Colorimetric and Fluorimetric Methods

One of the simplest methods of estimation of PolyPs in extracts is based on the assay of Pi, which is released from the PolyPs by hydrolysis with 1 M HC1 at 90 °C for 10 min. The Pi released under these conditions is defined as labile phosphorus . If the compounds containing organic labile phosphorus (i.e. nucleotide phosphates, sugar phosphates, etc.) were removed from the extracts by adsorption on Norit charcoal, the increase in Pj content after hydrolysis can be attributed to PolyP and pyrophosphate (PPi). Estimation of the PPj content (Mansurova, 1989) before hydrolysis may be needed in some cases for more precise calculations of the PolyP content. Pi may be determined by one of the well-known chemical methods (Fiske and Subarrow, 1925 Weil-Malerbe and Green, 1951). 

The method used for determination of PolyP, which is based on the Mn2+-induced quenching of the fluorescence of the calcium indicator Fura-2, has been described (Lorenz et al., 1997a). The effect of Mn2+ ions on the Fura-2 fluorescence is gradually removed in the presence of increasing PolyPs concentrations this allows the quantification of PolyPs isolated from tissues or cells. The described method has some advantages when compared with the conventional detection procedures based on the metachromatic effect. It can be applied to the determination of pyrophosphate, tripolyphosphate and other short-chain PolyPs not detectable by toluidine blue (Lorenz et al., 1997a). 

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Karam, H. El Kousy, N. Towakkol, M. Colorimetric and fluorimetric methods for the determination of some antihistaminics using acid dyes and charge transfer techniques. Anal. Lett. 1999,32, 79-96. 

Porstmann, B., Porstmann, T., Nugel, E., and Evers, U. (1985) Which of the commonly used marker enzymes gives the best results in colorimetric and fluorimetric enzyme immunoassays Horseradish peroxidase, alkaline phosphatase or P galactosidase. J. Immunol. Methods 79,27—37. 

There must be hundreds of visualization recipes, some of them highly ingenious, particularly where sequences of several colorimetric or fluorimetric methods have been elaborated to provide highly specific identification of certain compounds. These visualization methods have been gathered into various handbooks on paper and thin-layer chromatography [4, 11-14], and the wealth of applications they contain is far more abundant than can be included here. 

Those colorimetric methods developed at this time and found to be suitably sensitive and specific are the orcinol and resorcinol methods (see II. 1 below) and the periodic acid/thiobarbituric acid assay (see II. 2). These methods together with a recently introduced assay using methyl-3-benzothiazolinone-2-hydrazone and fluorimetric methods (II. 3 and II. 4, respectively) form the basis of currently used techniques. 

The methods of assay for aneurine in complex galenicals have been developed from the original colorimetric or fluorimetric methods necessary for the small concentrations present in natural sources microbiological methods can also be used for small amounts. For tablets and injection solutions where the amounts are greater Adamson and Handisyde have shown that a gravimetric technique is more accurate. Titration in non-aqueous solvents is also applicable (see p. 792). 

Snell, F.D. and Snell, C.T., Colorimetric Methods of Analysis, Including Photometric and Fluorimetric Methods, Vols I-IV, and various supplements up to Vol IV AAA, Van Nostrand Reinhold, (1919-1971). 

Assessing the resources available for method development should also be done before beginning a project. The resources available include not only HPLCs, detectors, and columns, but also tools for sample preparation, data capture and analysis software, trained analysts, and especially samples representative of the ultimate analyte matrix. Also, it should be considered whether a fast, secondary method of analysis can be used to optimize sample preparation steps. Often, a simple colorimetric or fluorimetric assay, without separation, can be used for this purpose. A preliminary estimate of the required assay throughput will help to guide selection of methods. 

The fluorimetric methods often offer improved specificity and sensitivity over colorimetric procedures and the quantitative assays for the aromatic amino acids tyrosine and phenylalanine illustrate this point. 

C. T. Yuen, P. R. N. Kind, R. G. Price, P. F. G. Praill, and A. C. Richardson, Colorimetric assay for N-acetyl-P-D-glucosaminidase (NAG) in pathological urine using the oo-nitrostyryl substrate the development of a kit and the comparison of the manual procedure with the automated fluorimetric method, Ann. Clin. Biochem., 21 (1984) 295-300. 

Several techniques have been developed for the determination of purine and pyrimidine derivatives in food sample and in particular for hypoxanthine quantification biosensors (220-223) and electrochemical methods making use of immobilized enzyme electrode (224 -227), electrochemical enzymatic-based HA methods (228,229), enzyme reaction with fluorimetric detection (230), radioimmunoassay (231), colorimetric methods (232), capillary electrophoresis (233), and TLC (234). Many HPLC methods have also been developed and are reported in Table 4 (235-247) the most recent ones are described next. 

Fluorimetric methods for the determination of amino acids are generally more sensitive than colorimetric methods. Fluorescamine (4-phenyl-spiro[furan-2(3H),l -phthalan]-3,3 -dione) and o-phthaldialdehyde (OPA) substances are used for protein analysis. Fluorescamine reacts with amino groups to form fluorophores that excite at 390 nm and emit at 475 nm (Weigele et al., 1972). Applications of fluorescamine include monitoring the hydrolysis of K-casein (Beeby, 1980 Pearce, 1979) and quantification of proteins, peptides, amino acids in extracts (Creamer et al., 1985). OPA produces fluorescence on reaction with 2-mercaptoethanol and primary amines, with strong absorption at 340 nm. Lemieux et al. (1990) claimed that this method was more accurate, convenient, and simple for estimating free amino acids than the TNBS, ninhydrin, or fluorescamine methods. 

Methods that utilized derivatives (DNP and 7-hydroxyquinoline) combined with colorimetric or fluorimetric detection were not specific for acrolein and consistently did not correlate with those obtained from bioassays. Certain direct methods of detection (nuclear magnetic resonance (NMR), fluorescence, and differential pulse polarography) gave the best correlation to the bioassay results (see discussion of analysis of environmental samples in Section 6.2). 

Pepsinogen. This precursor of the proteolytic enzyme pepsin is secreted by gastric parietal cells pepsinogen activities may be increased following peptic ulceration and by parasitic infections. The enzyme may be measured in plasma or gastric fluid using colorimetric, fluorimetric, or radioimmunometric methods and ELISA methods (Will et al. 1984 Ford et al. 1985 Tani et al. 1987 Lynch et al. 2004). 

More-specific methods are available for identifying and quantitating the typical, amino sugar component of heparin (and some heparan sulfate species), namely, 2-deoxy-2-sulfoamino-D-glucose. Most of these methods are based on conversion of these residues into 2,5-anhydro-o-mannose by deamination with nitrous acid (see Section VIII,2). The 2,5-anhydro-D-mannose residues may be determined either colorimetrically, " or fluorimetrically. ... 

Numerous techniques have been described to evaluate total tin on ashed or wet mineralized biological, food, or environmental media by colorimetric determination of tin(IV) with phenyl-fluorone [51-53], catechol violet [54-59], salicylaminothiophenol [60], hematoxylin [61], quercetin [62,63], dithizone [64], or dichloro-8-quinolinol [65]. In general, these analytical reagents are nonspecific and best suited to samples where tin values in materials are not lower than about 0.2 ppm. A better sensitivity may be obtained with fluorimetric methods using morin [66,67] or diacetylmonoxime nicotinylhydrazone [68]. 

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... 

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