Methods colorimetric

Tsubouchi reported a colorimetric procedure for the analysis of acetylcholine [33]. About 2-10 mL of a test solution containing acetylcholine is extracted into 1,2-dichloroethane in the presence of the ethanolic reagent (2mL at 5mM) and pH 10 0.1 M Na2B4O7-0.2 M Na2HPQ4 buffer 

Das and Abdulla reported a modified method for estimation of acetylcholine [36]. Acetylcholine reacts with hydroxylamine in alkaline medium to give hydroxamic acid, which produces a red-violet color when reacted with FeCl3. Absorbance measurements are made at 540 nm. The method can be applied to saliva samples stored at 0°C for as long as 72 h. 

Abernethy et al. reported an enzymatic method for erythrocyte acetylcholineesterase [37]. Haemolysate 50 pL was mixed with 67 mM phosphate buffer of pH 7.4 (2mL) and 21.5mM acetylcholine (50 pL), and the mixture incubated for 20 min at 37°C. A color reagent (500 pL) containing 4mM phenol, 0.8 mM aminoantipyrine, peroxidase (5 iUL), choline oxidase (4 L iU/L), and 0.86 mM physostigmine plus the buffer was added and, after 20 min the absorbance was measured at 500 nm. 

Sakai et al. reported a novel flow injection method for the selective spectrophotometric determination of acetylcholine using thermochro-mism of ion associates [38]. Samples (0.14mL) containing acetylcholine were injected into a flow injection system with a buffered (pH 11) carrier stream and a reagent stream (10 mM tetrabromo-phenolphthalein ethyl ester in dichloroethane) at 0.8 mL/min. The temperature of the flow cell was 45°C which reduced interference and improved recovery, and the detection was at 610 nm. 

Sakai et al. reported the use of batchwise and flow-injection methods for the thermo-spectrophotometric determination of acetylcholine and choline with tetrabromophenolphthalein ethyl ester [39]. An aqueous sample 

The colour reactions of phosgene constitute the most widely published methods for its determination. They are frequently reliable, and are often amenable to continuous monitoring of effluents and the workplace environment [1255]. The earliest methods based on colour changes of reagents were developed for the rapid detection of phosgene when used as a war gas [187b,642,643,1107,2024,2243]. 

Chemically labeling a protein always carries the possibility of disrupting its ligand-binding activity or blocking the epitope of the protein, thereby preventing secondary antibody attachment. Therefore, an alternative method to detect the bonnd proteins in their native form is sometimes preferred. 

Reducing sugars, including lactose, react on boiling with phenol (2.2) or anthrone (2.3) in strongly acidic solution (70%, v/v, H2SO4) to give a coloured solution. 

The complex with anthrone absorbs maximally at 625 nm. The concentration of lactose is determined from a standard curve prepared using a range of lactose concentrations. 

The method is very sensitive but must be performed under precisely controlled conditions. 

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In the DPD colorimetric method for the free chlorine residual, which is reported as parts per million of CI2, the oxidizing power of free chlorine converts the colorless amine N,N-diethyl-p-phenylenediamine to a colored dye that absorbs strongly over the wavelength range of 440-580 nm. Analysis of a set of calibration standards gave the following results... 

Color-forming reactions Color generation Colorimeters Colorimetric methods... 

In view of the chromophoric character of the elemental iodine itself, many colorimetric methods have been proposed for the deterrnination of inorganic iodine (88—92). These methods use the visible portion of the spectmm in reading iodine concentrations. In the visible range the extinction coefficient for iodine is not high enough to be used for minute quantities of iodine in water and other solvents (93). Higher sensitivities have been reported for elemental iodine in potassium iodide solutions in the ultraviolet (93,94). 

Titrations with dibutylamine [111-92-2] can also be used to determine the NCO content of isocyanates and prepolymers. Generally, an excess of amine in a suitable solvent such as chlorobenzene [108-90-7] is added to the sample. The resulting solution is allowed to react and the unreacted amine is back- titrated with dilute hydrochloric acid. For low NCO content levels, a colorimetric method is often used. The isocyanate-containing species is titrated with amine and the unreacted amine is deterrnined using malachite green [569-64-2]. 

Colorimetric Method. A finely powdered sample treated with sulfuric acid, hydrobromic acid [10035-10-6] and bromine [7726-95-6] gives a solution that when adjusted to pH 4 may be treated with dithi one [60-10-6] ia / -hexane [110-54-3] to form mercuric dithi2onate [14783-59-6] (20). The resultant amber-colored solution has a color iatensity that can be compared against that of standard solutions to determine the mercury concentration of the sample. Concentrations below 0.02 ppm have been measured by this method. 

Nickel also is deterrnined by a volumetric method employing ethylenediaminetetraacetic acid as a titrant. Inductively coupled plasma (ICP) is preferred to determine very low nickel values (see Trace AND RESIDUE ANALYSIS). The classical gravimetric method employing dimethylglyoxime to precipitate nickel as a red complex is used as a precise analytical technique (122). A colorimetric method employing dimethylglyoxime also is available. The classical method of electro deposition is a commonly employed technique to separate nickel in the presence of other metals, notably copper (qv). It is also used to estabhsh caUbration criteria for the spectrophotometric methods. X-ray diffraction often is used to identify nickel in crystalline form. 

Analytical and Test Methods. o-Nitrotoluene can be analyzed for purity and isomer content by infrared spectroscopy with an accuracy of about 1%. -Nitrotoluene content can be estimated by the decomposition of the isomeric toluene diazonium chlorides because the ortho and meta isomers decompose more readily than the para isomer. A colorimetric method for determining the content of the various isomers is based on the color which forms when the mononitrotoluenes are dissolved in sulfuric acid (45). From the absorption of the sulfuric acid solution at 436 and 305 nm, the ortho and para isomer content can be deterrnined, and the meta isomer can be obtained by difference. However, this and other colorimetric methods are subject to possible interferences from other aromatic nitro compounds. A titrimetric method, based on the reduction of the nitro group with titanium(III) sulfate or chloride, can be used to determine mononitrotoluenes (32). Chromatographic methods, eg, gas chromatography or high pressure Hquid chromatography, are well suited for the deterrnination of mononitrotoluenes as well as its individual isomers. Freezing points are used commonly as indicators of purity of the various isomers. 

A number of colorimetric methods are available (83) however, spectroscopic methods are generally preferred. 

Various colorimetric methods have been employed for measuring ozone residuals, although most of these ate susceptible to significant interferences (142). The indigo trisulfonate method (143), however, has been approved by the Standard Methods Committee of the American Pubfle Health Association (141) and the International Ozone Association for ozone residual measurement. 

There are many colorimetric methods used for trace analysis of peroxides using reagents such as ferrous ion, leuco base of methylene blue, yy -diphenylcarbohydrazide, titanium(IV), iodide ion, and Ai,A7-dimethyl- -phenylenediamine. The latter two are the most commonly used reagents... 

Contaminant by-products depend upon process routes to the product, so maximum impurity specifications may vary, eg, for CHA produced by aniline hydrogenation versus that made by cyclohexanol amination. Capillary column chromatography has improved resolution and quantitation of contaminants beyond the more fliUy described packed column methods (61) used historically to define specification standards. Wet chemical titrimetry for water by Kad Eisher or amine number by acid titration have changed Httle except for thein automation. Colorimetric methods remain based on APHA standards. 

More specifically, 2-aminophenol can be detected in solution using an iron(II) sulfate—hydrogen peroxide reagent (94). 3- Aminopheno1 has been analyzed colorimetrically by oxidation in base and subsequent extraction of a violet quinoneimide dye (95). A colorimetric method using... 

The sodium hydroxide is titrated with HCl. In a thermometric titration (92), the sibcate solution is treated first with hydrochloric acid to measure Na20 and then with hydrofluoric acid to determine precipitated Si02. Lower sibca concentrations are measured with the sibcomolybdate colorimetric method or instmmental techniques. X-ray fluorescence, atomic absorption and plasma emission spectroscopies, ion-selective electrodes, and ion chromatography are utilized to detect principal components as weU as trace cationic and anionic impurities. Eourier transform infrared, ft-nmr, laser Raman, and x-ray... 

Analytical Techniques. Sorbic acid and potassium sorbate are assayed titrimetricaHy (51). The quantitative analysis of sorbic acid in food or beverages, which may require solvent extraction or steam distillation (52,53), employs various techniques. The two classical methods are both spectrophotometric (54—56). In the ultraviolet method, the prepared sample is acidified and the sorbic acid is measured at 250 260 nm. In the colorimetric method, the sorbic acid in the prepared sample is oxidized and then reacts with thiobarbituric acid the complex is measured at - 530 nm. Chromatographic techniques are also used for the analysis of sorbic acid. High pressure Hquid chromatography with ultraviolet detection is used to separate and quantify sorbic acid from other ultraviolet-absorbing species (57—59). Sorbic acid in food extracts is deterrnined by gas chromatography with flame ionization detection (60—62). 

Sucrose quantitation has also been performed by colorimetric methods. However, in recent years, automated enzymatic analyzers and instmmental methods (eg, ion chromatography and hplc) have become increasingly popular, as they provide greater sensitivity and accuracy. Near infrared (nir) spectroscopy is currendy under evaluation as a tool for sucrose quantitation in sugar mills and food processing operations. 

Colorimetric Methods. Numerous colorimetric methods exist for the quantitative determination of carbohydrates as a group (8). Among the most popular of these is the phenol—sulfuric acid method of Dubois (9), which rehes on the color formed when a carbohydrate reacts with phenol in the presence of hot sulfuric acid. The test is sensitive for virtually all classes of carbohydrates. Colorimetric methods are usually employed when a very small concentration of carbohydrate is present, and are often used in clinical situations. The Somogyi method, of which there are many variations, rehes on the reduction of cupric sulfate to cuprous oxide and is appHcable to reducing sugars. 

Chloride. Chloride is common in freshwater because almost all chloride salts are very soluble in water. Its concentration is generally lO " to 10 M. Chloride can be titrated with mercuric nitrate. Diphenylcarbazone, which forms a purple complex with the excess mercuric ions at pH 2.3—2.8, is used as the indicator. The pH should be controlled to 0.1 pH unit. Bromide and iodide are the principal interferences, whereas chromate, ferric, and sulfite ions interfere at levels greater than 10 mg/L. Chloride can also be deterrnined by a colorimetric method based on the displacement of thiocyanate ion from mercuric thiocyanate by chloride ion. The Hberated SCN reacts with ferric ion to form the colored complex of ferric thiocyanate. The method is suitable for chloride concentrations from 10 to 10 M. 

Two colorimetric methods are recommended for boron analysis. One is the curcumin method, where the sample is acidified and evaporated after addition of curcumin reagent. A red product called rosocyanine remains it is dissolved in 95 wt % ethanol and measured photometrically. Nitrate concentrations >20 mg/L interfere with this method. Another colorimetric method is based upon the reaction between boron and carminic acid in concentrated sulfuric acid to form a bluish-red or blue product. Boron concentrations can also be deterrnined by atomic absorption spectroscopy with a nitrous oxide—acetjiene flame or graphite furnace. Atomic emission with an argon plasma source can also be used for boron measurement. 

Atomic absorption spectroscopy is an alternative to the colorimetric method. Arsine is stiU generated but is purged into a heated open-end tube furnace or an argon—hydrogen flame for atomi2ation of the arsenic and measurement. Arsenic can also be measured by direct sample injection into the graphite furnace. The detection limit with the air—acetylene flame is too high to be useful for most water analysis. 

Colorimetric methods always include hafnium. Most methods employ a separation step such as solvent extraction. The three reagents used successfully are 8-hydroxyquinoline (88), alizarin red S (89), and catechol violet (90). 

Colorimetric methods have led to the development of visual devices for measurement of benzene concentration. These visual detection tubes have been popular since the 1960s and have provided a simple and rehable method for evaluating ambient aromatic vapor contamination. These products are available from anumber of manufacturers such as Drager (Lubeck, Germany), Gastec (Tokyo, Japan), Kitagawa (Kawasaki, Japan), DuPont (Wilmington, Delaware, USA), and 3M (St. Paul, Minnesota, USA) (85). 

Colorimetric methods have been successfully used for determining trace amounts of ethanol. Ammonium hexanitratocerate(IV) has been used as a reagent (262) and for continuous automatic analysis. Alcohols form colored complexes with 8-hydroxyquinoline and vanadic compounds. The absorbance of these complexes, measured at 390 p.m has been used to provide an analytical procedure (263). 

Direct reading A sampling approach that provides immediate or very fast feedback such as a meter or colorimetric method. 

The extraction of alkaloids from jaborandi has been dealt with by Wurtzen, and the estimation of pilocarpine in the drug and its preparations by Jowett and by Bourcet and more recently by Taran and by Shupe.ii Elvidge has devised a colorimetric method based on Ekkert s test, < and a polarographic study has been made by Kirkpatrick. Tlie two first-pamed authors depend on the isolation of pilocarpine nitrate, which is also the basis of the process of manufacture described by Cheninitius.12... 

For the assay of ephedrine in the total alkaloids a colorimetric method based on the biuret reaction was used by Feng and Read and is described in detail by Feng. Krishna and Chose separated ephedrine and iji-ephedrine by treating the dry mixed hydrochlorides with dry chloroform in which the ephedrine salt is virtually insoluble and the -ephedrine salt soluble. ... 

The paper. strips used in the colorimetric method are impregnated with such dyes that the color of the test paper is dependent upon the pH of the medium in which the paper is placed. A standard color chart is supplied for comparison with the test strip. Test papers are available in a wide range type, which permits estimating pH to 0.5 units, and in narrow range papers, with which the pH can be estimated to 0.2 units. 

Consider now two solute species Bi and B3, between which no direct experimental comparison by the colorimetric method is possible, because their useful concentration ranges just fail to overlap. We can find an intermediate indicator solute B2, whose useful range partly overlaps that of B, and partly overlaps that of B3. Using B2, a relation between Bi and B3 may thus be established indirectly. In dilute solution this relation will be a simple one. We do not know enough about concentrated solutions to be in a position to say whether a similar relation should be expected. In the experiments to be described, the first aim was to obtain an answer to this question. 

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