Reagents, for derivatization

There are potentially viable reagents available that may be employed for the derivatization of compounds either for enhancing UV/visible radiation (called chromatags) or for reaction of non-fluorescent reagent molecules (called fluorotags) with solutes to yield fluorescent derivatives. 

Ninhydrin (a chromatag is commonly employed to yield corresponding derivatives of amino acids that show absorption specifically at about 570 nm as shown in the following reaction  

Dansyl Chloride (afluorotag) is invariably used to obtain fluorescent derivatives of proteins, amines and phenolic compounds, the excitation and emission wavelengths being 335 to 365 nm and 520 nm respectively. 

Some other important Fluorotags are stated below  

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Suitable reagents for derivatizing specific functional groups are summarized in Table 8.21. Many of the reactions and reagents are the familiar ones used in qualitative analysis for the characterization of organic compounds by physical means. Alcohols are converted to esters by reaction with an acid chloride in the presence of a base catalyst (e.g., pyridine, tertiary amine, etc). If the alcohol is to be recovered after the separation, then a derivative which is fairly easy to hydrolyze, such as p-nltrophenylcarbonate, is convenient. If the sample contains labile groups, phenylurethane derivatives can be prepared under very mild reaction conditions. Alcohols in aqueous solution can be derivatized with 3,5-dinitrobenzoyl chloride. 

Several reagents for derivatization of carbonyl groups are also available. Dansylhydrazine reacts with carbonyl groups in the presence of trichloroacetic acid, yielding highly fluorescent dansylhydrazones that exhibit, however, limited stability. In addition, fluorescent hydrazones can be produced from carbonyl-containing compounds after their reaction with 4-hydrazino-2-oxa-l,3-diazole or... 

Early applications of thin-layer chromatography in corticosteroid analysis were mainly based on use of phenylhydrazine in sulfuric acid as a spray reagent for derivatization of the analytes (529, 530). Later applications involved use of other spray reagents including phosphoric acid in methanol, tetrazolium blue. 

Cytochrome c, TPCK-trypsin, methionine-enkephalin, and amino acid standards were obtained from Sigma Chemical Co., St. Louis MO. Trifluoroacetic acid was from Pierce Chemical Co., Rockford IL. Borate buffer and AQC (AccQ-Fluor reagent) for derivatization were from Waters, Milford, MA. Other chemicals were HPLC or analytical grade. 

Zhoul, F. X., Thome, J. M., and Krull, I. S. 1992. Silica based, solid phase reagents for derivatizations in chromatography. Trends Anal. Chem., 11 80-85. 

Fluorescein isothiocyanate isomer 1 (FITC), which provides good sensitivity for primary and secondary amines, was chosen as a reagent for derivatization of dimethylamine (DMA) and other... 

After obtaining a stable baseline, prepare the reagents for derivatization (see Section 2.2., items 4 and 5). 

The number of reagents for derivatization of amino compounds is larger than for any other class of compounds. Two of the most useful reagents for precolumn fluorescence labeling m liquid chromatography are dansyl chloride and o-phthaldialdehyde (OPA). 

Acetyl chloride is not a widely used acetylation reagent for derivatization because, ideally, a base has to be included to remove the hydrochloric acid formed. Two examples are given, one using vapour phase acetylation. 

Principal physicochemical and gas chromatographic properties of some reagents for derivatization of carbonyl compounds are summarized in Table 4. 

The literature indicates that t-butyldimethylsilylimida-zole is probably the most popular reagent for derivatizing steroids, and it is often used in conjunction with t-butyldi-methylchlorosilane. These reagents form derivatives under relatively mild conditions (room temperature, reaction... 

K. Shimada, T. Oe, M. Tanaka and T. Nambara, Sensitive ferrocene reagents for derivatization of amines for high-performance liquid chromatography with electrochemical detection, J. Chromatogr., 1989, 487, 247-255. 

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