Colorimetric and Spectrophotometric Methods

There are several other colorimetric methods in the literature including measuring the pink colour flavonoids developed in the presence of magnesium and hydrochloric acid (Kwierty and Braverman 1959), the red colour developed by flavonoids when treated with l-nitroso-2-naphthol and nitric acid (Gengross and Renda 1966) and using an enzymatic colorimetric measurment with 2,2 -azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid) (Arnao et al. 1990). 

UV absorption can also be used to rapidly estimate flavonoid conent in samples comparable to the result obtained by the Davis method (Hendrickson and Edwards 1958). Sample is diluted/extracted with 2-propanol and filtered to obtain 

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The chief advantage of colorimetric and spectrophotometric methods is that they provide a simple means for determining minute quantities of substances. The upper limit of colorimetric methods is, in general, the determination of constituents which are present in quantities of less than 1 or 2 per cent. 

Reckendorf, Castringius and Spingler have utilized the colorimetric and spectrophotometric determination of nitrofurantoin in the urine and serum. Stone , and Puglisii have determined a trace of nitrofurantoin in milk by the colorimetric and spectrophotometric methods. Both procedures are based on the conversion of nitrofurantoin to 5-nitrofurfuraldehyde phenylhydrazone and are followed by the extraction and concentration on a chromatographic column. Final estimation depends upon development of a blue colour by the addition of hydramine base. Breinlich studied the titrimetric, spectrophotometric and chromatographic determinations of various nitrofurans and reported satisfactory results. 

If the absorbance increases by unity, the transmittance drops by a factor of ten. Equation (24.10) is an expression of the Beer-Lambert law, often called simply Beer s law. Beer s law is the basic equation for the various colorimetric and spectrophotometric methods of analysis. If Beer s law holds, then the absorbance is given by... 

The constant e is called the molar absorptivity when the concentration c is in moles per liter and b is in cm. The value of e is characteristic of the absorbing substance at a particular wavelength in a particular solvent and is independent of the concentration and of the path length, b. Equation 7.9 is a fundamental law on which colorimetric and spectrophotometric methods are based. It is known variously as... 

One of the reviews cited earlier enumerates spectrophotometric methods for the determination of cyanide up to 1962 [8]. Also, instrumental methods such as colorimetric and spectrophotometric methods, electroanalytical methods, catalytic methods, gas chromatography methods, radiochemical methods, and miscellaneous methods for determination of cyanide up to 1977 have been summarized by Williams [7]. The instrumental methods developed since 1977 are considered here. 

Most published quantitative data are based on isolation of the compounds, and thus represent minimum values. Colorimetric and spectrophotometric methods for quantitative determination of a few substances (without isolation) have been described. Ramaut et al. (1966) and Rundel (1969) determined usnic acid spectrophotometrically. Laasko and Gustafsson (1952) determined usnic acid as the FeClg-complex, and Jayasankar and Towers (1968) used the reaction product of usnic acid with Ehrlich s reagent for the determination. Determination methods for parietin (Hill and Woolhouse, 1966 Richardson, 1967) and atranorin (Vainshtein and Ravinskaya, 1971)... 

The methods most commonly used to detect hydrogen sulfide in environmental samples include GC/FPD, gas chromatography with electrochemical detection (GC/ECD), iodometric methods, the methylene blue colorimetric or spectrophotometric method, the spot method using paper or tiles impregnated with lead acetate or mercuric chloride, ion chromatography with conductivity, and potentiometric titration with a sulfide ion-selective electrode. Details of commonly used analytical methods for several types of environmental samples are presented in Table 6-2. 

Central ite 1 Analytical Procedures. Centr 1 can be detected by various colorimetric tests, such as described in Refs 1,6,12,24, 28 Sc 33. Some other qualitative tests are given in Ref 13. Quantitative detns of Centr 1 by bromination methods are described in Refs 4,5,7,9,10,14,16,17,20,27,34 Sc 35. Chromatographic and spectrophotometric methods are found in Refs-11,15,18,21,22,23,24, 29,30,32 Sc 33. Other quantitative methods, including polarographic, are in Refs 2,7a,20a,25, 26,27 Sc 30. X-ray diffraction spectra data are given in Ref 19. Detns of stabilizing action of Centr 1 by methods of Taliani and Thomas are discussed in Refs 3 8... 

W.Keyser, "Colorimetric Analysis , Chapman Sc Hall, London (1957) 7)D.F.Boltz, "Colorimetric Determination of Nonmetals , Wiley (1958) 8)E.B.Sandell, "Colorimetric Determination of Traces of Metals , Wiley, NY (1959 ) 9)Tintometer Ltd "Colormetric Chemical Analytical Methods , The Author, Salisbury, England (1959) 10)Vogel, InorgAnalysis (1961) 738-837 (Colorimetric and spectrophotometric analysis description of various colorimeters) ll)Pamphlets and catalogs of A.H.Thomas,... 

In addition to assay features already mentioned, other factors may influence the choice of assay by the user. In terms of sensitivity of the assay, the threshold of detection of lipase activity, using the procedures as described in this unit, is on the order of 10 2 U for titrimetry, 10H U for colorimetry, and 10 4 U for spectrophotometry (where U is the amount of enzyme required to yield 1 imol product per minute). The smallest amounts (volumes) of materials, including enzyme, are required for the spectrophotometric method, and progressively more material is required for the colorimetric and titrimetric methods. Unless a flow cell adapter is available, the spectrophotometric method is not suitable for analysis of particulate (immobilized) enzyme preparations, whereas the other assay procedures are. 

The choice of test equipment and methods has become extremely wide and, apart from large, integrated, electronic colorimetric and spectrophotometric instrumentation, field personnel can choose from miniburettes, direct-reading titrators (modified syringes), digital titrators, drop tests, tablet tests, permanent color standard comparators, indicator papers, portable colorimeters, immunoassays, etc. Today, field-test methods tend to be tailored by equipment manufacturers to their own analytical systems, and consequently the specified use of particular standard methods for the examination of water, from any one technology or official body, is probably not realistic. Rather it is the fitness-for-purpose rule that is more relevant. 

Spectrophotometric [colorimetric and gravimetric methods, e.g., with sodium diethylthiocarbamate (NaDTC)j, are still reliable and cheap, but neither very sensitive nor very specific methods for higher concentrations of copper in biological and environmental materials (Malvankar and Shinde 1991). 

Once in solution, the preferred method for measurement of boron is inductively coupled plasma atomic emission spectroscopy (ICP-AES) or inductively coupled plasma mass spectrometry (ICP-MS). The most widely used nonspectrophotometric method for analysis of boron is probably ICP-MS because it uses a small volume of sample, is fast, and can detect boron concentrations down to 0.15 pgL . When expensive ICP equipment is not available, colorimetric or spectrophotometric methods can be used. However, these methods are often subject to interference (e.g., nitrate, chloride, fluoride), and thus must be used with caution. Azomethine-H has been used to determine boron in environmental samples (Lopez et al. 1993), especially water samples. Another simple, sensitive spectrophotometric method uses Alizarin Red S (Garcia-Campana et al. 1992). 

Probably one of the most faseinating phenomena in science is the interactions of light, electromagnetic radiation, with matter. The essential nature of matter, the nature of the forces acting between the parts of atoms, between the atomic components of molecules, is revealed by such phenomena. It produces a cosmic arithmetic that relates the energy changes associated with changes in atomic and molecular structural states. These phenomena provide the basis for one of the most widely used methods of chemical analysis colorimetric and spectrophotometric analysis. 

Tannins, theaflavins, and thearubigens Tannins and theaflavins are pigmented products in black tea formed after oxidation and condensation of green tea flavanols during the fermentation process. These compounds are analyzed by spectrophotometric, colorimetric, and chromatographic methods, using LC and GC. 

In 1990, chromatographic methods accovmted for 95% of the tests used to assess purity in USP monographs. Spectrophotometric methods accounted for less than 4% of methods. The remaining 1% included titrimetric, nuclear magnetic resonance, colorimetric, and other methods. 

If there is enough substance available, the eluates of the scraped-off zones can be qualitatively and quantitatively examined by the usual spectrophotometric, colorimetric and fluorometric methods. 

Colorimetric. A sensitive method for the deterrnination of small concentrations of dissolved iron is the spectrophotometric deterrnination of the orange-red tris(1,10-phenanthroline)iron (IT) complex. Other substituted phenanthrolines can be even more sensitive. Only the inon(II) complexes of these Ligands are highly colored. The sample is first treated with an excess of reducing agent. The complexes are stable from pH 2 ndash 9 and analysis preferably is done at about pH 3.5. 

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. 

Only slightly less accurate ( 0.3—0.5%) and more versatile in scale are other titration techniques. Plutonium maybe oxidized in aqueous solution to PuO " 2 using AgO, and then reduced to Pu" " by a known excess of Fe", which is back-titrated with Ce" ". Pu" " may be titrated complexometricaHy with EDTA and a colorimetric indicator such as Arsenazo(I), even in the presence of a large excess of UO " 2- Solution spectrophotometry (Figs. 4 and 5) can be utilized if the plutonium oxidation state is known or controlled. The spectrophotometric method is very sensitive if a colored complex such as Arsenazo(III) is used. Analytically usehil absorption maxima and molar absorption coefficients ( s) are given in Table 10. Laser photoacoustic spectroscopy has been developed for both elemental analysis and speciation (oxidation state) at concentrations of lO " — 10 M (118). Chemical extraction can also be used to enhance this technique. 

Determination. Various classical techniques are used for the analysis of vanillin, including colorimetric, gravimetric, spectrophotometric, and chromatographic (tic, gc, and hplc) methods. The Food Chemical s Codex (FCC) prescribes infrared spectrophotometry for identifying and testing vanillin. However, more vanillin analyses are made by either gc or hplc. 

Tellurium and its compounds can be analysed by AA, ICP-AES and other spectrophotometric methods. Also, the metal can be identified by volumetric, gravimetric, and simple colorimetric measurements. 

The section Analysis starts with elemental composition of the compound. Thus the composition of any compound can be determined from its elemental analysis, particularly the metal content. For practically all metal salts, atomic absorption and emission spectrophotometric methods are favored in this text for measuring metal content. Also, some other instrumental techniques such as x-ray fluorescence, x-ray diffraction, and neutron activation analyses are suggested. Many refractory substances and also a number of salts can be characterized nondestructively by x-ray methods. Anions can be measured in aqueous solutions by ion chromatography, ion-selective electrodes, titration, and colorimetric reactions. Water of crystallization can be measured by simple gravimetry or thermogravimetric analysis. 

A sensitive spectrophotometric method based on the strong absorption of the aminochrome-sodium bisulfite addition products (see Section IV, F) at ca. 350 m/x. has been described recently by van Espen128and Oesterling and Tse 277-278 for determining total catecholamines. While not as sensitive as the fluorimetric procedures, this method is considerably more sensitive than the older colorimetric methods based on the visible absorption peak of the aminochromes. Also, it does not have many of the disadvantages (e.g. costly equipment and unstable blanks) often associated with fluorimetric techniques. The basic procedure can be satisfactorily applied to the differential determination of mixtures of adrenaline, noradrenaline, dopamine, metanephrine, and normetanephrine.178... 

Analytical methods employed in soil chemistry include the standard quantitative methods for the analysis of gases, solutions, and solids, including colorimetric, titrimetric, gravimetric, and instrumental methods. The flame emission spectrophotometric method is widely employed for potassium, sodium, calcium, and magnesium barium, copper and other elements are determined in cation exchange studies. Occasionally arc and spark spectrographic methods are employed. 

For the spectrophotometric method, there is no sample workup, allowing one to run -4 assays/hr. This can be increased to -16 to 100 or more samples/hr depending on equipment features and automation, such as multiple cuvette holders/changers and 96-well microplate readers. For the colorimetric procedure, sample workup requires -10 min/subsample, but several samples can be batch processed simulta-... 

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