INDIRECT C()I,()RIMETRIC ESTIMATION Ol CYSTEINE 309 o 600 l o X 400- -õ .::_ 200- ,,e-- ,i I I I I 2 3, 4 t I 5 Figure 2. Variation of the extinction with pH at 470 mft cystine being present. To make our method applicable to cysteine we had to study first the best conditions to ensure its oxidation with ferricyanide. It was found that the reaction takes place quantitatively in an alkaline medium (pH 10-11). Further we studied the absorption spectrum of the red color obtained at pH 1-2 when 0-dianisidine reacts with an excess of ferricyanic•e, and after reacting with cysteine our conclusion was that the presence of the products of oxidation does not modify the spectrum (Fig. 1). The same procedure carried out at pH 4 gave evidence that in the portion of the spectrum considered the extinction of the red color is always greater than the extinction of the bluish-green one. We examined the pH influence on the red color obtained (Fig. 2), its 500 0 0 400 X ..• 300 •zoo, ioo I I • I I I • 5 I0 20 3,0 40 50 120 150 rnJrt. Figure &--Variation with time of the extinction at pH 1.5 and 470 mu.

310 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS stability (Fig. 3) and the need to add zinc salts which after precipitating not only the ferrocyanide formed in the reaction but the traces that come as impurities with the added ferricyanide increase the oxidation potential of the latter and the sensitiveness of the reaction as well. Keeping in mind that in biochemical equilibria, as in every possible determination of cysteine, there is to be taken into account the presence of considerable amounts of cystine, we carried out a second lot of determina- tions with both compounds being present in a 50:1 cystine/cysteine concentration ratio. In such conditions we found that the extinctions obtained were identical with those read before the addition of cystine therefore it may be asserted that the extinction of the red color obtained does not depend on the presence of the products resulting from the oxidation of cysteine. 400 0 0 0 •00 = 200 '"0 • •oo o I I I I I 0.2 0.4 0.6 0.8 1.0 1.2 p.p.m. Figure 4.--Variation with cysteine concentration of the extinc- tion at pH 1.5 and 470 mia. The results obtained allow the application of a new method for the estimation of cysteine the procedure is easy even at concentrations as low as 0.8 •gm./ml. for which Beer's law is valid (Fig. 4). EXPERIMENTAl, Reagents (a) Use potassium ferricyanide, analytical grade, recrystallized many times from water and dried at 100øC. A solution is made containing 0.310 gin. of this salt per liter, which corresponds to 0.200 gin. of Fe(CN)•-a/1. (b) Cystine. 100 rag. are dissolved in one liter of water. (c) Cysteine. 100 rag. are dissolved in one liter of water. (d) Sulphuric acid, 2 N. (e) Zinc sulphate, 2 N. (f) o-Dianisidine 0.5 gin. of o-dianisidine is dissolved in 50 ml. of