Gas Chromatography - Quantitative Analysis > Derivatization > Page 56

Amino acids are more difficult to derivatize but can also be esterified in a comparable 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. Any excess methanol is then removed by evaporation in a stream of nitrogen. Any remaining water is removed by adding a little dichloromethane (ca 150 ml) and repeating the evaporation process. The derivative is in the form of the hydrochloride and the free base must be liberated without causing the ester to be saponified.

Another useful catalyst for esterification is thionyl chloride but the thionyl chloride must be purified by distilling from linseed oil before use. 10 to 50 mg of acid is placed in a stoppered vial and 200 ml of dry methanol is added and cooled in a solid carbon dioxide-acetone bath. 20 ml of thionyl chloride is added with shaking and the vial is the warmed to 40˚C and maintained at that temperature for two hours. The solution is evaporated to dryness in a stream of nitrogen.

The Lewis acid boron trifluoride or the equivalent reagent boron trichloride is also very useful for forming ester derivatives. Boron trifluoride is supplied as a 14% solution in methanol. Boron trifluoride catalyzed reactions are very fast and can be complete in a few minutes. 1 to 15 mg of the acid are placed in a vial fitted with a ground glass stopper and 1 ml of 14% boron trifluoride in methanol added. The mixture is heated on a water bath for 2 minutes and then cooled. The esters can be extracted with n-heptane with vigorous shaking. Care must be taken to extract all the derivative.

The complimentary form of derivatization would be the esterification of an involatile alcohol. The normal reagent used for this purpose is an acid anhydride which also removes the water as the esterification proceeds. There is some competition between the alcohol and the water for the anhydride if the conditions are optimized, the anhydride reacts preferentially with the water. 10 to 100 mg of acid are placed in a stopped vial and 1 molar equivalent of the alcohol is added together with 1.2 to 1.4 equivalents of trifluoroacetic anhydride. The mixture is warmed and the reaction proceeds rapidly to completion in about 10 minutes.