Phenylisothiocyanate Derivatization

PTC-AAA typically identifies the sixteen common amino acids in peptide/protein hydro-lysates, including proline. The derivatization reaction is relatively tolerant to variations in pH and molar ratios of reagents to sample, but a slow, variable degradation of the derivatives can occur in solution at room temperature. The derivatization protocol may also be successfully performed with ethanol in place of methanol, and triethylamine (TEA) substituted for N,N-diisopropylethylamine (DIEA), but resulting reagent by-product peaks will differ slightly from those shown in Figure 11.9.1 (Bidlingmeyer et al., 1984; Tarr, 1986).

The Basic Protocol describes a procedure for manual PTC derivatization. Alternatively, automatic PTC derivatization with on-line HPLC analysis can be performed with instrumentation marketed by PE Applied Biosystems. This instrumentation provides savings in time and labor as well as reduction in variability due to time-dependent degradation of the PTC derivatives. Automatic on-line vapor-phase HCl hydrolysis can also be performed with the PE Applied Biosystems model 420H hydrolyzer/derivatizer. Detailed comparisons of the performance of automatic and manual hydrolysis/PTC derivatization methods and applications of the automatic PTC-AAA technology are available elsewhere (West and Crabb, 1990, 1992).

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