Sodium Borate Buffer Research Articles

The role of pH change caused by the addition of water-miscible organic solvents in the destabilization of an enzyme

The possibility that a change in the pH of a buffer caused by addition of organic solvents can play a role in the destabilization of an enzyme in aqueous mixtures of polar solvents was studied. The magnitude of change in pH was dependent on the concentration of the organic solvent, the nature of the buffer ions, and the concentration and initial pH of the buffer. The decrease in pH of sodium borate buffer caused by the addition of polyhydroxy compounds was used to counteract the pH increase due to organic solvents. The stability and activity of trypsin, and β-galactosidases from Aspergillus oryzae and Escherichia coli in aqqueous NN-dimethyl formamide (DMF) with or without mannitol were compared with those at analogous pH values in aqueous solution. The stability and activity of β-galactosidase from A. oryzae in aqueous mixtures containing low concentrations of DMF paralleled those in buffers without DMF at comparable pH, whereas at higher concentrations of DMF the activity and stability were much lower. No such correlation was observed in the case of trypsin or β-galactosidase from E. coli. Therefore, at a lower concentration of organic solvents, the change in pH of the buffer plays a major role in affecting the stability and activity of A. oryzae β-galactosidase. As the concentration of the solvent increases, its direct interaction with the protein becomes the dominating factor in the denaturation of the enzyme.

Capillary electrophoresis of wheat gliadin: Initial studies and application to varietal identification

Wheat gluten proteins have important functional properties, and are reliable indicators of genotype. They are also very heterogeneous, demanding use of powerful analytical techniques. Gel electrophoresis methods are widely used for gluten analysis, but they are labor-intensive, and their data are not easily quantified. Methods of capillary electrophoresis (CE) are now available that promise to overcome these disadvantages. We here report successful application and optimization of two CE procedures for gliadin analysis. The first procedure used 0.06 mol/L sodium borate buffer, pH 9.0, containing 200 mL/L acetonitrile + 10 g/L sodium dodecyl sulfate; separations were excellent, but reproducibility was difficult to maintain. The second buffer, 0.1 mol/L phosphate, pH 2.5, containing a linear hydrophilic polymer, overcame this problem, providing separations of gliadins that are rapid, high-resolution, automatic, and easily quantified. CE analyses of gliadin also reliably differentiate varieties of all U.S. wheat classes. CE is thus a valuable technique for gliadin analysis, complementary to other methods of electrophoresis and chromatography. CE has much potential to become a routine tool for wheat varietal identification and classification, and for prediction of quality.

Enantiomeric separation of amino acids using micellar electrokinetic chromatography after pre-column derivatization with the chiral reagent 1-(9-fluorenyl)-ethyl chloroformate.

Direct enantiomeric separations of some racemic amino acids derivatized with 9-fluorenylmethyl chloroformate were obtained using cyclodextrin-modified micellar electrokinetic chromatography (CD/MEKC) with a buffer made up of 5 mM sodium borate (pH 9.2), 150 mM sodium dodecyl sulfate (SDS) and 40 mM gamma-CD. Alternatively, enantiomeric separations were also achieved indirectly using MEKC after pre-column derivatization with (+)-1-(9-fluorenyl) ethyl chloroformate (FLEC). Using either a 10 mM sodium phosphate (pH 6.8) or a 5 mM sodium borate buffer (pH 9.2), each of which contained 25 mM SDS and 10-15% of acetonitrile, FLEC-derivatized serine, alanine, valine, methionine, leucine, phenylalanine, tryptophan, and their diastereomeric pairs were all separated: the L-isomers migrated faster than the corresponding D-isomers. However, when (-)-FLEC was used for derivatization, the D-isomers migrated faster than the corresponding L-isomers. Also, the diastereomers of aspartic acid, glutamic acid, and proline were resolved using a 10 mM sodium citrate buffer (pH 4.4). Using KrF (248 nm) laser-induced fluorescence, the detection limit of (+)-FLEC derivatized DL-amino acids was obtained at the nM level, which was about 100 x more sensitive than UV absorption at 200 nm. Analyte concentrations as low as 3 x 10(-8) M (DL-Val) could be derivatized with (+)-FLEC.

Adsorption of ethyl xanthate anions on a silver electrode. An in-situ FTIR study

Abstract The adsorption of ethyl xanthatc ion (EX−) on a silver electrode in sodium sulphate, phosphate buffer (pH 6.8) and borate buffer (pH 9.2) solutions was studied in situ using Fourier transform IR reflection spectroscopy. The measurements were carried out using a thin-layer flow cell, which allows a continuous supply of the electroactive species into the thin layer. The voltammetric behaviour of the silver electrode in EX−-containing solution is characterized by the formation of silver ethyl xanthate (AgEX), which is preceded by a prewave due to chemisorption of ethyl xanthate. An IR band at 1220 cm −1, assigned to chemisorbed ethyl xanthate, was observed in spectra obtained in the chemisorption region. All IR bands characteristic of AgEX were observed in spectra of a silver electrode surface covered by a few monolayers of AgEX. The in-situ IR measurements also showed that the adsorption of EX− starts directly on the positive side of the potential of zero charge of silver (−0.7 V/SHE).

Enhanced Electrochromic Property of Nickel Hydroxide Thin Films Prepared by Anodic Deposition

Nickel hydroxide thin films were prepared onto transparent conductive tin oxide (NESA) substrates by potentiostatic electrolyses of a nickel ammine complex solution at various potentials (0.6 to 1.5 V vs. ). Nickel hydroxide thin film (F0.7) obtained at relatively lower anodic potential (0.7 V) showed enhanced electrochromism between colorless and dark brown in a sodium borate buffer solution at pH 12; the absorption spectrum in the colored (oxidized) state was broadened in the visible and near‐infrared region compared with the nickel hydroxide films prepared at the higher anodic potential (1.1 V). Characterization of the films revealed that crystal structure of F0.7 is assigned to , and that its electrochromism is based on the reversible oxidation to hexagonal structure. Composite nickel hydroxide film, i.e., by the electrolytic deposition at 1.1 V followed by that at 0.7 V, showed electrochromic property similar to F0.7 and its durability in repeated redox cycles were much improved in comparison with that of F0.7. Electrochromic properties in switching performance of this composite nickel hydroxide film were investigated.

Micellar electrokinetic chromatographic/ultraviolet diode array analysis of Eleutherococcus senticosus

AbstractThe main components of the roots of Eleutherococcus senticosus have been separated by capillary electrophoresis in the micellar mode. The best separation was achieved using a fused silica capillary tube and a 25 mM sodium borate buffer (pH 8.25) containing sodium dodecyl sulphate (SDS; 50 mM) at a constant voltage of 280 V/cm. The quantitative determination of eleutheroside B and E in E. senticosus extracts and formulations demonstrates the applicability of this method.

Determination of synthetic food dyes by capillary electrophoresis

A method for the determination of synthetic tar dyes used as food additives using capillary electrophoresis with photodiode-array detection was investigated. The dyes Erythrosine (R-3), Phloxine (R-104), Rose Bengal (R-105), Acid Red (R-106), Amaranth (R-2), New Coccine (R-102) and Allura Red AC (R-40) were separated on a capillary column (50 cm × 75 μm I.D.) and identified from the absorbance spectra of each peak. The electrophoresis buffer used was a mixture of 25 m M sodium phosphate buffer and 25 m M sodium borate buffer (1:1) (pH 8.0) containing 10 m M sodium dodecyl sulfate (SDS). Substitution of β-cyclodextrin for SDS in the electrolyte buffer was effective for the separation of R-2 and R-102. This modified method could be employed as an additional assay method for these two dyes.

Determination of metal ions complexed with 2,6-diacetylpyridine bis( N-methylenepyridiniohydrazone) by capillary electrophoresis

Metal ions were determined quantitatively and selectively by complexation with 2,6-diacetylpyridine bis( N-methylenepyridiniohydrazone) (H 2dapmp) and separation of the metal ion complexes formed using capillary zone electrophoresis. The effect of various alkyltrimethylammonium ions added to the running electrolyte on the migration behaviour of cationic chelate complexes was examined. As a result, some evidence was obtained that both electrophoretic migration and micellar partitioning play the major role in the separation mechanism. Optimum resolution of metal-H 2dapmp complexes was achieved with a 10 mM sodium borate buffer (pH 9.0) containing 75 mM tetradecyltrimethylammonium bromide and 10 mM sodium n-octanesulfonate as an ion-pairing counterion. With this micellar buffer system, it was possible to separate and determine up to fourteen metal ions in a single run within 12 min using fused-silica capillaries at 15 kV and direct on-column UV detection at 254 nm. Linear calibration ranges of more than two orders of magnitude and detection limits at mid-ppb levels are presented for the metal complexes of Cd(II), Co(II), Cu(II), Fe(III), Hg(II), Mo(VI), Sc(III), U(VI), V(V), Y(III) and Zn(II).

Validated micellar electrokinetic capillary chromatography method for quality control of the drug substances hydrochlorothiazide and chlorothiazide

A stability-indicating, quality control analysis method was developed and validated for the diuretic drug substances hydrochlorothiazide (HCTZ) and chlorothiazide (CTZ). Micellar electrokinetic capillary chromatography employing the anionic detergent sodium dodecyl sulfate at 30 mM in 20 mM sodium borate buffer pH 9.5 was utilized to separate and quantify the active drug substance HCTZ from CTZ and the common impurity, 4-amino-6-chloro-1,3-benzenedisulfonamide (DSA). A 100 μm I.D. uncoated fused-silica capillary was necessary to provide the sensitivity, i.e. 1 μg/ml, for quantification of the DSA impurity. In this study, the linearity, precision, selectivity, accuracy, reproducibility and limit of quantitation for the method were investigated for HCTZ, CTZ and DSA. As the first validation of a drug substance method by capillary electrophoresis in this laboratory, unusual care was taken to insure reliability and ruggedness with multiple instruments, capillaries and analysts. Precision and reproducibility in the range of 1% R.S.D. was achieved by controlling subtle injection factors. These included minimizing the time in which the capillary ends were not immersed in buffer or sample during the injection process and minimizing the number of assays for each anode or inlet buffer vial. Stacking induced by differences in ionic strength between sample and capillary buffer was reduced by using a sample buffer concentration similar to that of the capillary buffer. Although stacking accomplished by using lower sample buffer concentrations increased sensitivity, reproducibility was decreased. Achievement of the 1% R.S.D. precision level means that many quality control assays for drugs with good absorbance characteristics can be validated with HPLC reproducibility and CE efficiency. These micellar electrokinetic capillary chromatography methods conform to the USA and European Pharmacopoeial validation guidelines.

Separation of honey flavonoids by micellar electrokinetic capillary chromatography

The separation of thirteen flavonoids from honey by micellar electrokinetic capillary chromatography (MECC) is described. All the flavonoids were separated on a fused-silica column (75 cm × 75 μm I.D.) with 0.2 M sodium borate buffer (pH 8.0)-50 m M sodium dodecyl sulphate-10% methanol. These conditions were applied to the separation of flavonoids from lavender, rosemary, citrus and heather honey samples, to establish correlations between the flavonoid profiles and the botanical origin of honey. Citrus honey was characterized by the accumulation of hesperetin, lavender by luteolin, rosemary by 8-methoxykaempferol and heather by some unidentified flavonoids. The influence of the geographical origin on the honey flavonoid pattern was also studied by MECC. Honey samples from Spain, Mexico and Canada were analysed and no significant differences were found.

Speciation of organomercurials in marine samples using capillary electrophoresis

A rapid method for speciation and determination of organomercury compounds in biological samples of marine origin using Capillary Electrophoresis (CE) is reported. Organomercurials were extracted from the samples by means of the classical Westöö procedure thus giving organomercury-cysteine complexes which can be separated from each other by means of CE resulting in effective speciation. Electrophoretic separation was achieved in an open silica capillary tube at 15–18 kV using a 100m M sodium borate buffer (pH 8.35). All mercury species were distinctively separated within 12 min. Results are presented for the analysis of real marine samples and reference materials, and compared with those obtained by the GC commonly accepted procedure.

Borate ion-assisted stabilization of β-galactosidase from Aspergillus oryzae by polyhydroxy compounds in water-miscible organic solvents

The stability of β-galactosidase from Aspergillus oryzae in water-miscible organic solvents in different buffers at various pH values ranging from 4.6 to 8.0 was studied. The stability of the enzyme in all six organic solvents studied was dependent on pH and on the type of buffer ions present. At a given pH, destabilization by organic solvents was highest in sodium borate buffer. The destabilization of β-galactosidase by these solvents could be reversed by addition of sugars or polyhydroxy compounds exclusively in sodium borate, suggesting a role of borate ions in stabilization. A similar effect of addition of mannitol was observed on deactivation of β-galactosidase by N, N-dimethylformamide (DMF). Exclusively in sodium borate, at pH 8.0, the addition of mannitol (0.02 m) not only prevented the deactivation by DMF (8%, v/v) but increased the enzyme activity to the level at its optimum pH. Since β-galactosidase from Aspergillus oryzae is a glycoprotein, complexation of the borate ions to the carbohydrate part may result in change in protein conformation, which, without leading to denaturation or inactivation of the enzyme, may facilitate interaction of the organic solvents with the enzyme leading to its denaturation. Such a denaturation is probably prevented by addition of polyhydroxy compounds, which appear to compete favorably with the carbohydrate moiety of the protein in complexing with borate ions. This should result in the enzyme regaining its native conformation.

Clobazam and norclobazam quantitation in serum by capillary gas chromatography with electron-capture detection

We have developed a simple, rapid method for determination of the antiepileptic drug clobazam and its major active metabolite, norclobazam in serum. Serum (200 μL) made alkaline with sodium borate buffer is extracted with toluene. After evaporation of the organic layer and reconstitution with toluene, the extract is analyzed within 5 min by capillary gas chromatography (Hewlett Packard HP 5890 GC; 63Ni electron-capture detector; HP-5 column). Linear calibrations for clobazam and norclobazam (clobazam: r = 0.999; norclobazam: r = 0.997) have permitted automated integrator calculation of results. Imprecision and accuracy were evaluated using an in-house control. Intra- and interassay coefficients of variation (CV) were 6.2% ( n = 10) and 9.8% ( n = 21), respectively for clobazam and 5.7% ( n = 10) and 6.3% ( n = 21), respectively for norclobazam. The mean concentrations for clobazam and norclobazam in the control were 3.5 μmol/L and 8.7 μmol/L respectively, representing 106% and 100% of the weighted-in values. Simplicity, specificity, sensitivity, and rapidity are attributes that makes this assay suitabel for monitoring of clobazam and norclobazam in serum.

Determination and rotamer separation of enalapril maleate by capillary electrophoresis

Capillary electrophoresis (CE) was applied to the study of enalapril maleate, an inhibitor of angiotensin-converting enzyme. By the use of capillary zone electrophoresis, a method using sodium borate or sodium phosphate buffers (pH 8.25–9.7) as an electrolyte and using an external standard was developed for the determination of enalapril maleate at room temperature. At a method concentration of 0.2 mg/ml, the injection precision (R.S.D. = 0.62%, n = 10) and linearity of detector responses (range 50–150% of the method concentration, R 2 = 0.999) were satisfactory. The method precision was also satisfactory as demonstrated by the content uniformity assays of ten tables of vasotec (20 mg potency). The method is stability indicating and specific as enalapril can be well separated from its two degradates, enalaprilat (a hydrolysis product) and its diketopiperazine (a cyclization product). By the use of micellar elektrokinetic capillary chromatography, in which 40–100 m M sodium dodecyl sulfate were added to the electrolyte buffers to form micelles in capillary, the cis and trans rotamers of enalapril was separated at or below 20°C. As the temperature increased, the two peaks spread and coalesced to one peak above 40°C. The study demonstrates that CE can be effectively applied to both the determination and conformer separation of drugs in pharmaceutical research and development.

High-performance capillary electrophoresis of glycoproteins: The use of modifiers of electroosmotic flow for analysis of microheterogeneity

High-performance capillary electrophoresis (HPCE) is rapidly gaining acceptance as an analytical tool for the study of biological macromolecules. In the present study, the utility of HPCE for separation of glycoproteins is highlighted using a pure ovalbumin preparation. Ovalbumin, the 43-kDa glycoprotein of avian egg white, is known to be heterogeneous in nature with at least nine different carbohydrate structures having been identified on the single Asn residue. HPCE separation in an 87-cm capillary containing borate buffer and 1 m m putrescine resolves five major protein peaks in less than 30 min under nondenaturing conditions. This effect appears to be specific to glycoproteins since analysis of the nonglycosylated protein carbonic anhydrase under the same conditions showed no enhanced separation. The sodium borate buffer is proposed to play a key role in the separation by preferentially complexing with the diols of specific carbohydrate moieties on ovalbumin. Addition of putrescine enhances resolution by slowing bulk flow through the capillary and allowing electrophoretic separation of what is deduced to be closely related glycoforms of ovalbumin. Dephosphorylation of the ovalbumin with either calf intestinal alkaline phosphatase or potato acid phosphatase results in a shift of all peaks to a more rapid migration time and is consistent with a loss of negative charge. This suggests that all major ovalbumin isoforms are phosphorylated to the same degree and that heterology among ovalbumin isoforms resides solely in the carbohydrate structure. The enhanced resolution obtained with the employment of longer capillaries and modifiers of endo-osmotic flow was not restricted to ovalbumin since partial resolution of pepsin isoforms was observed under the same conditions.

A rapid, sensitive thin layer chromatography procedure for the detection of fumonisin B1 and B2.

A thin layer chromatography (TLC) method was developed for the detection of fumonisin B1 and B2 in corn and corn-based feedstuffs. Finely ground samples were extracted with acetonitrile:water (1:1), filtered, and applied to C18 cleanup columns. The columns were washed with 1% aqueous KCl followed by acetonitrile: 1% aqueous KCl (1:9), and the fumonisins were eluted with acetonitrile:water (7:3). The eluants were concentrated and spotted on reverse-phase C18 TLC plates along with fumonisin B1 and B2 standards, and the plates were developed in methanol: 4% aqueous KCl (3:2). The fumonisins were visualized by spraying the TLC plates successively with 0.1 M sodium borate buffer, fluorescamine, and 0.01 M boric acid. The plates were then dried and examined under longwave ultraviolet light. Fumonisin B1 and B2 appeared as bright yellowish-green fluorescent bands at Rfs of 0.5 and 0.1, respectively. The detection limit for the fumonisins on the TLC plate was 0.1 ppm in corn. Recoveries from spiked samples averaged greater than 80%. The identification of the fumonisins was confirmed by hydrolyzing the parent compounds of B1 and B2 to their respective C22 amino-alcohols and reexamining by TLC with the same visualizing reagents. This procedure was used to survey 193 corn samples collected from University of Missouri test plots in 1990 for fumonisin B1. Fumonisin B1 was detected in 15% of the corn samples.

Purification and properties of the elastase from Aspergillus fumigatus.

Elastase, a potential virulence factor from the opportunistic pathogen Aspergillus fumigatus, was purified 220-fold from culture broth by fast-performance liquid chromatography employing anion exchange (Q Sepharose fast flow), cation exchange (S Sepharose fast flow), and gel filtration (Superose 12). Purified to near homogeneity, the elastase had an apparent molecular mass of 32 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (silver stain) but a mass of about 19.1 kDa as determined by gel filtration on Superdex 75. The elastase is not glycosylated and is positively charged at neutral pH, having a pI of 8.75. Inhibition by 0.2 mM phenylmethylsulfonyl fluoride (100%) and 0.21 mM leupeptin (60%) implies that the elastase is a serine protease. However, the enzyme is also inhibited by 5 mM EDTA (100%) and 10 mM 1,10-orthophenanthroline (30%), suggesting a requirement for divalent cations. The enzyme acts optimally at pH 7.4 and 45 degrees C in 50 mM sodium borate buffer, but in Tris HCl, the pH optimum shifts to 8.8.

Separation and sensitive determination of i-urobilin and 1-stercobilin by high-performance liquid chromatography with fluorimetric detection

i-Urobilin and 1-stercobilin were separated by high-performance liquid chromatography on a reversed-phase octadecylsilane-bonded column and detected fluorimetrically through formation of phosphor with zinc ions in the eluent. The separation and the intensity of the fluorescence response were affected by concentrations of zinc acetate and sodium borate buffer, pH and methanol content in the eluent. The optimal eluent used consisted of 0.1% zinc acetate in 75 m M boric acid buffer (pH 6.0)—methanol (25:75). The detection limit was 0.2 μg/l for both i-urobilin and 1-stercobilin (signal-to-noise ratio 2), which makes the method 250–2500 times more sensitive than conventional methods.

Corrosion Studies of Rapidly Solidified Magnesium Alloys

Effects of alloying elements on the corrosion resistance of mostly binary rapidly solidified (RS) magnesium alloys were examined using electrochemical techniques. Uniform corrosion rates were measured at room temperature in a pH 9.2 sodium borate buffer using electrochemical impedance spectroscopy (EIS). Comparisons of electrochemically and gravimetrically determined corrosion rates of bulk magnesium and magnesium alloys showed that the charge transfer resistance could be used to accurately measure the corrosion rates of these materials provided the attack was indeed uniform. The correlation broke down for pure magnesium, which suffered a nonuniform attack due to the undermining of small particles. Corrosion rate measurements of binary alloys showed that aluminum was the only element which caused a decrease in the corrosion rate of magnesium; the corrosion rate decreased with increasing aluminum content. Low concentrations of zinc and lithium resulted in alloys with corrosion rates slightly higher than that of the pure magnesium. Anodic polarization scans were used to compare as‐cast and rapidly solidified commercial ternary alloy AZ61 in borate (pH 9.2) and carbonate/bicarbonate (pH 10.0) buffer solutions containing 100 ppm, 1000 ppm, and 3.5 weight percent sodium chloride. The RS AZ61 formed a more protective film than the cast material under these conditions.

Ion Chromatography of Soluble Cr(III) and Cr(VI)

Ion chromatography coupled with a conductivity detector was used to investigate the analysis of Cr(III) and Cr(VI) in aqueous samples. An IC methodology for Cr(III) was developed using a cation column and an eluent containing tartaric acid, ethylenediamine, and acetonitrile at pH 2.9. The detection limit of this method can reach 0.1 ppm level with good precision. Several operational parameters were evaluated during the regular use of the method. Comparison of the IC method with AA method showed good agreement between the two methods. The anion exchange column was used for Cr(VI) determination. The best results were obtained with an eluent containing sodium gluconate, borate buffer, glycerin, and acetonitrile. The retention time for the Cr207 2 - sample was 11 min. and the calibration curve was linear between 1.0 and 100 ppm.