Methods of Biochemical Analysis, Volume 16 by David (Editor); Whitehead, J. K.; Strehler, Bernard L.;

By David (Editor); Whitehead, J. K.; Strehler, Bernard L.; Schwartz, Morton Glick


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Partial reoxidization of the tritiated material removes labile tritium. This operation enables the derivative to be extracted and purified more easily. Location and counting is also simplified. h. 2,4-Dinitrofl~orobenzene-~H and -14C (DNFB) The application of this reagent to derivative analysis wm first described by Beale and Whitehead (10) for the determination of amino acids in protein hydrolysates and for the estimation of N-terminal amino acids of peptides. These authors (10) describe the preparation of both the 14C- and 3H-reagents, the former by nitration of chlorobenzene-14C followed by halogen exchange with potassium fluoride, and the latter from bromobenzene by a similar method.

The GLC separation was effected on a 6 ft X % in. coiled-glass column packed with 2% SE30 on 30-60 mesh Chromosorb-W. The other conditions were: flash hgater, 280°C; column, 245°C; cell, 260°C; argon carrier gas 20 psi, flow rate 50 ml/min. The gas chromatograph was coupled to an automatic fraction collector in which the column effluent corresponding to the aldosterone peak was condensed on p-terphenyl crystals which were then dissolved in a liquid scintillator and counted (see Table 111, no. 20).

In order to attain the necessary purity of a derivative in the most efficient way, it is often necessary to modify the molecule chemically. The introduction of a new physical property will often shorten the number of chromatographic steps necessary to obtain the radiochemically pure derivative. ISOTOPE DERIVATIVE METHOD IN BIOCHEMICAL ANALYSIS 35 (2) Second Derivative. Steroid thiosemicarbazones can be acetylated to the 2,4-diacetyl derivatives (133). These compounds are more polar than the nonacetylated thiosemicarbazones, which in turn chromatograph more slowly than the parent steroids.

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