bluuebunny/crossref_metadata_2025_split huggingface dataset:
Influence of transference number on migration and deformation of concentration boundaries in capillary zone electrophoresis
Authors: Štědrý Milan , Popelka Štěpán , Gaš Bohuslav , Kenndler Ernst 
Published: May 2005

AbstractThe change of the position and shape of a concentration boundary existing in the background electrolyte in capillary zone electrophoresis is teated theoretically. Such boundaries may occur when the ionic concentration, c, of the sample differs from that of the background electrolyte. Both mathematical treatment and numerical simulation lead to the result that when an (initially symmetrical) concentration boundary migrates in the capillary it becomes nonsymmetrical. The reason for this is the nonsymmetry of the function, which is obtained as the difference of the transference number (which is a function depending on c) and its linear approximation at the average value of the concentration. Numerical simulations for capillary zone electrophoresis with LiI or potassium benzorate as a hypothetical background electrolyte show the formation of asymmetrical plugs for samples of more diluted electrolyte solutions.
Flowable networks as DNA sequencing media in capillary columns
Authors: Menchen Steve , Johnson Ben , Winnik Mitchell A. , Xu Bai 
Published: May 2005

AbstractA novel class of materials that self‐assemble in water into equilibrium network structures with a well‐defined mesh size consist of polyethylene glycols (PEG's) end‐capped with micelle‐forming fluorocarbon tails. These micellar systems form flowable aqueous gel‐like networks that permit electrophoretic DNA sequencing in capillary columns. The gels have unusual rheological properties, including netword breakdown under shear, resulting in plug flow that allows colums refill with complete ejection of byproducts of the previous sequencing analysis. In this system, DNA fragment electrophoretic mobilities are unaffected by the hydrophobicity of the polymer tails. Low molecular weight (M) PEG chains (M 8000) show catastrophic resolution loss for DNA fragments larger than 100 bases due to band broadening. For a longer PEG segment (M 35000) separating the end groups, band broadening occurs for DNA fragments larger than 300 bases, implying that the PEG segment length controls the mesh size in the equilibrium network structure. Optimum sequencing results were obtained from a 6% solution of a 1:1 mixture of C6F13 end‐capped‐ and C8F17 end‐capped PEG 35000. The resolution limit of fluorescent‐dye‐labeled sequencing products in this formulation was 450 bases in 75 μm capillaries at 200 V/cm.
Characteristics of single‐stranded DNA separation by capillary gel electrophoresis
Authors: Kamahori Masao , Kambara Hideki 
Published: May 2005

AbstractThe optimization of electrophoretic conditions for fast separation of singlestranded DNA by capillary gel electrophoresis was investigated. Mobilities and band broadening of the DNA fragments were measured for a 4%T, 5%C gel at 100–300 V/cm. The resolution of DNA peaks was evaluated for band spacing and band width. Band spacing decreased with increasing electric field strengths as well as DNA fragment size; resolution for large DNA fragments also decreased at higher electric field strengths. The measured mobilities in a 4%T, 5%C gel were analyzed by the repration theory and compared with those in a 9%T, 0%C matrix. The reptation plots indicate that the onset of reptation in a 4%T, 5%C gel occurs at a DNA size of about 200 bases and about 150 bases in a 9%T, 0%C matrix. The 4%T, 5%C gel proved superior to the 9%T, 0%C matrix with respect to resolution and analysis time. The optimum electric field strength and migration distance for specific DNA fragment length (200, 400 and 700 bases) in the shortest time were calculated from band spacing and the measured band width for the 4%T, 5%C gel at Rs = 0.5. Under optimized conditions, analysis of DNA fragments up to 200, 400 and 700 bases could be carried out in about 5, 50, and 230 min, respectively.
Negative staining with zinc‐imidazole of gel electrophoresis‐separated nucleic acids
Authors: Hardy Eugenio , Pupo Elder , Casalvilla Racmar , Sosa Angela E. , Trujillo Luis E. , López Eduardo , Castellanos‐Serra Lila 
Published: May 2005

AbstractZinc and imidazole salts were applied for the detection of nucleic acids on either polyacrylamide or agarose gels. After electrophoresis, polyacrylamide gels are washed in distilled water to remove most of the residual electrophoresis reagents, then incubated in 10 mM zinc sulfate for 10 min, and subsequently immersed in 0.2 M imidazole for 3 min. As a result, zinc salts precipitate on the gel surface, except in the positions occupied by nucleic acids, which appear as transparent, colorless bands. Staining of nucleic acids on agarose gels can be performed by incubation in 40 mM zinc sulfate for 10 min, followed by immersion in 0.2 M imidazole for 5 min to form a deep white‐stained background. On soaking in 2 M imidazole for 45 min, the imidazole‐induced zinc precipitate is removed from the positions where nucleic acids are located resulting in a negative image of colorless and transparent nucleic acid bands against a white background. The sensitivity of this stain ranges from 5 to 7 ng/band for small (from 1 to 0.2 kbp) DNA, from 7.8 to 13 ng/ band for different 22‐base oligonucleotides, from 62 to 125 ng/band for large (from 20 to 2 kbp) DNA, and is 1 μg/band for human peripheral‐blood monocyte RNA. After chelation of zinc with EDTA, the nucleic acids can be quantitatively recovered from the gel. The principal advantage of this technique over ethidium bromide staining is evident for preparative purposes. Using zinc‐imidazole in the detection of purified pBACIB.1 (2.8 kbp) plasmid DNA and antiHBsAg single chain Fv antibody fragment (0.7 kbp) DNA, followed by elution from gel slices, ligation and transformation of competent E. coli XL‐1 Blue cells, the number of transformants notably increased from 280 (obtained with conventional ethidium bromide staining plus UV‐irradiation at 312 nm) to 10000.
Direct determination of glutathione in human blood by micellar electrokinetic chromatography: Simultaneous determination of lipoamide and lipoic acid
Authors: Pañak Karina C. , Ruiz Oscar A. , Giorgieri Sergio A. , Díaz Luis E. 
Published: May 2005

AbstractA reproducible, rapid procedure for the determination of glutathione in human blood by micellar electrokinetic chromatography has been developed. Whole blood samples were deproteinized with 5% w/v sulfosalicylic acid (final concentration). After centrifugation, the supernatant was directly injected for analysis, without further derivatization. Separations were performed by using an uncoated capillary of 30 cm effective length and 50 μm internal diameter (ID), 50 mM Tris‐HCl, 30 mM sodium dodecyl sulfate (SDS), pH 7.00, as running buffer, and 10–20 kV. On‐line detection was carried out at 214 nm and a detection limit in the range of femtomoles was achieved. Under the same experimental conditions, we resolved a mixed standard solution containing glutathione in its oxidized and reduced forms, lipoamide and α‐lipoic acid. The corresponding migration times were reproducible. The present method allows rapid determination of these compounds, which play a critical role in oxidative stress, in cellular defense against injurious agents and whose levels are related to the toxicology and metabolism of several toxins and drugs, such as antineoplastic agents.
Identification of human myocardial proteins separated by two‐dimensional electrophoresis using an effective sample preparation for mass spectrometry
Authors: Otto Albrecht , Thiede Bernd , Müller Eva‐Christina , Scheler Christian , Wittmann‐Liebold Brigitte , Jungblut Peter 
Published: May 2005

AbstractPeptide mass fingerprinting is a powerful tool for the identification of proteins separated by two‐dimensional gel electrophoresis (2‐DE). The identification of in‐gel digested proteins by peptide mass fingerprinting was significantly improved in comparison to blot‐digests by using a peptide‐collecting device. This device allows the effective purification and concentration of enzymatic digests of low‐intensity spots without expensive equipment and is described in detail. Sensitivity in the fmol range was demonstrated by unequivocal identification of bovine serum albumin after sodium dodecyl sulfate — polyacrylamide gel electrophoresis. Furthermore the high performance liquid chromatography pattern of in‐gel digests indicated a 2‐ to 3‐fold higher yield of the separated peptides. Therefore, a higher amount of the peptides was available to perform N‐terminal sequencing. The identification of 16 proteins from a high‐resolution 2‐DE gel map of human myocardium tissue has been achieved by means of this technique. Three of these proteins were associated with changes in spot intensity with dilated cardiomyopathy.
Efficiency of separation of DNA mutations by constant denaturant capillary electrophoresis is controlled by the kinetics of DNA melting equilibrium
Authors: Khrapko Konstantin , Coller Hilary , Thilly William 
Published: May 2005

AbstractConstant denaturant capillary electrophoresis (CDCE) separation takes place in the heated portion of the capillary where faster‐moving, unmelted DNA fragments are in equilibrium with slower‐moving, partially melted forms. Within a certain temperature range, the position of the melting equilibrium and thus the average electrophoretic mobility of each mutant is different. The resulting difference in mobility allow sequences containing single base pair point mutations to be separated from each other. We report the results of experiments in which we explored the rules defining separation efficiency by varying the parameters of CDCE. We discovered an unusual peak broadening mechanism. In contrast to most other DNA electrophoresis systems, peak width in CDCE steadily decreases with the square root of the separation speed. Moreover, the peak width displays a sharp maximum at a specific temperature. To account for these observations, we use a model which describes CDCE separation as a random walk. According to this model, peaks in CDCE are broad because the kinetics of the melting equilibrium are slow and there‐fore the number of random walk steps represented by melting/renaturation transitions is relatively small. In addition to providing a satisfactory interpretation of the data, the model also predicts that separation efficiency will increase as the ionic strength of the running buffer is increased and as the concentration of denaturant in the buffer is decreased. These predictions were verified and were used to establish conditions for high‐resolution CDCE suitable for separating complex mixtures of single base pair mutants.
Linearity evaluation in absorbance detection: The use of light‐emitting diodes for on‐capillary detection in capillary electrophoresis
Authors: Macka Miroslav , Andersson Per , Haddad Paul R. 
Published: May 2005

AbstractA model which takes into account both stray light and polychromatic light was used to predict and evaluate linearity in on‐capillary detection in capillary electrophoresis (CE). According to the model the stray light is the major factor which determines linearity under typical CE operating conditions. By calculating theoretical absorbance versus concentration plots, the influence of different levels of stray light and polychromatic light on linearity is demonstrated. Experimentally, six light‐emitting diodes (LEDs) in the range from 563 to 654 nm were examined as light sources for on‐capillary detection in CE. Fitting theoretical curves to measured linearity plots enabled determination of the values of both effective path lenght and stray light for a particular detection system. The detector linearity for the four LEDs was compared to mercury and tungsten lamps used with interference filters. For potassium permanganate as the test compound, the linear range for a 563 nm LED was two times greater than that for a mercury lamp operated at 546 nm. The relatively poor linearity of the mercury lamp detector is explained by its high level of stray light. The noise of the LED563‐based detector was the same as for the mercury lamp, whereas the other LEDs of higher light intensity gave approximately half the noise of the mercury lamp. The lowest noise level of 3 × 10−5 AU was obtained for the LED at 554 nm (determined at a detector time constant of 0.1 s).
A practical procedure for the determination of association constants of the analyte‐chiral selector equilibria by capilllary zone electrophoresis
Authors: Fanali Salvatore , Boček Petr 
Published: May 2005

AbstractA practical procedure is proposed for the determination of association constants and mobility of the associate of a solute with a chiral selector in chiral separations by capillary zone electrophoresis. The procedure is based on the measurement of the effective mobility of a solute at zero and two different nonzero concentrations of the chiral selector. Simple explicit formulas have been derived in order to calculate the required data. The essence of the procedure is that all mobility data are adjusted to the background electrolyte (BGE) without chiral selector, serving as the viscosity reference. A simple procedure is described for measuring the viscosity of the operational electrolytes directly with the commercial capillary electrophoresis instrumentation, and Walden's rule has been utilized for adjusting the experimental mobility data to constant reference viscosity. The use of the procedure is exemplified by a separation of D,L‐tryptophan in BGE containing α‐cyclodextrin as chiral selector.
Enantioseparation of amino acids and dipeptides using vancomycin as chiral selector in capillary electrophoresis
Authors: Wan Hong , Blomberg Lars G. 
Published: May 2005

AbstractVancomycin was applied as chiral selector for the enantiomeric separation of derivatized amino acids and dipeptides. The influence of vancomycin concentration, pH and presence of 2‐propanol in the buffer were examined in order to find optimal separation conditions. Optimization was by factorial design. Further, chiral separation of derivatives prepared with three different reagents was compared. These reagents were 9‐fluorenylmethyl chloroformate (FMOC), 2‐(9‐anthryl)ethyl chloroformate (AEOC) and dansyl chloride (dansyl). Optimum resolution was at high vancomycin concentrations, while optimum efficiency was at low vancomycin concentrations. As a consequence of the very high enantioselectivity of vancomycin, the vancomycin concentration below the amount necessary for maximal resolution can be used. Separation efficiency was relatively low, and this could be attributed to adsorption of the selector at the capillary wall. Three factors led to decreased adsorption: application of a pH above the zero mobility pH value, low vancomycin concentrations and the presence of 2‐propanol. For amino acids, the resolutions of the different derivatives were: dansyl > AEOC > FMOC, while for dipeptides, the highest selectivity was with AEOC.
Characterization of the stereoselective metabolism of methadone and its primary metabolite via cyclodextrin capillary electrophoretic determination of their urinary enantiomers
Authors: Lanz Matthias , Thormann Wolfgang 
Published: May 2005

AbstractUsing capillary zone electrophoresis with a phosphate buffer at pH 3, containing 4.3 nM hydroxypropyl‐β‐cyclodextrin as chiral selector, the simultaneous separation of the enantiomers of methadone and its primary metabolite, 2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine (EDDP), is reported. After solid‐phase extraction and analysis of the reconstituted extracts in a 60 cm fused‐silica capillary of 50 μm ID within about 25 min, the mean S/R enantiomeric ratio of methadone in the urines of eight patients undergoing methadone therapy is shown to be 0.653 (range: 0.502–0.842). The mean enantiomeric ratio for the metabolite is 0.630 (range: 0.517–0.729). These data document the stereoselectivity of the methadone metabolism. Finally, the EDDP/methadone ratio is shown to vary between 0.357 and 2.917 with a mean value of 1.731. The capillary electrophoretic assay described is simple, inexpensive and neither requires any sample derivatization, nor large amounts of organic solvents or expensive separation columns.
Pore size of agarose gels by atomic force microscopy
Authors: Pernodet Nadine , Maaloum Mounir , Tinland Bernard 
Published: May 2005

AbstractThe pore size of agarose gel in water at different concentrations was directly measured using atomic force microscopy (AFM). The experiment was specially designed to work under aqueous conditions and allows direct observation of the “unperturbed” gel without invasive treatment. The pore size a as a function of gel concentration C shows a power law dependence a ˜ C−γ, where γ lies between the prediction of the Ogston model for a random array of straight chains, 0.5, and the value predicted by De Gennes for a network of flexible chains, 0.75. We confirm that gels present a wide pore size distribution and show that it narrows as the concentration increases.
Application of two‐dimensional gel electrophoresis to Prunus armeniaca leaf and bark tissues
Authors: Faurobert Mireille 
Published: May 2005

AbstractThe protein composition of Prunus armeniaca bark and leaf tissues was investigated by two‐dimensional gel electrophoresis. Three different extraction procedures were tested in order to obtain reproducible gels with numerous spots of high intensity. The best results were achieved with extraction in Tris‐buffer in the presence of a nonionic detergent, reducing agents, and polyphenol oxidase inhibitors. As many as 744 protein spots were resolved from leaf tissues. The patterns exhibited well‐focused spots, with apparent molecular masses ranging from 19 to 90 kDa and isoelectric point from 4.5 to 8.5. The Tris extraction buffer was also the most appropriate for cortical tissue analysis.
Analysis of domoic acid isomers in seafood by capillary electrophoresis
Authors: Zhao Jian‐Ying , Thibault Pierre , Quilliam Michael A. 
Published: May 2005

AbstractMethods for the analysis of domoic acid (DA) based upon capillary electrophoresis (CE) combined with UV absorbance detection were investigated. DA could be analyzed using bare fused‐silica capillaries in either the cationic or anionic mode with acidic or basic buffer systems, respectively. Highest performance, in terms of both separation efficiency and analysis time, was achieved with phosphate or borate buffers at a pH of approximately 9. The addition of (3‐cyclodextrin to the borate buffer permitted a separation of DA and several of its isomers (isodomoic acids) that was superior to that achieved with liquid chromatography (LC). The optimum background electrolyte for the separation was 22.5 mM sodium tetraborate at pH 9.2 with 20 mM (3‐cyclodextrin. In addition, an extraction and clean‐up procedure was developed and tested with mussels, clams and anchovies. Aqueous methanol extraction of samples followed by a tandem strong anion and strong cation exchange clean‐up provided an extract that was completely compatible with CE analysis. A mass detection limit of 3 pg of DA injected and a method detection limit of 150 ng/g in tissues could be achieved. Comparison with LC showed that comparable precision and accuracy could be attained by the two techniques.
Reproducibility of polypeptide spot positions in two‐dimensional gels run using carrier ampholytes in the isoelectric focusing dimension
Authors: Lopez Mary F. , Patton Wayne F. 
Published: May 2005

AbstractThe reproducibility of complex protein patterns in two‐dimensional (2‐D) gels run with carrier ampholytes in the first dimension has been investigated. Two different laboratories collaborated in the study and 18 or 19 gels were run in each laboratory for comparison. The electrophoresis chemicals, running devices, and samples were standardized in both labs. The resulting 37 gels were scanned with a charge‐coupled device (CCD) camera and spots were located, counted, quantified, and matched using a commercially available image analysis system. Subsequently, the reproducibility of spot position was determined. To perform the statistical analysis, the test gels were initially each matched to a master references gel. Next, three sets of 12 gels (the image analysis software database could analyze only 12 gels at a time) were analyzed and the isoelectric point (pI) and molecular weight (Mr) positional variation of all the spots that matched across the gels in each set was determined. The resulting statistical analysis indicates very high reproducibility of the carrier ampholyte technique.
Construction of HSC‐2DPAGE: A two‐dimensional gel electrophoresis database of heart proteins
Authors: Evans Guy , Wheele Colin H. , Corbett Joseph M. , Dunn Michael J. 
Published: May 2005

AbstractThe dissemination of information relating to the characterisation of proteins from two‐dimensional electrophoresis (2‐DE) gel databases is essential for their effective utilisation in the study of protein expression in cell biology. Since the inception of the World Wide Web and the pioneering development of SWISS‐2DPAGE as a tool for retrieving information on proteins separated by 2‐DE, the Internet has become the method of choice for disseminating and accessing information on 2‐DE protein databases. At Harefield we have established HSC‐2DPAGE which is an advanced interface for accessing protein databases relating to heart disease. The Web site currently includes databases of proteins from human, dog and rat ventricular tissue and a human endothelial cell line. The databases are searchable individually or as a whole by remote keyword searches. Each database is represented by both synthetic (computer generated) and real (scanned gel) clickable images upon which characterised protein spots are highlighted by hyperlinked symbols. The database conforms to all the rules proposed for federated 2‐DE protein databases and individual protein entries are linked to other protein databases such as SWISS‐PROT by active cross‐references. This paper describes the construction of HSC‐2DPAGE, its maintenance, and access via the Internet.
Acute‐phase proteins in perinatal human plasma
Authors: Liberatori Sabrina , Bini Luca , De Felice Claudio , Magi Barbara , Marzocchi Barbara , Raggiaschi Roberto , Pallini Vitaliano , Bracci Rodolfo 
Published: May 2005

AbstractPlasma from eight newborns (4 pre‐term and 4 full‐term) with early‐onset (<72 h) sepsis and six apparently healthy controls was analyzed. The presence of spots identified as haptoglobin and serum amyloid A protein was the electrophoretic result most consistently associated with disease. Time course monitoring showed rises, peaks and declines of spot intensity as expected for acute‐phase proteins induced by transient stimuli. Haptoglobin β chains appear to be undersialated in pre‐term newborns, whereas post‐translational modifications of α chains and serum amyloid A protein are similar to those observed in adults. The undersialation of β chain and occurrence of α chain phenotypes different from those found in maternal serum indicate that perinatal haptoglobin originates from neonatal synthesis.