Acylation Reactions

Acylation is also used to form volatile derivatives of highly polar and involatile organic materials. In addition to improving volatility, acylation reduces the polarity of the substance and, thus, improves the quality of the separation by reducing peak tailing. As a result amide esters are usually well separated with symmetrical peaks. By inserting protecting groups into the molecule, acylation also improves the stability of those compounds that are inherently thermally labile. Acylation can also render extremely polar materials such as sugars amenable to separation by GC and are a practical alternative to the silyl derivatives. Acylation is used to provide derivatives of amines, amides, alcohols, thiols, phenols, enols, and glycols. A typical example of acylation is the reaction acetyl chloride with an alcohol.

R–CO–Cl + R'–O–H = R–CO–O–R' + HCl

The acid (ca 5 mg) is dissolved in 5 ml of chloroform together with 0.5 ml of acetic anhydride and 1 ml of acetic acid and held for 2-16 hours at 50°C. The excess reagents are removed under vacuum and the residue dissolved in chloroform and injected directly onto the column. Sodium acetate can be used as an alternative to acetic acid (i.e. 0.3 ml of acetic anhydride and 12 mg of sodium acetate) Derivatization is carried out at 100°C for about an hour, excess reagent is removed by evaporation and the residue taken up in a suitable solvent for analysis. A reagent that might appear useful is acetyl chloride but, the removal of the hydrochloric acid, formed during acylation, is considered a problem. Anhydrides are usually employed for acylation unless special circumstances demand alternative reagents. An excellent handbook, describing a wide range of procedures used to produce derivatives for chromatographic analysis, has been compiled by Blau and Halket (15).

In LC, derivatization is largely used to improve the detection limits of a specific substance. There a number of reagents that link specific chemical groups to the solute molecule, e.g., UV chromophores or fluorophores. Each reagent class comprises groups based on the chemical nature of the solutes with which they react (e.g. fluorescent reagents that derivatize acids, alcohols, amino acids, etc.).

Two UV absorbing reagents used to derivatize amino groups are benzoyl chloride and m–toluoyl chloride. Both reagents add a benzene ring to the molecule that contributes a strong UV absorbing chromaphore. The p–nitrobenzoyl chloride reagent imparts an even stronger UV chromaphore than the simple aromatic ring and may be preferred for this reason. The reaction is fairly rapid and can take place at room temperature. Pyridine is usually employed to scavenge the hydrochloric acid that is released. Similar reagents are the toluene-sulphonyl chloride and the benzene sulphonyl chloride, which both produce sulfonamides with the amines. As an example, gentamicin, a polyfunctional amino compound, can be analyzed by reverse phase chromatography after labeling with benzene sulphonyl chloride, the reaction is completed in 10 minutes at 75°C. 1-Fluoro-2,4-dinitrobenzene is also used for derivatizing amino compounds (e.g. the amino glycosides such as neomycin, fortimicin, amikacin, tobramicin, gentamicin and sissomicin).