Derivatization
Derivatization procedures are employed in both GC, LC and in TLC. In GC, derivatization is mostly carried out to render the solutes volatile so that they can be separated by a gaseous mobile phase. In LC, derivatization is mostly used to add UV chromaphores, fluorophores etc. to solute molecules to aid detection. Before LC had been developed to its present efficient level, sample derivatization was carried out merely to make gas chromatography amenable to the analysis of involatile and strongly polar materials As GC was the more accurate technique it became the preferred technique. However, when LC became as accurate and dependable as GC, volatile substances suitable for separation by GC, were then derivatized to make them detectable by LC. Unfortunately, it was found that LC requires difficult and expensive solvent disposal and as GC has no waste disposal problems, derivatization is now being used again to make solutes volatile and amenable to analysis by GC. Examples of highly polar, involatile materials that need derivatization are organic acids, amides, polyhydroxy compounds and amino acids etc. Such materials are either esterified, silanated or acetylated, but there are a number of different methods used for synthesizing each of the three classes of derivative.
Esterification
Acids can be esterfied by treating them with an alcohol in the presence of an inorganic acid to catalyze the reaction. Hydrochloric acid is the favored catalyst as it is sufficiently strong and can be removed easily. Sulfuric acid is less favored as it is difficult to remove and can cause charring. Other esterification catalysts are trifluoroacetic acid, dichloroacetic acid, benzene sulphonic acid, p-toluene sulphonic acids and suphuryl and thionyl chloride. A volatile acid such as hydrochloric acid or thionyl chloride is recommended. However, the derivative must not be too volatile, or it will be lost while removing the excess alcohol and the hydrochloric acid or thionyl chloride. One or two milligrams of the sample in a small vial are heated with 125 ml of either methanol or ethanol that contains 3M hydrochloric acid at 65°C for 35 minutes. The alcohol is removed with a stream of nitrogen leaving the residual ester. The derivatized ester must be sufficiently high boiling to be sure that none is lost in the alcohol removal.
Amino acids, although more difficult to derivatize than most simple acids, can also be esterified in a similar manner. A few milligrams of the amino acid mixture is mixed with 2 ml of 4M alcoholic methanol and heated at 70°C for 2 hours. The excess methanol is removed by evaporation in a stream of nitrogen. Entrained water can be removed by the addition of methylene dichloride (ca 150 ml) and then removing the solvent by evaporation. The derivative is the hydrochloride of the amino acid, and thus the free base must be released without saponifying the ester to facilitate separation on the GC column.
