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PubMed2006

Influence of calcium and phosphorus, lactose, and salt-to-moisture ratio on Cheddar cheese quality: pH buffering properties of cheese.

The pH buffering capacity of cheese is an important determinant of cheese pH. However, the effects of different constituents of cheese on its pH buffering capacity have not been fully clarified. The objective of this study was to characterize the chemical species and chemical equilibria that are responsible for the pH buffering properties of cheese. Eight cheeses with 2 levels of Ca and P (0.67 and 0.47% vs. 0.53 and 0.39%, respectively), residual lactose (2.4 vs. 0.78%), and salt-to-moisture ratio (6.4 vs. 4.8%) were manufactured. The pH-titration curves for these cheeses were obtained by titrating cheese:water (1:39 wt/wt) dispersions with 1 N HCl, and backtitrating with 1 N NaOH. To understand the role of different chemical equilibria and the respective chemical species in controlling the pH of cheese, pH buffering was modeled mathematically. The 36 chemical species that were found to be relevant for modeling can be classified as cations (Na+, Ca2+, Mg2+), anions (phosphate, citrate, lactate), protein-bound amino acids with a side-chain pKa in the range of 3 to 9 (glutamate, histidine, serine phosphate, aspartate), metal ion complexes (phosphate, citrate, and lactate complexes of Na+, Ca2+, and Mg2+), and calcium phosphate precipitates. A set of 36 corresponding equations was solved to give the concentrations of all chemical species as a function of pH, allowing the prediction of buffering curves. Changes in the calculated species concentrations allowed the identification of the chemical species and chemical equilibria that dominate the pH buffering properties of cheese in different pH ranges. The model indicates that pH buffering in the pH range from 4.5 to 5.5 is predominantly due to a precipitate of Ca and phosphate, and the protonation equilibrium involving the side chains of protein-bound glutamate. In the literature, the precipitate is often referred to as amorphous colloidal calcium phosphate. A comparison of experimental data and model predictions shows that the buffering properties of the precipitate can be explained, assuming that it consists of hydroxyapatite [Ca5(OH)(PO4)3] or Ca3(PO4)2. The pH buffering in the region from pH 3.5 to 4.5 is due to protonation of side-chain carboxylates of protein-bound glutamate, aspartate, and lactate, in order of decreasing significance. In addition, pH buffering between pH 5 to 8 in the backtitration results from the reprecipitation of calcium and phosphate either as CaHPO4 or Ca4H(PO4)3.

Buffers↗
PubMed1976

Effect of ethanesulfonic acid buffers and pH on the accumulation of a nervous system-specific protein (S-100) and a glial-enriched enzyme in a clonal line of rat astrocytes (C6).

Stationary phase cultures of a clonal line of rat astrocytes (C6) were maintained at pH values ranging from 6.0 to 8.4 using media buffered with various combinations of organic buffers or graded concentrations of bicarbonate ion at a constant CO2 tension. The accumulation of a soluble acidic protein unique to the nervous system (S-100) in media buffered with organic buffers was optimal in the pH range 6.4 to 6.8, significantly more acid than that optimal for cell growth (pH 7.0 to 7.8). Cells maintained in CO2-bicarbonate-buffered media exhibited a higher and less marked pH optimum for S-100 protein accumulation and a lower efficiency of accumulation of the protein. These data suggest that the organic buffer ions themselves, apart from their function as buffers, are influencing the accumulation of S-100. The specific activity (assayed at the enzymatic pH optimum) of a membrane-bound enzyme enriched in glial cells and myelin, 2',3'-cyclic nucleotide 3'-phosphohydrolase, was markedly pH-dependent. The optimal pH range was 6.4 to 6.7 in organic buffer controlled media. In CO2-bicarbonate controlled media the optimal pH range was only slightly higher (pH 6.6 to 7.0), but the specific activities were reduced relative to organic buffer-grown cells. The structural relationship of some of the aminoethanesulfonic acid buffers used in these experiments to certain compounds of neurochemical interest (such as taurine and alpha-flupenthixol) is noted.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1997

In vitro transcorneal permeation of ketorolac tromethamine from buffered and unbuffered aqueous ocular drops.

In vitro transcorneal permeation of ketorolac tromethamine from 0.5% w/v solutions containing equimolar (0.02 M) concentrations of citrate (pH 6.5), phosphate (pH 6.5 and 7), citrate-phosphate (pH 7) and borate (pH 7) buffers was studied using goat cornea. Cumulative % permeation was maximum with phosphate buffered drops of pH 6.5. The effect of pH and ionic strength on permeation of ketorolac tromethamine from buffered (phosphate) drops was next investigated. Cumulative % permeation of ketorolac tromethamine from buffered drops was pH dependent being maximum at pH 4.5. Adjustment of ionic strength of drops to 0.2 resulted in decreased permeation of drug. Permeation of ketorolac tromethamine from unbuffered drops of varying pH and ionic strength 0.2 was also pH dependent and was maximum at pH 4.5. Buffered drops of pH between 4.5-5.5, ionic strength 0.2, provided better permeation of drug compared to unbuffered drops of same pH and ionic strength. Above pH 6.5 unbuffered drops showed better permeation than buffered drops. Increase in molarity of phosphate buffer (pH 4.5) used in making drops, between 0 to 0.15 M increased permeation. Aqueous drops of ketorolac tromethamine formulated in 0.15 M phosphate buffer of pH 4.5 and ionic strength 0.2 showed maximum cumulative % permeation in vitro. Considering lacrimation induced drug loss in vivo, by buffer of high concentration, ketorolac tromethamine drops formulated in buffer of low molarity, pH 4.5 and ionic strength 0.2 appear suitable.

Animals↗
PubMed2002

Effects of liquid chromatography mobile phase buffer contents on the ionization and fragmentation of analytes in liquid chromatographic/ionspray tandem mass spectrometric determination.

The effects of liquid chromatography mobile phase buffer contents on the ionization and fragmentation of drug molecules in liquid chromatographic/ionspray tandem mass spectrometric (LC/MS/MS) determination were evaluated for simvastatin (SV) and its hydroxy acid (SVA). The objective was to improve further the sensitivity for SV by overcoming the unfavorable condition caused by the formation of multiple major adduct ions and multiple major fragment ions when using ammonium as LC mobile phase buffer. Mobile phases (70:30 acetonitrile-buffer, 2 mM, pH 4.5) with buffers made from ammonium, hydrazine or alkyl (methyl, ethyl, dimethyl or trimethyl)-substituted ammonium acetate were evaluated. Q1 scan and product ion scan spectra were obtained for SV in each of the mobile phases under optimized conditions. The results showed that, with the alkylammonium buffers, the alkylammonium-adducted SV was observed as the only major molecular ion, while the formation of other adduct ions ([M + H](+), [M + Na](+) and [M + K](+)) was successfully suppressed. On the other hand, product ion spectra with a single major fragment ion were not observed for any of the alkylammonium-adducted SVs. The affinity of the alkylammoniums to SV and the basicity of the alkylamines are believed to be factors influencing the formation and abundance of molecular and fragment ions, respectively. Methylammonium acetate provided the most favorable condition among all the buffers evaluated and improved the sensitivity several-fold for SV in LC/MS/MS quantitation compared with that obtained using ammonium acetate buffer. Better precision for SV in both Q1 and SRM scans was observed when using methylammonium buffer compared with those using ammonium buffer. The mobile phase buffer contents did not seem to affect the ionization, fragmentation and chromatography of SVA. The results of this evaluation can be applied to similar situations with other organic molecules in ionspray LC/MS/MS determination.

Anticholesteremic Agents↗
PubMed1999

pH and buffer capacities of apoplastic and cytoplasmic cell compartments in leaves.

After opening the stomata in CO(2)-free air, darkened leaves of several plant species were titrated with CO(2) at concentrations between 1 and 16%, in air in order to reversibly decrease cellular pH values and to calculate buffer capacities from pH changes and bicarbonate accumulation using both gas-exchange and fluorescence methods for analysis. After equilibration with CO(2) for times ranging between 4.4 and 300 s, fast CO(2) release from bicarbonate indicated catalysis by highly active carbonic anhydrase. Its time constant was below 2.5 s. Additional CO(2) was released with time constants of about 5, 15 and approximately 300 s. With CO(2) as the acidifying agent, calculated buffer capacities depend on assumptions regarding initial pH in the absence of an acid load. At an initial stroma pH of 7.7, the stromal buffer capacity was about 20 mM pH-unit(-1 )in darkened spinach leaves. At an initial pH of 7.5 it would be only 12 mM pH-unit(-1), i.e. not higher than expected solely on the basis of known stromal concentrations of phosphate and phosphate esters, disregarding the contribution of other solutes. At a concentration of 16%, CO(2) reduced the stromal pH by about 1 pH unit. Buffering of the cytosol was measured by the CO(2)-dependent quenching of the fluorescence of pyranine which was fed to spinach leaves via the petiole. Brief exposures to high CO(2) minimized interference by effective cytosolic pH regulation. Cytosolic buffering appeared to be similar to or only somewhat higher than chloroplast buffering if the initial cytosolic pH was assumed to be 7.25, which is in accord with published cytosolic pH values. The difference from chloroplast pH values indicates the existence of a pH gradient across the chloroplast envelope even in darkened leaves. Apoplastic buffering was weak as measured by the CO(2)-dependent quenching of dextran-conjugated fluorescein isothiocyanate which was infiltrated together with sodium vanadate into potato leaves. In the absence of vanadate, the kinetics of apoplastic fluorescence quenching were more complex than in its presence, indicating fast apoplastic pH regulation which strongly interfered with the determination of apoplastic buffering capacities. At an apoplastic pH of 6.1 in potato leaves, apoplastic buffering as determined by CO(2) titration with and without added buffer was somewhat below 4 mM pH-unit(-1). Thus the apoplastic and cytosolic pH responses to additions of CO(2 )indicated that the observed cytoplasmic pH regulation under acid stress involves pumping of protons from the cytosol into the vacuole of leaf cells, but not into the apoplast.

Journal Article↗
PubMed1995

In vitro effects of bicarbonate- versus lactate-buffered continuous ambulatory peritoneal dialysis fluids on peritoneal macrophage function.

At present, lactate is the most commonly used buffer in peritoneal dialysis fluids (PDFs). The high lactate concentration in combination with low original pH was demonstrated to suppress phagocytic function. We evaluated the in vitro effects of a newly formulated bicarbonate-buffered PDF containing glycylglycine (BiGG15 and BiGG40; Pierre Fabre Medicament, Castres, France) on peritoneal macrophage (PMO) function, and compared them with those of equiosmolar lactate-buffered PDF (1.5% and 4.25% glucose; pH 5.4 and pH 7.4) and control buffer. Peritoneal macrophages were isolated from the effluents of 10 continuous ambulatory peritoneal dialysis patients and tested for luminol- and lucigenin-enhanced chemiluminescence, superoxide (O2-) generation measured by cytochrome c reduction, killing capacity, and phagocytosis after incubation in the PDF used. Exposure of PMO to lactate-buffered PDF with an original pH of 5.4 resulted in a significant suppression of all PMO functions measured, compared with bicarbonate- and lactate-buffered PDFs with a pH of 7.4. At physiological pH (7.4), chemiluminescence generation of PMO exposed to BiGG15/40 was significantly higher compared with the corresponding equiosmolar lactate-buffered PDF (1,992 +/- 858 x 10(3) cpm/10(4) cells v 856 +/- 398 x 10(3) cpm/10(4) cells; P < 0.004). O2- generation, killing capacity, and phagocytosis were not significantly different after PMO exposure to bicarbonate compared with exposure to lactate-buffered PDF with a neutral pH. Irrespective of the buffer used, high-osmolality PDFs suppressed PMO function significantly more than low-osmolar PDFs. In conclusion, bicarbonate-buffered PDFs are less detrimental to PMO function than lactate-containing PDFs; these preliminary in vitro results need to be confirmed in vivo.

Bicarbonates↗
PubMed1985

Clinical buffering of metabolic acidosis: problems and a solution.

Traditionally sodium bicarbonate has been the buffer of first choice in the treatment of metabolic acidosis. This treatment, however, involves risks of developing a hyperosmolar state, a high sodium concentration in the blood, increased arterial carbon dioxide tension and, as a result of the latter, intracellular and intracerebral acidosis and also cerebral oedema. The buffering effect occurs slowly and as a consequence of this, and of the titration curve of sodium bicarbonate, overcorrection of metabolic acidosis is often seen. Tris buffer was introduced as an alternative and has been claimed to solve most of these problems, but on the other hand it entails a very high risk of peripheral venous thrombosis and thrombophlebitic lesions owing to its local irritative effect. In order to overcome these disadvantages a new mixture of Tris, acetate, bicarbonate and phosphate has been designed. In the studies described it was shown to have an adequate buffering effect and to provide a solution to most of the problems connected with buffering of metabolic acidosis. The new Tris buffer mixture has a buffering effect in blood equivalent to 0.5 mol/l sodium bicarbonate, although its sodium content has been decreased to one-third of pure sodium bicarbonate. Its administration also results in predictable buffering in cerebrospinal fluid and skeletal muscle. In a clinical study it was demonstrated that the new Tris buffer mixture results in sufficient and adequate buffering without significant side-effects.

Acetates↗
PubMed1992

Calcium gradients and buffers in bovine chromaffin cells.

1. Digital imaging and photometry were used in conjunction with the fluorescent Ca2+ indicator, Fura-2, to examine intracellular Ca2+ signals produced by depolarization of single adrenal chromaffin cells. 2. Depolarization with a patch pipette produced radial gradients of Ca2+ within the cell, with Ca2+ concentration highest in the vicinity of the plasma membrane. These gradients dissipated within a few hundred milliseconds when the voltage-gated Ca2+ channels were closed. 3. Dialysis of Fura-2 into the chromaffin cell caused concentration-dependent changes in the depolarization-induced Ca2+ signal, decreasing its magnitude and slowing its recovery time course. These changes were used to estimate the properties of the endogenous cytoplasmic Ca2+ buffer with which Fura-2 competes for Ca2+. 4. The spatially averaged Fura-2 signal was well described by a model assuming fast competition between Fura-2 and an endogenous buffer on a millisecond time scale. Retrieval of calcium by pumps and slow buffers occurs on a seconds-long time scale. No temporal changes indicative of buffers with intermediate kinetics could be detected. 5. Two independent estimates of the capacity of the fast endogenous Ca2+ buffer suggest that 98-99% of the Ca2+ entering the cell normally is taken up by this buffer. This buffer appears to be immobile, because it does not wash out of the cell during dialysis. It has a low affinity for Ca2+ ions, because it does not saturate with 1 microM-Ca2+ inside the cell. 6. The low capacity, affinity and mobility of the endogenous Ca2+ buffer makes it possible for relatively small amounts of exogenous Ca2+ buffers, such as Fura-2, to exert a significant influence on the characteristics of the Ca2+ concentration signal as measured by fluorescence ratios. On the other hand, even at moderate Fura-2 concentrations (0.4 mM) Fura-2 will dominate over the endogenous buffers. Under these conditions radiometric Ca2+ concentration signals are largely attenuated, but absolute fluorescence changes (at 390 nm) accurately reflect calcium fluxes.

Adrenal Glands↗
PubMed2004

The concept of "buffering" in systems and control theory: from metaphor to math.

The paradigm of "buffering" is used increasingly for the description of diverse "systemic" phenomena encountered in evolutionary genetics, ecology, integrative physiology, and other areas. However, in this new context, the paradigm has not yet matured into a truly quantitative concept inasmuch as it lacks a corresponding quantitative measure of "systems-level buffering strength". Here, I develop such measures on the basis of a formal and general approach to the quantitation of buffering action. "Systems-level buffering" is shown to be synonymous with "disturbance rejection" in feedback-control systems, and can be quantitated by means of dimensionless proportions between partial flows in two-partitioned systems. The units allow either the time-independent, "static" buffering properties or the time-dependent, "dynamic" ones to be measured. Analogous to this "resistance to change", one can define and measure the "conductance to change"; this quantity corresponds to "set-point tracking" in feedback-control systems. Together, these units provide a systematic framework for the quantitation of buffering action in systems biology, and reveal the common principle behind systems-level buffering, classical acid-base buffering, and multiple other manifestations of buffering.

Acid-Base Equilibrium↗
PubMed1995

Fast, high-resolution (capillary) electrophoresis in buffers designed for high field strengths.

Capillary electrophoresis in conventional buffers and in 50 microns capillaries permits field strengths as high as 300-500 V/cm with acceptably low thermal zone deformation. However, still higher field strengths (up to at least 2000 V/cm) can be applied without a decrease in resolution if the experiments are performed in the buffers described in this paper. Characteristic of these buffers is their low electrical conductivity and yet satisfactory buffering capacity accomplished either (i) by selecting buffer constituents of relatively high molecular weight and small net charge or (ii) by fractionation of carrier ampholytes (originally introduced for isoelectric focusing experiments) into a series of narrow pH range fractions and using these fractions as buffers, or (iii) by selecting an ampholyte with two acidic groups and one basic group (or one acidic group and two basic groups) and with a pI value close to two of its pK values. In such buffers, aromatic carboxylic acids and proteins used as model substances could be analyzed rapidly. For instance, albumin and transferrin were separated at 30,000 V (1.99 microA) in 15 cm long fused silica capillaries (50 microns ID) within 40 s and the carboxylic acids within 25 s. The resolution was similar to that obtained at standard voltage (5000 V; 0.33 microA), but the analysis time was reduced sixfold. Although not verified experimentally we also suggest the use of relatively high-molecular-weight polyoxyethylene derivatized with one acidic group (for instance, boric acid) and one basic group (an amine), both having the same pK value, which should afford both a very high buffering capacity and very low electrical conductivity (at low buffer concentrations).

Buffers↗
PubMed1989

Cytotoxicity and effect on mutagenicity of buffers in a microsuspension assay.

We have examined the effects of three buffers--Vogel-Bonner minimal E (VBM) and 0.15 and 0.015 M sodium phosphate, pH 7.4--and two concentrations of overnight cells (5x and 10x) on the mutagenicity of two pure compounds and two complex mixtures in strain TA98 using a modification of a microsuspension assay developed by Kado et al. (Mutation Research 121:25-32, 1983). The assay was performed by adding 50 microliters of cell concentrate of an overnight culture of TA98 resuspended in the appropriate buffer; 50 microliters of the same buffer or S9 mix; and 2 microliters of mutagen or dimethyl sulfoxide to a 1-dram vial or 13 x 150-mm test tube. The suspension was incubated for 90 min at 37 degrees C, top agar was added, and the contents poured onto a bottom agar of minimal medium. Cell concentration (5x vs. 10x) had little effect on the mutagenic potencies of the agents tested. The mutagenic potencies of the direct-acting agents (1-nitropyrene and diesel exhaust) were consistently lower with 0.15 M buffer compared to the mutagenic potencies of these agents with the other two buffers. The three buffers gave similar results for the indirect-acting agents (2-aminoanthracene and environmental tobacco smoke). The 0.15 M buffer was considerably cytotoxic in the absence of S9, which may explain why the direct-acting agents were less potent with this buffer compared to the other buffers. VBM was also somewhat cytotoxic, but this did not appear to affect the mutagenic potencies of the agents studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Anthracenes↗
PubMed1991

Effect of aging on the buffering capacity of fast-twitch skeletal muscle.

The effects of aging on the in situ buffering capacity of fast-twitch muscle fibers was examined in the tibialis anterior (TA) and extensor digitorum longus (EDL) muscles of specific pathogen free Fischer 344 rats. Muscles were electrically stimulated with trains of impulses lasting 100 ms at a frequency of 80 Hz. Trains were delivered at a rate of 1 Hz for 1 min with the hindlimb circulation occluded. Muscle hydrogen ion (H+) release during stimulation was estimated from the accumulation of metabolites. The free [H+] was measured using an homogenate technique. Muscle buffering capacity (Slykes) was estimated as delta mmol H+/l muscle water/delta pH unit. Muscle pH was unaffected by age both at rest and following stimulation in the TA and EDL. H+ release and buffering capacity were significantly reduced in aged TA muscle but unaffected by age in the EDL. Reduced buffering through metabolic processes accounted for only a small portion of the lower buffering capacity in aged TA. Most of the decrease in buffering capacity appeared to be due to reduced protein buffering. Therefore, aged TA muscle was less able to buffer a given H+ load when compared to adult controls. A more rapid accumulation of H+ during intense stimulation may lead to a earlier onset of fatigue in the aged muscle. It is not clear why the EDL buffering capacity was unaffected by age when the fiber mass profiles of the EDL and TA muscles appear similar (approximately 80% fast glycolytic fibers). It is possible that alterations in activity patterns with aging could have differential effects on the two muscles. Detailed activity pattern and fiber mass analyses are required in adult and aged EDL and TA muscles of Fischer 344 rats to answer this question.

Aging↗
PubMed1984

The effect of acetylsalicylate on aggregation and arachidonate metabolism by human platelets suspended in plasma or buffer.

Acetylsalicylate inhibits prostaglandin and thromboxane production by human platelets suspended in plasma or buffer. Acetylsalicylate inhibits arachidonate-induced aggregation of human platelets suspended in plasma, but the effect of acetylsalicylate on arachidonate-induced aggregation of human washed platelets in buffer has not been reported. We have therefore studied this in relation to arachidonate metabolism in human platelets suspended in plasma or buffer. Platelets suspended in plasma and in buffer were both prepared from each donor, who had not taken acetylsalicylate or like-acting drugs for at least 7 days. Acetylsalicylate was 1500 times less potent in inhibiting arachidonate-induced aggregation in buffer (IC50 = 27.3 +/- 7.5 (s.e.m.)mM) than it was in plasma (IC50 = 18.3 +/- 6.0 microM); whereas it was only 4 times less potent in inhibiting thromboxane production in buffer (IC50 = 110 +/- 51.0 microM) than in plasma (IC50 = 25.3 +/- 8.9 microM). The acetylsalicylate concentration required to inhibit aggregation in buffer was sufficient to inhibit 12-hydroxyeicosatetraenoic acid production whereas the concentration that inhibited thromboxane production in buffer was not. These results indicate that arachidonate-induced aggregation of platelets in buffer may depend on product(s) of lipoxygenase rather than of cyclooxygenase, and is hence insensitive to inhibition by acetylsalicylate compared with arachidonate-induced aggregation of platelets in plasma.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1989

The biological activity of adrenaline after injection through an intravenous cannula containing alkaline buffer.

Since it is known that alkaline pH inactivates adrenaline it has been recommended that this drug not be administered in the same i.v. line as alkaline buffer solutions during cardiopulmonary resuscitation. In order to test the validity of this statement a simulation model of the clinical situation was designed where the biological activity of adrenaline was measured in anesthetized rats after having been mixed with alkaline buffer solution contained in a standard i.v. cannula. The biological activity of adrenaline was measured by comparing the blood pressure response after repeated administration of a test (adrenaline + alkaline buffer) and control (adrenaline + normal saline) solution to a rat which had previously received a ganglion-blocking agent. Two alkaline buffer solutions, sodium bicarbonate and Tris buffer mixture were tested. These resulted in a decrease of the biological effect of adrenaline to 77 +/- 6 and 82 +/- 9% of control values, respectively. If however, adrenaline mixed with Tris buffer mixture was injected into a recipient of phosphate buffer (pH 7.40 and buffer capacity equal to human blood) as much as 94 +/- 17% of its activity remained. The results lead us to suggest that, in the cardiopulmonary resuscitation situation, adrenaline may well be given via a cannula containing alkaline buffer solution without significant interference with its effect.

Animals↗
PubMed2004

Influence of a buffered solution on the adsorption isotherm and overloaded band profiles of an ionizable compound.

The overloaded band profiles and the adsorption isotherms of propranolol were acquired at 23 degrees C, on the endcapped C18-Kromasil stationary phase, using two aqueous solutions of methanol as the mobile phase. The first solution contained 40% methanol and no buffer. The second contained an aqueous acetate buffer at C(buffer) = 0.20 M and pH = 5.9. In both cases, 33 isotherm data points were acquired by frontal analysis (FA), to achieve an accurate description of the isotherms in the concentration range between 1.54 x 10(-3) and 1.54 x 10(-1) mol/l of propranolol. The isotherms obtained were best described by a bi-Langmuir and a bi-Moreau isotherm model, depending on whether the mobile phase was buffered or not. This shows that the adsorption of propranolol takes place on two different types of sites, a behavior similar to the one already observed with phenol and caffeine on the same column. The presence of the buffer in the mobile phase drastically changes the adsorption mechanism of propranolol. Weak adsorbate-adsorbate interactions (two and three times RT on the low- and the high-energy sites, respectively) take place in the absence of buffer but vanish when the mobile phase is buffered. As expected, the adsorption constants on the abundant low-energy sites with or without buffer are comparable because the mobile phase composition was adjusted to give similar retention times in the two cases. On the other hand, the adsorption of propranolol on the high-energy sites is stronger in presence of the buffer. The difference probably comes from ion-pair formation in the adsorbed phase between the propranolol cation and the acetate anion. The change in total saturation capacity of the adsorbent (22%) compared to that for phenol is explained by the difference in methanol content of the mobile phase.

Adsorption↗
PubMed2005

Buffer system for the separation of neutral and charged small molecules using micellar electrokinetic chromatography with mass spectrometric detection.

An organic buffer system will be discussed that is suitable for the separation of neutral as well as charged molecules be means of micellar electrokinetic chromatography (MEKC). The buffers are based on the combination of a long chain alkyl acid, such as lauric acid with ammonium hydroxide or an organic base such as tris-hydroxymethylaminomethane (Tris). The resulting buffer system is able to separate neutral compounds based on its micellar properties. These buffers exhibit much reduced conductivity compared to traditional MEKC buffers, such as sodium dodecylsulfate (SDS), which contain inorganic salts. They also have inherent buffer capacity at high pH resulting from the basic buffer component, which in our studies had pK values from about 8-11. The separations that were observed showed high efficiency with plate counts in many cases above 500,000 plates per meter. The reduced conductivity allowed for the application of much higher electric fields, resulting in very fast analysis times. Alternatively, an increase in detection sensitivity could be achieved, as the reduced conductivity allowed for the use of capillaries with lager internal diameters. Combinations of different alkyl acids and organic bases provided for significant flexibility in selectivity tuning. Finally, the fact that the organic micellar buffer systems discussed here do not contain inorganic ions, allows for coupling with mass spectrometric (MS) detection. The possibility of MS detection combined with the high speed in analysis that can be obtained using these organic buffer systems, could make this approach an interesting option for high throughput analysis of combinatorial libraries.

Buffers↗
PubMed2006

Characterization of discontinuous buffer junctions using pH indicators in capillary electrophoresis for protein preconcentration.

An effective sample preconcentration technique for proteins and peptides was recently developed using capillary electrophoresis (CE) with discontinuous buffers [C.A. Nesbitt, J.T.-M. Lo, K.K.-C. Yeung, J. Chromatogr. A 1073 (2005) 175]. Two buffers of different pH created a junction to trap the sample molecules at their isoelectric points and resulted in over 1000-fold preconcentration for myoglobin within 30 min. To study the formation of pH junctions in CE, a pH indicator, bromothymol blue, is used in this work to reveal the pH changes at the discontinuous buffer boundary. Bromothymol blue (BTB) exhibits a drastic change in its visible absorption spectrum (300-600 nm) going from the acidic to basic pH conditions, and is therefore ideal for visualizing the changes in pH at the junctions created by various buffer combinations. Preconcentration of myoglobin was performed in discontinuous buffers containing BTB. Major differences in the BTB absorption profiles were identified from buffer systems that differ significantly in preconcentration performance, which in turn, allowed for the identification of ideal buffers for sample preconcentration. Up to 2000-fold preconcentrations of myoglobin were achieved in the buffer systems studied in this work. In addition, the role of the electroosmotic flow (EOF) on the preconcentration performance was investigated. A low EOF was found to be desirable, as the pH junction could stay longer in the capillary for accumulation of proteins. The pH junction also displayed characteristics to resist bandbroadening. Potential laminar flow resulted from the mismatched residual EOFs under the two pH conditions within the discontinuous buffers appeared to have minimal effect on the preconcentration. In fact, external applied pressure can be used to control the migration of the pH junction without compromising the protein preconcentration.

Animals↗
PubMed1998

Laboratory diagnosis of Acanthamoeba keratitis using buffered charcoal-yeast extract agar.

PURPOSE: To evaluate the use of buffered charcoal-yeast extract agar for the isolation of Acanthamoeba from clinical specimens. METHODS: We retrospectively reviewed laboratory records of patients with ocular acanthamebic infection from October 1993 to September 1997 to compare the recovery of Acanthamoeba from clinical specimens inoculated onto various media. We then compared the experimental recovery of 10 corneal isolates of Acanthamoeba on buffered charcoal-yeast extract and blood agars. RESULTS: Paired data for buffered charcoal-yeast extract and blood agars were available from 24 cultures performed in 13 cases of ocular acanthamebic infection. Acanthamebic trails were detected on both buffered charcoal-yeast extract and blood agars in nine cultures, only on buffered charcoal-yeast extract agar in nine cultures, and only on blood agar in one culture (P = .027). In the experimental study, all 10 clinical isolates produced trails on buffered charcoal-yeast extract agar, and the mean recovery after 10 days of incubation ranged from 38% to 95% of the original inoculum number. For seven of the 10 isolates, more than 70% of the original inoculum was recovered on buffered charcoal-yeast extract agar. Only two of the 10 strains produced persistent trails on the blood agar, and the mean recoveries after 10 days of incubation were 0.67% and 1.17%. Recovery was significantly better on buffered charcoal-yeast extract agar than blood agar (P < or = .0003). CONCLUSION: Buffered charcoal-yeast extract agar is an excellent commercially available culture medium for the recovery of Acanthamoeba.

Acanthamoeba↗