Generation Of pH Gradient Research Articles

Fundamental formulation of electrokinetic extraction of contaminants from soil

Electro-osmosis and ionic migration are the basic cleanup mechanisms in the electrokinetic extraction of contaminants from fine-grained soils. These are coupled flows as the flows of fluid and contaminants are driven by an externally applied electrical gradient. Moreover, other electrochemical reactions will occur simultaneously during the process. The most pronounced effect is the generation of pH gradient in the soil. The change of pH in the pore fluid can have a significant impact on the degree of sorption and desorption of chemicals on soil particle surfaces, complexes formation and precipitation of chemical species, and dissociation of organic acids; thus affecting the feasibility and efficiency of the cleanup technique tremendously. An attempt is made to formulate the coupled flows of ionic contaminants and the resulting change of pH in the pore fluid during the electrokinetic extraction process. The coupled flows of contaminants are formulated by the formalism of nonequilibrium thermodynamics. The pH is determined as a function of time and space by maintaining electrical neutrality throughout the system all the times. A numerical model NEUTRAL is developed to simulate the processes. The good agreement between computed and experimental results published in the literature indicates that the approach is a valid step toward a better understanding of the physics and chemistry involved during electrokinetic treatment of contaminated soils. Key words : electrokinetics, in situ remediation, contaminated soil, coupled flows, electrochemistry, nonequilibrium thermodynamics.

Numerical studies of multicomponent chromatography using pH gradients

AbstractA general numerical method is developed for multicomponent chromatography for the case where a pH gradient occurs and several buffering species are present that become adsorbed together with the components being separated through an ion‐exchange mechanism. Acid‐base equilibrium relations are used to determine the ionic compositions in the liquid and adsorbed phases and are solved using a single‐variable Newton's method. The differential material‐balance relations are integrated numerically using the method of characteristics. The transport relations are incorporated using a matrix analog of the linear‐driving‐force approximation. The resulting method accounts for the adsorption of each ionic form of each buffering species, for multicomponent diffusional interactions arising from induced electric fields, for volume and concentration overloading of proteins, and for changes in the adsorption capacity caused by pH variations. Numerical calculations illustrate factors govening the selection of the adsorbent and buffer components for use in separating mixtures of proteins using retained, internally generated pH gradients.

Extracellular pH regulation in microdomains of colonic crypts: effects of short-chain fatty acids.

It has been suggested that transepithelial gradients of short-chain fatty acids (SCFAs; the major anions in the colonic lumen) generate pH gradients across the colonic epithelium. Quantitative confocal microscopy was used to study extracellular pH in mouse distal colon with intact epithelial architecture, by superfusing tissue with carboxy SNARF-1 (a pH-sensitive fluorescent dye). Results demonstrate extracellular pH regulation in two separate microdomains surrounding colonic crypts: the crypt lumen and the subepithelial tissue adjacent to crypt colonocytes. Apical superfusion with (i) a poorly metabolized SCFA (isobutyrate), (ii) an avidly metabolized SCFA (n-butyrate), or (iii) a physiologic mixture of acetate/propionate/n-butyrate produced similar results: alkalinization of the crypt lumen and acidification of subepithelial tissue. Effects were (i) dependent on the presence and orientation of a transepithelial SCFA gradient, (ii) not observed with gluconate substitution, and (iii) required activation of sustained vectorial acid/base transport by SCFAs. Results suggest that the crypt lumen functions as a pH microdomain due to slow mixing with bulk superfusates and that crypts contribute significant buffering capacity to the lumen. In conclusion, physiologic SCFA gradients cause polarized extracellular pH regulation because epithelial architecture and vectorial transport synergize to establish regulated microenvironments.

Towards new formulations for polyacrylamide matrices: N-acryloylaminoethoxyethanol, a novel monomer combining high hydrophilicity with extreme hydrolytic stability.

Matrices for electrokinetic separations, based on a unique class of mono- and disubstituted (on the amido nitrogen) acrylamides such as e.g., N-acryloylaminoethoxyethanol (AAEE) and acrylamido-N,N-diethoxyethanol, offer the following advantages: (i) strong resistance to alkaline hydrolysis (most zone separations occurring at basic pH values), (ii) high hydrophilicity and (iii) greater porosity, due to the higher molecular weight of the monomers. When compared with conventional poly(acrylamide), a poly(AAEE) matrix, when subjected to mild alkaline hydrolysis (0.1 N NaOH, 70 degrees C) appears to be 500 times more stable. Such stability is also confirmed under strong alkaline hydrolysis (1 N NaOH, 100 degrees C) as well as under mild and strong acidic hydrolysis. Mildly hydrolyzed poly(AAEE) matrices still perform extremely well in both conventional isoelectric focusing and immobilized pH gradients, techniques which are quite sensitive to traces of acrylate in the polymer coil. Conversely, mildly hydrolyzed poly(acrylamide) matrices, when used in isoelectric focusing, generate pH gradients between pH 4 and 5, having an inflection point (pH 4.6) equivalent to the pK value of acrylic acid. This novel class of monomers shows great promise for future applications in all electrokinetic methodologies.

Ampholyte‐free isoelectric focusing of proteins in cone shaped capillaries

AbstractA simple method of generating pH gradients for use in capillary isoelectric focusing was described and evaluated experimentally. In the proposed scheme, the cone shaped capillary filled with a conventional buffer is used to produce an effect of nonuniform heating which then generate temperature and corresponding pH gradients along the capillary axis. Concentration and separation of proteins by applying a simple isoelectric focusing system based on this principle is demonstrated. An inexpensive absorption imaging detection system is used to continuously monitor progress of the separation and concentration processes.

Protons, pumps, and potentials: Control of cytochrome oxidase

Cytochrome c oxidase oxidizes cytochrome c and reduces molecular oxygen to water. When the enzyme is embedded across a membrane, this process generates electrical and pH gradients, and these gradients inhibit enzyme turnover. This respiratory control process is seen both in intact mitochondria and in reconstituted proteoliposomes. Generation of pH gradients and their role in respiratory control are described. Both electron and proton movement seem to be implicated. A topochemical arrangement of redox centers, like that in the photosynthetic reaction center and the cytochrome bc1 complex, ensures charge separation as a result of electron movement. Proton translocation does not require such a topology, although it does require alternating access to the two sides of the membrane by proton-donating and accepting groups. The sites of respiratory control within the enzyme are discussed and a model presented for electron transfer and proton pumping by the oxidase in the light of current knowledge of the transmembranous location of the redox centers involved.

Computer-controlled generation of pH gradients in capillary zone electrophoresis

A new capillary zone electrophoresis (CZE) apparatus was developed with the possibility of computer-controlled pH-gradient elution. The details of the apparatus are described together with an example showing the analysis of aspartame, benzoate and caffeine in diet Pepsi.

Automatic titrations in unsegmented flow systems based on variable flow-rate patterns: Part 1. Principles and applications to acid-base titrations

Automatic acid-base titrations were carried out by generating pH gradients in a continuous-flow manifold. A programmable, computer-controlled pump was used to produce variable, reproducible flow-rate patterns that were used to increase gradually the titrant concentration in the flow system. A colorimetric indicator reagent allowed the spectrophotometric detection of the equivalence point. Strong and weak acids, both alone and in mixtures, and two protons of the same acid were titrated with a relative standard deviation of 1.8%. The procedure was applied to the analysis of various real samples.

Adaptation by Streptococcus mutans to acid tolerance.

Our previous continuous culture studies with strains of Streptococcus mutans have indicated that the organism has the capacity of adapt to growth in acidic environments. This study was undertaken to examine this question in more detail. S. mutans Ingbritt and the phosphotransferase system (PTS)-defective mutant, S. mutans DR0001/6, were grown in continuous culture at pH 7.5 and 5.5 or 5.1, and the pH optimum for glucose uptake and glycolysis and the capacity of the cells to generate pH gradients were determined over the pH range 4.5 to 8.0 with steady state, washed cells. In addition, the proton permeability of the cells was measured over the pH range by an acid pulse technique. The results indicate that the pH optimum for glucose uptake by S. mutans Ingbritt grown at pH 7.5 was 7.5 and this optimum shifted to 7.0 and 6.0 for cells grown at pH 5.5 and 5.1, whereas with the S. mutans DR0001/6, the optimum shifted from 7.5 to 7.0 for the pH 5.5 cells. A similar shift in the pH optimum for glycolysis was observed for the 2 strains, and this was particularly pronounced for cells incubated with glucose in the presence of gramicidin to dissipate proton gradients. The capacity of the cells to generate pH gradients was related to their metabolic activity, and although larger gradients were not formed by the pH 5.5 cells, these cells were nevertheless capable of maintaining gradients at a lower pH; S. mutans DR0001/6 generated 2-fold larger pH gradients at pH 5.5 than S. mutans Ingbritt.(ABSTRACT TRUNCATED AT 250 WORDS)

Isoelectric focusing field-flow fractionation : Experimental study of the generation of pH gradient

Isoelectric focusing field-flow fractionation is a method for the separation of ampholytes using, in addition to the electric field and pH gradient, the flow of the carrier liquid through the fractionation channel as the third active separating factor. Flow action permits a decrease in the channel dimension in the direction of the electric field. It makes it possible to decrease the absolute value of the voltage while maintaining a high field strength and results in a lower Joule heat production. Moreover, the laminar flow of the carrier liquid stabilizes the pH gradient against convection. A channel was designed from which ampholyte samples can be taken at twelve different positions. pH value of the samples taken were measured with the aid of a capillary pH electrode. The pH dependence of the applied voltage, flow-rate of ampholyte solutions and concentrations of the solutions of ampholyte and electrode electrolytes were measured at different channel positions. It follows from the measurements performed so far that the pH gradient generation is sufficiently fast and reproducible for use in isoelectric focusing field-flow fractionation.

A vesicular preparation enriched in basolateral membranes from cow ciliary body epithelium.

A procedure is described for the preparation of a tissue fraction enriched in basal-lateral membranes of the epithelium of ciliary body dissected from abattoir-derived cow eyes. Microscopical methods established the viability of the epithelial cells in the dissected tissue. A two-step homogenization procedure was employed. First, mild trituration with a Potter-Elvehjem tissue grinder was used to release the epithelial cells with minimal disintegration of the stroma and muscle components. Then, complete vesiculation of the released material was induced with a Dounce homogenizer. This material was fractionated through sequential differential and (Ficoll 400) equilibrium density gradient centrifugation steps. The procedure led to the isolation of a fraction enriched 16.8 +/- 5.5 (+/- SE, n = 5) fold in Na(+)+K(+)-ATPase activity with respect to the initial homogenate. Using a method to artificially generate pH gradients it was demonstrated that the preparation contained sealed vesicles and that the membranes of these vesicles exhibited a K+ selectivity consistent with a plasma membrane origin.

Cyclosporin A and vehicle toxicity in primary cultures of rabbit renal proximal tubule cells

The capability of cyclosporin to produce direct injury to primary proximal tubular renal cells was studied. These cells, when grown on Millicell inserts, retain the functional polarity of the proximal tubule, i.e., generate a transepithelial pH gradient (apical compartment acidic) that is reversibly blocked by amiloride addition only if it is added to the apical compartment. Administration of ouabain to the basal compartment also blocks the generation of the transepithelial pH gradient. Additionally, the cells were more responsive to parathyroid hormone (PTH), a proximal tubule characteristic, than to arginine vasopressin (AVP), a distal tubule characteristic. The following substances were tested for their effect on the capacity of these cells to generate a pH gradient: Sandimmune, the commercial form of cyclosporin A; the free form of the drug; Cremophor EL, the vehicle used in the commercial preparation; and ethanol, the vehicle used to dissolve the free form. Sandimmune, at 25-50 microM, inhibited the generation of the pH gradient within 24 h. Surprisingly, Cremophor also blocked the development of a pH gradient, although somewhat less effectively. In contrast, 10 microM cyclosporin, regardless of the form tested, had no effect for up to 96 h. These findings show that cyclosporin, in the form of Sandimmune, has a direct toxic effect on these cells; they also suggest that the vehicle, Cremophor, may contribute to the well-established nephrotoxicity of cyclosporin A.

On-line precolumn photochemical generation of pH gradient: micro-high-performance liquid chromatography of methotrexate and its impurities

On-line UV irradiation of a flowing mobile phase that contains formate buffer and hydrogen peroxide results in a significant increase in pH owing to the chain reaction to carbonic acid. This reaction was used for pH gradient formation in micro-high-performance liquid chromatography. The gradient was started by switching on a photoreactor which was inserted in front of the injection valve. The shape of the pH gradient was affected by covering part of the PTFE capillary from UV light. The range between the initial and final pH values corresponds to the range of p K a values of formic and carbonic acid. The use of photochemical pH control for the separation of methotrexate and its impurities without buffer mixing is illustrated. The reproducibility of retention times was better than 1% and of peak heights better than 25%. The detection limit for methotrexate was 5 pmol at 315 nm.

Substituted thieno[3,4-d]imidazoles, a novel group of H +/K +-ATPase inhibitors. Differentiation of their inhibition characteristics from those of omeprazole

The action of the H +/K +-ATPase inhibitors, Hoe 731 and S 4216, both thieno-imidazole derivatives, was compared with that of the benzimidazole derivative, omeprazole. In intact, gastric membrane vesicles under conditions shown to result in acidification of the vesicle interior, Hoe 731 and S 4216 inhibited H +/K +-ATPase activity with an IC 50 value of about 1.0 μM. In the absence of a generated pH gradient the respective IC 50 values were 5.5 and 2.1 μM. In contrast, omeprazole inhibited the enzyme only in the presence of proton accumulation (IC 50: 0.7 μM). The inhibitory action of omeprazole on H +/K +-ATPase-mediated proton transport was prevented by the membrane permeable mercaptane, dithioerythritol, but not by the membrane impermeable, mercaptane glutathione, whereas both mercaptanes were able to prevent the effect of Hoe 731 and S 4216. These results indicate that the thienoimidazoles react with intravesicular (luminal) and extravesicular (cytosolic) SH groups of the H +/K +-ATPase, whereas omeprazole interacts uniquely with luminal SH groups of the enzyme. In isolated parietal cells all drugs caused a concentration-dependent inhibition of HCl production, as measured by [ 14C]aminopyrine uptake, during histamine and dibutyryl-cAMP stimulation. The IC 50 value was 0.1 μM for Hoe 731 and omeprazole and 0.4 μM for S 4216 after 30-min incubation. The inhibitory action of Hoe 731 and S 4216 faded with increasing incubation time, whereas omeprazole caused an unchanged inhibition over the entire 120-min incubation period. We suggest that several factors, e.g. weaker chemical stability of the drugs or perturbation of cellular glutathione levels, may be responsible for the fading inhibitory action of thienoimidazoles in the parietal cell.

By 1023/SK&F 96022: Biochemistry of a novel (H + + K +-ATPase inhibitor

The mechanism by which the substituted benzimidazole sulphoxide BY 1023/SK&F 96022 inhibited the (H + + K +-ATPase, the enzyme responsible for hydrogen ion secretion in the stomach, was studied in a variety of in vitro preparations. In gastric preparations that were capable of active hydrogen ion transport with consequent lumenal acidification, BY 1023/SK&F 96022 inhibited with high potency and in a time-dependent manner consistent with the acid-induced conversion of the parent benzimidazole sulphoxide to a covalent inhibitor (cyclic sulphenamide). The following ic 50 values were obtained for the inhibition of aminopyrine accumulation: intact gastric glands stimulated with 1 mM dibutyryl cAMP, 1.0 μM; permeabilized gastric glands stimulated with 5mM ATP, 0.42 μM; intact gastric vesicles stimulated with 150 mM KC1, 9 μM valinomycin and 2mM MgATP, 3.5 μM. In a preparation that could not generate pH gradients, lyophilized gastric vesicles at pH 7.4, BY 1023/SK&F 96022 inhibited K +-stimulated ATPase activity with relatively low potency, 70 μM, indicating its good chemical stability at neutral pH. As assessed by ATPase inhibition, this stability was three times greater than that of omeprazole. Inhibition by BY 1023/SK&F 96022 was not reversed by dilution in either permeabilized gastric glands or intact gastric vesicles. Inhibition could, however, be completely reversed by subsequent incubation with 20 mM β-mercaptoethanol (intact gastric glands) or 100 mM dithiothreitol (intact gastric vesicles) suggesting a disulphide link between inhibitor and enzyme. The concentration of glutathione needed to protect against inhibition by BY 1023/SK&F 96022 was 10,000 times higher in intact, compared with lyophilized, gastric vesicles indicating an interaction with the lumenal (extra-cellular) face of the (H ++ K +-ATPase. BY 1023/SK&F 96022 and omeprazole were also found to inhibit acidification in purified kidney lysosomes with ic 50 values of 194 and 75 μM, respectively. Protection by 10 μM glutathione suggested that this did not result from intralysosomal activation of these inhibitors. Thus, BY 1023/SK&F 96022 has the combined properties of good chemical stability at neutral pH and effective conversion to the cyclic sulphenamide at acidic pH. In this way the activation to the cyclic sulphenamide may be optimally restricted to the parietal cell canaliculus.

Electrofocusing of acid phosphatases from frog liver, using an immobilized pH gradient

Isoelectric focusing on carrier ampholyte-containing immobilized pH gradient gels was applied (i) to gels submerged in silicone oil on a Peltier cooled apparatus, (ii) to the separation of the higher molecular weight (HMW, Mr 140,000) and the lower molecular weight (LMW, Mr 38,000) acid phosphatases (AcPases) from frog livers. (i) Electrofocusing was conducted on gels submerged under silicone oil cooled and stirred on a Peltier-thermoregulated horizontal gel support plate. This procedure aimed at a) improving the temperature control of the gel by direct contact of coolant with the gel surface, and thus at being able to focus at the maximal field strength and consequently highest resolution; b) preventing evaporation from the gel and c) excluding atmospheric carbon dioxide. Silicone oil submersion did not abolish water loss from the gel into the electrolyte strips during isoelectric focusing, or a rippled gel surface. Absence of water exudation on the ripples noted previously by Atland [1] was observed. (ii) The electrofocusing of AcPases on immobilized pH gradients yielded patterns which remained stationary as a function of time, by contrast to previous analyses on carrier ampholyte generated pH gradients. The total number of enzymatically active components found in the enzyme preparations from different stages of purification and in the isolated HMW and LMW AcPases was 18. The HMW and LMW AcPases focused in characteristic pH ranges and exhibited qualitative and quantitative pattern differences. Their band patterns add up to that of a crude preparation containing both enzymes. Neither polyacrylamide gel electrophoresis (PAGE) at any nondenaturing pH, nor isoelectric focusing in carrier ampholytes with pattern changes due to the pH gradient drift were able to yield that result.

Interaction of the vitamin D‐binding protein (groupspecific component) and its ligand 25‐hydroxyvitamin D3: Binding differences of the various genetic types disclosed by isoelectric focusing

The three common variants of the vitamin D binding protein, also known as group specific component (Gc), namely types 1S, 1F and 2, as well as some rare variants were studied by thin-layer polyacrylamide gel isoelectric focusing in a pH 4.5-5.4 carrier ampholyte generated pH gradient, additionally containing N-(2-acetamido)-2-aminoethanesulfonic acid (ACES). Prior to isoelectric focusing, whole serum or purified preparations of the vitamin D binding protein were incubated with 25-hydroxycholecalciferol at various ligand/protein ratios. Binding differences were found for the anodal and cathodal isoforms of Gc 1 variants and also for various allelic types. Isoforms with higher isoelectric points generally had a lower affinity for the ligand than the variants with lower isoelectric points.

Light-dependent pH gradients are generated in liposomes containing ferrocyanide

Ferrocyanide solutions absorb light energy in a photochemical reaction that causes cyanide radicals to be lost from the iron complex. Because cyanide is a weak acid (p K a = 9.2), at pH ranges between 6 and 9 it associates with protons, thereby causing the pH of its solutions to increase. The pH change is proportional to the photon flux and concentration of ferrocyanide. For instance, the pH of an unbuffered 1.0 mM potassium ferrocyanide solution increases from 6 to 9 within seconds after illumination. We have encapsulated ferrocyanide in liposomes, and demonstrated that light-dependent pH gradients were readily produced, alkaline inside. This preparation offers a convenient system for generating pH gradients across lipid bilayer membranes. It also provides a useful model for simulating primitive bioenergetic systems relevant to light energy transduction by early forms of life.

Application of immobilized pH gradient isoelectric focusing to forensic hemogenetics: a survey on a three year experience with the transferrin (TF) and alpha 1-antitrypsin (PI) systems.

The subtypes of transferrin (TF) and alpha 1-antitrypsin (PI), first discovered using isoelectric focusing, are now mostly determined in immobilized pH gradient gels. We report on our experience in the parentage expertise with both polymorphisms over a period of three years. The complexity of the technology was compensated by the fact that most subtypes of TF and PI could be more reliably recognized. The PI alleles PI*M1, M2, M3, S, F, T, and Z and TF alleles TF*C1, C2 and C3, and in addition four further rare TF alleles were observed. The allele frequencies from non-related individuals did not deviate from the Hardy-Weinberg equilibria and corresponded well to known frequencies from West Germany and other Caucasoid populations. With the TF system 36 accused men, and with the PI system 54 were excluded from paternity from a total of 344 (TF) respectively 347 (PI) cases. From the data presented here isoelectric focusing in immobilized pH gradient gels appears to be a major improvement over carrier ampholyte generated pH gradients in the distinction of TF and PI phenotypes.

Generation of pH gradient across the rabbit collecting duct segments perfused in vitro

Ability of pH gradient generation was examined in three segments of rabbit collecting duct, cortical collecting duct (CCD), outer medullary collecting duct (OMCD) and inner medullary collecting duct (IMCD). These segments were perfused in vitro, and the steady-state luminal pH in stop-flow condition (pHs) and the electrochemical potential difference of H+ (EH) were determined using double-barreled liquid membrane pH microelectrode punctured into the lumen. In CCD, pHs was 7.71 +/- 0.08 in normal rabbits, 7.72 +/- 0.08 in DOCA-treated rabbits and 7.27 +/- 0.05 in starved rabbits, while peritubular fluid was kept at pH 7.5 EH (positive value means H+ accumulation in the lumen above electrochemical equilibrium) was -15.3 +/- 5.2, -31.3 +/- 3.7 and 10.3 +/- 3.1 mV, respectively. Peritubular acidification (peritubular pH 6.8) by reducing HCO3-concentration decreased pHs, but increased its negativity of EH in all groups. In OMCD pHs was 6.57 +/- 0.08 in normal, 6.58 +/- 0.11 in DOCA-treated and 6.47 +/- 0.12 in starved animals. EH was 54.5 +/- 4.6, 57.7 +/- 6.8 and 64.2 +/- 6.9 mV, respectively. Peritubular acidification lowered pHs further, 5.51 +/- 0.07, 5.67 +/- 0.16 and 5.41 +/- 0.19, respectively. EH was enhanced in all groups. In IMCD pHs was 7.36 +/- 0.04 with EH being 6.8 +/- 2.9 mV, and peritubular acidification did not generate pH gradient. These data suggest that the generation of a steep acid pH gradient is mainly due to OMCD. Luminal alkalinization predominated in CCD except in starved rabbits. IMCD did not generate appreciable pH gradient.(ABSTRACT TRUNCATED AT 250 WORDS)