Amino Acids

For amino acids, derivatization is indispensable for analysis by GC since they all exist in the zwitterion form. Some also contain other polar functionalities, including hydroxyl, thiol and imino groups. The different reactivities of these groups greatly complicate their concurrent derivatization. Silylation offers the best approach for a single-step attachment of the same tag to all these functional groups.

The most successful attempt to generate a single product is by silylation with MTBSTFA to form TBDMS derivatives. Reaction conditions (heating at 150°C for 2.5 h) were developed for the reproducible derivatization of amino acids in high yield. TMS derivatives of the common amino acids, except ar-ginine, can also be prepared with BSTFA under similar conditions.

An alternative method of derivatization of amino acids entails first esterification and then acylation to produce various N-acyl alkyl esters (Figure 2). The most widely used of these combinations is the

Figure 2 Separation of N, O-heptafuorobutyryl amino acid iso-butyl ester derivatives obtained from silkworm t-RNA after deacylation and analysed with FID. GC conditions: 25 m x 0.4 mm i.d. capillary column coated with 5% Chromosorb Rand 15% OV-101 SCOT column; carrier gas, hydrogen at a flow rate of 3mLmin~1; make-up gas, nitrogen at a flow rate of 30mLmin~1; hydrogen flow rate, 27mLmin~1; air flow rate, 350mLmin~1; temperatures: detector, 320°C; no inlet heater block; column, 80°C programmed at 4°Cmin~1. Pulse interval, 15 |is; attenuation, 2 x 102; sample size, 20 |L. Peaks: 1, Alanine; 2, glycine; 3, valine; 4, threonine; 5, serine; 6, leucine; 7, isoleucine; 8, norleucine (I.S.); 9, proline; 10, methionine; 11, aspartic; 12, glutamic acid; 13, lysine; 14, tyrosine; 15, arginine. (Reproduced with permission from Chauhan J and Darbre A (1982) Determination of amino acids by means of glass capillary gas-liquid chromatography with temperature-programmed electron-capture detection. Journal ofChromatography 236: 151.

N-trifluoroacetyl-«-butyl ester (TAB) derivative. Esterification is performed by one of the methods described above and acylation by heating the dried product with trifluoroacetic anhydride. The selectivity of the NP detector can be exploited to monitor amino acids in the presence of interfering matrices, particularly lipids.

Enantiomeric resolution has been achieved with a chiral aliphatic alcohol and an achiral acylating agent such as N-trifluoroacetyl chloride. Alternatively, the amino group has been converted to dia-stereomeric amides, ureas, thioureas and isoindoles.

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