B.—CHEMISTRY 53 
ANTHOCYANINS AS INDICATORS AND THE CAUSES OF VARIATIONS OF 
: CoLours OF FLOWERS 
(With Mrs. G. M. Rosinson). 
The amphoteric character of the anthocyanins accounts for the exhibi- 
tion of a wide variety of colours in a range of solutions of graded pH, and 
this method (demonstration), using buffered solutions, can be employed 
for the characterisation of anthocyanidins and anthocyanins. Under the 
specified conditions the results are fully reproducible and the pH values 
have been controlled by electrical methods as well as by the use of indi- 
cators. Thus, if the pH of an acid cyanin solution is increased until the 
violet tone matches that of an alkaline cyanin solution, the pH of which 
is decreased in order to reach the same condition, then the pH of the violet 
solution will be found to be 7:0-9-0, depending on the shade of violet 
produced. Cyanin is red in solutions of pH 3-0 or less, violet at pH 8°5 
and blue at pH 11-0. The red, violet and blue forms are the oxonium salt 
(XXIV), the colour-base (XXVI) and the salt of the colour-base (X XVII). 
HOA Ov, at HOO yy io La PBe 
c=¢7)=0 e=¢ =0 
| tal ee | 
C-0-C,H,,0; C-0-C,H,0; 
/ ay 
C.H,,0,0 & C,H,,0;"0 
H 
(XXVI) (XXVII) 
(There is no evidence in regard to the assumed position of the quinonoid 
group and the acidic hydroxyl.) 
Now cyanin was isolated by Willstatter and his colleagues from the 
blue cornflower and from the red rose, and it seemed quite a simple step 
to assume that the cell-sap in the cornflower was alkaline and that in the 
rose acid, particularly in view of the fact that the absorption spectra of the 
coloured aqueous extracts correspond with these conditions. 
It has indeed been generally assumed that the indicator colour of the 
anthocyanin will give a measure of the pH of the cell-sap, but unfortunately 
this method cannot be relied upon for several reasons. In the first place 
there is a glaring anomaly in the fact that direct measurement by electrical 
methods (glass electrode as arranged by Mrs. Kerridge) shows that the 
cell-saps are all well on the acid side of the neutral point. Thus the 
conventional view for red flowers may well be correct, but some special 
circumstances must be invoked in the case of blue flowers. 
Turning at once to the blue cornflower (the cultivated annual kind), 
a blue filtered extract made with distilled water was found to be sufficiently 
acid to turn blue litmus red. Using 3 g. of petals in 14 c.c. of distilled 
water (pH 6-3 owing to dissolved CO,), the pH was 4:9. (These 
quantities were used throughout the experiments and the use of larger 
relative quantities of the petals did not alter the pH appreciably.) Addi- 
tion of a buffered solution of pH 4-4 did not affect the colour, but the 
colour changed to violet when the B.D.H. Universal Buffer, pH 9:0, 
was added. It was at once apparent that the only simple explanation is 
