Phenolic groups, detection

Chemical attachment of a detectable component to an oligonucleotide forms the basis for constructing a sensitive hybridization reagent. Unfortunately, the methods developed to crosslink or label other biological molecules such as proteins do not always apply to nucleic acids. The major reactive sites on proteins involve primary amines, sulfhydryls, carboxylates, or phenolates— groups that are relatively easy to derivatize. RNA and DNA contain none of these functionalities. 

The Folin-Ciocalteu assay is the most widely used method to determine the total content of food phenolics (Fleck and others 2008). Folin-Ciocalteu reagent is not specific and detects all phenolic groups found in extracts, including those found in extractable proteins. A disadvantage of this assay is the interference of reducing substances, such as ascorbic acid (Singleton and others 1999). The content of phenolics is expressed as gallic acid or catechin equivalents. 

An interesting effect of Ca2+ ions on the autoxidation of hydroxy-1,4-naphthoquinone, which may have a bactericidal function in sea urchins, has been reported. Autoxidation resulted in the detection of a semiquinone, which was accompanied by only minimal oxygen uptake. The presence of Ca2+ resulted in a massive enhancement in the rate of 02 uptake. Potentiometric titrations revealed that Ca2+ ions, by forming complexes with the compound, lowered the pKa values of its phenolic groups, thereby generating the oxidation-prone phenolate anions. Autoxidation was also facilitated by the spin stabilisation effect of the Ca2+ ions on the naphthosemiquinone.121 This effect may be of importance in mammalian cells, where quinones induce elevations in the free Ca2+ concentration. 

This type of derivatization seems promising for the detection of drugs and metabolites with phenolic groups. A recent application on the determination of chlorophenols in surface water showed that the dansyl derivatives of phenols are readily convertible to the highly fluorescent dansyl-OH and dansyl-OCHs products after postchromatographic irradiation (281). Fluorescence gain factors of up to 8000-fold were obtained for chlorophenol derivatives with a low native fluorescence. 

High-performance LC is also a suitable method for separating BHA isomers. Commercially available BHA is a mixture of two positional isomers, an approximately 85 15 ratio of 3-BHA and 2-BHA. The former is approximately 2.4 times more effective as a food antioxidant than is 2-BHA, and half as effective as an inhibitor of benzo(a)pyrene-induced for stomach neoplasia in mice. For the separation, Ansari (116) used isocratic elution with 7% of 2-propanol in hexane on a Pirkle Type I-A column packed with 5- 01 y-aminopropyl packing, modified with lV-3,5-dinitrobenzoyl derivative of D-phenylglycine. Column eluent was monitored at 288 nm, with a detection limit between 1 and 2 ng. Under these conditions, isomers were separated without derivatization, where the phenolic group of 3-BHA was sterically hindered by an o-rm-butyl group and therefore could not interact with stationary phase that resulted in its rapid elution. 

When an inorganic phosphate buffer or sodium hydroxide is present, without imidazole, only weakly acidic phenol of pKa 11.6 can be detected. Since the phenolic group is adjacent to anionic sulfonates of the AOT, ionization is weakened. / -Nitrophenol in the water pool is influenced primarily by electrostatic interaction between molecular imodazole (Im) and the anionic surfactant group. When excess imidazole (but not methanol or n-butanol) is added to the water pool, a fraction of the interfacial /mitrophenol is displaced into the water pool. 

If any salicylic acid remains unreacted, its presence can be detected with a 1% iron(III) chloride solution. Salicylic acid has a phenol group in the molecule. The iron(III) chloride gives a violet color with any molecule possessing a phenol group (see Experiment 30). Notice the aspirin no longer has the phenol group. Thus a pure sample of aspirin will not give a purple color with 1% iron(III) chloride solution. 

The compound l-fluoro-2,4-dinitrobenzene (FDNB) reacts with free amino, imidazole, and phenolic groups at neutral to alkaline pH to yield the corresponding, colored dinitrophenyl (DNP) compounds. Thus, FDNB will react with the free, unprotonated a-amino groups on amino acids, as well as with the side chains of lysine, histidine, and tyrosine (Fig. 6-1). Dansyl chloride is another compound that is known to react with the unprotonated, N-terminal amino groups of peptides. De-rivatization of peptides with this compound yields fluorescent products that provide a very sensitive method of detection of the amino acid derivatives (Fig. 6-2). 

Imprinted polyurethanes as coatings for optrodes have been used for the detection of solvent vapours in air (see chapter 20). One per cent of substituted 3,3-diphenylphthalide as indicator has been intercalated within the polymer. The phthalide forms a highly coloured planar carbenium ion by interaction with an acidic component and a subsequent cleavage of the lactone ring - the unreacted phenolic groups in a polyurethane provide enough acidity for this reaction (Fig. 21.4). The incorporation of analytes reduces the acidity and the back-reaction... 

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