Dansyl derivatization

HPTLC analysis by dansyl derivatization [247] as opium in plant extract by TLC [565. p. 

As early as 1995, twelve dansyl-derivatized amino acids were resolved by... 

The dansyl derivatization has been extensively studied to label a- or 8-amino groups. DNS derivatives are formed within 2 min and are detected by either UV or fluorescence. A typical example of a separation of dansyl amino acids is illustrated in Fig. 3. 

Roos, R.W. Medwick, T. Application of dansyl derivatization to the high pressure liquid chromatographic identification of equine estrogens. J.ChromatogrSci., 1980, 18, 626-630... 

Tris(2,2 -bipyridyl)ruthenium(n) (Rulbyp) ) is used as the electrogenerated CL (ECL) reagent for the amino compounds in the LC-CL detection system. The CL reaction is supposed to proceed as follows initially, the oxidation of Rulbypls to Ru(byp) is performed at the electrode surface, and then Ru(byp) reacts with amines to emit light at 620 nm. The ECL intensity is proportional to the amount of amines, and the order of increasing CL intensities is tertiary > secondary > primary amines. Dansyl derivatization of primary, secondary, and tertiary amines are used to increase the sensitivity. DNS-Glu can be determined by ECL with the detection limit of 2 pmol. 

Source From Optimization of dansyl derivatization and chromatographic conditions in the determination of neuroactive amino acids of biological samples, in Clin. Chim. Acta. ... 

Lamshoeft M, Grobe N, Spiteller M (2011) Picomolar concentrations of morphine in human urine determined by dansyl derivatization and liquid chromatography-mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 879(13-14) 933-937... 

Grosvenor and Gray (1990) synthesized 2,4-dinitrophenylpyridium and used it as a colorimetric or fluorometric detection reagent for amino acids. Mueller and Eckert (1989) quantified glycine betaine in plants by use of two ion-exchange columns and silica gel TLC. Surgova et al. (1990) separated histamine from polyamines and determined it by spectrodensitometric TLC after dansyl derivatization. 

Righetti and co-workers [11] were one of the first to demonstrate the utility of classical isoelectric focusing for the chiral separation of small molecules in a slab gel configuration. In their system, dansylated amino acids were resolved enan-tiomerically through complexation with (i-cyclodextrin. Preferential complexation between the cyclodextrin and the derivatized amino acid induced as much as a 0.1 pH unit difference in the pK s of the dansyl group. 

An excellent discussion on derivatization techniques has been given by Lawrence (17) including a detailed discussion on pre-column derivatization (18) and post-column derivatization (19). Probably, the more popular procedures are those that produce fluorescing derivatives to improve detector sensitivity. One of the more commonly used reagents is dansyl chloride (20), 5-dimethylamino-naphthalene-1-sulphonyl chloride (sometimes called DNS-chloride or DNS-C1). The reagent reacts with phenols and primary and secondary amines under slightly basic conditions forming sulphonate esters or sulphonamides. 

Perhaps most encouraging in these discoveries was the observation that NDA/CN worked equally well for derivatization of dipeptides and higher homologues of the primary amino acid series. Again, a stable, fluorescent, isolatable derivative was obtained. One of the most important initial findings was the high fluorescence efficiency of the CBI adduct (12). Tables 1 and 2 list the efficiencies for a representative group of mono-, di-, and tripeptides and a limited comparison of the CBI efficiencies with the more traditional OPA (8) and dansyl (9) derivatives, respectively. 

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 is the most widely used of the derivatizing reagents. It forms derivatives with primary and secondary amines readily, less rapidly with phenols and imidazoles, and very slowly with alcohols. The reaction medium is usually an aqueous-organic sixture (e.g., 1 1 acetone-water) adjusted to a pH of 9.5-10. Dansyl chloride has two major application areas. It is used to determine small amounts of amines, amino acids and phenols, as... 

El, E2 El, E2, 17ot-E2, EE2, E3 Cleaning SPE and LC SBSE Centrifugation, lyophilization, grinding, sieving USE Cleanup SPE, derivatization with dansyl chloride, LLE with hexane, silica gel column chromatography... 

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],... 

Amines are another important group of analytes. Mellbin and Smith [72] compared three different fluorescent reagents, dansyl chloride, 4-chloro-7-nitrobenzo-1,2,5-oxadiazole, and o-phthaldialdehyde, for derivatization of alkylamines. The dansyl tag was found to be the most effective. Hamachi et al. [73] described the application of an HPLC-POCL method for determination of a fluorescent derivative of the synthetic peptide ebiratide. Another comparative study was done by Kwakman et al. [74], where naphthalene-2,3-dialdehyde and anthracene-2,3-dial-dehyde were evaluated as precolumn labeling agents for primary amines. The anthracene-2,3-dialdehyde derivatives were not stable, especially in the presence of hydrogen peroxide, and the POCL detection of these derivatives was therefore... 

Table 4 illustrates the use of the CAR technique to develop CL kinetic-based determinations for various analytes in different fields. As can be seen, the dynamic range, limit of detection, precision, and throughput (—80-100 samples/ h) are all quite good. All determinations are based on the use of the TCPO/ hydrogen peroxide system by exception, that for p-carboline alkaloids uses TCPO and DNPO. A comparison of the analytical figures of merit for these alkaloids reveals that DNPO results in better sensitivity and lower detection limits. However, it also leads to poorer precision as a result of its extremely fast reactions with the analytes. Finally, psychotropic indole derivatives with a chemical structure derived from tryptamines have also been determined, at very low concentrations, by CAR-CLS albeit following derivatization with dansyl chloride. 

Enhancements in the sensitivity with which amino acids containing a primary amine group can be determined have been achieved by derivatization. Chen and Sato [37] reported derivatization with divinyl-sulfone-reduced limits of detection by several orders of magnitude, while Lee and Nieman [38] reported derivatization with dansyl-chloride-reduced limits of detection by a factor of three. 

Figure 14 Representative fluorescence derivatization reagents used for PO-CL detection. DNS-CI, dansyl chloride DBD-F, 4-(A,A-dimethylaminosulphonyl-7-fluoro-2,l, 3-benzoxadiazole NDA, naphthalene-2,3-dicarboxaldehyde DNS-H, dansyl hydrazine DBD-H, 4-(iV,iV-dimethylaminosulphonyl-7-hydrazino-2,l,3-benzoxadiazole).

Most HPLC instruments monitor sample elution via ultraviolet (UV) light absorption, so the technique is most useful for molecules that absorb UV. Pure amino acids generally do not absorb UV therefore, they normally must be chemically derivatized (structurally altered) before HPLC analysis is possible. The need to derivatize increases the complexity of the methods. Examples of derivatizing agents include o-phthaldehyde, dansyl chloride, and phenylisothiocyanate. Peptides, proteins, amino acids cleaved from polypeptide chains, nucleotides, and nucleic acid fragments all absorb UV, so derivatization is not required for these molecules. 

A comparative study was made of the RP-HPLC analysis of free amino acids in physiological concentrations in biological fluids, with pre-column derivatization by one of the four major reagents o-phthalaldehyde (73) in the presence of 2-mercaptoethanol, 9-fluorenylmethyl chloroformate (90), dansyl chloride (92) and phenyl isothiocyanate (97, R = Ph) (these reagents are discussed separately below). Duration of the analysis was 13-40 min. Sensitivity with the latter reagent was inferior to the other three however, its use is convenient in clinical analysis, where sample availability is rarely a problem. The derivatives of 73 were unstable and required automatized derivatization lines. Only 92 allowed reliable quantation of cystine. All four HPLC methods compared favorably with the conventional ion-exchange amino acid analysis188. 

Nonchiral columns can be used with nonchiral derivatization for better detectability of the analytes, using chiral modifiers of the carrier solvent. For example, RP-HPLC resolution of dansylated (92) D,L-amino acids using L-phenylalanine-Cu(II) complex... 

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