Treatment of Amino andor Hydroxy Groups

Reactive anhydrides listed in Table 1 are frequently employed for this purpose. Acetic anhydride, which has the additional advantage of reacting in aqueous media, has proved its usefulness for esterification of phenolic hydroxyl groups. However, its lower reactivity and the lower volatility of the (per)acetylated forms prevents its wider use.

Acylation of amino acid butyl or isobutyl esters with TFA or HFBA carried out at 100°C or more, usually in the presence of a solvent such as dichloro-methane after evaporation of the first esterification medium, is one of the best-established procedures of the 1960s and 1970s. All protein amino acids, including arginine, which is the most difficult amino acid to derivatize and determine by GC, have been successfully analysed and quantitated. Experience gained in the derivatization of amines, alcohols and acids is valuable for procedures for amino acids. A study of these approaches affords a basic knowledge of commonly occurring derivatization problems.

Acid halides have been employed for acylation less often. Except for PFB-Cl, which has even been used for acylation of amino acid isobutyl esters, the use of other acid chlorides is rather rare. However, surprisingly good results have been reported for cat-echolamines treated with HFB-Cl, which results in ready acylation of the phenolic, amino and alcoholic groups of analytes in an aqueous buffer. Unlike other chlorides, which are almost explosively reactive with water, HFB-Cl appears to be more stable towards hydrolysis. Many applications, aimed at the trace analysis of amines, phenols and alcohols with ECD by acylation with haloanhydrides are outside the

Table 2 Comparison of the reaction conditions for HFBA- and the ECF-induced esterification of carboxylic acids

HFBA-cataIyseda

ECF-cataIysedb

Medium Alcohol Reagent Base

Reaction time

Yield

Extraction

Chloroform

HFBA (six-fold volume) None Pyridine (25%)

30 min 4 min (boiling)

Not given

Chloroform versus aq. HCl and NaOH

Chloroform EtOH (1 %) ECF (1 %) Pyridine (2%) Few seconds > 90%

Hexane versus aq. NaHCO3

Water-acetonitrile (1 : 1) EtOH (30%) ECF (5%) Pyridine (8%)

aEdman DC and Brooks JB (1983) Gas-liquid chromatography—frequency pulse-modulated electron-capture detection in the diagnosis of infectious diseases. JournalofChromatography 274: 1-25.

bHusek P, Rijks JA, Leclercq PA and Cramers CA (1990) Fast esterification of fatty acids with alkyl chloroformates. Optimization and application in gas chromatography. Journal ofHigh Resolution Chromatography 13: 633-638.

scope of this article; for details the reader should consult the Blau and Halket book or the papers by Poole (see Further Reading).

The amino group is best derivatized in aqueous media with chloroformates. Catecholamines are treated with MCF; if the alcoholic OH group is present it is silylated or acetylated in addition. During this step the MCF-alkylated phenolic hydroxyls reconvert to O-TMS or O-acetyl esters and only the amino group retains the alkyl group from the MCF. On well-deactivated capillaries the analysis succeeds even without this extra treatment. When tertiary amines are treated with chloroformates, the smallest alkyl group attached to the nitrogen undergoes a displacement and the amine is thus transformed to a carba-mate. Hydrolysis of the latter yields a secondary amine, which is then exposed for further derivatiz-ation.

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