Dimethylaminonaphthalene sulphonyl

Precolumn derivatization For separation by reversed-phase chromatography, precolmnn derivatization is a necessity. The derivatization step increases not only detectability of the analyte but also its hydrop-hobicity, which makes the separation of the amino acids by reversed-phase chromatography possible. Several precolmnn derivatization reagents are available. These include OPA, naphthalenedialdehyde (NDA), dimethylaminonaphthalene sulphonyl chloride (DANSYL), phenyl isothiocyanate (PITC), and N-(9-fluorenyl)methoxycarbonate (FMOC). 

Dansyl chloride (dimethylaminonaphthalene-5-sulphonyl chloride) will react with free amino groups in alkaline solution (pH 9.5-10.5) to form strongly fluorescent derivatives (Figure 10.14). This method can also be used in combination with chromatographic procedures for amino acid identification in a similar manner to the FDNB reagent but shows an approximately 100-fold increase in sensitivity. This makes it applicable to less than 1 nmol of material and more amenable for use with very small amounts of amino acids liberated after hydrolysis of peptides. The dansyl amino acids are also very resistant to hydrolysis and they can be located easily after chromatographic separation by viewing under an ultraviolet lamp see Procedure 10.1. 

Synthesis of tritium-labelled 1-N-dimethylaminonaphthalene-5-sulphonyl-d,l-coniine-3H (Dns-coniine 3H)... 

Rosier and Van Peteghem (1988) described a rapid method for the extraction, derivatization, and determination by HPLC of the 5-dimethylaminonaphthalene-l-sulphonyl (dansyl chloride) derivatives of putrescine, cadaverine, histamine, spermidine, and spermine from fish. Comparison of this procedure to earlier methods reflected considerable reduction in the time needed for sample preparation (from approx 8 to 0.5 hr) and cost (use of water and methanol instead of acetonitrile). 

Rosier, J., and Van Peteghem, C. (1988). A screening method for the simultaneous determination of putrescine, cadaverine, histamine, spermidine and spermine in fish by means of high pressure liquid chromatography of their 5-dimethylaminonaphthalene-l-sulphonyl derivatives. Zeitsch. Leb. Enters. Forsch. 186, 25-28. 

Dansyl chloride (DNS-Cl) (l-dimethylaminonaphthalene-5-sulphonyl-chloride). This reagent was originally introduced into protein chemistry for end-group analysis over twenty years ago (Gray and Hartley, 1963) and has been widely used because of the simplicity of the reaction and its ability to react with both primary and secondary amines, unlike OPA and fluram. Furthermore, in contrast to other fluorescent reagents, the dansyl derivatives are stable to acid hydrolysis, and can therefore be used in N-group labelling before hydrolysis. HPLC separations of dansyl derivatives have recently been published (Tapuhi et al., 1981). Sensitivity of detection is at the low picomole level. The sensitivity is limited because of the side-reactions which can occur with lysine and, to a lesser extent, histidine and tyrosine. 

Similarly, phosphohpids containing primary amino groups can be derivatized with DNS-Cl (l-dimethylaminonaphthalene-5-sulphonyl chloride). DNS derivatives are highly fluorescent and, using an excitation wavelength of 342 nm and emission wavelength of 500 nm, a lower limit of detection of about 20 pmol has been achieved (Chen et al., 1981). 

A very sensitive fluorimetric nethod (16) based on the reaction of clonidine hydrochloride with l-dimethylaminonaphthalene-5-sulphonyl chloride (dan-syl chloride) to give a highly fluorescent derivative. 

Dimethylamino-l-naphthylisothiocyanate [224], 4-(benzyloxycarbonylaminomethyl)phenylisothiocyanate [225] and 4-(dimethylaminonaphthalene-l-sulphonyl-amino)phenylisothiocyanate [226, 227] have been introduced in order to improve the sensitivity of the Edman method. These reagents are used in the same way as the other isothiocyanates, and allow end-group determinations in the picomole-range. 

The separation and quantitative determination of glucosamine and galacto-samine at the nanogramme level has been reported by prior reaction with a suitable sulphonyl chloride, followed by h.p.l.c. The use of toluene-p-sulphonyl chloride was more rapid (10 min) but less sensitive (0.4-50 /Ug) than that using 5-dimethylaminonaphthalene 1-sulphonyl chloride (0.02-2 jUg in 25 min). ... 

Improved methods for end group analysis have become available, notably the use of dansyl chloride (5-dimethylaminonaphthalene-l-sulphonyl chloride) for labelling iV-terminal amino acids and the Edman technique. Dansyl-amino acids fluoresce strongly in ultraviolet light and can be detected in very small amounts so that the method is much more sensitive than Sanger s original DNP technique. 

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