T H E INFLUENCE OF T H E pH UPON T H E ULTRAVIOLET ABSORPTION SPECTRA OF CERTAIN CYCLIC COMPOUNDS BY WILHELM STEXSTROM AND MELVIN REINHARD*
Most cyclic compounds in water solutions give absorption bands in the ultraviolet. While studying this absorption for a mixture of amino acids we noticed that the absorption band shifted towards longer wave-lengths when the mixture was made alkaline1 and therefore, decided to see how the change was related to the pH for tyrosine and tryptophane. For this investigation we used a Hilger ultraviolet spectroscope size C and a Hilger sector photometer. Tryptophane did not show any marked change in the absorption curve whether it was made strongly acid with HC1 or strongly alkaline with NaOH. Tyrosine on the other hand, showed a decided shift of its absorption band when it was made alkaline with NaOH. (These observations are in accordance with the findings of P. A. Kober2.) One important difference between these two amino-acids is that tyrosine has a hydroxyl group in the benzene ring while tryptophane has no such hydroxyl group. In order to find out if it was this group that was responsible for the difference in behavior just mentioned, we examined phenylalanine which differs from tyrosine only by not having the hydroxyl-group in the benzene ring. This substance did not show any shift of the absorption band when it was made alkaline. On the other hand the simplest compound with such a hydroxylgroup, phenol, gave the shift when made alkaline. if
It therefore, seems reasonable to conclude that this shift is related to the hydroxyl group in the benzene ring. The curves for phenol before and after the shift are shown in Fig. I A and B, those for tyrosine in Fig. 2 A and B. "From the State Institute for the Study of blalignant Disease, Buffalo, N. Y., Burton T. Simpson, M.D., Director. 'Stenstrom and Reinhard: Ultraviolet absorption spectra of blood serum and certain Amino Acids (not yet published). J. BIOI.Chem. 25. Biol. Chem. 22, 440 (1915).
1478
WILHELM STENSTROM
AND MELVIN REINHARD
In order to answer the questions: when does this shift take place and does it take place suddenly for a certain alkalinity or gradually when the alkalinity is increased, it was necessary to determine the absorption for different concentrations of the hydrogen ions. The pH was measured with the colorimetric method with phenolsulphophtalein, phenolphthalein and tropaeolin 0 indicators1. It would require a very great number of exposures to plot the I
FIG. I
A . Phenol in water. p < H4 R. Phenol in water XaOH.pH 1 0 . 5
+
FIG.2 A. Tyrosine in water p H < 4. B.-Tyrosineinwater XaOH pH
+
12
7
complete curve for each step of pH and therefore, we decided to see how the wave-length changed a t the points a,b and c (see Figs. I and 2 ) when the extinction coefficient (E) was kept equal to 1350 for phenol, equal to 1600 for tyrosine and equal to 700 for resorcinol which we also examined in this way. Fig. 3 gives the relation between the wave-length (ordinate) and the pH for phenol, Fig. 4 for tyrosine and Fig. 5 for resorcinol. The shift is very marked at a and c whereas at b it is small and irregular. The irregularities may however, be due to errors of the measurements. The shift starts a t “a” a t a considerably lower value of pH than at c. For phenol it starts at “a” for about pH = 8 . 7 and the sharp incline is over at pH = 9. The curve still slopes slightly at least up to pH = 11. At c the sharp incline starts at pH = 9. It is of interest GO note that the dissociation constant for phenol is + about I . 3 X IO-'^. [HI [Phenol] = 1.3 X 1 0 - l ~[Phenol]. Thus the number of phenol ions must increase from about 1% to roYc of the phenol molecules while the pH changes from 8 t o 9. At pH = I O there ought to be about the same number of phenol ions as of phenol molecules and for higher pH the number of phenol molecules becomes smaller and smaller. If we assume that in Fig. I curve A is produced by the phenol molecules and curve B by the ‘Clark: “The Determination of Hydrogen Ions.”
INFLUENCE O F pH UPON
ULTPAVIOLET ABSORPTION
I479
phenol ions then the shift is explained. The curves in Fig. I have a sharp incline at a and a small change in wave-length changes the absorption greatly. Therefore, a small addition of ions will increase the absorption or (which is the same) will give the same absorption a t a longer wave-length than before. At c the extinction coefficient is only 50% greater for the ion than for the molecule a,nd consequently no change can be noticed there until the relative number of ions has gone up considerably. In order to explain the shift in the curve for tyrosine in a similar way we must assume that the dissociation starts at the carboxyl group and later on also takes place a t the hydroxyl group. The affinity constant for tyrosine is 4 X 1o-O while the shift starts first
PHfNOL
E=1350
FIG.3
FIG.4
a t pH = g (a) and ends at pH = I 1.5 (c). If T is the concentration of tyrosine molecules and ions ionized at the carboxyl group only and T" the con-
T T for pH = I 1.5 (Judging from the rela-
must be of centration of tyrosine ions ionized at the hydroxyl group then yJ
the order of I O for pH = 9 and 1/10 tion between the absorption curve and the concentration of phenol ions and phenol molecules.) Therefore, the second affinity constant for tyrosine ought to lie between I O - ~ O and IO-^^; nearer 1 0 - l ~ .
I n order to test this theory further we examined the absorption of metaand para-dihydroxybenzene (resorcinol and hydroquinone) paraoxybensoic acid and salicylic acid, paraoxybenzaldehyde and benzoic acid at different pH. All of these compounds gave a shift, when made alkaline enough except benzoic acid which does not contain the hydroxyl group (Hydroquinone was broken down.)
I 480
WILHELM STENSTROM
Resorcinol 3.6X10-l~ I3 2350 2601 2570 2670 2830 3040 840 840
Affinity Const. PH X at a
b C
Extinc. coeff.
AND MELVIN REINHARD
Hydroquinone
Paraoxybenzoic acid. 1.1 x 10-10 2.9 X IO-^ I3 I3 2 3go molecule 2190? 2242 2640 destroyed 2345 2540 2740 3010 30i0 3770 5800 5800 840 840 tf
:)!I H
H
H
HC
0
c0
H
Salicylic acid
I.OXIO-~ I X IO+
Affinity Const. PH
X at a b c Extinc. coeff.
Paraoxy benzaldehyde
I3
Off
Benzoic acid 6 X IO-^
2570
I3 2488 2870
3070 5100
3580 5100
I3 2535 2615 2800 800
H
If our assumption is correct then it should be possible to determine the hydrogen ion concentration between certain limits for solutions of compounds with hydroxyl groups in the benzene ring with help of such curves as are shown in Figs. 3, 4 and 5, and also to determine the affinity constants in respect to the hydroxyl group. A curve giving the shift for blood serum corresponding to the curve for tyrosine is reproduced in Fig. 6. It is of interest to notice that the shift starts first at pH = 12 in spite of the fact that it is probably the tyrosine
INFLUENCE O F pH UPON ULTRAVIOLET ABSORPTION
1481
constituent of the proteins which is responsible for this shift. It seems therefore, as if the tyrosine were joined in the proteins in such a way that the complete ionization a t the hydroxyl group cannot take place until a pH = 1 2 has been reached. Summary I. It has been shown that the absorption bands in the ultraviolet somewhere between z zoo and 3 600 A are dependent upon the hydrogen ion concentration for the following compounds : phenol, tyrosine, resorcinol, paraoxybenzoic acid, salicylic acid and paraoxybenzaldehyde in water solutions. It
I
IO PH ~€so/?c//voL € = 700 FIG. 5
FIG.
6
seemed to be independent of the pH within the experimental error for benzoic acid , phenylalanine and tryptophane in water solutions. The relation between the structure of the band and the hydrogen ion 2. concentration can be outlined briefly thus: The band will shift towards longer wave-lengths and increase in intensity when a certain alkalinity has been reached by adding YaOH (other alkalies seem to have the same effect) to the water solution of the compound. 3 . It seems to be compounds with a hydroxyl group in the benzene ring which show this shift. The affinity constant for this group in the compound seem to be related to the critical value of pH for the shift. 4. The relation between the pH and the wave-length for which a certain extinction coefficient is obtained has been determined and plotted for phenol, tyrosine and resorcinol (Figs. 3, 4 and 5.) 5 . The shift can be explained as the change from a curve characteristic of the molecule to one curve characteristic of the compound ionized at the carboxyl group. 6 . A curve showing the shift for blood serum has been plotted.
