Subject colorimetric

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. 

The method, obviously, is subjective, the precision and speed of the match depending upon the observer and his experience. Results on foods have usually been expressed in terms of color disks, which are different for each product and which must be carefully standardized. [Conversions to standard colorimetric systems of notation can be made (12), provided suitable colorimetric data are available for the disks used.] Furthermore, instruments suitable for the most precise work by this method are not at the present time commercially available. 

Colorimetric procedures used In steroid assays are often subject to drug Interference. In the determination of 17-Ketosterolds by the Zimmerman reaction, drugs with the 17-Keto basic structure such as ascorbic acid, morphine and reserplne will cause Increased values. In the determination of 17,21 -dlhydroxysterolds by the Porter-Sllber reaction the dlhydroxy-acetone chain Is the reactive unit. Drugs like meprobamate, chloral hydrate, chloropromazlne and potassium Iodide will Interfere with this reaction and cause elevated values. In the colorimetric determination of vanlllylmandellc acid (VMA) by a dlazo reaction, drugs like methocarbamol and methyl dopa cause... 

Instrumental color measurements eliminate subjectivity, are more precise, take less time, and are simpler to perform. However, to evaluate instrumental results properly, the physics of the measurement processes must be considered. Three types of color measurement instruments are used for food the monochromatic colorimeter, the tristimulus colorimeter, and the colorimetric spectrophotometer. 

DFDT has been subjected to the usual analysis for its halogen content, but no specific method has been worked out for its determination, nor has an investigation been made of its response to the known colorimetric reactions of DDT. 

Introduction of microbiological methods for the determination of amino acids made possible the estimation of the amount of both free and combined amino acids in urine. Dunn et al. (D4), Thompson and Kirby (Tl), Eckhard and Davidson (El), and Woodson et al. (W3) estimated the amount of amino acids liberated in the course of acid or, as in the case of tryptophan determination, alkaline hydrolysis. Microbiological and colorimetric methods used for the determination of certain amino acids present very little opportunity for evaluating the proper quantitative relations between free and combined amino acids, since under the applied condition both combined and free amino acids are equally involved in the reaction. In 1949 Albanese et al. (A3) applied such methods to the quantitative determination of free and combined amino acids in the nondiffusible fraction of urine, and subjected the procedures to broad criticism from just this point of view. 

Rapid progress has been reported in the development of methods for sulfonamide residues in tissues, milk, and eggs since the subject was reviewed by Horwitz ( ) in 1981, The colorimetric method of Tishler et al (j ) has in the past been used to detect violative levels of sulfonamide residues in animal tissues. The lack of specificity and the variable background levels produced by this method have been discussed by Horwitz ( ), Matusik et al (15), and Lloyd et al (16), Recently, a number of specific chromatograpiic methods have been described for determination of residues of a variety of sulfonamides, These are summarized in Table 1 and suggest that HPLC is emerging as the method of choice followed by GLC and TLC methods. 

A. Analysis of Wastewater and Natural Waters. The presence of certain anions in wastewater effluents can cause deterioration of natural water systems. Phosphorous and nitrogen can be present in several chemical forms in wastewaters. Phosphorous is usually present as phosphate, polyphosphate and organically-bound phosphorus. The nitrogen compounds of interest in wastewater characterization are ammonia, nitrite, nitrate and organic nitrogen. Analyses are often based on titrimetric, and colorimetric methods (3). These methods are time consuming and subject to a number of interferences. Ion Chromatography can be used to determine low ppm concentrations of these ions in less than thirty minutes with no sample preparation. 

Colorimetric assays used in endocrinological procedures are also often subject to drug interference. We have observed an interesting interference in a patient with carcinoid. The patient excreted 400 mg of 5-hydroxyindoleacetic acid (5-HIAA) and when a vanillylmandclic acid (VMA) determination was performed by a nonspecific diazo method, the value was reported to be 375 mg. The catecholamines were just above normal. There was an immediate suggestion that the patient also had a pheochromocytoma. However, when a specific chromatographic VMA method was used, the value was found to be within normal limits. Subse-... 

Samples of metal complexes isolated from the final solutions were subjected to microanalysis (for carbon, hydrogen, oxygen, and sulfur). Metals were determined colorimetrically by the following methods— copper as the complex formed with sodium diethyl dithiocarbamate (6) cobalt as the nitroso-R salt complex (7) nickel as the dimethylglyoxime complex (4). 

Table 3.7.2 Biotinidase activity in plasma from normal subjects, patients with different forms of biotinidase deficiency and in obligate heterozygotes (parents) measured at 30°C by colorimetric assay using two different substrate concentrations. Km Michaelis-Menten constant, n number of individuals ... 

A number of methods are available for following lipase activity. Although numerous modifications and variations have been introduced, the basic methods are (1) titration of the liberated fatty acids, (2) changes in surface tension, (3) colorimetric determination of the fatty acids, (4) use of gas-liquid chromatography, and (5) use of radioactive substrates. Kuzdzal-Savoie (1980) has reviewed the subject. 

Figure F3.3.3 Comparison of subjective sensory assessment (A percent discoloration) and objective colorimetric evaluation (B hue angle). Beef was obtained from cattle supplemented with 0 (E-0), 500 (E-500) or 2000 (E-2000) IU a-tocopherol acetate per head per day. The a-tocopherol demonstrated a color preservation effect. Hue angle was calculated as [tan-1(b7a )] (36072rc). Standard error bars are indicated. Adapted from Chan et al. (1995), with permission from the Institute of Food Technologists.

Nitrite is determined separately by the colorimetric method on another aliquot of the sample not subjected to Cd reduction. Subtract N02 -N value to determine the concentration of N03-N in the sample. 

Hair dyes must meet a number of conditions related to their end use. Color can be assessed by colorimetry [49], The limits of precision are set by the substrate on which the measurement is performed. Studies on test subjects are difficult because of the uneven natural hair color and the background color of the scalp. Tresses are hard to prepare at a constant quality level. Measurements on wool cloth give reproducible results, but for oxidation dyes the shades are not identical to those produced on hair. Colorimetric methods are therefore useful only for comparative measurements on the same object, for example, in lightfastness tests. Because hair must be redyed after four to six weeks due to growth, the fastness required of hair dyes is generally less than that needed for textiles. However, stability is still a problem with many indo dyes (see Section 5.4.3). Some of them... 

Perhaps the most exacting TLC studies were carried out by Simon et al43. phenytoin was extracted frcm plasma, concentrated by evaporation, and subjected to TLC on silica gel plates. After the phenytoin spot was scraped off, the colorimetric procedure of Dill et al4 was applied directly to the scrapings. Phenytoin levels of 5 ug/ml or more could be recognized by visual inspection of the plates under ultraviolet light, and serum levels of 1 ug/ml were detectable with the colorimetric procedure. 

Determination of basal biophysiological parameters may identify subjects with sensitive skin. Earlier studies have shown that increased skin susceptibility has been correlated with an increased basal TEWL, 1 41 skin surface pH,42 and fair skin complexion (measured by chromametric L values),43 whereas no relationship was shown for basal skin thickness, skin blood flow, sebum excretion, and skin hydration.29 However, a recent study by Seidenari et al. utilizing multiple bioengineering techniques showed significant correlations only for capacitance and colorimetric a values.44... 

Methods of Analysis of Pesticides. The extracted residue obtained after isolation from tissues and other biological materials is subjected to qualitative and quantitative determination of the pesticides. Sometimes, the amount of material available is so small that the colorimetric and other allied methods cannot be successfully applied as some of the residue is likely to be lost during the purification technique. Furthermore, these purification techniques required for spectrophotometry, colorimetry, and other sophisticated instrumental methods are appreciably time consuming. Therefore, other techniques were sought for the quantitative determination of pesticides. Thin layer chromatographic (TLC) techniques were found to be most suitable for toxicological analysis of pesticides. Randerath(16j stated that... 

Leaf discs have commonly been used for bioassays to determine if herbicides inhibit photosynthesis (Table 16.2). The simplest leaf-disc bioassay uses small discs cut from fully expanded cucumber or pumpkin cotyledons, floated in the light on a phosphate buffered medium containing suspected photosynthesis inhibitors.115 Qualitatively, if photosynthesis is inhibited, the leaf disc sinks. There are several variations of this method that can provide quantitative data. Evolution of O2 in the test solution can be measured with an oxygen electrode, CO2 induced pH changes colorimetrically determined with bromothymol-blue, or electrolyte leakage monitored with a conductivity meter. Leaf strips, algae, isolated chloroplasts, and duckweed (Lemna minor) have been used as test subjects. Although the bioassays presented in Table 16.2 are fairly easy to perform, few allelochemicals have been tested as possible inhibitors of photosynthesis. Many... 

These AAS methods replaced colorimetric determinations such as alpha-benzildioxime or dimethylglyoxime (6,13) that are subject to positive interferences from copper and cobalt, as well as iron and aluminum (Table VII ). 

Methyl Esters. These long-chain acyl esters are subjected to thin-layer chromatography, colorimetric analysis, and gas-liquid chromatography, coupled with mass spectrometry, as described in Chapter 4. These assays will provide information on the ester/P molar ratio (based on starting phosphate value it should be 2.0) and on the composition and relative distribution of fatty acyl residues. 

The purification of many enzymes has been achieved by use of gel-filtration chromatography, ion-exchange chromatography, or other chromatographic procedures. Eluates from such columns may be analyzed for carbohydrate and protein content, as well as for enzymic activity. Such analyses have revealed that, for glycoenzymes, the enzymic activity, the carbohydrate content, and the protein content are distributed identically in the eluates from the columns. Results of this type of experiment for ribonuclease B are shown in Fig. 1. In these experiments,8 ribonuclease B and a hydrolyzate of the enzyme were subjected to gel filtration on Sephadex, and the eluates were analyzed for protein and for carbohydrate by appropriate colorimetric methods. A control experiment containing ribonuclease A and D-man-nose was treated similarly. The data in Fig. 1 show that the carbohydrate component in the unhydrolyzed sample of ribonuclease B... 

Even today, despite enormous progress made in recent years, the numerical expression of a sensory perception is still subject to certain preconditions, not least because the standard colorimetric systems in use still have faults and limitations. Unfortunately, many manufacturers of colorimeters contribute to the uncertainty with impressive software and by claiming a degree of accuracy to so many decimal places that simply is not possible. Users often trust blindly in the manufacturer s claims, although any number of good books are available that describe the problems of colorimetry. 

Free formaldehyde is reacted with acetylacetone in the presence of an excess of an ammonium salt to form the yellow fluorescent compound, 3,5-diacetyl-1,4-dihydrolutidine and subsequently determined spectrophotometrically in methods A-E (14). In these methods, the test sample must be colorless and free from other carbonyl compounds. Some other derivatives have been used to analyze formaldehyde. For example, formaldehyde was reacted with sodium 4,5-dihydroxy-2,7-naphthalene disulfonate in sulfuric acid solution to yield a purple color (580 nm) and then subjected to colorimetric analysis. A purple-colored pararosaniline derivative was used to analyze formaldehyde in air (15). Air sample was passed through an aqueous solution which contained 0.4% of 3-methyl-2-benzothiazolone hydrazone hydrochloride and then a dye produced was determined at 635 or 670 nm (16). Molecular sieve (1.6 mm pettet) was used to trap formaldehyde in air samples. The formaldehyde... 

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