Alcohols fluorescent derivatives

Reports on the use of fluorescent derivatives abound (5). Some reagents have become widely used. The dansyl group is probably the most thoroughly studied. Dansyl chloride has been widely used as a fluorescent derivatizing reagent for HPLC (6,7). It reacts readily with primary and secondary amino groups (7) and with phenols (8), but forms derivatives of alcohols very slowly (9). The lower detection limit for dansyl derivatives of aliphatic amines is in the range of 300 femtomoles per injection. 

This fluorescent acid chloride can be used to form derivatives of alcohols, amines, and phenols. Using these fluorescent derivatives, an analysis of a series of n-alcohols from Ci to C4 was developed. A chromatogram produced by this technique is shown in Figure 3. Derivatives were also formed from ammonia, dimethylamine, and phenol. A derivative was formed from pentachlorophenol but was not fully characterized. The quantum yields of fluorescence of the alcohol derivatives of V were lower than those of the alkyl halide derivatives of III. 

It is sometimes possible to improve detection by changing the pH of the eluent, or by the use of photochemical reactions. The common barbiturates used in therapy are weak acids that are easily separated in their acid (unionised) forms. Because the conjugate bases are much stronger chromophores than the acids, barbiturates have been detected by post-column mixing with a pH 10 borate buffer followed by uv absorption at 254 nm. An example of the second approach is the detection of cannabis derivatives in body fluids involving the conversion of cannabis alcohols to fluorescent derivatives on subjecting the column effluent to intense uv radiation. 

Used for the precolumn preparation of fluorescent derivatives of primary and secondary alcohols and phenols Reference 23... 

Analytical Methods.—Convenient new methods are described for the estimation of steroidal alcohols and ketones as fluorescent derivatives. 1-Dimethylamino-naphthalene-5-sulphonyl chloride [ dansyl chloride 550)] reacts with steroidal alcohols to provide fluorescent esters which are suitable for thin-layer chromatography.The corresponding sulphonyl hydrazine [ dansyl hydrazine (551)] provides similar fluorescent derivatives of steroidal ketones. A fluorescent... 

Alcohols may be converted into benzoate esters (Fitzpatrick and Siggia 1973) or into fluorescent derivatives by reaction with 9-fluorenylmethyl chloroformate (Miles and Moyne 1988). 

If a compound is nonfluorescent, it may be converted to a fluorescent derivative. For example, nonfluorescent steroids may be converted to fluorescent compounds by dehydration with concentrated sulfuric acid. These cyclic alcohols are converted to phenols. Similarly, dibasic acids, such as malic acid, may be reacted with j8-naphthol in concentrated sulfuric acid to form a fluorescing derivative. White and Argauer have developed fluorometric methods for many metals by forming chelates with organic compounds (see Ref. 23). Antibodies may be made to fluoresce by condensing them with fluorescein isocyanate, which reacts with the free amino groups of the proteins. NADH, the reduced form of nicotinamide adenine dmucleotide, fluoresces. It is a product or reactant (cofactor) in many enzyme reactions (see Chapter 24), and its fluorescence serves as the basis of the sensitive assay of enzymes and their substrates. Most amino acids do not fluoresce, but fluorescent derivatives are formed by reaction with dansyl chloride. 

Diazomethyl-7-methoxycoumarin is a rather stable reagent which allows convenient derivative formation carboxylic acids form esters by heating in acetonitrile. With alcohols, fluorescent ethers are obtained in dichlo-romethane in the presence of HBF4 as catalyst [477]. 

Analytes containing hydroxyl groups, such as phenols, glycols, and alcohols, can be converted with 3.5-dinitrobenzoyl chloride [276] or dabsyl chloride [277] - [279] into compounds that ab.sorb UV or visible light. Fluorescent derivatives can be obtained with 7-(chlorocarbonyl) methoxy-4-methylcoumarin [280]. 

Note The pre- and post-treatment of the chromatograms with the basic tri-ethylamine solution, which can be replaced by an alcoholic solution of sodium hydroxide [1,4] or a phosphate buffer solution pH = 8.0 (c = 0.2 mol/1) [5], serves to stabilize the fluorescence of the amino derivatives [2]. A final spraying with methanolic hydrochloric acid (chci = 5 mol/1) or 70% perchloric acid renders the detection reaction highly specific for histamine [4] and for catecholamines and indolamines [5]. 

The identification of anionic polyfacrylic acid) sizes can be carried out by staining with a fluorescent cationic dye (Cl Basic Orange 14) followed by spectroscopic measurement of excitation wavelength and fluorescence emission [195,196]. Such methods can also be used (with Cl Basic Orange 14 or Cl Basic Red 1) to detect and estimate carboxymethylcellulose, poly(vinyl alcohol) and starch derivatives [197]. 

One of the interesting features in the structure-photophysical property relationship of fluorescein is that the quantum yield of fluorescein increases under the basic condition. Therefore, many of fluorescein derivatives have been used as pH sensors to measure intracellular pH due to their pH-responding photophysical property [53]. Although fluorescein itself is slightly fluorescent in alcoholic solutions, the addition of alkali (pH > 8) to the fluorescein solution produces the very intense fluorescent alkali salt. The salt form of fluorescein... 

The second family ofxanthene dyes is fluorescein and its derivatives. Fluorescein itself is only slightly fluorescent in alcohol solution. In contrast, the alkali salt obtained by addition of alkali exhibits the well-known yellow-green fluorescence characteristic of the fluorescein dianion (uranin). Fluorescein and its derivatives, e.g. eosin Yand erythrosin Y, are known to be very sensitive to pH and can thus be used as pH fluorescent probes (see Chapter 10). 

Chlorine spray is used to identify peptides and amino acid derivatives. [231 A blue color is given by almost all compounds that contain NH groups. Alcohols and acids interfere with the test and should be removed prior to application of the spray. The test fails when dioxane is used to develop the plate or when certain fluorescent indicators are present. Several protocols have been described for this test.14 These protocols vary in the reagents that are used to generate the reactive chlorine and the iodine-starch reagent that is used for visualization. 

Change-transfer complexes of solute-alcohol stoichiometry 1 2 have been reported by Walker, Bednar, and Lumry3 for indole and certain methyl derivatives (M) in mixtures of associating solvents n-butanol and methanol (Q) with n-pentane these authors introduced the term exciplex to describe the emitter of the red-shifted structureless fluorescence band which increases in intensity with the alcohol content of the mixed solvent. The shift of the exciplex band to longer wavelengths as the solvent polarity is increased, described by Eq. (15), confirms the dipolar nature of the complex that must have the structure M+Q2. No emission corresponding to the 1 1 complex is observed in these systems which indicates (but does not prove) that the photo-association involves the alcohol dimer. The complex stoichiometry M+Q determined from (Eqs. 9, 10, and 12)... 

Riboflavin (vitamin B2 6.18) consists of an isoalloxazine ring linked to an alcohol derived from ribose. The ribose side chain of riboflavin can be modified by the formation of a phosphoester (forming flavin mononucleotide, FMN, 6.19). FMN can be joined to adenine monophosphate to form flavin adenine dinucleotide (FAD, 6.20). FMN and FAD act as co-enzymes by accepting or donating two hydrogen atoms and thus are involved in redox reactions. Flavoprotein enzymes are involved in many metabolic pathways. Riboflavin is a yellow-green fluorescent compound and, in addition to its role as a vitamin, it is responsible for the colour of milk serum (Chapter 11). 

Tetracycline antibiotics are closely related derivatives of the polycyclic naphtha-cenecarboxamide. They are amphoteric compounds with characteristic dissociation constants corresponding to the acidic hydroxyl group at position 3 (pK about 3.3), die dimethylamino group at position 4 (pK, about 7.5), and the hydroxyl group at position 12 (pK about 9.4). In aqueous solutions of pH 4-7, tetracyclines exist as dipolar ions, but as the pH increases to 8-9 marked dissociation of the dimethylamine cation occurs. They are soluble in acids, bases, and alcohols but are quite insoluble in organic solvents such as chloroform. Their ultraviolet spectra show strong absorption at around 270 and 360 nm in neutral and acidic solutions. Tetracyclines are readily transformed into fluorescent products in the presence of metal ions or under alkaline conditions. 

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