D&C RED NO. 9 ANALYSIS AND PURIFICATION 279 i Fl©t©ntion tim© {minut©$} Figure 5. Gas chromatogram of chloroform extract from Red Lake C Amine. Column: 5% OV-17 on Uniport HP 80-100 mesh, 2 mm i.d. x 1 m 80-180øC (4øC/min.) Carrier gas: N2, 30 ml/min. Detector: NP-FID, 32 x 10 2. QUANTIFICATION OF UNSULFONATED AROMATIC AMINES IN RED LAKE C AMINE Three kinds of typical aromatic amines in Red Lake C Amine were quantitatively analyzed using the procedure shown in Figure 6. The analytical results are in Table IV. All six samples of Red Lake C Amine contained three aromatic amine impurities. Aniline (P-0A) was below 1 ppm in all samples, while 3-chloro-p-toluidine (P-12A) and 4-chloro-m-toluidine (P-15A) were present from 1.2 to 15.4 ppm. Some variation in the amounts of aromatic amines was observed between manufacturers and production batches. These results suggested that subsidiary colors contained in D&C Red No. 9 were due to aromatic amines in Red Lake C Amine used as the raw material for synthesis. 3. PURIFICATION OF RED LAKE C AMINE Red Lake C Amine was dissolved by heating in sodium hydroxide solution. Activated carbon ("Shirasagi," Takeda Pharmaceutical Co.) was added to the solution, and it was filtered. Hydrochloric acid was added to the flitrate to crystallize Red Lake C Amine which was separated by filtration. The filter cake of Red Lake C Amine was washed and dried. Each aromatic amine in the purified Red Lake C Amine produced by this procedure was below 0.5 ppm.

280 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS IRed Lake C Alaine1.00g I IWeigh exactly) •Dissolve with 2% aq. NaOH. 60ml on water bath Transfer the solution into 100ml separating funnel I Extract with 25ml chloroform (Repeat three times) I ! I IAqueous layer I IChloroform layer I I Dehydration through sodium sulfate anhydrous column • Add 1.00ml n-tetradeca Concentrate with rotary evaporator I IHP'C-vl Column: Nucleosil-50, 4mm i.d.x 25cm, Temp.: R.T. Eluent : 20% ethyl acetate/n-hexane, 2.0ml/min. Detector: (i)UV (254nm), 0.02 AUFS (•)FL (Ex----300nm. Eu----330nm), Sen.----3 Inj. vol. : 25/zl Figure 6. Flow chart for determination of aromatic amines in Red Lake C Amine. RELATIONSHIP BETWEEN AROMATIC AMINES IN RED LAKE C AMINE AND SUBSIDIARY COLORS IN D&C RED NO. 9 In order to understand the quantitative relation between aromatic amines in Red Lake C Amine and the subsidiary colors in D&C Red No. 9 and to determine the purity of Red Lake C Amine, D&C Red No. 9s were synthesized with purified Red Lake C Amine and with Red Lake C Amine to which 1, 5, 10, and 50 ppm of three kinds of Table IV Determination of Aromatic Amines in Commercial Red Lake C Amines (ppm) Samples P- 0A P-12A P-15A Manufacturer D 0.13 9.3 7.1 Manufacturer D 0.15 2.8 5.4 Manufacturer D 0.37 6.9 7.0 , Manufacturer E 0.15 15.4 102 Manufacturer F 0.19 9.5 15.3 Manufacturer G 0.27 1.6 1.2 P-OA' Aniline P-12A: 3-Chloro-p-toluidine P-15A: 4-Chloro-m-toluidine