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PubMed1998

Factors influencing the choice of buffer in background electrolytes for indirect detection of fast anions by capillary electrophoresis.

The suitability of relatively slow (low absolute value of mobility) coanionic buffers in background electrolytes (BGEs) for indirect photometric detection of anions by capillary electrophoresis was investigated. As a model system, 2-(cyclohexylamino)ethanesulfonic acid (CHES) was used to buffer the indirect detection electrolyte of sodium chromate. CHES (PKa 9.55) is a zwitterionic molecule carrying a net negative charge depending on the pH (effective charge -0.5 at pH = pKa). Within its useful pH buffering range CHES acted as a competing probe coanion. System peaks were induced which had deleterious effects on the detection sensitivity of slow to medium mobility anions. The mobility of the system peak was determined by the effective mobility of CHES, both of which increased with increasing pH. The peaks of analytes that migrated near or on the system peak were distorted and lost all quantitative properties. Analytes that migrated after the system peak either were not detected or reversed their responses. Analytes that migrated well before the system peak were unaffected. Consequently, the suitability of slow coanionic buffers is limited either to (i) fast anions or, (ii) a pH range much below the PKa, where the buffering capacity is not optimal.

Anions↗
PubMed2004

Performance of throughout in-capillary derivatization capillary electrophoresis employing an on-line sample and run buffer loading device.

We report on the effect on performance of varying the length of the capillary during throughout in-capillary derivatization (TICD) capillary electrophoresis (CE). Performance was evaluated by on-line coupling with a sample and CE runbuffer loading device that was newly introduced for this study. The device was assembled with a low cost using two 5 mm inner diameter (ID) disposable polyethylene syringes. First, a sequence was manually formed consisting of a 200 microL run buffer solution plug, a 100 microL sample plug and another 200 microL run buffer solution plug. Each plug was separated from its neighbor by a 100 microL air plug. When each plug reached the injection point where both a platinum-wire anode and the end of the separation capillary tube were located, 340 V/cm separation voltage (electrophoresis voltage) and 34 V/cm injection voltage were applied to the capillary for 3 s. Then the analytes were derivatized during migration in 50 microm ID capillaries filled with 2 mM o-phthalaldehyde (OPA)/N-acetylcysteine (NAC) in a 20 mM phosphate-borate buffer (pH 10), followed by separating and detecting of OPA derivatives by absorbance of 340 nm. Derivatization, separation, and detection were performed systematically using capillaries which varied in length from 5 to 80 cm. In the case of TICD-CE of a mixture containing 1 mM aspartic acid (Asp) and 20 mM m-nitorophenol (MNP) as a test solution, it was determined that peak area and peak width ratios of Asp to MNP did not depend on capillary length. Enantiomeric separations of DL-alanine (Ala) and Asp were examined using a run buffer consisting of a 45 microM beta-cyclodextrin (CD)-2 mM OPA/NAC-20 mM phosphate-borate buffer (pH 10). Even though the resolution of these enantiomeric pairs decreased with decreasing capillary length, as expected, the peaks corresponding to both enantiomeric amino acids were identified even when a 5 cm capillary was used. An 8-component amino acid mixture was also tested with 5 cm and 10 cm capillaries.

Aspartic Acid↗
PubMed2004

Effect of surfactant concentration and buffer selection on chromatographic figures of merit in chiral microemulsion electrokinetic chromatography.

The enantiomeric resolution of 15 different pharmaceutical compounds was explored using chiral microemulsion electrokinetic chromatography (MEEKC). The microemulsion employed was comprised of the chiral surfactant dodecoxycarbonylvaline (DDCV), 1-butanol, and ethyl acetate, at an initial composition of 1% w/v:1.2% v/v:0.5% v/v, respectively. The effect of varying the background buffer composition, voltage, and ultimately the surfactant concentration and/or aggregate phase ratio were examined. Changing from a zwitterionic buffer ((2-[2-amino-2-oxoethyl)amino]ethanesulfonic acid, ACES) to the same concentration of phosphate buffer improved the efficiency and decreased overall analysis time, but also resulted in a decrease in chiral resolution. Furthermore, using phosphate buffer while simultaneously increasing the percent DDCV from 1 to 4% increased the efficiencies from a range of 34,000 to 59,000 N/m to a range of 160,000 to 400,000 N/m. While the enantioselectivities did not change significantly, the improvement in efficiencies, elution range, and retention factors provided an increase in both resolution and the number of enantiomers that were separated. Using an optimized microemulsion comprised of phosphate buffer and 4% DDCV, chiral separation was achieved for all 11 pairs of enantiomers, with a resolution ranging from 0.90 to 4.71. Moreover, the average resolution doubled in going from nonoptimized to optimized conditions for five of the eleven compounds. Finally, a comparison was made of the effect of increasing only the surfactant concentration by a factor of 4 versus increasing the overall composition (or phase ratio) by a factor of 4. Ultimately, the microemulsion containing 4% DDCV provided a larger elution range, greater resolution, and more optimal retention than that provided by the 4x phase increase.

Buffers↗
PubMed2006

Enhanced binding and conformational selectivity in affinity capillary electrophoresis using a water-soluble resorcin[4]arene as intrinsic buffer and electrokinetic host.

Affinity capillary electrophoresis (ACE) is a widely used technique for quantifying non-covalent molecular interactions that is dependent on the specific buffer conditions selected. In this study, dynamic 1:1 host-guest inclusion complexation involving a charged resorcin[4]arene with a group of neutral corticosteroids was examined by ACE, where the macrocycle serves as both an intrinsic buffer and electrokinetic host. It was determined that over a 200% enhancement in the apparent binding constant (KB) was realized by ACE when using the host as an intrinsic buffer at pH 7.5 relative to an extrinsic sodium phosphate buffer system, which was also confirmed by 1H-NMR experiments. This report also revealed improved selectivity mediated by the discrete conformational properties of the complex among similar corticosteroid guests, as reflected by the complex mobility (mu(ep, AC)) or the relative change in complexation-induced mobility (1-mu(ep, AC)/mu(ep, C)). This latter property is a unique feature of ACE reflective of hydrodynamic size selectivity involving bulky guests or substrates. 1H-NMR and computer molecular modeling provided complementary information regarding the relative orientation, conformation and overall molecular area of the complex. The coupling of thermodynamic (KB) and electrokinetic (mu(ep, AC)) factors associated with complex formation in buffered solutions that minimize the effects of extrinsic electrolytes serves to enhance enthalpy-driven molecular recognition processes by ACE.

Adrenal Cortex Hormones↗
PubMed1981

An in-vitro study of the effect of buffer on the degradation of poly(glycolic acid) sutures.

The tensile strengths of poly(glycolic acid) (PGA) sutures immersed in buffered and unbuffered aqueous media were compared. The media used were an unbuffered physiological saline solution (pH = 5.0) and a phosphate-buffered physiological saline solution (pH = 7.4). PGA samples were immersed for various periods in each medium, and kept at 37 +/- 1 degree C in a constant temperature oven. The tensile strengths of the specimens were tested immediately after removal from the medium. Stress-strain curves of the specimens were expressed in terms of the stress unit "tenacity," commonly used in the study of fibrous polymers; it is an appropriate unit for materials of fibrous nature. These stress-strain curves were investigated as functions of buffering and duration of immersion. Degradation reduced the tensile strength of PGA more in the buffered saline solution than in the unbuffered. This higher rate of degradation in the buffered solution might be due to the presence of Na2HPO4, which removed the degradation products, shifted the reaction toward increased hydrolosis, and accelerated the loss of tensile strength in the PGA. A continuous decrease in the pH of the unbuffered solution supports this explanation. Tied-chain segments of macromolecules, a theory widely used in the study of mechanical strength of fibrous polymer may be the key to a comprehensive description of the degradation phenomenon of PGA.

Biodegradation, Environmental↗
PubMed1990

Inhibition of late endosome-lysosome fusion: studies on the mechanism by which isotonic-K+ buffers alter intracellular ligand movement.

Incubation of alveolar macrophages or hepatocytes in media in which Na+ is replaced by K+ ("isotonic-K buffer") inhibited the movement of internalized ligand from late endosomes to lysosomes (Ward et al.: Journal of Cell Biology 110:1013-1022, 1990). In this study we investigate the mechanism responsible for the isotonic-K+ block in movement of ligand from late endosomes to lysosomes. We observed that iso-K+ inhibition of endosome-lysosome fusion is not unique to alveolar macrophages or hepatocytes but can be seen in a variety of cell types including J774 and Hela cells. The inhibition in intracellular ligand movement was time dependent with the maximum change occurring after 60 minutes. Once established the inhibition resulted in a prolonged and apparently permanent decrease in vesicle movement. Cells were able to recover from the effects of iso-K+ buffers over a time course of 5-10 minutes when placed back in Na(+)-containing media. The effect of iso-K+ buffers was independent of intracellular pH changes and appeared to involve cell swelling. When cells were incubated in iso-K+ buffers under conditions in which cell volume changes were reduced, intracellular ligand movement approached normal levels. Such conditions included replacing Cl- with the less permeant anion gluconate, and by addition of sucrose to isotonic-K+ buffers. Analysis of the mechanism by which changes in cell volume could alter intracellular movement ruled out changes in cyclic nucleotides, Ca2+, or microtubules. These results suggest that changes in cell shape or volume can alter intracellular transport systems by novel routes.

Animals↗
PubMed2006

Effect of buffer species on the thermally induced aggregation of interferon-tau.

It is now becoming apparent that a common pathway of protein aggregation involves the unimolecular structural rearrangement from the native state to a slightly expanded aggregation-competent species. It is the goal of this study to understand the aggregation and the effects of buffer on the stability of IFN-tau. In this study, the thermally-induced aggregation of interferon-tau (IFN-tau) is described. By monitoring the aggregation rate in the presence of increasing amounts of sucrose, the relative change in surface area (Deltas) for conversion to the aggregation-competent state can be determined. Under conditions of pH 7 and in 20 mM buffer, the protein displays different aggregation rates depending on the nature of the buffer species. The protein aggregates mostly quickly in phosphate buffer, slower in the presence of Tris and slowest in the presence of histidine. The largest value for Deltas occurs for the histidine-containing samples, where aggregation proceeds via a slightly expanded aggregation competent state with a surface area increase of 7.6%. Furthermore, it appears that histidine binds to the native state of IFN-tau, thereby stabilizing the native state and retarding aggregation. Measurement of the second virial coefficient, B(22), for different formulations indicates that inclusion of histidine has only a small effect on repulsion between protein molecules, suggesting that colloidal stabilization is not the dominant mechanism for stabilization of IFN-tau. This study represents the first detailed biophysical study of specific buffer-induced stabilization, resulting in shifting the equilibrium towards the native state and away form the expanded aggregation-competent species.

Animals↗
PubMed1983

Influence of food on aspirin absorption from tablets and buffered solutions.

After a standard meal, 12 normal volunteers received three aspirin dosage forms in a single-dose, complete crossover study. The three dosage forms were an unbuffered tablet, an effervescent solution with 16 meq of buffer, and an effervescent solution with 34 meq of buffer. Plasma and urine aspirin, salicylic acid, and salicyluric acid were measured for 10 hr. Significant differences in the absorption kinetics of aspirin were observed, with aspirin from the two solutions being absorbed faster than from the tablet. Urine pH and renal clearance for all three acid compounds were influenced by the buffer during the first 2 hr only. Area under the curve (AUC) and urine accumulation comparisons suggest that 15-20% more aspirin reaches the general circulation after the tablet, but that the total salicylate absorbed is not different. Comparison with an earlier study indicates the solution with 34 meq of buffer is virtually unaffected by the presence of the meal while the solution with 16 meq buffer and the tablet are more slowly absorbed in the nonfasted state.

Adult↗
PubMed1985

Dissolution of carboxylic acids. III: The effect of polyionizable buffers.

The dissolution behavior of three carboxylic acids of variable aqueous solubility but with approximately equal pKa values into aqueous buffered solutions has been studied as a function of pH and of buffer properties. The dissolution from constant-surface-area compressed disks of benzoic acid, 2-naphthoic acid, and indomethacin into solutions of constant ionic strength (mu = 0.5 with potassium chloride) and constant pH (maintained by pH stat) at 25 degrees C using a rotating disk apparatus was evaluated. Models for dissolution of these weak acids into diprotic and triprotic buffering media are developed to predict the flux of the acid as a function of bulk solution pH and the physical and chemical properties of the buffer and acid. The models assume that mass transfer can be represented by a single second order diffusive term and that instantaneous equilibrium between all reactive species exists. Values of flux and pH at the solid-liquid interface are calculated and the fluxes compared to experimentally determined values. Reasonable correlation was found between values predicted by the models and experimental flux values. Major influences on model accuracy are the Ka and physical properties of the buffer.

Benzoates↗
PubMed2002

Rabbit cloning: improved fusion rates using cytochalasin B in the fusion buffer.

This study investigated the possibility of producing rabbits from embryos obtained by transfer of somatic cell nuclei; Muscle biopsies were taken from the upper part of the hind limb of fetuses at 24 days of gestation. Fetal fibroblasts were cultured in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% FCS at 38 degrees C in 5% CO(2) in air. Cells were passaged at least three times; for 5 days prior to nuclear transfer, cells were cultured in 0.5% FCS medium to induce them into G(0). These cells then were placed into the perivitelline space of enucleated rabbit MII stage oocytes. Fusion media were Ca/Mg-containing 0.3 M mannitol (Buffer 1) or 0.3 M mannitol plus 7.5 microg/ml cytochalasin B (CB) (Buffer 2). Fusion was studied with 6 protocols: culture of donor cell-cytoplast complexes (CCCs) in TCM199 for 0, 30, or 60 min after micromaniupulation and electrical stimulation in Buffer 1 or Buffer 2. Fusion rates were 57.4, 42.9 and 52.8%, respectively, in groups 0 + B1, 30 + B1, and 60 + B1, significantly lower than the rates in groups 0 + B2, 30 + B2 and 60 + B2 (75.9, 75.7, and 76.4%, respectively) (P < 0.05). Thus, CB in electrical fusion buffer improved the fusion rate. The percentages of blastocyst formation were 40% in 0 + B2 and 37.1% in 0 + B1 groups, significantly higher (P < 0.05) than those in 30 + B1 (20%), 30 + B2 (15.6%), 60 + B1 (13%) and 60 + B2 (8.3%). A total of 653 cloned embryos were at 1-cell, 2-4-cell, or morula/blastocyst stages were transferred to 44 mated or non-mated synchronized recipients. No cloned embryos developed to term.

Animals↗
PubMed1994

On-line buffer removal and fraction selection in gradient capillary high-performance liquid chromatography prior to electrospray mass spectrometry of peptides and proteins.

The technique for on-line removal of buffers and for fraction selection to eliminate source clogging and suppression of electrospray ionization by unwanted constituents consisted of: (i) selecting the initial mobile phase composition to retain analytes on top of a capillary high-performance liquid chromatography (HPLC) column; (ii) washing the buffer isocratically to waste, using a two-way micro dump valve; (iii) starting the gradient and switching the dump valve to introduce analytes into the source. Peptide and protein mixtures were prepared in 0.05-0.5 M phosphate and 0.55 mM tris-based buffers, and water. After buffer removal, chromatograms and electrospray spectra were indistinguishable from those of aqueous controls, down to 1 pmol (acidic fibroblast growth factor) consumed, and up to 78 kDa (bovine transferrin) molecular weight. Aided by the dump valve, 100 fmol of angiotensin could be fractionated and identified on the slope of a 10 x 10(6) fmol of leucine enkephalin HPLC peak. On-line buffer removal and fraction selection eliminate the need for additional preparation steps than can lead to excessive sample losses.

Buffers↗
PubMed1993

Nonurea sodium dodecyl sulfate-polyacrylamide gel electrophoresis with high-molarity buffers for the separation of proteins and peptides.

A sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a discontinuous buffer system for separation of both peptides and proteins, which is a modification of the Laemmli system, is described. In the modified procedure, twofold higher concentrations of buffers in the separating gel and the running buffer solution allow superior resolution for peptides as small as M(r) 5000. The resolution of peptides was dependent on salt concentrations in the systems in which sodium chloride was partially substituted for Tris-HCl buffer and buffer concentrations were varied. In the stacking gel of the modified procedure, detection of peptides and SDS demonstrated a sharp stack of peptides at the trailing edge of the SDS stack. On the other hand, this SDS stack included peptides, forced them to diffuse, and produced a broad starting zone under Laemmli conditions. In addition, following expansion of the SDS stack impaired peptide resolution further in the separating gel. Accordingly, the different interaction with the SDS stack in the stacking process was found to produce different resolution of peptides in the electrophoretic procedures. The modified conditions have potential to provide a superior alternative to the Laemmli system for analysis of various proteins.

Buffers↗
PubMed1993

Isoelectric focusing: pH gradients established with simple buffers and a cation-selective membrane.

A method for isoelectric focusing is presented in which pH gradients are established with simple buffer systems in a thin granulated gel bed. The gel bed is supported by a cation-selective membrane over a buffer-filled channel with a sloping bottom. The component of the electric field parallel to the gel and membrane surfaces that results from this arrangement may be considered fixed at each position in the gel bed but increases with the decreasing depth of the buffer filled channel below. This feature, together with the ion-selective properties of the supporting membrane, allows the electric field to maintain a shallow pH gradient by adjusting the composition of the buffer at each position in the gel bed. Ribonuclease, myoglobin, beta-lactoglobulin, and ovalbumin, representing proteins with a wide distribution of isoelectric points, were focused in the buffers glycine, imidazole, Mes, and acetic acid, respectively. Focused protein fractions were rerun on conventional polyacrylamide-carrier ampholyte gels.

Buffers↗
PubMed2000

Polymerization of deoxy-sickle cell hemoglobin in high-phosphate buffer.

Deoxy-sicklecell hemoglobin (HbS) polymerizes in 0.05 M phosphate buffer to form long helical fibers. The reaction typically occurs when the concentration of HbS is about 165 mg/ml. Polymerization produces a variety of polymorphic forms. The structure of the fibers can be probed by using site-directed mutants to examine the effect of altering the residues involved in intermolecular interactions. Polymerization can also be induced in the presence of 1.5 M phosphate buffer. Under these conditions polymerization occurs at much lower concentrations (ca. 5 mg/ml), which is advantageous when site-directed mutants are being used because only small quantities of the mutants are available. We have characterized the structure of HbS polymers formed in 1.5 M phosphate to determine how their structures are related to the polymers formed under more physiological conditions. Under both sets of conditions fibers are the first species to form. At pHs between 6.7 and 7.3 fibers initially form bundles and then crystals. At lower pHs fibers form macrofibers and then crystals. Fourier transforms of micrographs of the polymers formed in 1.5 M phosphate display the 32- and 64-A(-1) periodicity characteristic of fibers formed in 0.05 M phosphate buffer. The 64-A(-1) layer line is less prominent in Fourier transforms of negatively stained fibers formed in 1.5 M phosphate possibly because salt interferes with staining of the fibers. However, micrographs and Fourier transforms of frozen hydrated fibers formed in high and low phosphate display the same periodicities. Under both sets of reaction conditions HbS polymers form crystals with the same unit cell parameters as Wishner-Love crystals (a = 64 A, b = 185 A, c = 53 A). Some of the polymerization intermediates were examined in the frozen-hydrated state in order to determine whether their structures were significantly perturbed by negative staining. We have also carried out reconstructions of the frozen-hydrated fibers in high and low phosphate to compare their molecular coordinates. The helical projection of the reconstructions in low phosphate shows the expected 14-strand structure. In high phosphate the 14-strand fibers are also formed and their molecular coordinates are the same (within experimental error) as those of fibers formed in 0.05 M phosphate. In addition, the reconstructions of high-phosphate fibers reveal a new minor variant of fiber containing 10 strands. The polymerization products in 1.5 M phosphate buffer were generally indistinguishable from those formed in 0.05 M phosphate buffer. Micrographs of frozen hydrated specimens have facilitated the interpretation of previously published micrographs using negative staining.

Biopolymers↗
PubMed1994

Buffer-stimulated citrate efflux in Penicillium simplicissimum: an alternative charge balancing ion flow in case of reduced proton backflow?

Organic acids excreted by filamentous fungi may be used to win metals from industrial secondary raw materials. For a future commercial use a high production rate of organic acids is necessary. The conditions under which the commercially used fungus Aspergillus niger excretes high amounts of citric acid can not be maintained in metal leaching processes. However, Penicillium simplicissimum showed an enhanced citric acid efflux in the presence of an industrial filter dust containing 50% zinc oxide. Because Good buffers of high molarity were able to mimic the effect of zinc oxide, the high buffering capacity of zinc oxide and not an effect of the zinc ions was held responsible for the enhanced citric acid efflux. The presence of ammonium and trace elements reduced this buffer-stimulated citric acid efflux, whereas the plant hormone auxine canceled this reduction. This citric acid efflux was influenced by a depolarization of the membrane: the freely permeable compound tetraphenylphosphoniumbromide decreased the citric acid efflux, without decreasing intracellular citric acid or consumption of glucose and oxygen. Vanadate, an inhibitor of the plasma membrane H(+)-ATPase also reduced the buffer-stimulated citric acid efllux. The role of the efflux of citrate anions as an alternative charge balancing ion flow in case of impaired backflow of extruded protons because of a high extracellular buffering capacity is discussed.

Biological Transport↗
PubMed1993

Calcium buffering in bursting Helix pacemaker neurons.

Bursting pacemaker neurons of the snail Helix pomatia were voltage-clamped and Ca currents in response to depolarizing steps were recorded. Simultaneously, changes in intracellular Ca concentrations were measured using the fluorescent dye fura-2 and a highly sensitive digital camera. Ca influx through voltage-gated channels induced a spatially non-uniform increase in intracellular Ca. The Ca signals decayed with a time constant of about 5 s. By increasing the concentration of the indicator dye, its Ca-buffering capacity was enhanced and Ca transients in response to depolarization were diminished. Thereby, the endogenous Ca buffer capacity could be determined and was calculated to be about 480 buffered ions for every free Ca ion. The buffer capacity did not vary significantly with the amount of Ca influx within the range tested, suggesting that the buffer is not saturated at Ca concentrations of up to 1 microM.

Animals↗
PubMed1977

Influence of inorganic phosphate and organic buffers on cephalosporin production by Streptomyces clavuligerus.

A high concentration of potassium phosphate (75--100 mM) stabilized pH and supported extensive growth of Streptomyces clavuligerus in a chemically defined medium; such a concentration also inhibited cephalosporin production. Although Tris buffer was found to have detrimental effects on growth and antibiotic production, 3-(N-morpholine)-propane sulfonate (MOPS) or 2-(NP-morpholine)-ethane sulfonate (MES) buffer provided a nontoxic buffering system. In the presence of MOPS buffer, cephalosporin production was optimal at 25 mM phosphate, whereas higher concentrations of phosphate progressively inhibited antibiotic production up to 85% without modifying the pH pattern. MOPS buffer can be used to conduct fermentations at a relatively constant pH value in shake flasks.

Alkanesulfonates↗
PubMed1991

The pharmacokinetics of aspirin in rats and the effect of buffer.

Aspirin (acetylsalicyclic acid) was administered to rats intravenously, orally, and intraintestinally at different doses or in different dosage forms. The distribution and elimination kinetics of aspirin in rats following intravenous administration were best described by a two-compartmental open system and were dose independent up to 15 mg/kg. The terminal elimination half-life following intravenous dosing (10 mg/kg) was 3.36 +/- 0.85 min (n = 15) with the clearance being 8.40 +/- 1.24 L/(kg.hr). Intravenous distribution and elimination kinetics of aspirin in rats were not influenced by an orally administered buffered solution with a buffer capacity of 0.933 mEq ANC (acid neutralizing capacity) per kg of body weight. However, this orally buffered solution did change the gastrointestinal absorption kinetics of aspirin in rats. The absolute bioavailable dose of aspirin was 56.6 +/- 10.4% (n = 6) following its administration in an unbuffered solution while it was only 31.8 +/- 8.0% (n = 6) following administration in the buffered solution. The corresponding values of the absolute bioavailable doses were 43.4 +/- 3.7% and 25.5 +/- 1.8% following intraintestinal administration. The lower systemic availability of aspirin in the presence of buffer is attributed to a greater fraction of the administered dose becoming available for absorption from the intestine where the extraction efficiency is higher than that in the stomach.

Administration, Oral↗