UV-absorbing Derivatives

Department of Clinical Biochemistry, King s College School of Medicine and Dentistry, London 

MRC Toxicology Unit, Medical Research Council Laboratories, Woodmansteme Road, Carshalton, Surrey 

REAGENTS FOR AMINES AND AMINO 2. REAGENTS FOR CARBOXYLIC ACIDS 164 

2 Arylsulphonyl chlorides 160 after conversion into acyl chlorides 167 

Edited by K. Blau and ]. M. Halket 1993 John Wiley Sons Ltd 

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Derivatization of primary and secondary amines using 9-fluorenylmethyl chloroformate to form a nonpolar, uv-absorbing derivative has been reported (90,91). Amphetamine and catecholamine were used as probes to evaluate this procedure. The derivatives were well behaved and allowed separation in a short time. 

Supercritical fluid chromatography (sfc) combines the advantages of gc and hplc in that it allows the use of gc-type detectors when supercritical fluids are used instead of the solvents normally used in hplc. Carbon dioxide, n-petane, and ammonia are common supercritical fluids, For example, carbon dioxide employed at 7.38 MPa (72 9 atm) and 31.3°C has a density of 448 g/mL. Derivatization of primary and secondary amines using 9-fluorenylmethy] chlorofomiate to form a nonpolar, uv-absorbing derivative lias been reported. 

In liquid chromatography, derivatization for detection enhancement is frequently needed, since no universal, sensitive, and simple-to-operate detector exists (the preparation of UV-absorbing derivatives is essential to obtain the sensitivity required for samples in the nanogram range). Since most fatty acids do not absorb UV radiation (at least not in the wavelength ranges of most commercial UV monitors), detection of quantities in the 1-ng range can be difficult. Tag-... 

The post-column derivatization of amino acids by the ninhydrin technique is a well known method for routine analysis of amino acids [7-9]. The amino acids are usually separated by ion-exchange chromatography and then converted into UV-absorbing derivatives for quantitation. The ninhydrin reaction is often used for TLC detection of amino acids and proteins. 

The formation of highly UV-absorbing derivatives of estrogens with azobenzene-4-sulfonyl chloride [29] has been examined for analysis in biological extracts. The derivatives are separated by TLC arid are quantitated by direct densitometry of the chromatoplates. 

The use of benzoylation for the formation of UV-absorbing derivatives of non-UV absorbing hydroxy steroids has been reported for the analysis of these compounds by liquid chromatography [33]. This reaction may prove useful for the separation of steroids which cannot be separated as their DNPH derivatives. Molar absorptivities of many steroid benzoates are normally greater than 100001/mole cm at 230 nm. 

Shaikh, B., Pontzer, N. J., Molina, J. E., and Kelsey, M. I., Separation and detection of UV-absorbing derivatives of fecal bile acid metabolites by bigh-performance liquid chromatography. Anal. Biochem. 85, 47-55 (1978). 

Coloured and UV-absorbing derivatives are prepared for chromatography to improve the detectivity of compounds which do not possess a chromophore or fluorophore. 

Honda et al. [142] reported that l-phenyl-3-methyl-5-pyrazolone reacted with reducing carbohydrates aintost quantitatively to yield strongly UV-absorbing derivatives. This reaction differs from other derivatization reactions via the hydroxy group of carbohydrates, such as benzoylation or, p-bromobenzoylation, in that it gives no stereoisomers. This method is especially useful for the analysis of component monosaccharides of gly-... 

Precolumn derivatization of sugar alcohols with a chromophoric reagent, such as phenylisocyanate, benzoate, and 2,4-dinitrobenzoate allows the use of inexpensive UV detectors. p-Nitrobenzoyl chloride was found to be a rapid and quantitative derivatizing agent for sugar alcohols since strong UV-absorbing derivatives at 260 nm are produced. 

One advantage of the method is that any impurities not converted to UV-absorbing derivatives are not detected, so cannot obscure the separations. On the other hand, it is likely again that the principal use of such methods will be for small-scale preparative separations of single components for structural analysis or for liquid scintillation counting. The technique may also prove invaluable for determination of the fatty acid composition of seed oils containing thermally labile fatty acids, such as those with cyclopropene moieties [987]. GC is likely to remain the method of choice for analytical... 

A procedure for the release of the diacylglycerol moieties from galactosyidiacylglycerols (including sulfoquinovosyidiacylglycerols) has been described, involving periodic acid oxidation in methanol followed by incubation with 1,1-dimethylhydrazine [382]. The diacylglycerols are then converted to UV-absorbing derivatives for separation by means of HPLC [475], but equally it should be possible to prepare TMS or BDMS ethers and use the GC conditions developed for the analysis of the equivalent compounds released from phospholipids (see Section C above). 

By converting diacylglycerols prepared from phospholipids to UV-absorbing derivatives, it has proved possible to use the high sensitivity and specificity of spectrophotometric detection in the... 

TLC is rarely applied to determination of fatty acids, since HPLC and GC (usually of methyl esters) give more precise quantitative information. However, reversed-phase TLC is suitable for separation by acyl chain length, with the higher acids having shorter Rf values. Sensitive detection is a problem, but may be handled by making UV-absorbing derivatives, e.g., by tiiethylamine-catalyzed reaction with bromoacetophenone (32). 

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