Dansyl derivatives

Acids can also be converted to fluorescent dansyl derivatives The reaction of Cg to C24 fatty acids with dansyl semipiperazide or semicadavende provides an excellent example (Fig 34) [87] Odd-numbered and unsaturated fatty acids [88] and propionic, sorbic and benzoic acid [89] can be detected in the same manner... 

Liquid paraffin estnol as dansyl derivative 10-fold dipping solution, 67% m n-hexane [247]... 

Liquid paraffin morphine as dansyl derivative stabilization dipping solution, 20 /o m n-hexane [291]... 

A CSP with a smaller (i-cyclodextrin moiety (seven glucose units) immobilized on silica gel (ChiraDex ) is able to separate the dansyl-derivatives [5-(dimethy-lamino)-naphthalin-l-sulfonylchloride] of amino acids [26]. 

If reaction time is kept short, however, the derivatization process can be intercepted at the mono-adduct form, which is sufficiently fluorescent for assay purposes. It should be noted that the fluorescence efficiencies of the CBI adducts are relatively insensitive to the water content of the solvent mixture (11,12) in contrast with earlier reports on the dansyl derivatives, which lose an order of magnitude of efficiency in aqueous-based solvent systems(9). 

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. 

Dansyl chloride does have some drawbacks when used with reverse-phase HPLC. One of these is that the quantum yield of fluorescence varies greatly with the polarity of the solvent for dansyl derivatives (10,11). As the polarity of the solvent increases, the... 

The fluorescent signal will change with variation in quantum yield of fluorescence and with molar absorptivity. Not only do fluorescence quantum yields vary with the different dansyl derivatives formed, but so do the molar absorptivities (12). Another problem is exemplified by the 30-nm difference in the emission maxima of the dansyl derivatives of phenol and 2,4,5-trichlorophenol (13). 

It would be convenient if the quantum yields and molar absorptivities were constant within a set of derivatives. Finally, the fluorescent signal from dansyl derivatives decreases in acidic solution (13) due to protonation of the dimethylamino group. 

The dansyl derivative 9-azidononyl-5-(dimethylamino)naphthalene-l-sulfonate 35 was used by Yi and collaborators [91] as an azido-fluorescent label in a tandem method of sulfonium alkylation and click chemistry for the modification of biomolecules. Fluorescent labeling of a protein was successfully carried out after simple incubation of BSA with sulfonium salt 36 followed by azido-containing fluorophore 35, at room temperature. 

The amino acid analyser using fluorescamine as the detecting reagent has been used to measure 250 picomoles of individual amino acids routinely [262], and dansyl derivatives have been detected fluorometrically at the 10 15 M level [260]. Where the amounts of amino acid are high enough, the fluorescamine method, with no concentration step, can be recommended for its simplicity. At lower concentrations, the dansyl method, with an extraction of the fluorescent derivatives into a non-polar solvent, should be more sensitive and less subject to interferences. For proteins and peptides, the fluorescamine method seems to be the most sensitive available method. 

Kwakman et al. [65] described the synthesis of a new dansyl derivative for carboxylic acids. The label, N- (bromoacetyl)-A -[5-(dimethylamino)naphthalene-l-sulfonyl]-piperazine, reacted with both aliphatic and aromatic carboxylic acids in less than 30 min. Excess reagent was converted to a relatively polar compound and subsequently separated from the derivatives on a silica cartridge. A separation of carboxylic acid enantiomers was performed after labeling with either of three chiral labels and the applicability of the method was demonstrated by determinations of racemic ibuprofen in rat plasma and human urine [66], Other examples of labels used to derivatize carboxylic acids are 3-aminoperylene [67], various coumarin compounds [68], 9-anthracenemethanol [69], 6,7-dimethoxy-l-methyl-2(lH)-quinoxalinone-3-propionylcarboxylic acid hydrazide (quinoxalinone) [70], and a quinolizinocoumarin derivative termed Lumarin 4 [71],... 

Procedure 10.1 Preparation of dansyl derivatives of amino acids for sub e quest separation by thin layer chromatography... 

The use of dansyl derivatives is not recommended for routine analysis of free amino acids but is very suitable in the identification of an unknown amino acid that has been selectively extracted from the original sample and is present in small quantities. Both kinds of derivative can be easily separated by chromatography or electrophesis and no locating reagent is required for either because the DNP derivatives are themselves yellow in colour and the dansyl derivatives are fluorescent. 

Others have reported the use of fluorescent dansyl derivatives for the identification of 17a-oestradiol from serum samples (O Figure 2-2) (Nozaki et al., 1988). The derivatization of the keto functional group of particular steroids (ketosteroids) can also be achieved by hydrazone formation (O Figure 2-3) the most... 

Carboxylic acids, dansyl derivates of amino acids Aminopropyl derivate of the ergot alkaloid (+ )-terguride Methanol—acetate, 205 pH range 3.8—5.5... 

V- Much effort has been expended in the development of more sensitive methods for the analysis and detection of catecholamines. They have been analyzed as the dansyl derivatives (376) or after precolumn derivati-zation with o-phthalaldehyde (377, 378). Postcolumn derivatization followed by fluorometric analysis have been described in which the fluoro-phore was formed with o-phthalaldehyde (379) or with 9,10-dimethoxyanthracene-2-sulfonate as the ion-pair (380). Several laboratories have shown the sensitivity and specificity in electrochemical detection methods (381 -383). 

These techniques have been used successfully in the micro-Zdman degradation of the enzyme mouse sarcoma dihydrofolate reductase to obtain the amino acid sequence of the first 25 amino acids 455). Similarly, RPC has been used in coqjunction with the automated Edman technique for sequencing 32 residues of myoglobin 456). Methionine and its oxidation products, methionine sulfoxide and methionine sulfone, in methionine fortified foods have been analyzed as their dansyl derivatives 457). Lysine has been determined as its dansyl derivative in a study in which the stability of lysine in fortified wheat flour was evaluated (458). 

Pyrroline (12i), found in the desert locust and the Mediterranean fruit fly (Table II), has been identified by formation of the I-dimethylaminonaph-thalene-5-sulfonyl (dansyl) derivative (106) or the colored adduct with o-ami-nobenzaldehyde, in addition to gas chromatography-mass spectrometry (102). The 1-pyrrolines (12) have been prepared by treatment of their parent pyr-... 

Nelson et al. [60] published a method for estrone and estradiol measurement using the dansyl derivatives with analysis by APCI. After adding di-estrone and ck-estradiol to 0.5 ml serum, the steroids are extracted with 6 ml methylene chloride. After drying the solvent, 50 pi of sodium bicarbonate and (100 mmol/1, pH 10.5) 50 pi of dansyl chloride (1 g/1) are added. The samples can be injected after heating at 60 C for 1 min. 

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. 

Draw a picture in your notebook of the polyamide thin-layer plate exposed under UV light after each of the two or three solvent developments. These pictures should look similar to Figure E2.7. Three fluorescent areas should be evident after solvent 2 however, better separation is achieved by solvent 3. A blue fluorescent area at the bottom of the plate is dansic acid (DNS-OH), which is a hydrolysis product of dansyl chloride. A blue-green fluorescent spot about one-third to one-half up the plate is dansyl amide (DNS-NH2), which is produced by reaction of dansyl chloride with ammonia. A third spot, which usually fluoresces green, is the dansyl derivative of the NH2-terminus amino acid. Note the positions of the standard dansyl amino acids and compare with the unknown. What is the identity of the NH2-terminal amino acid Are any other fluorescent spots evident on the plate Using polarity or nonpolarity, try to explain the position of each molecule on the thin-layer plate. 

Dansyl chloride and phenylisothiocyanate (PITC) are the derivatizating agents most used in UV detection. Dansyl chloride reacts with the primary and secondary amino groups of peptides in a basic medium (pH 9.5), forming dansylated derivatives that are very stable to hydrolysis but are photosensitive. The derivatives are detectable in UV at 254 nm and by fluorescence. Dansyl sulfonic acid is formed as a by-product of the reaction, and excess reagent reacts with the dansyl derivatives to form dansyl amide the conditions of derivatization must therefore be optimized in order to avoid the formation of such by-products to the extent possible. The conditions of the reaction with dansyl chloride and of the separation of the derivatives thus formed have been thoroughly studied (83,84). Martin et al. (85) carried out derivatization using an excess concentration of dansyl chloride of 5 -10-fold in a basic medium (lithium carbonate, pH 9.5) in darkness for 1 h. 

Hwang et al. developed a rapid and sensitive HPLC-UV method for the analysis of nine derivatized BAs, with benzoyl chloride as the derivatization agent. The reaction is faster than with tosyl chloride and leads to stable products with shorter elution times than do dansyl derivatives. The amines were previously extracted after acidification with TCA. The method was applied to detect BAs in fried marlin fillet, implicated in a food poisoning incident (in Taipei City in 1996) and indicated that a high level of His (84.1 mg/100 g) was present in the sample (80). 

Fig. 2 Reversed-phase HPLC chromatogram of the dansyl derivatives of amines from a sample of spoiled table olives. Peak identities 1, putrescine 2, cadaverine 3, 1,7-diaminoheptane (IS) 4, tyramine. (From Ref. 19.)...

The application of fluorogenic labeling to the determination of some organophosphate insecticides has been attempted [178,179]. Fenthion (0,0-dimethyl 0-[(4-metiiylthio)-m-tolyl] phosphorothioate), Ruelene (0-2-chloro-4-ferf.-butylphenyl O-methyl methyl-phosphoramidate), GC 6506 [dimethyl p-(methylthio)phenyl phosphate] and several other compounds which yield phenols on hydrolysis have been examined. The limits of detection for some of these labeled derivatives have been reported to be in the low nanogram range. The organophosphate Proban [0,0-dimethyl 0-(p-sulphamoylphenyl) phosphorothioate] has been determined directly without hydrolysis by dansylation of the free amino group of the molecule [180]. The derivative exhibited blue fluorescence, as compared to yellow for phenol and alkylamine dansyl derivatives. 

Tag Name Molecular Weight Formula Mol. Wt. of Dansyl Derivative... 

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