Bromothymol Research Articles

Optical ph sensing material prepared from doped sol-gel film for use in acid-base titration

An optical pH sensing material has been prepared in this study by using sol-gel technique. Bromothymol blue, bromophenol blue and thymol blue were chosen in this study as acidbase indicators for strong acid-strong base, strong acid-weak base and weak acid-strong base titration, respectively. The results show that these indicators could be successfully entrapped inside the sol-gel film and still maintain its chemical behaviour as in solution. The entrapped acid-base indicators respond well to any pH changes and could be used to determine the end-point of the acid-base titration.

Tris(2-chloroethyl)amine isomerization and subsequent spectrophotometric determination using sulfonephthaleins

In a polar environment, tris(2-chloroethyl)amine molecule undergoes isomerization when forming N,N-bis(2-chloroethyl)aziridinium cation as a reactive intermediate. New methods were developed to spectrophotometrically determine tris(2-chloroethyl)amine in the form of aziridinium cation extraction using 4 sulfonephthaleins—bromothymol blue, thymol blue, bromoxylenol blue, and bromocresol green. The developed methods, reflecting potent electrophilic properties of the analyte, are based on the formation of extractable ion pairs between the aziridinium cation and a quinoid anion form of a sulfonephthalein. Chloroform was used as the solvent for extraction from the water phase. The conditions of the methods were optimised by determining the suitable pH (8.5) of a buffer and the concentration of sulfonephthaleins as reagents. The dependence of the reaction time in the water phase was found to be 10 min. The composition of the ion pairs was found to be 1:1 by in all cases and the conditional extraction constant of the complexes were calculated. The detection and determination limits of separate procedures were ascertained. Best results (detection limit 3.5 µg ml−1 and determination limit 11.6 µg ml−1) were obtained using bromothymol blue. The methods were empirically compared with a group spectrophotometric method to determine alkyl halides using the alkalized water–ethanol solution of thymolphthalein. Relatively low interferences of other nitrogen mustards and sulfur mustard were recorded. The interaction mechanism—ion association—was validated.

Ionic liquid-based slab optical waveguide sensor for the detection of ammonia in human breath

HypothesisA novel slab optical waveguide sensor exploiting an ionic liquid (trihexyl(tetradecyl)phosphonium chloride)-based film can be used to detect gaseous ammonia. Bromothymol blue (BTB) incorporated into the ethyl cellulose–supported ionic liquid film can absorb the evanescent wave generated in the sensor and act as an indicator. The diffusion of gaseous ammonia across the sensing film is thus expected to change the absorbance of bromothymol blue, providing the basis for sensitive ammonia detection. ExperimentsThe above sensor was exposed to different concentrations of ammonia gas, showing fast and reproducible responses and being superior to previously reported waveguiding-based sensors. Moreover, the fabricated system was used to detect ammonia in the breath of human volunteers. FindingsUnder optimal conditions, gaseous ammonia was accurately detected in the range of 100–1800 ppb, with a detection limit of 69 ppb (3σ/s). The developed sensor was re-usable due to exhibiting reversible gas sorption/desorption dynamics and was successfully applied to the determination of ammonia content in human breath.

Mice pathology study of toxA and exoS genes bearing Pseudomonas aeruginosa isolated from bovine Sub-clinical mastitis in West Begal with their Antibiogram

Sub-clinical Mastitis is one of the major problems in India which is among the largest milk producing countries in the world, hampering national economy. Among 422 bovine milk samples screened from different cattle farms of West Bengal with history of infection and drop in milk yield by on-spot bromothymol blue (BTB) test, 371 samples were considered for collection and secondary screening by somatic cell count study. The collected milk samples showed significantly high average SCC value of 3.26-4.88 lakh cells/ml of milk which was indicative of infection. Samples (23, 6.53%) were found to be positive for Pseudomonas sp. showing distinct bluish-green pigmentation on cetrimide agar, typical results on morphological and in biochemical characterization. Presence of 19 (5.39%) Pseudomonas aeruginosa isolates were confirmed by fluorescent technique for detection of characteristic blue-green fluorescence by the pigment pyoverdin. PCR of these positive isolates revealed that 11 isolates were positive for toxA and 6 isolates for exoS genes with 2 isolates possessing both the genes. In Mice pathogenicity test, highest fatality (100%) was shown by the isolates with both the genes followed by isolates with toxA (66.67%) and exoS (41.67%) respectively with successful re-isolation of P. aeruginosa from the dead mice. Post-mortem examination of the dead mice showed marked haemorrhages on liver, blackish discoloration of intestines and haemorrhages on lungs. Antibiogram revealed that the isolates were highly sensitive to drugs like amikacin (89.47%), imipenem (84.21%), meropenem (78.95%) and cefoperazone-sulbactam (84.21%) but resistant to tetracycline (68.42%), piperacillin (63.16%), oxacillin (52.63%), mecilinam (47.37%) and others.

Phenotypic and molecular assessment of chickpea rhizobia from different chickpea cultivars of India.

In the present study, heterogeneity in natural chickpea rhizobia populations associated with 18 different chickpea (Cicer arientinum) cultivars of India was investigated. Physiological diversity of 20 chickpea rhizobia was characterized based on phenotypic parameters such as Bromothymol blue (BTB) test, pH, temperature and salinity tolerance. Based on response to BTB test and pH tolerance, all chickpea rhizobia were further divided into slow growers/alkali producers (14 isolates) and fast growers/acid producers (6 isolates). The temperature (upto 40°C) and salinity (NaCl) tolerance (upto 6%) tests provided a wide description of physiological diversity among the rhizobial isolates. The intrinsic antibiotic resistance of each isolate against 14 different antibiotics distinguished all chickpea rhizobia into five clades at the level of 80% similarity coefficient. Further, based on UPGMA phylogeny of carbon utilization profile, all isolates were dispersed into six clusters at the level of 85% similarity coefficient, which indicated a remarkable variability among the rhizobia. The evaluation of nodule-forming efficiency of all isolates revealed that the isolate ACR15 was more competent for nodule formation than all other isolates. The representative strain from each carbon metabolic cluster was further subjected for molecular identification through 16S rRNA gene characterization. Neighbour-joining method-based phylogeny of 16S rRNA gene sequence revealed a high degree of species diversity among the isolates. Further, the prominent nodule-forming isolate such as ACR15 was identified as Mesorhizobium ciceri, while other isolates showed similarity with other species of Mesorhizobium genus. The present study contributed to the knowledge that besides M. ciceri and M. mediterraneum, chickpea can also be nodulated by many other native chickpea rhizobia which indicates the impact of exploration of promising native populations. These findings may support the further investigation of symbiotic as well as stress responsive genes of chickpea rhizobia leading to develop more effective inoculant strains for wide agricultural applications.

A new pH indicator dye-based method for rapid and efficient screening of l -asparaginase producing microorganisms

l-asparaginase is a pharmaceutically and industrially important enzyme as it has potential to treat different cancers and inhibit acrylamide formation in fried and baked food products. In the present study, an attempt was made to screen for new and novel l-asparaginase producers using a widely applied phenol red and bromothymol blue (BTB)1 dye-based plate assay. Screening of four different soil samples for l-asparaginase producers resulted in the isolation of three new potential l-asparaginase producing bacteria. These three strains identified (by 16S rRNA sequencing) as a Pseudomonas resinovorans strain IGS-131, a Bacillus safensis strain IGS-81, and a Glutamicibacter arilaitensis strain ICS-13 with enzyme activities of 10.91 IU/ml, 6.65 IU/ml, and 1.47 IU/ml, respectively. These three strains of bacteria have not been reported as l-asparaginase producers previously. Also, we developed a new pH indicator dye-based plate assay for the screening of l-asparaginase producers after testing eight different pH indicator dyes. This cresol red dye-based method gave a better differentiable zone of hydrolysis and consistent results as compared to previously reported phenol red and BTB-based plate assay. It was also found to be efficient in comparison to all other dyes studied. It produced a bright yellow color at acidic pH (5.5) and turned into a dark red or maroon color when pH was increased (above 7.5). This finding is expected to make screening of all kinds of l-asparaginases more comfortable, rapid, and efficient.

Synthesis of pH indicators for Cerenkov imaging by electrophilic substitution of bromine by fluorine in an aromatic system

Direct electrophilic fluorination using molecular fluorine gas is used in organic synthesis to create novel fluorine-containing compounds with potential beneficial activity that could not be obtained by nucleophilic substitution. In this paper, we report a novel electrophilic substitution of bromine by fluorine in an aromatic system. The mechanism of this type of fluorination was explored using the reaction between bromothymolsulfonphthalein (Bromothymol Blue) and dilute fluorine gas under acidic conditions. Substitution occurs in the bromine atoms located in the ortho-position relative to the hydroxyl group. A similar electrophilic fluorination of thymolsulfonphthalein (Thymol Blue) leads to a substitution of hydrogen atoms in the same position (ortho to hydroxyl). NMR spectroscopy was used to confirm the fluorination sites. NMR spectra of thymolsulfonphthalein and its derivatives under basic conditions can be explained by considering the absence of resonance between the two phenolic rings. Both dibromothymol blue and fluorobromothymol blue intermolecularly attenuate Cerenkov radiation selectively near their maximum absorbance in a pH dependent manner.

Detection and characterization of pathogenic Pseudomonas aeruginosa from bovine subclinical mastitis in West Bengal, India.

Aim:Subclinical mastitis in bovines is mainly responsible for the huge economic loss of the dairy farmers, of which Pseudomonas aeruginosa is one of the causative agents. The study was aimed at a screening of suspected milk samples from different cattle farms of West Bengal for detection and confirmation of P. aeruginosa strains followed by their characterization.Materials and Methods:Around 422 milk samples were screened from different dairy farms primarily by on-spot bromothymol blue (BTB) test and then in the lab by somatic cell counts (SCC) to finally consider 352 samples for detection of P. aeruginosa. Selective isolation and confirmation of the isolates were done using selective media, viz., cetrimide and Pseudomonas agar followed by confirmation by fluorescent technique. Molecular characterization of the strains was done by polymerase chain reaction for the presence of toxA (enterotoxin A, 352 bp) and exoS (exoenzyme S, 504 bp) genes.Results:Approximately, 371 (87.9%) samples were positive in on-spot BTB test among which 352 (94.8%) samples revealed high SCC values (more than 3 lakh cells/ml) showing infection when screened. Among these, 23 (6.5%) samples yielded typical Pseudomonas sp. isolates out of which only 19 (5.4%) isolates were confirmed to be P. aeruginosa which showed characteristic blue-green fluorescence due to the presence of pigment pyoverdin under ultraviolet light. Out of these 19 isolates, 11 isolates were positive for toxA, 6 isolates for exoS, and 2 for both these pathogenic genes.Conclusion:Approximately, 5.4% cases of bovine subclinical mastitis infections in South Bengal were associated with P. aeruginosa which possess pathogenic genes such as toxA (63.2%) and exoS (36.8%).

Development of a robust system for high-throughput colorimetric assay of diverse amino acid decarboxylases

Abstract Amino acid decarboxylases catalyze decarboxylation of amino acids into amines that possess wide industrial applications. As key enzymes in biobased production of industrially important amines such as cadaverine, putrescine and β-alanine, lysine decarboxylase, ornithine decarboxylase and aspartic acid decarboxylase have attracted increasing attention. To develop enzyme variants with superior catalytic properties, there is a great need for high-throughput assay of these decarboxylases. Here we report the development of assays based on the color change of pH indicator – chlorophenol red (CPR) or bromothymol blue (BTB) – in decarboxylation reactions, in which one proton was consumed per carboxylic group decarboxylated resulting in an increase in pH. First, two buffer-indicator pairs, 4-morpholineethanesulfonic acid (MES)-CPR and 3-morpholinopropanesulfonic acid (MOPS)-BTB, were chosen on the basis of their similar pKa values at approximately pH 6.0 and 7.0, both of which are physiologically relevant. Next, the effects of buffer strength and indicator concentration on absorbance changes were examined in assay mixtures with NaOH titration, which mimicked proton consumption in decarboxylation reactions. Finally, high-throughput quantification of lysine decarboxylase, ornithine decarboxylase and aspartic acid decarboxylase was achieved using a microplate format. These results suggest that our indicator assay system may have potential applications for screening diverse decarboxylases.

Functionalized Ionic Microgel Sensor Array for Colorimetric Detection and Discrimination of Metal Ions.

A functional ionic microgel sensor array was developed by using 1-(2-pyridinylazo)-2-naphthaleno (PAN)- and bromothymol blue (BTB)-functionalized ionic microgels, which were designed and synthesized by quaternization reaction and anion-exchange reaction, respectively. The PAN microgels (PAN-MG) and BTB microgels (BTB-MG) were spherical in shape with a narrow size distribution and exhibited characteristic colors in aqueous solution in the presence of various trace-metal ions, which could be visually distinguished by the naked eye. Such microgels could be used for the colorimetric detection of various metal ions in aqueous solution at submicromolar levels, which were lower than the U.S. Environmental Protection Agency standard for the safety limit of metal ions in drinking water. A total of 10 species of metal ions in aqueous solution, Ba2+, Cr3+, Mn2+, Pb2+, Fe3+, Co2+, Zn2+, Ni2+, Cu2+, and Al3+, were successfully discriminated by the as-constructed microgel sensor array combined with discriminant analysis, agglomerative hierarchical clustering, and leave-one-out cross-validation analysis.

Fluorescent gold nanoclusters as pH sensors for the pH 5 to 9 range and for imaging of blood cell pH values

The authors describe the use of gold nanoclusters (AuNCs) with a diameter of ~2 nm for fluorescent sensing of pH values in the range from 5 to 9. The AuNCs were synthesized in the presence of bovine serum albumin (BSA) which acts as both a reducing agent and capping agent. The resulting AuNCs were characterized in terms of size and surface chemistry using TEM and FTIR. The BSA-capped AuNCs display red luminescence, with excitation/emission peaks at 470/640 nm, which is strongly modulated by the pH indicator bromothymol blue (BTB). The effect depends mainly on an inner filter effect due to spectral overlap between the absorption BTB and the emission of the AuNCs. The pH nanosensor responds to pH values in the range from 5 to 9 which is the so-called physiological pH range. The method was applied to detect changes in the pH values that occur after the death of red blood cells. Such pH changes are considered as a potential forensic marker for estimating the time passed since death. The results show the BTB-BSA-AuNC system to be capable of detecting respective intracellular pH changes.

Determination of equilibrium structures of bromothymol blue revealed by using quantum chemistry with an aid of multivariate analysis of electronic absorption spectra

The pH dependent chemical structures of bromothymol blue (BTB), which have long been under controversy, are determined by employing a combined technique of multivariate analysis of electronic absorption spectra and quantum chemistry. Principle component analysis (PCA) of the pH dependent spectra apparently reveals that only two chemical species are adequate to fully account for the color changes, with which the spectral decomposition is readily performed by using augmented alternative least-squares (ALS) regression analysis. The quantity variation by the ALS analysis also reveals the practical acid dissociation constant, pKa′. The determination of pKa′ is performed for various ionic strengths, which reveals the thermodynamic acid constant (pKa=7.5) and the number of charge on each chemical species; the yellow form is negatively charged species of −1 and the blue form that of −2. On this chemical information, the quantum chemical calculation is carried out to find that BTB molecules take the pure quinoid form in an acid solution and the quinoid-phenolate form in an alkaline solution. The time-dependent density functional theory (TD-DFT) calculations for the theoretically determined chemical structures account for the peak shift of the electronic spectra. In this manner, the structures of all the chemical species appeared in equilibrium have finally been confirmed.

Preferential adsorption of various anionic and cationic dyes from aqueous solution over ternary CuMgAl layered double hydroxide

To investigate the effect of Cu on structural and adsorption properties of CuMgAl LDH towards the efficient removal of organic dye contaminants from aqueous solution, a series of CuMgAl LDHs with different molar ratio of Cu/Mg were synthesized via co-precipitation method. The synthesized materials were characterized by using various techniques such as powder X-ray diffraction (PXRD), Fourier transformed infrared (FTIR), thermogravimetric analysis (TGA), scanning electron microscopy (SEM), N2 adsorption–desorption, transmission electron microscopy (TEM) and diffuse-reflectance UV–vis (UV-DRS) spectroscopy. The presence of Cu greatly enhanced the adsorption capacity of the adsorbents for efficient removal of organic dye pollutant from aqueous solution. The adsorption performance of the Cu-rich LDH was examined for preferential adsorption of various anionic and cationic dyes such as methyl orange, bromothymol blue, erichrome black-T, Congo red, methylene blue and rhodamine B from aqueous solution. The anionic dyes were adsorbed in higher amount over the ternary LDH compared to that of cationic dyes. The influence of various reaction parameters like contact time, adsorbent dosage, initial dye concentration and solution pH on adsorption process was studied thoroughly. The adsorption process was analyzed by using various isotherm models and the Langmuir isotherm model showed the best fitting to the isotherm data. The kinetics study showed that the adsorption process followed second order kinetic model. The successful interaction of dye with the adsorbent surface was studied by FTIR spectroscopy. The adsorbent was stable and active upto three successive cycles.

Mass Spectra of Sulfonephthalein pH Indicator Dyes and Their Impurities.

Sulfonephthalein pH indicator dyes have historically been noted for impurities originating from the synthesis process, which lead to batch to batch differences in pH measurements. Uncertainties up to 0.1 pH units have been attributed to impurities in these reagents. In an effort to understand the extent of impurities in these dyes and the potential impact on the expanded uncertainty of spectrophotometric pH measurements, we have examined seven commercially available sulfonephthalein pH indicators using liquid chromatography-mass spectrometry (LC-MS): bromocresol green (BCG), bromocresol purple (BCP), bromothymol blue (BTB), cresol red (CR), meta-cresol purple (MCP), phenol red (PR), and thymol blue (TB). Peaks eluting from the LC were subjected to fragmentation by mass spectrometry. The resulting dataset will aid in efforts to improve the synthesis or purification of these dyes.

Phenotyping Groundnut Rhizobia Native to Phosphorus Deficient Soils in Western Kenya

Most soils in western Kenya are characterized by high acidity level and phosphorus (P) deficiency, which affect nodulation and nitrogen fixation of groundnut (Arachis hypogaea L.). This study aimed at characterising rhizobia capable of nodulating groundnut in P deficient soils in Western Kenya. Sixty four isolates out of the 68 were confirmed to be rhizobia due to their ability to nodulate groundnut. Ninety six percent of the isolates exhibited semi-globose to globose colony shape on yeast extract mannitol agar (YEMA). Groundnut was nodulated by both fast and slow growing rhizobia isolates with 81% being fast growers. Fifty one isolates representing 75% produced acid on YEMA medium supplemented with bromothymol blue (BTB). The isolates varied in their response to pH with 39 and 61 growing at pH 4.0 and 5.5, respectively. All the isolates grew at pH 7.0 and 8.5. YEMA medium containing glucose, sucrose, starch and citrate had 64, 61, 56 and 5 isolates growing, respectively. Sixty four isolates exhibited clear zone of solubilization on medium containing dicalcium phosphate as source of inorganic phosphate. Solubilization index (SI) varied from 1.1 to 6.8. Fast-growing rhizobia isolates N01, B02, I06, Q01, F05, C02, E01, Q03, I01 and B01 recorded the highest solubilization index of 3.8, 4.5, 4.6, 4.6, 4.7, 5.0, 5.1, 6.1, 6.1 and 6.8, respectively. Groundnut rhizobia showed variation in their potential to solubilize inorganic phosphate and effectively nodulate the host. The most promising isolates from this study would be used as bio-fertilizer upon further validation in the greenhouse and field.

A flow-based platform for measuring the acidity parameters in wine

The present work describes a valuable tool for winemaking industry to measure the acidity parameters with rapid response, simple sample handling, with no or minimal pre-treatment. Thus, a sequential injection analysis (SIA) system with spectrophotometric detection was used as platform for the development of methodologies for the quantification of volatile and total acidity in wine samples. Both procedures make use of the same colour reagent, bromothymol blue (BTB) that changes its intrinsic colour in the presence of the acidity compounds. The volatile acidity value was attained with the separation of the volatile fraction of acids by means of a membrane separation technique, a gas-diffusion unit. For the total acidity value, the sample was merged with the colour reagent on the way towards detection. The fixed acidity is a result of the difference from the total and the volatile acidity.The presented tool displayed a low sample and reagent consumption, 346 and 102µL of sample and 37 and 32µg of BTB, for the volatile acidity (VA) and for the total acidity (TA), respectively. The observed limits of detection and quantification were 0.03 and 0.01gL−1 (VA) and 0.09 and 0.02gL−1 (TA) with high determination rates, 35 (VA) and 62 (TA) determinations per hour. The proposed system was successfully applied to the quantification of volatile, total and fixed acidity in white table wine samples. The obtained results were in good agreement with the ones obtained by the reference methods.

Unravelling the effects of mobile phase additives in supercritical fluid chromatography. Part I: Polarity and acidity of the mobile phase

The mobile phases employed in current supercritical fluid chromatography (SFC) are usually composed of a mixture of pressurized carbon dioxide and a co-solvent. The co-solvent is most often an alcohol and may contain a third component in small proportions, called an additive (acid, base or salt). The polarity of such mobile phase compositions is here re-evaluated with a solvatochromic dye (Nile Red), particularly to assess the contribution of additives. It appears that additives, when employed in usual concentration range (0.1% or 20mM) do not modify the polarity in the immediate environment of the probe.In addition, the combination of carbon dioxide and an alcohol is known to form alkoxylcarbonic acid, supposedly conferring some acidic character to SFC mobile phases. Direct measurements of the apparent pH are impossible, but colour indicators of pH can be used to define the range of apparent pH provided by carbon dioxide-alcohol mixtures, with or without additives. Five colour indicators (Thymol Blue, Bromocresol Green, Methyl Red, Bromocresol Purple, and Bromothymol Blue) were selected to provide a wide range of aqueous pKa values (from 1.7 to 8.9). UV–vis absorption spectra measured in liquid phases of controlled pH were compared to those measured with a diode-array detector employed in SFC, with the help of chemometric methods. Based on these observations, it is concluded that the apparent pH range in carbon dioxide-methanol mobile phases is close to 5. Increasing the proportion of methanol (in the course of a gradient elution for instance) causes decreasing apparent pH. Strong acids can further decrease the apparent pH below 1.7; strong bases have little influence on the apparent pH, probably because, in this range of concentrations, they are titrated by alkoxylcarbonic acid or form ion pairs with alkoxycarbonate. However, bases and salts could stabilize the acidity in the course of gradient runs.

Improved mesostructure by incorporating surfactant on thin film to develop an advanced optical fiber pH sensor with a temperature cross sensitivity feature

A new optical fiber pH sensor based on bromothymol blue (BTB) thin film with temperature cross-sensitivity has been proposed in this paper. The BTB thin film was prepared by depositing a thin layer of a solution containing tetraethyl orthosilicate (TEOS) and a BTB pH indicator in the presence of surfactants, namely cetyltrimethyl ammonium bromide (C19H42BrN, CTAB), by sol–gel technology on an unclad multimode fiber (MMF) surface. The number of layers and the deposition length of the thin film were varied, and the power transmission versus pH variation was studied. The concentration of the surfactant was increased to understand the effect of increasing porosity in the sol–gel matrix to achieve improved pH sensitivity. A straightforward way to utilize the temperature cross-sensitivity feature of the optical fiber pH-sensitive device has been introduced to develop a high sensitivity temperature sensor. A sensitivity of 79.96 nW pH−1 was obtained by a 20-layer thin-film coated sensor in the pH range of 3–12.

Effect of Dye Structure on the Photodegradation Kinetic Using TiO<sub>2</sub> Nanoparticles

In this study the effect of pH, adsorption behavior and the chemical struc-tures of two dyes (Methyl Orange and Bromothymol Blue) on the photo-degradation rate constant, was investigated. Adsorption isotherm shows that the adsorption amount of dyes on TiO2 surface is highly related to the pH of the solution and to the pKa of each dye. In acidic medium the adsorption percentage of Methyl Orange on TiO2 surface was 76% facing 5% for Bromothymol Blue. The kinetic study shows compatibility between the degradation rate constant and the adsorption percentage on the surface. In basic medium the adsorption percentage of Methyl orange and Bromothymol Blue is similar while the degradation rate of Methyl orange is two times faster than that of Bromothymol Blue which reveals the role of chemical structure in the photodegradation rate.

Novel Spectrophotometric Method Development for the Estimation of Boceprevir in Bulk and in Pharmaceutical Formulations

A New, Simple, Rapid and economical extractive spectrophotometric methods were developed for the determination of Boceprevir (anti-retroviral drug). Boceprevir is a direct acting protease inhibitor for the treatment of hepatitis C. It also has two isomers in which the S isomer is more active than the R-isomer. The methods were based on the formation of color chromogens with Bromo cresol green, Bromo thymol blue, Bromo phenol blue and Methyl orange indicator. The extractive spectrophotometry was carried out with phthalate buffer and chloroform. The absorbances of the chromogens were measured at 410 nm and 415 nm against the corresponding reagent blank. The proposed methods have been successfully applied to the bulk drug. The method has been statistically evaluated and was found to be precise and accurate.