bluuebunny/crossref_metadata_2025_split huggingface dataset:
A new type of pH‐ and detergent‐stable coating for elimination of electroendosmosis and adsorption in (capillary) electrophoresis
Authors: Hjertén Stellan , Kubo Kanenobu 
Published: May 2005

AbstractWe describe a method to coat fused silica, quartz, or glass electrophoresis tubes with hydrophilic polymers, such as methyl cellulose or dextran, in order to eliminate electroendosmosis and adsorption of solutes onto the tube wall. The coating was not split off upon exposure to a 5% solution of sodium dodecyl sulfate (SDS) at pH around 12 during a period of sixteen days or to 0.5 M HCI (pH 0.3) during 10 days. The high stability of the coating, which is attached to the silica surfaceviaSi‐O‐Si‐C bonds, permits electrophoresis experiments at extreme (besides neutral) pH values and allows short washes of the electrophoresis tubes between the runs with 0.05 M sodium hydroxide (pH 12.7) containing 5% SDS, and with 1 M HCI. These washing solutions release efficiently those solutes which have a tendency to adsorb onto the coating, which is a prerequisite for reproducible runs. The quality of the coating was strongly affected by the surface structure of the commercial fused silica tubing, which may vary from batch to batch. Attempts to standardize the surface structure of any tubing by treatments with NaOH and HCI to obtain a coating, the quality of which is independent of the original surface structure, were not successful. Therefore, it may be necessary to modify certain details of the coating procedure for each new batch of tubing to obtain optimum stability. Alternative coating procedures are therefore described, one of which is of general applicability (see last paragraph in Section 4).
Studies in capillary zone electrophoresis with a postcolumn multiple capillary device for fraction collection and stepwise increase in electroosmotic flow during analysis
Authors: Nashabeh Wassim , Smith Joel T. , Rassi Ziad El 
Published: May 2005

AbstractA new approach involving the stepwise increase in electroosmotic flow during analysis in capillary zone electrophoresis is introduced and evaluated in the rapid separations of proteins and peptides. The stepwise increase in electroosmotic flow is based on the principle of coupled capillaries in series having different flow characteristics, a concept that was introduced recently by our laboratory. To produce stepwise changes in electroosmotic flow during analysis, a post‐column multiple capillary device, which allowed the switching between several coupled capillary systems, was designed and constructed in‐house. The utility of the multiple capillary device was also demonstrated and extended to fraction collection of separated analytes in short capillary segments. The fraction collection in capillaries facilitated the quantitative transfer of the collected fractions to high performance liquid chromatography (HPLC) for further analysis or to mass spectrometry (MS) for structural determination. The off‐line combination of capillary zone electrophoresis with HPLC or MS utilized commercial instruments without the need of expensive interfacing designs.
Use of small‐diameter capillaries for increasing peptide and protein detection sensitivity in capillary electrophoresis‐mass spectrometry
Authors: Wahl Jon H. , Goodlett David R. , Udseth Harold R. , Smith Richard D. 
Published: May 2005

AbstractThe use of small ID capillaries is shown to provide a substantial increase in sensitivity for capillary electrophoresis‐electrospray ionization/mass spectrometry (CE‐ESI/MS). In a comparison using capillaries ranging from either 100 to 10 μm or 50 to 5 μm ID and chemically modified with aminopropylsilane, a 25‐ to 50‐fold increase in sensitivity was observed for both peptide and protein mixtures. This enhanced solute sensitivity allowed the detection of approximately 150 attomoles of melittin (2845 Da) with selected ion monitoring and 600 attomoles of carbonic anhydrase (29157 Da) while scanning for CE‐MS with a quadrupole mass spectrometer. For the protein mixture, mass spectra of sufficient quality for precise molecular weight determination (⩽ 0.05%) were obtained for 600 attomole injections using a 5 μm ID capillary. The increase in sensitivity with small capillary diameters can be primarily attributed to a reduced mass flow rate of buffer and other background constituents into the electrospray source, which allows for greater sample ionization efficiency. A model that qualitatively accounts for the results is presented, but quantitative agreement is precluded due to difficulties in accounting for contributions due to a liquid sheath flow used with the electrospray source. The model accounts for the observation that the ESI/MS appears to function as a concentration‐sensitive detector under many conditions using large‐diameter capillaries. A transition occurs, however, to a regime where the ESI/MS functions as a mass flow‐sensitive detector for small‐diameter capillaries, where the ESI current is limited by the rate of delivery to the ESI source of charge carrying species in solution. These results suggest peptide and protein analysis at low attomole and subattomole levels should be obtainable with alternative types of mass spectrometers.
Using capillary electrophoresis in the optimization of a carboxypeptidase Y catalyzed transpeptidation reaction
Authors: Vinther Anders , Adelhorst Kim , Kirk Ole 
Published: May 2005

AbstractA peptide amide, R‐Arg‐NH2, was produced by carboxypeptidase Y (CPDY)‐catalyzed transpeptidation of a peptide, R‐Ala‐OH in presence of a large excess of Arg‐NH2. Baseline separation of R‐Ala‐OH and R‐Arg‐NH2 was achieved by free solution capillary electrophoresis (CE) analysis. With CE the reactions could be closely followed with an analysis frequency of 3–6 h−1. Due to a low consumption of sample per CE analysis (1–5 nL introduced, 5–6 μL in the sample vial), the reactions were performed in 100 and 250 μL volumes. Consequently, the optimization experiments consumed limited amounts of enzyme and substrate only. At optimized experimental conditions approximately 90% conversion of the starting peptide, R‐Ala‐OH, to R‐Arg‐NH2 was achieved.
A model for the mobility of single‐stranded DNA in capillary gel electrophoresis
Authors: Luckey John A. , Smith Lloyd M. 
Published: May 2005

AbstractThe electrophoretic mobility of single‐stranded DNA in denaturing polyacrylamide gel‐filled capillaries is analyzed as a function of the applied electric field. The resultant mobility plots are complex functions of the fragment size and electric field. Traditionally, these plots are separated into three mobility regimes corresponding to Ogston sieving, reptation without stretching and reptation with stretching theories. However, none of these theories accurately models the variations in mobility that we observe with electric field strength. As a result, we propose a modification of the Ogston sieving theory which accounts for the stretching of migrating DNA molecules in the direction of the electric field. This theory assumes that the applied electric field in conjunction with the gel matrix distorts the DNA, altering the effective size of the migrating molecule. The stretched DNA offers a smaller cross‐section to the gel pore and thus sieves as though it were a smaller molecule. In this modified Ogston theory, the electrophoretic mobility depends only on the applied electric field, the size of the fragment, and constants which are independent of size and field strength. The modified Ogston equation accurately predicts the mobilities of DNA fragments in all three mobility regimes9 providing a single, simple model to account for all of the observed behavior.
Capillary zone electrophoresis of large DNA
Authors: Guszczynski Tadeusz , Pulyaeva Helena , Tietz Dietmar , Garner Mark M. , Chrambach Andreas 
Published: May 2005

AbstractCapillary zone electrophoresis (CZE) of DNA 23.1 to 48.5 kb in length in polyacrylamide solutions of several concentrations provides evidence for polymer concentration and DNA length‐dependent stretching and orientation of these species and suggests an effective separation at a polymer concentration of about 0.6%. Applying a 0.1 % polyacrylamide concentration to the λ‐phage DNA ladder, at least 5 components are separated; separation improves with lowering of the field strength to 2 V/cm and, correspondingly, extended duration of CZE. Saccharomyces pombe chromosomal DNA separates into 3 major components on CZE at high field strength (270 V/cm) in 0.9% polyacrylamide solution, confirming a previous finding made on electrophoresis in a 1.1 mm ID tube at low field strength. However, the finding is limited to one source of the DNA plug, and the chromosomal identity of the components remains unknown. Methodological problems in the CZE of large DNA relate to the need for extended duration of pressure injection if absorbance detection is applied, the need to define the starting zone after extended pressure injection, the need to melt and digest agarose plugs prior to loading, and related needs for thermostating of the sample chamber and for software compatible with low voltage operation.
Capillary electrophoresis/mass spectrometry determination of inorganic ions using an ion spray‐sheath flow interface
Authors: Huggins Thomas G. , Henion Jack D. 
Published: May 2005

AbstractThe determination of inorganic cations and anions by capillary electrophoresis/mass spectrometry (CE/MS) is reported using an ion spray‐sheath flow interface coupling. A twelve‐component synthetic mixture of cations which included the positive ions of K, Ba, Ca, Mn, Cd, Co, Pb, Cr, Ni, Zn, Ag, and Cu was loaded into the capillary column at levels ranging from 30 to 300 pg, separated by CE, and detected by indirect UV and in the full‐scan (m/z 35–450) positive ion CE/MS mode using an aqueous buffer containing 30 mM creatinine and 8 mM α‐hydroxyisobutyric acid, pH 4.8. Creatinine forms adducts with the cations which are observed in the gas phase and requires rather high (120 electron volts) declustering energy to dissociate. This produces a reduction in charge state to form the free, singly charged, inorganic cations which are observed in the mass spectra. CE/MS analysis of an aqueous acidic extract of used aircraft engine oil revealed high levels of lead as well as lower levels of chromium and nickel. CE‐indirect UV analysis of a synthetic mixture containing 300 pg each of 11 inorganic ions, which included the anions of Br, Cl, NO2, NO3, S2O3, N3, SCN, SO4, SeO4, oxalate, and MoO4, is shown. The running buffer which affected this separation contained 5 mM ammonium dichromate, 10 mM ammonium acetate, and 20 mM diethylenetriamine at pH 9.3. Although indirect UV detection revealed good separation of these anions, CE/MS analysis of this mixture was complicated by interfering ion current signals from the cluster ions formed by the interaction between the additives and the analytes. Thus only three of these singly charged anions, e.g. Br, SCN, and HSeO4, could be satisfactorily detected in this mixture by CE/MS.
Acetylator phenotyping via analysis of four caffeine metabolites in human urine by micellar electrokinetic capillary chromatography with multiwavelength detection
Authors: Guo Ruichen , Thormann Wolfgang 
Published: May 2005

AbstractThe simultaneous analysis of 5‐acetylamino‐6‐formylamino‐3‐methyluracil (AFMU), 5‐acetylamino‐6‐amino‐3‐methyluracil (AAMU), 1‐methylxanthine (1X) and 1‐methyluric acid (1U), the four urinary metabolites of caffeine employed for acetylator phenotyping in man, by micellar electrokinetic capillary chromatography (MECC) is reported. Using on‐column fast scanning multiwavelength detection is shown to permit unambiguous identification of AAMU, AFMU, 1X and 1U in the three‐dimensional electropherograms obtained with both direct urine injection or application of extracted urine. The phenotype status of a number of healthy volunteers, some with known acetylator status, were determined using peak height ratios of AFMU/1X, (AFMU + AAMU)/1X and compared to those obtained by high performance liquid chromatography (HPLC) employing the AFMU/1X peak height ratio. Another set of MECC data was obtained after deliberate conversion of AFMU to AAMU, the results being expressed by AAMU/1X, AAMU/(1U + 1X) or AAMU/(AAMU + 1U + 1X) ratios. The data suggest that most reliable data are obtained when phenotyping is conducted under conditions of complete conversion of AFMU to AAMU. Phenotyping employing caffeine and analysis of its urinary metabolites by MECC is shown to be simple and, with automated instruments, potentially suitable for routine use and large‐scale population analysis. The agreement of MECC data obtained from three different instruments demonstrates the high level of maturity of this emerging technology.
On the use of dimensionless parameters in acid‐base theory. III. The molar buffer capacity of trivalent protolytes
Authors: Rilbe Harry 
Published: May 2005

AbstractThe general equation for the buffer capacity of trivalent weak acids is derived by way of the mean valence. It is valid for trivalent bases and ampholytes as well. Special interest is devoted to protolytes with symmetrically spaced pK values since both phosphoric acid and citric acid have this property. The symmetry implies a mathematical simplification which makes it possible to deduce the locations and magnitudes of the maxima and minima. Thus it is shown that the buffer capacity curve has three maxima and two minima for ΔpK values bigger than 1.29.
Quantitative analysis of gel electrophoretograms by image analysis and least squares modeling
Authors: Vohradský Jiří , Pánek Josef 
Published: May 2005

AbstractA computer‐aided quantitative method for a complex analysis of gel electrophoretograms is presented. The analysis consists of several steps: (i) determination of the background image by methods of mathematical morphology and its subtraction from the gel image, (ii) selection of an appropriate part of the gel lane including curved lanes and lanes with a nonuniform width, (iii) computation of the lane densitogram by averaging several lane‐parallel scans, (iv) decomposition of the lane densitogram into component bands using a data selecting algorithm and Marquardt's minimizer. Several different functions for component bands are utilized. It is shown that the densitogram can be decomposed into component bands with reasonable accuracy only if an appropriate model function is chosen. The algorithms are tested on several different gel electrophoretograms which show typical features as a nonuniform background, curved lanes, an asymmetrical band shape and a superposition of small bands on the shoulders of big ones. It is shown that overlapped bands are best approximated by an asymmetrical Gaussian curve and an asymmetrical Gauss‐Cauchy function. Linear response to the serial dilution of the protein sample is tested.
Towards stoichiometric silver staining of proteins resolved in complex two‐dimensional electrophoresis gels: Real‐time analysis of pattern development
Authors: Rodriguez Lewis V. , Gersten Douglas M. , Ramagli Louis S. , Johnston Dennis A. 
Published: May 2005

AbstractToward the ultimate goal of deriving quantitative protein data from silver‐stained proteins resolved in complex two‐dimensional electrophoresis gels we have performed computerized real‐time analysis during pattern protein “silver stain” development. Three points emerged from this study: (i) Development time is a far more important variable than originally envisioned. (ii) Previous studies which, on the basis of a single development time, sought to relate silver stain slope and threshold properties to amino acid composition are non‐informative since it will be shown that slope values change markedly with development time, (iii) The combination of real‐time analysis and total protein load consideration allows, for the first time, protein “spots” to be grouped into categories, which in turn aid in quantification.
Characterization of proteins by capillary electrophoresis in fused‐silica columns: Review on serum protein analysis and application to immunoassays
Authors: Chen Fu‐Tai A. , Sternberg James C. 
Published: May 2005

AbstractProtein mixtures can be characterized in terms of their separations by capillary electrophoresis (CE). The separation of proteins by CE is performed in untreated fused‐silica columns. Model proteins and complex protein mixtures with pI values ranging from 4.0 to 11.0 are separated in such columns in less than 10 min in the presence of phosphate buffer with a pH between 4.0 and 9.0. The application of CE separation procedures for routine analysis of protein in serum, urine, and cerebrospinal fluid in borate‐based buffer is also demonstrated. The detection of protein in CE is usually based on the intrinsic ultraviolet (UV) absorbance of the peptide bond at or near 200 nm, which provides a detection limit of about 10−5 M. The same protein separation procedures can also be applied to immunochemical reaction systems in which one component is labeled. Thus, an antigen analyte, or the antibody to the analyte, may be labeled with a fluor and detected by laser‐induced fluorescence (LIF). With a fluorescent‐labeled reactant, the use of LIF detection further extends the detection limit to 10−11 M. The CE separation technique for proteins provides a means to separate the bound and free species of the labeled antigen or antibody without the use of a solid support. The application of these separation techniques in conjunction with laser‐induced fluorescence detection to make possible the homogeneous immunochemical measurement of species at concentrations in the range of 10−9 to 10−10 M is shown.
Application of dynamic capillary isoelectric focusing to the analysis of human hemoglobin variants
Authors: Molteni Sarah , Frischknecht Hannes , Thormann Wolfgang 
Published: May 2005

AbstractCapillary isoelectric focusing (CIEF) with electroosmotic zone displacement of normal and pathological hemoglobins (Hb) is reported. CIEF is performed in untreated, open‐tubular, fused‐silica capillaries of 75 μm internal diameter using methylcellulose for dynamic column conditioning. After direct injection of hemolysates mixed with carrier ampholytes, high resolution separation of Hb variants, including Hb A1c, A, F, D, S, E and A2, is obtained, this permitting unambiguous characterization of Hb patterns of normal adults, newborns, patients with diabetes, different hemoglobinopathies and thalassemia syndromes. Qualitatively, the CIEF data compare well with those obtained by gel isoelectric focusing and high‐performance liquid chromatography. CIEF is demonstrated to be a simple, rapid and fully instrumental approach to Hb analysis. Run times of less than 20 min make CIEF an attractive method for routine Hb investigations and screening programs.
Separation and sequencing of familiar and novel murine proteins using preparative two‐dimensional gel electrophoresis
Authors: Merrick B. Alex , Patterson Rachel M. , Witcher Lora L. , He Chaoying , Selkirk James K. 
Published: May 2005

AbstractStrategies are needed for rapid protein isolation in order to identify disease‐related proteins and facilitate the design of oligonucleotides for further molecular inquiry. In our laboratory, C3H10T1/2 murine fibroblasts have been found to express a variety of proteins in various subcellular fractions which are relevant to experimental transformation and carcinogenesis. Preparative two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) procedures were developed to identify major cytoplasmic proteins by electroblotting and microseqencing. Isoelectric focusing tube gels were enlarged to 6 mm ID to accommodate larger protein loads at 0.5 to 2 mg protein. Separated proteins were electrotransferred from 6 mm thick slab gels onto 0.22 μ polyvinylidene difluoride membranes. Nearly 100 prominent blotted proteins were stained with Coomassie Brilliant Blue between pI 4.5–7.0 and 18–106 kDa and, of these, 27 prominent and well‐resolved proteins were selected for sequencing. Sequences of 14 to 24 amino acid residues in length were obtained from 11 proteins which were identified from computerized databases. Some of these identified proteins had structural or enzymatic functions while others had only recently been discovered, including a newly reported Hsp 70 class member and a novel calcium‐binding protein, reticulocalbin. The new heat shock protein has a molecular mass of 75 kDa and has been designated as Grp75, PBP74, CSA or p66mol‐1 in mice and humans with purported roles in transformation and antigen processing. Reticulocalbin is an endoplasmic reticular protein which contains six domains of the EF‐hand motif associated with high‐affinity calcium‐binding proteins. It may be involved in protein transport and luminal protein processing. In addition, sequences of 5 to 11 residues in length were also obtained from six other unidentified proteis. Thus, we have found that preparative 2‐D PAGE serves as a powerful one‐step purification method for protein isolation and characterization from an important in vitro murine model for the study of carcinogenesis.
Improved separation of diastereomeric derivatives of enantiomers by a physical network of linear polyvinylpyrrolidone applied as pseudophase in capillary zone electrophoresis
Authors: Schützner Wolfgang , Caponecchi Gabriele , Fanali Salvatore , Rizzi Andreas , Kenndler Ernst 
Published: May 2005

AbstractDiastereomeric analytes were separated using capillary zone electrophoresis with polyvinylpyrrolidone as polymeric additive to the buffer solution. As test substances derivatives of D‐ and L‐ α‐amino and α‐hydroxy acids formed by reaction with (+)‐O,O′‐diacetyl‐ and (+)−O,O′‐dibenzoyl‐L‐tartaric anhydride, respectively, were used. The physical network formed by the linear polymer is supposed to act as a kind of pseudophase. It was found that the network affects the mobility of diastereomeric compounds to a different extent, enchancing the selectivity of the system. In nearly all cases of aromatic acids the diastereomer carrying the D‐amino acid was more strongly retained than the L‐isomer, as opposed to the situation with aliphatic acids.
About some aspects of the use of charged cyclodextrins for capillary electrophoresis enantioseparation
Authors: Chankvetadze Bezhan , Endresz Gabriele , Blaschke Gottfried 
Published: May 2005

AbstractFree capillary zone electrophoresis with the negatively charged polyanion of the β‐cyclodextrin sulfobutyl ether (SBE‐β‐CD) as a chiral additive was used for the resolution of basic racemic drugs. High enantioselectivity was established for some racemic compounds using extremely low (micromolar) concentrations of the chiral additive. The dependencies of the migration times and the selectivity of the enantioseparation on the concentration of the chiral additive and the pH of the run buffer were studied. Examples of the chiral separation in counter‐current flows of discrete zones of the chiral selector and the racemic compound as well as separation of the neutral racemic compound thalidomide in a micellar electrokinetic chromatography‐like modus were demonstrated using SBE‐β‐CD.
Enantiomeric separation of salbutamol and related impurities using capillary electrophoresis
Authors: Rogan Manus M. , Altria Kevin D. , Goodall David M. 
Published: May 2005

AbstractThe enantiomeric resolution of salbutamol and its chiral and achiral related impurites is investigated using capillary electrophoresis. The effects of nine varieties of cyclodextrin, cyclodextrin concentration and organic modifier concentration were studied in an attempt to resolve all possible analytes in a complex mixture of salbutamol‐related solutes. Eleven components including three enantiomeric pairs were baseline resolved using 112 mM dimethyl‐β‐cyclodextrin at pH 2.5 in a citric acid/phosphate buffer. Both methanol and iso‐propyl alcohol at up to 20% were found to have a deleterious effect on the separation. Binding constants and mobility values for the free and complexed forms for each solute were determined. The results are interpreted by considering the physical properties of the molecules under the conditions employed and a rationale proposed for the underlying basis for chiral and achiral selectivity.