001

Capillary electrophoresis using a surfactant-treated capillary coupled with offline matrix-assisted laser desorption ionization mass spectrometry for high efficiency and sensitivity detection of proteins.

A method of combining capillary electrophoresis (CE) using a surfactant-modified capillary with matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) is described for protein analysis. The CE-MALDI-MS coupling is based on CE fraction collection of nanoliter volume samples in less than 5 microl of dilute acid. This offline coupling does not require any special instrumentation and can be readily performed with commercial instruments. Protein adsorption during CE separation is prevented by coating the capillary with the surfactant didodecyldimethylammonium bromide. This surfactant binds strongly with the capillary wall, hence it does not desorb significantly to interfere with subsequent MALDI-MS analysis. It is shown that the use of a dilute acid for CE fraction collection is advantageous in lowering the detection limit of MALDI-MS compared to using an electrophoretic buffer. The detection limit for proteins such as cytochrome c is 23 fmol injected for CE, or 1.2 fmol spotted for MALDI-MS. This sensitivity is comparable to alternative CE-MALDI-MS coupling techniques using direct CE sample deposition on the MALDI target. In addition, the fraction collection approach has the advantage of allowing multiple reactions to be carried out on the fractioned sample. These reactions are very important in protein identification and structure analysis.

Cytochrome c Group↗

PubMed2002

Interaction between netropsin and double-stranded DNA in capillary zone electrophoresis and affinity capillary electrophoresis.

Capillary zone electrophoresis (CZE) and affinity capillary electrophoresis (ACE) were applied to study the interaction between netropsin and a 14mer double-stranded DNA (dsDNA). The use of a polyacrylamide coated capillary can suppress the electroosmotic flow (EOF) and the adsorption of DNA onto the wall. Better analysis of the DNA was achieved in a coated capillary upon Tris-acetate. In CZE, the peak width broadened due to the affinity interaction between dsDNA and netropsin. In ACE, o-toluic acid, a negatively charged molecule was used as the indicator to monitor the changes of EOF when netropsin was added to the running buffer. The 14mer dsDNA showed different mobilities upon various concentrations of netropsin due to the affinity interaction between the dsDNA and netropsin. The binding constants of this interaction were (1.07 +/- 0.10) x 10(5) M(-1) calculated from CZE and (4.75 +/- 0.30) x 10(4) M(-1) from ACE using a Scatchard plot. The binding stoichiometry was 1:1 calculated from CZE which was superior to ACE in this study.

Base Sequence↗

PubMed2003

Use of short-end injection capillary packed with a glycopeptide antibiotic stationary phase in electrochromatography and capillary liquid chromatography for the enantiomeric separation of hydroxy acids.

A new chiral stationary phase (CSP) was prepared by reacting MDL 63,246 (Hepta-Tyr), a glycopeptide antibiotic belonging to the teicoplanin family, with 5-microm diol-silica particles. The CSP mixed with 5-microm amino silica particles (3:1) was packed into 75-microm fused-silica capillaries for only 6.6 cm and used for electrochromatographic experiments analyzing several hydroxy acid enantiomers. A reversed electroosmotic flow carried both analytes and mobile phase towards the anode in a short time (1-3 min), being baseline resolved all the studied analytes. In order to achieve the fastest enantiomeric resolution of the studied hydroxy acids, the effect of several experimental parameters such as mobile phase composition (organic modifier type and concentration, pH of the buffer and ionic strength), capillary temperature and applied voltage on enantioresolution factor, retention time, enantioselectivity were evaluated. The packed capillary column allowed the separation of mandelic acid enantiomers in less than 72 s with resolution factor Rs=2.18 applying a voltage of 30 kV and eluting with a mobile phase composed by 50 mM ammonium acetate (pH 6)-water-acetonitrile (1:4:5, v/v). The CSP was also tested in the capillary liquid chromatography mode resolving all the studied enantiomers applying 12 bar pressure to the mobile phase [50 mM ammonium acetate (pH 6)-water-methanol-acetonitrile, 1:4:2:3, v/v)], however, relatively long analysis times were observed (12-20 min).

Acids↗

PubMed1997

On-line post-capillary affinity detection of immunoglobulin G subclasses and monoclonal antibody variants for capillary electrophoresis.

Human immunoglobulin G (IgG) subclasses each play a unique role in an immune response to foreign antigens. Three of the human IgG subclasses have distinct electrophoretic mobilities and are resolved by capillary zone electrophoresis (CZE). A post-capillary reactor is constructed to allow on-line addition of fragment B (of protein A)-fluorescein to form affinity complexes with separated IgG subclasses. Post-capillary affinity detection provides selective identification of human IgG subclasses and illustrates the effect of affinity binding constant on detection sensitivity. Additionally, post-capillary affinity detection for CZE facilitates rapid and selective heterogeneity analysis of mouse monoclonal anti-(human-alpha 1-antitrypsin) and anti-human follicle stimulating hormone in complex sample matrices. A constant mobility difference is observed between the antibody isoforms, likely the result of charge heterogeneity due to deamination, degradation or variation in sialic acid content.

Animals↗

PubMed1998

Application of capillary zone electrophoresis-inductively coupled plasma mass spectrometry and capillary isoelectric focusing-inductively coupled plasma mass spectrometry for selenium speciation.

The high resolution potential of capillary electrophoresis (CE) makes CE techniques valuable for separations of selenium species, both, organic and inorganic. Such effective separations of charged species are possible using the CZE mode (Z = zone). Very powerful separations can be achieved by capillary isoelectric focusing, when analysing molecules showing different isoelectric points (pI) values, such as organic Se species. Inductively coupled plasma (ICP) MS is an element-specific multi-element detector, providing extremely low detection limits. The combination of CE with ICP-MS promises a powerful tool for metal speciation. Therefore, an online hyphenation of CE [using capillary zone electrophoresis (CZE) or capillary isoelectric focusing (cITP) model] with ICP-MS, which was developed earlier, was modified and applied to selenium speciation. Separation was differentiated from the detection step during hyphenation. This resulted in short separation times (10 min) and a subsequent detection step lasting 100 s. Firstly, a CZE method was applied, providing a separation of six Se species of interest in one run [Se(IV), Se(VI), selenium carrying glutathione (GSSeSG), selenomethionine (SeM), selenocystine (SeC), selenocystamine (SeCM)]. This CZE method used an alkaline background electrolyte (Na2CO3/NaOH). The Se species were separated sufficiently from each other. Detection limits were calculated as 10 or 20 micrograms Se l-1 for inorganic Se species and 30-50 micrograms Se l-1 for organic Se species. The method was applied to standard mixtures and body fluids like human milk and serum. Secondly, a cIEF separation (pH range 2-10) was employed for organic Se species only in parallel. Detection limits were around 10-30 micrograms Se l-1. The method was applied to standard mixtures and body fluids like human milk and serum.

Electrophoresis, Capillary↗

PubMed1999

Noncompetitive immunoassays using protein G affinity capillary chromatography and capillary electrophoresis with laser-induced fluorescence detection.

A new and simple approach to perform immunoassay using protein G affinity capillary chromatography and laser-induced fluorescence detection was described. A noncompetitive assay for monoclonal anti-bovine serum albumin (BSA) was used to test the performance of the system. Fluorescein isothiocyanate labeled BSA was used as a tracer to determine anti-BSA in pM level. Capillaries with inner diameter of 150 microns were packed with recombinant protein G-bound perfusive support. The packed capillary was used to capture the immunocomplexes, which were subsequently desorbed by 100 mM glycine (pH 9.0). Open tube capillary electrophoresis-based immunoassay (CEIA) for anti-BSA was also performed. Using standard samples, calibration curves for anti-BSA was established in both assays. Compared with CEIA, this system improved the concentration sensitivity for about 100-fold due to the pre-concentration of immunocomplex in the protein G column, while the mass sensitivity was similar in the two methods.

Antibodies, Monoclonal↗

PubMed2000

Dynamically-coated capillaries allow for capillary electrophoretic resolution of transferrin sialoforms via direct analysis of human serum.

Transferrin sialoforms with fewer than three sialic acid residues (carbohydrate deficient transferrin; CDT) have been implicated as a marker of certain liver pathologies. Transferrin sialoforms in human sera from alcoholic and non-alcoholic patients was analyzed by capillary electrophoresis (CE) using diaminobutane (DAB) to dynamically-coat the capillary wall to minimize protein-wall interactions. Using a DAB concentration of 3 mM, transferrin sialoforms were adequately resolved to allow for direct detection of CDT without extensive treatment of the sera. Serum immunoglobulins, which migrated close to the CDT region, were removed via subtraction with protein A, enhancing the detection of CDT. The reproducibility of sialoform separation in dynamically-coated capillaries was found to be acceptable with run-to-run relative standard deviation values of 0.15% for a sample on a given day and 0.29+/-0.06% for four samples day-to-day. These results suggest that dynamic-coating approaches may provide a simple alternative to the use of covalently-coated capillaries for the CE separation of complex samples.

Alcoholism↗

PubMed1997

Capillary electrophoresis of peptides and proteins at neutral pH in capillaries covalently coated with polyethyleneimine.

Two procedures for the derivatization of the inner wall of fused-silica capillaries for the analysis of peptides and proteins by capillary electrophoresis (CE) at neutral pH are presented. In the first procedure, polyethyleneimine (PEI) is covalently attached to the capillary wall. In the second procedure, PEI is additionally cross-linked. We present analysis of standard peptides and proteins by CE using the coated capillaries. These coatings will have application for the separation of protein complexes at neutral pH, prior to analysis by electrospray mass spectrometry.

Buffers↗

PubMed2000

Reversed-phase capillary liquid chromatography coupled on-line to capillary electrophoresis immunoassays.

Capillary reversed-phase liquid chromatography (RPLC) was coupled on-line to competitive capillary electrophoresis immunoassay (CEIA) to improve concentration sensitivity of the competitive CEIA and to provide a means for detecting multiple species that cross-react with antibody. A competitive CEIA for glucagon was used for demonstration of this technique. Five-microliter samples were injected onto a 4-cm-long by 50-micron-i.d. RPLC column. Sample was desorbed by gradient elution, mixed on-line with fluorescently labeled glucagon and anti-glucagon, incubated in a continuous-flow reaction capillary, and analyzed by capillary electrophoresis with flow-gated injection and laser-induced fluorescence detection. Electrophoretic analysis of the reactor stream was performed every 1.5 s, allowing nearly continuous monitoring of the RPLC separation. Preconcentration achieved by RPLC allowed improvement in the detection limit from 760 to 20 pM. Addition of the RPLC column also allowed multiple cross-reactive species to be differentiated by first separating them chromatographically and then detecting them with the immunoassay. The technique was used to measure glucagon secretion from single islets of Langerhans and to differentiate cross-reactive forms of glucagon with one assay.

Animals↗

PubMed1997

Noncovalent polycationic coatings for capillaries in capillary electrophoresis of proteins.

The adsorption of proteins with net positive charges (pI > pH) on the walls of fused-silica capillaries is a common problem in the analysis of proteins by capillary electrophoresis. This paper explores the use of polycationic polymers as noncovalent coatings to limit this problem. The behavior of three sets of proteins was compared using uncoated and coated capillaries: (i) a protein charge ladder obtained by acetylation of lysozyme (EC 3.2.1.17); (ii) a protein charge ladder obtained by acetylation of carbonic anhydrase II (EC 4.2.1.1); (iii) a test panel of proteins with a range of values of molecular weight and pI. Four polycationic polymers were examined: polyethylenimine (PEI; MWav = 15000), Polybrene (MWav = 25000), poly(methoxyethoxyethyl)ethylenimine (MWav = 64000), and poly(diallyldimethylammonium chloride) (MWav = 10000). Detection of proteins with high pI was readily achieved using the first three of these polycationic polymer coatings but not with the poly(diallyldimethyl-ammonium chloride). Examination of the stability of these coatings indicates that they are robust: the change in electroosmotic flow was less than 10% for 25 replications of the same separations, using capillaries coated with PEI or Polybrene. This study demonstrates that the charge ladder obtained by acetylation of lysozyme is a good model with which to test the efficiency of polycationic coatings. A study of the electrophoretic mobilities of the members of this charge ladder at pH 8.3 determined the effective charge of lysozyme (Zp(0) = +7.6 +/- 0.1) and established the acidity of the alpha-ammonium group of lysozyme (pKa = 7.8 +/- 0.1). Results from the test panel of proteins suggest that protein adsorption is mainly driven by electrostatic interactions.

Acetylation↗