Development and validation of an alternative titration method for the determination of sulfate ion in indinavir sulfate

A simple and rapid precipitation titration method was developed and validated to determine sulfate ion content in indinavir sulfate raw material. 0.1 mol L-1 lead nitrate volumetric solution was used as titrant employing potentiometric endpoint determination using a lead-specific electrode. The United States Pharmacopoeia Forum indicates a potentiometric method for sulfate ion quantitation using 0.1 mol L-1 lead perchlorate as titrant. Both methods were validated concerning linearity, precision and accuracy, yielding good results. The sulfate ion content found by the two validated methods was compared by the statistical t-student test, indicating that there was no statistically significant difference between the methods.

Fast determination of minoxidil by photometric flow titration

A photometric flow titration based on the redox reaction between KMnO4 and minoxidil is described. The best titration results were observed at 3.20 x 10-4 mol L-1 KMnO4 and 1.00 x 10-3 mol L-1 minoxidil, using the minoxidil solutions as titrant. The flow rate was fixed at 17 mL min-1 and the titrant was added to the system in aliquots of 500 µL, the color changes were monitored at 550 nm. The method was applied to commercial samples and compared with the results from a chromatographic procedure. Recoveries from 97.6 to 102.8 % were observed depending on the sample. Comparison with the chromatographic procedure reveled relative errors of 3.5 - 4.0 %.

A direct potentiometric titration study of the dissociation of humic acid with selectively blocked functional groups

A direct potentiometric titration method was applied to commercial and soil humic acids in order to determine their carboxyl and phenol group concentrations and apparent and intrinsic pK. In that context, acid-base properties of humic acids are interpreted by selective blocking of carboxylic and phenolic groups by esterification and acetylation. Differences in underivatized and derivatized HA's acid-base properties are ascribed to carboxyl and phenol groups influence on total humic acidity. Potentiometric data were treated with the modified Henderson-Hasselbalch equation. Infra red results, the acidic group contents and the average values of apparent and intrinsic pK for underivatized and derivatized HAs confirmed the selectivity of esterification derivatization method. After blocking of the functional groups, the values of acidic group contents decreased, while the value of apparent pK increased after derivatization. Phenol groups cannot be specifically identified by the acetylation method, due to low selectivity of the acetylation method.

Determination of Alkalinity and dissociation constants of high salinity waters: use of F5BC titration function

Measurements of parameters expressed in terms of carbonic species such as Alkalinity and Acidity of saline waters do not analyze the influence of external parameters to the titration such as Total free and associated Carbonic Species Concentration, activity coefficient, ion pairing formation and Residual Liquid Junction Potential in pH measurements. This paper shows the development of F5BC titration function based on the titrations developed by Gran (1952) for the carbonate system of natural waters. For practical use, samples of saline waters from Pocinhos reservoir in Paraiba were submitted to titration and linear regression analysis. Results showed that F5BC involves F1x and F2x Gran functions determination, respectively, for Alkalinity and Acidity calculations without knowing "a priori" the endpoint of the titration. F5BC also allows the determination of the First and Second Apparent Dissociation Constant of the carbonate system of saline and high ionic strength waters.

pH titration of native and unfolded ß-trypsin: evaluation of the D D G0 titration and the carboxyl pK values

The stabilizing free energy of ß-trypsin was determined by hydrogen ion titration. In the pH range from 3.0 to 7.0, the change in free energy difference for the stabilization of the native protein relative to the unfolded one (D D G0 titration) was 9.51 ± 0.06 kcal/mol. An isoelectric point of 10.0 was determined, allowing us to calculate the Tanford and Kirkwood electrostatic factor w. This factor presented a nonlinear behavior and indicated more than one type of titratable carboxyl groups in ß-trypsin. In fact, one class of carboxyl group with a pK = 3.91 ± 0.01 and another one with a pK = 4.63 ± 0.03 were also found by hydrogen ion titration of the protein in the folded state

In situ enzyme immunoassay for titration of a Brazilian hepatitis A virus strain (HAF-203)

Hepatitis A virus (HAV) replicates relatively slowly in cell culture without a cytopathic effect, a fact that limits the use of tissue culture assays. The radioimmunofocus assay is the standard method for HAV titration, although it is labor intensive and requires the use of radioisotopes. A simple, rapid and objective infectivity assay based on an in situ enzyme immunoassay (EIA) is described here for a Brazilian cell culture-adapted HAV strain (HAF-203). The assay uses a peroxidase-labeled polyclonal antibody to fixed monolayers as an indicator of infection. EIA may be completed within 7 days using serial 5-fold dilutions of the virus, yielding a titer of 5.024 log 50% tissue culture infective dose (TCID50)/ml for HAF-203. This technique had a detection limit of 1.1 log TCID50/ml and the specificity was demonstrated by detecting no reaction on the columns of uninfected wells. The reproducibility (with intra- and inter-assay coefficients of variation ranging from 1.9 to 3.8% and from 3.5 to 9.9%, respectively) and quantitation of the assay were demonstrated by close agreement in virus infectivity titers among different assays of the same amount of virus and between assays of different amounts of virus. Furthermore, this assay does not require the use of radiolabeled antibodies. We describe here an efficient EIA that is highly reproducible and that could be used to monitor HAV growth in cell culture and to determine the quantity of HAV antigen needed for diagnostic assays. This is the first report of the infectious titer of the Brazilian cell culture-adapted HAV strain (HAF-203).

Primary hemocyte culture of Penaeus monodon as an in vitro model for white spot syndrome virus titration, viral and immune related gene expression and cytotoxicity assays

Immortal cell lines have not yet been reported from Penaeus monodon, which delimits the prospects of investigating the associated viral pathogens especially white spot syndrome virus (WSSV). In this context, a method of developing primary hemocyte culture from this crustacean has been standardized by employing modified double strength Leibovitz-15 (L-15) growth medium supplemented with 2% glucose, MEM vitamins (1 ), tryptose phosphate broth (2.95 g l 1), 20% FBS, N-phenylthiourea (0.2 mM), 0.06 lgml 1 chloramphenicol, 100 lgml 1 streptomycin and 100 IU ml 1 penicillin and hemolymph drawn from shrimp grown under a bio-secured recirculating aquaculture system (RAS). In this medium the hemocytes remained viable up to 8 days. 5-Bromo-20-deoxyuridine (BrdU) labeling assay revealed its incorporation in 22 ± 7% of cells at 24 h. Susceptibility of the cells to WSSV was confirmed by immunofluoresence assay using a monoclonal antibody against 28 kDa envelope protein of WSSV. A convenient method for determining virus titer as MTT50/ml was standardized employing the primary hemocyte culture. Expression of viral genes and cellular immune genes were also investigated. The cell culture could be demonstrated for determining toxicity of a management chemical (benzalkonium chloride) by determining its IC50. The primary hemocyte culture could serve as a model for WSSV titration and viral and cellular immune related gene expression and also for investigations on cytotoxicity of aquaculture drugs and chemicals

Probing protein−tannin interactions by isothermal titration microcalorimetry

Isothermal titration microcalorimetry (ITC) has been applied to investigate protein−tannin interactions. Two hydrolyzable tannins were studied, namely myrabolan and tara tannins, for their interaction with bovine serum albumin (BSA), a model globular protein, and gelatin, a model proline-rich random coil protein. Calorimetry data indicate that protein−tannin interaction mechanisms are dependent upon the nature of the protein involved. Tannins apparently interact nonspecifically with the globular BSA, leading to binding saturation at estimated tannin/BSA molar ratios of 48:1 for tara- and 178:1 for myrabolan tannins. Tannins bind to the random coil protein gelatin by a two-stage mechanism. The energetics of the first stage show evidence for cooperative binding of tannins to the protein, while the second stage indicates gradual saturation of binding sites as observed for interaction with BSA. The structure and flexibility of the tannins themselves alters the stoichiometry of the interaction, but does not appear to have any significant affect on the overall binding mechanism observed. This study demonstrates the potential of ITC for providing an insight into the nature of protein−tannin interactions.

Probing protein-tannin interactions by isothermal titration microcalorimetry

Isothermal titration microcalorimetry (ITC) has been applied to investigate protein-tannin interactions. Two hydrolyzable tannins were studied, namely myrabolan and tara tannins, for their interaction with bovine serum albumin (BSA), a model globular protein, and gelatin, a model proline-rich random coil protein. Calorimetry data indicate that protein-tannin interaction mechanisms are dependent upon the nature of the protein involved. Tannins apparently interact nonspecifically with the globular BSA, leading to binding saturation at estimated tannin/BSA molar ratios of 48:1 for tara- and 178:1 for myrabolan tannins. Tannins bind to the random coil protein gelatin by a two-stage mechanism. The energetics of the first stage show evidence for cooperative binding of tannins to the protein, while the second stage indicates gradual saturation of binding sites as observed for interaction with BSA. The structure and flexibility of the tannins themselves alters the stoichiometry of the interaction, but does not appear to have any significant affect on the overall binding mechanism observed. This study demonstrates the potential of ITC for providing an insight into the nature of protein-tannin interactions.

Probing protein-tannin interactions by isothermal titration microcalorimetry

Isothermal titration microcalorimetry (ITC) has been applied to investigate protein-tannin interactions. Two hydrolyzable tannins were studied, namely myrabolan and tara tannins, for their interaction with bovine serum albumin (BSA), a model globular protein, and gelatin, a model proline-rich random coil protein. Calorimetry data indicate that protein-tannin interaction mechanisms are dependent upon the nature of the protein involved. Tannins apparently interact nonspecifically with the globular BSA, leading to binding saturation at estimated tannin/BSA molar ratios of 48:1 for tara- and 178:1 for myrabolan tannins. Tannins bind to the random coil protein gelatin by a two-stage mechanism. The energetics of the first stage show evidence for cooperative binding of tannins to the protein, while the second stage indicates gradual saturation of binding sites as observed for interaction with BSA. The structure and flexibility of the tannins themselves alters the stoichiometry of the interaction, but does not appear to have any significant affect on the overall binding mechanism observed. This study demonstrates the potential of ITC for providing an insight into the nature of protein-tannin interactions.

Isothermal titration calorimetry study of epicatechin binding to serum albumin

The interaction of epicatechin with bovine serum albumin (BSA) was studied by isothermal titration calorimetry. The binding constant (K) and associated thermodynamic binding parameters (n, Delta H) were determined for the interaction at three solution concentrations of BSA using a binding model assuming independent binding sites. These data show weak non-covalent binding of epicatechin to BSA. The interaction energetics varied with BSA concentration in the calorimeter cell, suggesting that the binding of epicatechin induced BSA aggregation. The free energy (Delta G) remained constant within a range of 2 kJ mol(-1) and negative entropy was observed, indicating an enthalpy driven exothermic interaction. It is concluded that the non-covalent epicatechin-BSA complex is formed by hydrogen bonding. (c) 2006 Elsevier B.V. All rights reserved.

A unified titration formula

In a number of standard titrations, a volume of a monoprotic base MOH at a specific concentration is added to a volume of a monoprotic acid HA at a specific concentrations. Four different types of titration are possible, depending on whether the acid and base are strong or weak. A single unifying formula covering all four cases has been determined.