In work described elsewhere methods have been developed for the determination of amines occurring in small amounts in body fluids . These methods depended on preparation and spectrophotometric estimation of dinitrophenyl (DNP) derivatives of the amines . In the present paper are described methods for the paper chromatographic separation of DNP-amines .
EXPERIMENTAL Preparation of dinitrophenyl derivatives of amines
For preparation of authentic specimens, the method of GRASSMANN, HORMAN AND ENDRES' was used, except for tryptamine . The results are summarised in Table I and Table II . DNP-tyramine (Table I) was recrystallised from its solution in ethanolchloroform . The DNP-derivatives of amines listed in Table II were extracted with ethyl acetate and all, except DNP-t4-mercaptoethylamine, were precipitated from solution in ethanol-chloroform . DNP-f3-mercaptoethylamine was precipitated from its solution in monochlorobenzene .
DNP-tryptamine was prepared as follows : 30 mg of tryptamine hydrochloride were mixed with 50 ml of NaHCO 3Na2CO 3 buffer (approx . pH to), and x ml of a solution of I-fluoro-2,4-dinitrobenzene (3 % v/v) . The mixture was heated at 6o °for 2o min and then 20 ml of o.2 N NaOH in 6o % dioxan were added . After heating for x h, the volume of the mixture was made to 2oo ml with water and DNP-tryptamine was extracted with chloroform . The chloroform extract was evaporated to dryness and the residue was dissolved in ethanol and crystallised .
The method of isolation of DNP-derivatives of amines in body fluids is described elsewhere .
Preparation of reversed phase papers (a) Silicone-treated papers . A strip of Whatman No . x paper (17 x 56 cm) was placed in a shallow tray (I8 x 6o cm), containing a 5 % solution of silicone fluid (M-S . 1107) in cyclohexane . The solution was allowed to flow over the paper, by gently