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Buffer System (buffer + system)
Selected AbstractsChiral CE of aromatic amino acids by ligand-exchange with zinc(II),L -lysine complexELECTROPHORESIS, Issue 15 2007Li Qi Abstract A novel method of chiral ligand-exchange CE was developed with either L - or D -lysine (Lys) as a chiral ligand and zinc(II) as a central ion. This type of chiral complexes was explored for the first time to efficiently separate either individual pairs of or mixed aromatic amino acid enantiomers. Using a running buffer of 5,mM ammonium acetate, 100,mM boric acid, 3,mM ZnSO4·7H2O and 6,mM L -Lys at pH,7.6, unlabeled D,L -tryptophan, D,L -phenylalanine, and D,L -tyrosine were well separated, giving a chiral resolution of up to 7.09. The best separation was obtained at a Lys-to-zinc ratio of 2:1, zinc concentration of 2,4,mM and running buffer pH,7.6. The buffer pH was determined to have a strong influence on resolution, while buffer composition and concentration impacted on both the resolution and peak shape. Boric acid with some ammonium acetate was an adoptable buffer system, and some additives like ethylene diamine tetraacetic acid capable of destroying the complex should be avoided. Fine-tuning of the chiral resolution and elution order was achieved by regulating the ratio of L -Lys to D -Lys; i.e. the resolution increased from zero to its highest value as the ratio ascended from 1:0 to 1:infinitive, and L -isomers eluted before or after D -isomers in excessive D - or L -Lys, respectively. [source] Integration of continuous-flow sampling with microchip electrophoresis using poly(dimethylsiloxane)-based valves in a reversibly sealed deviceELECTROPHORESIS, Issue 14 2007Michelle W. Li Abstract Here we describe a reversibly sealed microchip device that incorporates poly(dimethylsiloxane) (PDMS)-based valves for the rapid injection of analytes from a continuously flowing stream into a channel network for analysis with microchip electrophoresis. The microchip was reversibly sealed to a PDMS-coated glass substrate and microbore tubing was used for the introduction of gas and fluids to the microchip device. Two pneumatic valves were incorporated into the design and actuated on the order of hundreds of milliseconds, allowing analyte from a continuously flowing sampling stream to be injected into an electrophoresis separation channel. The device was characterized in terms of the valve actuation time and pushback voltage. It was also found that the addition of sodium dodecyl sulfate (SDS) to the buffer system greatly increased the reproducibility of the injection scheme and enabled the analysis of amino acids derivatized with naphthalene-2,3-dicarboxaldehyde/cyanide. Results from continuous injections of a 0.39,nL fluorescein plug into the optimized system showed that the injection process was reproducible (RSD of 0.7%, n,=,10). Studies also showed that the device was capable of monitoring off-chip changes in concentration with a device lag time of 90,s. Finally, the ability of the device to rapidly monitor on-chip concentration changes was demonstrated by continually sampling from an analyte plug that was derivatized upstream from the electrophoresis/continuous flow interface. A reversibly sealed device of this type will be useful for the continuous monitoring and analysis of processes that occur either off-chip (such as microdialysis sampling) or on-chip from other integrated functions. [source] Determination of ethyl sulfate , a marker for recent ethanol consumption , in human urine by CE with indirect UV detectionELECTROPHORESIS, Issue 23 2006Francesc A. Esteve-Turrillas Abstract A CE method for the determination of the ethanol consumption marker ethyl sulfate,(EtS) in human urine was developed. Analysis was performed in negative polarity mode with a background electrolyte composed of 15,mM maleic acid, 1,mM phthalic acid, and 0.05,mM cetyltrimethylammonium bromide (CTAB) at pH,2.5 and indirect UV detection at 220,nm (300,nm reference wavelength). This buffer system provided selective separation conditions for EtS and vinylsulfonic acid, employed as internal standard, from urine matrix components. Sample pretreatment of urine was minimized to a 1:5 dilution with water. The optimized CE method was validated in the range of 5,700,mg/L using seven lots of urine. Intra- and inter-day precision and accuracy values, determined at 5, 60, and 700,mg/L with each lot of urine, fulfilled the requirements according to common guidelines for bioanalytical method validation. The application to forensic urine samples collected at autopsies as well as a successful cross-validation with a LC-MS/MS-based method confirmed the overall validity and real-world suitability of the developed expeditious CE assay (sample throughput 130 per day). [source] A new multiphasic buffer system for benzyldimethyl- n -hexadecylammonium chloride polyacrylamide gel electrophoresis of proteins providing efficient stackingELECTROPHORESIS, Issue 2 2006Michael L. Kramer Dr. Abstract Acidic PAGE systems using cationic detergents such as benzyldimethyl- n -hexadecylammonium chloride (16-BAC) or CTAB have proven useful for the detection of methoxy esters sensitive to alkaline pH, resolving basic proteins such as histones and membrane proteins. However, the interesting phosphate-based system suffered from poor stacking, resulting in broadened bands and long running times. Therefore, a new 16-BAC PAGE system based on the theory of moving boundary electrophoresis with properties comparable to the classical SDS-PAGE system was designed. As a result a new multiphasic analytical 16-BAC PAGE system providing efficient stacking and significantly shorter running times is presented here. It is based on acetic acid and methoxyacetic acid as common ion constituents. This PAGE system takes advantage of the additional counterstacking effect due to a cross boundary electrophoresis system resulting from the selected buffer constituents. Furthermore, the concentration of 16-BAC was optimized by determining its previously unknown CMC. Due to efficient focusing of the introduced tracking dye, methyl green, termination of electrophoresis can now be more easily followed as compared to the Schlieren line. [source] Capillary electrophoresis-laser induced fluorescence-electrospray ionization-mass spectrometry: A case studyELECTROPHORESIS, Issue 7-8 2005Carolin Huhn Abstract The simultaneous hyphenation of capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection and electrospray ionization-mass spectrometry (ESI-MS) as a novel combined detection system for CE is presented. ,-Carbolines were chosen as model analytes with a forensic background. Nonaqueous CE as well as conventional CE with an aqueous buffer system are compared concerning efficiency and obtainable detection limits. The distance between the optical detection window and the sprayer tip was minimized by placing the optical cell directly in front of the electrospray interface. Similar separation efficiencies for both detection modes could thus be obtained. No significant peak-broadening induced by the MS interface was observed. The high fluorescence quantum yield and the high proton affinity of the model analytes investigated resulted in limits of detection in the fg (nmol/L) range for both detection methods. The analysis of confiscated ayahuasca samples and ethanolic plant extracts revealed complementary selectivities for LIF and MS detection. Thus, it is possible to improve peak identification of the solutes investigated by the use of these two detection principles. [source] Chiral ion-exchange capillary electrochromatography of arylglycine amides with dextran sulfate as a pseudostationary phaseELECTROPHORESIS, Issue 4-5 2005Yi Chen Abstract A low-cost tunable chiral ion-exchange capillary electrochromatographic method has been developed for the separation of arylglycine amide racemic mixtures with dextran sulfate (DS) as an anionic and chiral pseudostationary phase and Tris-tartrate as a buffer system. The concentrations of DS and Tris had opposite influences on retention and resolution and could serve as ideal factors to finely tune the running speed and chiral resolution. Tartrate and pH largely impact the separation but pH should be confined within 3.0,5.5, only suitable for coarse tuning, while tartrate was preserved as the key buffering reagent, normally maintained at 40 mmol/L. With a working system composed of 0.1,1.0% DS, 20,60 mmol/L Tris, and 40 mmol/L tartrate at pH 3.50,4.50, the enantioresolution of arylglycine amides was shown to be dependent on their chemical structure: The chiral resolution increased when the hydrogen at the ,-amino group or at the p -position of phenyl ring was replaced by other larger group(s) but the resolution decreased when the group at the o- or m- site on the phenyl ring was enlarged. Further, the electronegative substitute of -Cl had larger resolution increment than methyl or methoxy at the position p- of phenyl ring but much lower increment at position m- . It is possible to well explain the resolution variation phenomenon by considering the group resistance and the variation of hydrogen-bonds formed inside the amino amides and between the solutes and DS. The amido group was shown irreplaceable to have chiral resolution with DS alone as an ionic and chiral pseudostationary phase. [source] Capillary electrophoresis as a probe of enantiospecific interactions between photoactive transition metal complexes and DNAELECTROPHORESIS, Issue 15 2003James P. Schaeper Abstract Recently, we have demonstrated the capacity to separate chiral transition metal (TM) complexes of the type [M(diimine)3]n+ using CE buffers containing chiral tartrate salts. In separate work, several chromium(III)- tris -diimine complexes in particular have been shown to bind enantioselectively with calf-thymus (CT) DNA, and a qualitative assessment of the relative strength and enantiospecificity of this interaction is of significant interest in the characterization of these complexes as potential DNA photocleavage agents. Here, we describe two convenient approaches to investigate such binding behavior using chiral CE. For complexes with lower DNA affinities exhibiting primarily surface binding, DNA itself is used as the chiral resolving agent in the electrophoretic buffer. In this approach, resolution of the TM complexes into their , and , isomers is achieved with the isomer eluting later exhibiting superior binding affinity toward DNA. For more strongly bound TM complexes containing ligands known to intercalate with DNA, the [Cr(diimine)3]3+ complexes are preincubated with oligonucleotide and subsequently enantiomerically resolved in a dibenzoyl- L -tartrate buffer system that facilitates analysis of the unbound TM species only. Differences in isomer binding affinity are distinguished by the relative peak areas of the ,- and ,-isomers, and relative binding strengths of different complexes can be inferred from comparison of the total amount of unbound complex at equivalent DNA/TM ratios. [source] A versatile electrophoresis system for the analysis of high- and low-molecular-weight proteinsELECTROPHORESIS, Issue 11 2003Christophe Tastet Abstract A new, versatile, multiphasic buffer system for high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins in the relative molecular weight range of 300,000,3000 Da is described. The system, based on the theory of multiphasic zone electrophoresis, allows complete stacking and destacking of proteins in the above Mr range. The buffer system uses taurine and chloride as trailing and leading ion, respectively, and Tris, at a pH close to its pKa, as the buffering counterion. Coupled with limited variation in the acrylamide concentration, this electrophoresis system allows to tailor the resolution in the 6,200 kDa Mr range, with minimal difficulties in the post electrophoretic identification processes. [source] Ammonium fluoride extraction for determining inorganic sulphur in acid forest soilsEUROPEAN JOURNAL OF SOIL SCIENCE, Issue 2 2000J. Prietzel Summary Current methods for determining inorganic sulphur (S) in aerated mineral soil horizons often result in underestimates. To overcome this defect we developed a new method combining a batch extraction with 0.5 m NH4F solution at a soil:solution ratio of 1:5 with a subsequent analysis of the mobilized SO42, by ion chromatography. The ammonium fluoride extraction enables us to characterize inorganic sulphate in non-calcareous forest soils. It is more efficient than conventional procedures in which inorganic S is extracted with phosphate or bicarbonate solution. In contrast to the extraction with strongly alkaline reagents (NaOH, KOH, LiOH), the NH4+,NH3 buffer system in NH4F prevents the pH of the suspension from exceeding 9.0 and thus the undesired conversion of organic S into SO42, by auto-oxidation and hydrolysis of ester sulphate. In a comparison we demonstrated that the inorganic S in six German forest soils is underestimated by up to 50% or 200 kg S ha,1 in the uppermost 60 cm, if it is assessed by extraction with 0.016 m KH2PO4 or 0.5 m NaHCO3 instead of 0.5 m NH4F. Conversely, the pool of ester sulphate is overestimated almost threefold. [source] Fractionation of Caseins by Anion-exchange Chromatography Using Food-grade BuffersJOURNAL OF FOOD SCIENCE, Issue 5 2003K.N. Turhan ABSTRACT : Caseins prepared by microfiltration of bovine skim milk were fractionated using anion-exchange chromatography. Laser densitometry of electrophoresis gels was shown to be sufficiently quantitative to perform accurate mass balance calculations detailing the fate of each casein fraction. L-cysteine was successfully used as a reducing agent instead of traditional toxic agents, such as dithiothreitol or ,-mercaptoethanol, enabling development of the first food-grade buffer system for casein fractionation. More salt was required for elution of the casein fractions having a greater charge: ,s -casein > ,-casein > ,-casein. Increasing flow rate decreased the extent of separation. Use of smaller beads was suggested as a method to maintain separation at increased flow rate. [source] Direct STR Amplification from Whole Blood and Blood- or Saliva-Spotted FTA® without DNA Purification,JOURNAL OF FORENSIC SCIENCES, Issue 2 2008Su Jeong Park Ph.D. Abstract:, The DNA purification step has been thought to be essential for typing of STR DNA. However, this process is time-consuming, and there is a risk of unexpected cross-contamination during purification. We report a new method for direct short tandem repeat (STR) amplification using a newly developed direct PCR buffer, AnyDirectÔ, which can amplify STR loci from whole blood and blood- or saliva-spotted FTA® cards without DNA purification. The autosomal and Y chromosomal STR loci were analyzed for whole blood and blood or saliva spots of random individuals, followed by comparison of the results with those of corresponding purified DNA. The results from whole blood and blood spots showed perfect concordance with those from purified DNA without allele or locus drop-out. However, in the case of saliva spots, no amplification or locus drop-out was observed in some of the samples, which offers a topic for further study. Additionally, some commercial hot-start DNA polymerases other than AmpliTaq Gold® DNA polymerase were also found to be compatible with this buffer system. Therefore, this direct PCR buffer was demonstrated to be useful for fast forensic DNA analysis or criminal DNA databases for which there is no need to store DNA samples. [source] PEG enhances viral clearance on ceramic hydroxyapatiteJOURNAL OF SEPARATION SCIENCE, JSS, Issue 23-24 2009Mark A. Snyder Abstract Viral clearance across ceramic hydroxyapatite (CHTÔ) was examined in two elution systems: sodium chloride and sodium chloride plus poly(ethylene glycol) (PEG). In both cases clearance of xenotropic murine leukemia virus was significant (3,4,log) while that of minute virus of mice varied between 1.7 and 2.7,log; in addition, the addition of PEG to the elution buffer enhanced viral clearance. The data are in agreement with the previous results and demonstrate that additional clearance can be obtained by adding PEG to a ceramic hydroxyapatite buffer system. [source] Capillary electrophoresis with capacitively coupled contactless conductivity detection for low molecular weight organic acids in different samplesJOURNAL OF SEPARATION SCIENCE, JSS, Issue 18 2007Wai Siang Law Abstract CE with capacitively coupled contactless conductivity detection (CE-C4D) was explored and validated for the identification and quantification of organic acids in various types of samples. The analyses were performed under optimized conditions, using a buffer system composed of 20 mM MES-histidine (His), pH 6.0, 0.1 mM CTAB, 0.025% HP-,-CD, and 10% methanol. The investigation included a study of the effects of buffer pH, concentration of CTAB, type and concentration of organic additives, on the migration behavior, resolution and selectivity of the organic acids. The intra- and interday RSDs (n = 6) obtained for migration time and peak area were typically in the range of 0.12,2% and 0.5,4%, respectively. Linearity, detection limits, and repeatability were evaluated. In order to evaluate the application potential of the developed method, real samples from different sources were analyzed. The results demonstrate that CE-C4D is a versatile tool for analyzing organic acids in beverages, Chinese herbal medicines (CHM) and plants as it allows for their detection, identification, and quantification. [source] Indirect laser-induced fluorescence detection of diuretics separated by capillary electrophoresisJOURNAL OF SEPARATION SCIENCE, JSS, Issue 5 2006Xiuhan Yang Abstract Indirect LIF detection was applied to the detection of four acidic diuretics separated by CZE. Semiconductor laser was employed to provide the stable excitation of 473 nm. With an optimized electrophoretic buffer system which contained 5 mM of triethylamine, 0.1 ,M of fluorescein, and 5% of n -butanol, fast separation of four diuretics (ethacrynic acid, chlorthalidone, bendroflumethiazide, and bumetanide) can be performed within 3 min with the detection limits of 0.2,2 ,g/mL. The impacts of buffer components including the concentrations of the electrolytes, fluorescence probe, and the organic additives were demonstrated. The method was applied for the detection of diuretics in urine. As an alternative way for the fast analysis of diuretics, this indirect detection method provided the technical support for future microchip performances, in which diuretics may be detected in the microchip by the common LIF detector without derivatization. [source] Determination of dissociation constants of ten alkaloids by capillary zone electrophoresisJOURNAL OF SEPARATION SCIENCE, JSS, Issue 6-7 2003Suxuan Gong Abstract The dissociation constants of ten alkaloids, berbamine, (+)-tetrandrine, thalifaretine, thalifaricine, northaltive, matrine, sophocarpine, sophoridine, oxymatrine, and oxysophocarpine isolated from herbal medicines Thalictrum and Sophoraflavescens, were determined by capillary zone electrophoresis (CZE). The analysis time for each alkaloid was within 10 min under the following experimental conditions: acetate (pH 3.81,5.55) and phosphate buffer (pH 6.05,9.29) with ionic strength set at 0.03, applied voltage 30 kV, UV detection at 210 nm, capillary temperature 25°C. Effective mobility for each alkaloid and corresponding pH were fitted to an equilibrium expression by using nonlinear regression as well as linear regression, showing that the two models give comparable results for all studied alkaloids. The correlation coefficients were between 0.987 and 1.00 in the linear regression model, and between 0.978 and 1.00 in the nonlinear model. The measured pKa values for the ten alkaloids were from 5.7 to 7.8. The pKa values changed with the addition of organic additive to the buffer system. [source] Norepinephrine Uptake Sites in the Locus Coeruleus of Rat Lines Selectively Bred for High and Low Alcohol Preference: A Quantitative Autoradiographic Binding Study Using [3H]-TomoxetineALCOHOLISM, Issue 5 2000Bang H. Hwang Background: The locus coeruleus (LC) is the largest norepinephrinergic cell group in the central nervous system and contains a high density of norepinephrine (NE) uptake sites. Alcohol-preferring (AP) rats and high,alcohol-drinking (HAD) rats are selectively bred for high alcohol preference, whereas alcohol-nonpreferring (NP) rats and low,alcohol-drinking (LAD) rats are bred for low alcohol preference. However, it is unknown whether NE uptake sites in the LC are associated with alcohol preference in AP and HAD rats when compared with their respective control rats, NP and LAD rats. This study was designed to examine this question. Methods: Animals were decapitated and brains were removed, frozen with dry ice powder, and stored in a deep freezer. The LC tissue blocks were cut into 14 , cryostat sections, collected on glass slides, and incubated with 0.6 nM [3H]-tomoxetine in 50 mM Tris-HCl buffer system. For nonspecific binding, 1 ,M desipramine was added to the radioactive ligand. Sections were rinsed, quickly dried, and processed for quantitative autoradiography. In addition, galanin content in the LC was also studied. Results: The LC possessed a high density of [3H]-tomoxetine binding sites. There were fewer tomoxetine binding sites (fmol/mg protein) in the AP rats (433.0 ± 8.1) than in the NP rats (495.6 ± 3.7). HAD rats (386.5 ± 13.2) also possessed fewer tomoxetine binding sites than LAD rats (458.7 ± 10.1). Galanin content in the LC was similar between AP and NP rats and between HAD and LAD rats. Conclusions: Because both AP rats and HAD rats were selectively bred for alcohol preference, the finding of consistently low levels of [3H]-tomoxetine binding in the LC of these two lines of rats with high alcohol preference suggests that down-regulation of NE transporters in the LC of AP and HAD rats may be associated with alcohol-seeking behavior. A possible involvement of the coerulear NE uptake sites in depression is also discussed. Galanin in the LC may not relate to alcohol preference. [source] Grass-Shrub Riparian Buffer Removal of Sediment, Phosphorus, and Nitrogen From Simulated Runoff,JOURNAL OF THE AMERICAN WATER RESOURCES ASSOCIATION, Issue 5 2007Kyle R. Mankin Abstract:, Riparian buffer forests and vegetative filter strips are widely recommended for improving surface water quality, but grass-shrub riparian buffer system (RBSs) are less well studied. The objective of this study was to assess the influence of buffer width and vegetation type on the key processes and overall reductions of total suspended solids (TSS), phosphorus (P), and nitrogen (N) from simulated runoff passed through established (7-year old) RBSs. Nine 1-m RBS plots, with three replicates of three vegetation types (all natural selection grasses, two-segment buffer with native grasses and plum shrub, and two-segment buffer with natural selection grasses and plum shrub) and widths ranging from 8.3 to 16.1 m, received simulated runoff having 4,433 mg/l TSS from on-site soil, 1.6 mg/l total P, and 20 mg/l total N. Flow-weighted samples were collected by using Runoff Sampling System (ROSS) units. The buffers were very efficient in removal of sediments, N, and P, with removal efficiencies strongly linked to infiltration. Mass and concentration reductions averaged 99.7% and 97.9% for TSS, 91.8% and 42.9% for total P, and 92.1% and 44.4% for total N. Infiltration alone could account for >75% of TSS removal, >90% of total P removal, and >90% of total N removal. Vegetation type induced significant differences in removal of TSS, total P, and total N. These results demonstrate that adequately designed and implemented grass-shrub buffers with widths of only 8 m provide for water quality improvement, particularly if adequate infiltration is achieved. [source] Biomass recycling from a riboflavin cultivation with B. subtilis: Lysis, extract production and testing as substrate in riboflavin cultivationBIOTECHNOLOGY & BIOENGINEERING, Issue 6 2006Karlheinz Bretz Abstract Autolysis of riboflavin-producing B. subtilis can be induced by pH, lack of carbon source, and the buffer system. Stress factors like temperature shift or oxygen dearth enhance the autolysis process. After cultivation of a riboflavin-producing strain, the pH of the whole culture broth was adjusted to 6.5,7.5. At a temperature of 40°C, autolysis started after 1 h. Adding a defined amount of commercially available endo- and exo-proteases enhanced both auto- and proteo-lysis. Optimization of endo- and exo-protease concentrations and of the time increased the degree of proteolysis. Additionally, the amount of DNA and Protein trapped in the riboflavin crystals could be significantly reduced by autolysis. After autolysis, the cultivation broth was centrifuged and the supernatant was cross-flow filtrated with a cut off of 10 kDa. Using this autolysate instead of yeast extract as a medium component for riboflavin production with B. subtilis, a riboflavin yield of 77% was obtained in comparison with the standard cultivation on yeast extract. © 2006 Wiley Periodicals, Inc. [source] High-performance affinity chromatography with immobilization of protein A and L-histidine on molded monolithBIOTECHNOLOGY & BIOENGINEERING, Issue 5 2002Quanzhou Luo Abstract Reactive monoliths of macroporous poly(glycidyl methacrylate- co -ethylene dimethacrylate) have been prepared by "in-situ" copolymerization of the monomers in the presence of porogenic diluents. Protein A and L-histidine were immobilized on the monoliths directly or through a spacer arm, respectively. The properties of these two kinds of affinity columns were characterized, and the results showed that the columns with coupling of ligands by a spacer arm have some extent of non-specific adsorption for bovine serum albumin. The affinity column based on the monolithic polymer support provided us with good hydrodynamic characteristic, low flow resistance, and easy preparation. These two affinity columns were used for the purification of immunoglobulin G from human serum. The purity of the purified IgG was detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The stability of the protein A affinity column was investigated, and its performance remained invariable after half a year. The effects of the nature and the pH of the buffer system on the adsorption capacity of human IgG on histidyl affinity column were also investigated. The protein A affinity column is favorable for rapid analysis of human IgG samples. In contrast, the advantages of mild elution conditions, high stability, as well as low cost provide the histidyl column further potential possibility for fast removal of IgG from human plasma in clinical applications. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 481,489, 2002. [source] pH measurement and a rational and practical pH control strategy for high throughput cell culture systemBIOTECHNOLOGY PROGRESS, Issue 3 2010Haiying Zhou Abstract The number of therapeutic proteins produced by cell culture in the pharmaceutical industry continues to increase. During the early stages of manufacturing process development, hundreds of clones and various cell culture conditions are evaluated to develop a robust process to identify and select cell lines with high productivity. It is highly desirable to establish a high throughput system to accelerate process development and reduce cost. Multiwell plates and shake flasks are widely used in the industry as the scale down model for large-scale bioreactors. However, one of the limitations of these two systems is the inability to measure and control pH in a high throughput manner. As pH is an important process parameter for cell culture, this could limit the applications of these scale down model vessels. An economical, rapid, and robust pH measurement method was developed at Eli Lilly and Company by employing SNARF-4F 5-(-and 6)-carboxylic acid. The method demonstrated the ability to measure the pH values of cell culture samples in a high throughput manner. Based upon the chemical equilibrium of CO2, HCO, and the buffer system, i.e., HEPES, we established a mathematical model to regulate pH in multiwell plates and shake flasks. The model calculates the required %CO2 from the incubator and the amount of sodium bicarbonate to be added to adjust pH to a preset value. The model was validated by experimental data, and pH was accurately regulated by this method. The feasibility of studying the pH effect on cell culture in 96-well plates and shake flasks was also demonstrated in this study. This work shed light on mini-bioreactor scale down model construction and paved the way for cell culture process development to improve productivity or product quality using high throughput systems. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Poly(methylmethacrylate) and Topas capillary electrophoresis microchip performance with electrochemical detectionELECTROPHORESIS, Issue 16 2005Mario Castaño-Álvarez Abstract A capillary electrophoresis (CE) microchip made of a new and promising polymeric material: Topas (thermoplastic olefin polymer of amorphous structure), a cyclic olefin copolymer with high chemical resistance, has been tested for the first time with analytical purposes, employing an electrochemical detection. A simple end-channel platinum amperometric detector has been designed, checked, and optimized in a poly-(methylmethacrylate) (PMMA) CE microchip. The end-channel design is based on a platinum wire manually aligned at the exit of the separation channel. This is a simple and durable detection in which the working electrode is not pretreated. H2O2 was employed as model analyte to study the performance of the PMMA microchip and the detector. Factors influencing migration and detection processes were examined and optimized. Separation of H2O2 and L -ascorbic acid (AsA) was developed in order to evaluate the efficiency of microchips using different buffer systems. This detection has been checked for the first time with a microchip made of Topas, obtaining a good linear relationship for mixtures of H2O2 and AsA in different buffers. [source] Capillary electrophoresis analysis of glucooligosaccharide regioisomersELECTROPHORESIS, Issue 6 2004Gilles Joucla Abstract Complex gluco-oligosaccharide mixtures of two regioisomer series were successfully separated by CE. The gluco-oligosaccharide series were synthesized, employing a dextransucrase from Leuconostoc mesenteroides NRRL B-512F, by successive glucopyranosyl transfers from sucrose to the acceptor glucose or maltose. The glucosyl transfer to both acceptors, occurring through the formation of ,1,6 linkages, differed for the two series only in the glucosidic bond to the reducing end namely ,1,6 or ,1,4 bond for glucose or maltose acceptor, respectively. Thus, the combination of the two series results in mixed pairs of gluco-oligosaccharide regioisomers with different degrees of polymerization (DP). These regioisomer series were first derivatized by reductive amination with 9-aminopyrene-1,4,6-trisulfonate (APTS). Under acidic conditions using triethyl ammonium acetate as electrolyte, the APTS-gluco-oligosaccharides of each series were separated enabling unambiguous size determination by coupling CE to electrospray-mass spectrometry. However, neither these acidic conditions nor alkaline buffer systems could be adapted for the separation of the gluco-oligosaccharide regioisomers arising from the two combined series. By contrast, increased resolution was observed in an alkaline borate buffer, using differential complexation of the regioisomers with the borate anions. Such conditions were also successfully applied to the separation of glucodisaccharide regioisomers composed of ,1,2, ,1,3, ,1,4, and ,1,6 linkages commonly synthesized by glucansucrase enzymes. [source] Polyacrylamide gel electrophoresis followed by sodium dodecyl sulfate gradient polyacrylamide gel electrophoresis for the study of the dimer to monomer transition of human transthyretinELECTROPHORESIS, Issue 14 2003Klaus Altland Abstract Familial amyloidotic polyneuropathy (FAP) is caused by mutations which destabilize transthyretin (TTR) and facilitate the aggregation into extracellular amyloid fibrils preferentially in peripheral nerve and heart tissues. Therapeutic and preventive trials for FAP at the plasma TTR level require a careful study of the destabilization of TTR under variable conditions. We have developed a simple double one-dimensional (D1-D) electrophoretic procedure with polyacrylamide gel electrophoresis (PAGE) followed by sodium dodecylsulfate (SDS) gradient PAGE to study the dimer to monomer transition. TTR is first isolated by PAGE from other plasma proteins. The gel strip containing the TTR fraction is incubated in 2% SDS under varying conditions of temperature, buffer composition, pH, and additives like urea and/or a sulfhydryl-reactive agent, followed by SDS-gradient PAGE for the separation of TTR dimers and monomers. We demonstrate that an unidirectional dimer to monomer transition of normal TTR is achieved at 70,80°C in neutral to mild alkaline buffers or at 37°C and slightly acidic pH (6,7). Addition of urea favors the transition into monomers. Amyloidogenic mutations like amyloidogenic TTR (ATTR)-V30M or ATTR-I107V favor the transition into monomers in buffer systems close to the physiological pH of human plasma. We conclude that this finding has to be considered by any hypothesis on ATTR-derived amyloidogenesis. [source] Stability and detection of ,-pinene oxide in aqueous culture mediumENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 9 2000Kimberly K. Kajihara Abstract Methane consumption by methanotrophic bacteria was previously shown to be temporarily inhibited by ,-pinene. Based on literature considerations, loss of inhibition may be due to bacterial degradation of the monoterpene to ,-pinene oxide, an anticipated metabolite. However, since ,-pinene oxide is unstable in aqueous media, detection of its production by methanotrophs or other bacteria is problematic. Therefore, we used gas chromatography-mass spectrometry analysis to study the chemical breakdown of ,-pinene oxide in various buffer systems (Tris[hydroxymethyl]am inomethane, 3-[N-morpholino]propanesulfonic acid, phosphate; pH 7-9) suitable for bacterial whole-cell and cell-free experiments. In every case, aqueous phase ,-pinene oxide was unstable and its disappearance was accompanied by the appearance of five decomposition products in a characteristic fingerprint that was in part buffer dependent. However, this fingerprint was adequately stable in phosphate buffer such that its appearance could be used to infer the intermediacy of ,-pinene oxide if produced by the bacteria at or near their optimal pH. [source] SMAP-29 has two LPS-binding sites and a central hingeFEBS JOURNAL, Issue 4 2002Brian F. Tack The CD spectra of SMAP-29, an antimicrobial peptide from sheep, showed disordered structure in aqueous buffers, and significant helicity in membrane-like environments, including SDS micelles, lipopolysaccharide (LPS) dispersions, and trifluoroethanol buffer systems. A structure determined by NMR in 40% perdeuterated trifluoroethanol indicated that residues 8,17 were helical, residues 18,19 formed a hinge, and residues 20,28 formed an ordered, hydrophobic segment. SMAP-29 was flexible in 40% trifluoroethanol, forming two sets of conformers that differed in the relative orientation of the N-terminal domain. We used a chromogenic Limulus assay to determine the EC50 of the peptide (the concentration that bound 50% of the added LPS). Studies with full-length and truncated SMAP-29 molecules revealed that each end of the holopeptide contained an LPS-binding domain. The higher affinity LPS-binding domain was situated in the flexible N-terminal portion. LPS binding to full-length SMAP-29 showed positive cooperativity, so the EC50 of the peptide (2.6 µm) was considerably lower than that of the individual LPS-binding domains. LPS-binding studies with a mixture of truncated peptides revealed that this cooperativity was primarily intramolecular (i.e. involving the N- and C-terminal LPS-binding sites of the same peptide molecule). CAP-18[106,142], an antimicrobial cathelicidin peptide of rabbits, resembled SMAP-29 in that it contained N- and C-terminal LPS-binding domains, had an EC50 of 2.5 µm, and bound LPS with positive cooperativity. We conclude that the presence of multiple binding sites that function cooperatively allow peptides such as SMAP-29 and CAP-18 to bind LPS with high affinity. [source] Measurement of dissociation constants (pKa values) of organic compounds by multiplexed capillary electrophoresis using aqueous and cosolvent buffers,JOURNAL OF PHARMACEUTICAL SCIENCES, Issue 7 2008Marina Shalaeva Abstract Evaluation of a multiplexed capillary electrophoresis (CE) method for pKa measurements of organic compounds, including low solubility compounds, is presented. The method is validated on a set of 105 diverse compounds, mostly drugs, and results are compared to literature values obtained from multiple references. Two versions of the instrument in two different labs were used to collect data over a period of 3 years and inter-laboratory and inter-instrument variations are discussed. Twenty-four point aqueous and mixed cosolvent buffer systems were employed to improve the accuracy of pKa measurements. It has been demonstrated that the method allows direct pKa measurements in aqueous buffers for many compounds of low solubility, often unattainable by other methods. The pKa measurements of compounds with extremely low solubility using multiplexed CE with methanol/water cosolvent buffers are presented. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:2581,2606, 2008 [source] Improvements for the visualization of low-molecular weight protein and peptides of human tears using MALDIPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 17 2008Terry Nguyen-Khuong Abstract Many low-molecular weight proteins and peptides in human tears are potentially bioactive proteins but the range and number of these is yet to be fully characterized. A number of different sample preparation techniques were used to maximize the visualization of peptides from reflex tears. Samples were pretreated using precipitation and filtration techniques prior to analyses using MALDI-TOF MS. Peptides were searched for between 700 to 4000 m/z. Sample dilution in several different buffer systems followed by filtration with a 30-kDa cutoff filter and C18 reverse phase microcolumn purification produced significantly (p = 0.049) more peaks in tears than other methods used to prepare tears prior to MALDI-TOF MS. This study has established a technique for optimizing the visualization of naturally occurring peptides in tears. [source] Linear Expression of the Mathematical Relationship between Electroosmotic Mobility and Buffer ConcentrationCHINESE JOURNAL OF CHEMISTRY, Issue 1 2006Xu Xu Abstract It was believed that electroosmotic mobility ,eo is inversely proportional to the square root of the ionic strength I. But the linear relationship for regression analysis was expressed differently in different papers. The paper studied the linear expression of the mathematical relationship between ,eo and c (background buffer concentration) by mathematical transform and real experimental data. ,eo values of fused silica capillary were determined in four buffer systems. Their experimental conditions were controlled carefully for decreasing temperature difference ,T and pH difference ,pH in 50 µm ID capillary, in which no double layer overlap existed. The linear relationship between the reciprocal of electroosmotic mobility and the square root of concentration (or ionic strength) was derived by mathematical method. The regression analysis of experimental data was shown to well correspond to the relationship. The constants in regression equation could be well defined and the calculated results were acceptable. [source] In-vitro and in-vivo studies of cefpirom using bile salts as absorption enhancersJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 12 2003Yahya Mrestani ABSTRACT Cephalosporins have to be administered by injection because of the poor intestinal absorption of the orally delivered drugs. Because of the obvious drawbacks of drug delivery by injection, the development of alternatives with enhanced oral bioavailability is receiving much attention in pharmaceutical research. Cefpirom (Cp) is a new semi-synthetic amino-2-thiazolyl-methoxyimino cephalosporin that has been substituted in position 3 with a cyclopenteno-pyridinium group in order to create a zwitterionic compound. It exhibits highly hydrophilic properties, as shown from its extremely low partition coefficient, and therefore its lipophilicity was increased using bile salts. The effect of this on the partition coefficients determined in the n-octanol/buffer system was confirmed using an in-vitro transport model with artificial and biological membranes. The pharmacokinetic properties of Cp were investigated in rabbits after intraduodenal administration with and without bile salts. Furthermore, the physiological compatibility of the bile salts was investigated using active D-glucose transport. [source] | |||||||||||||||||||