414
E.. B. HUGHES, H. H. G. JELLINEK, AND B. A . AMBROSE
The authors wish to express their thanks to Dr. L. H. Lampitt and Mr. F. R. Jones, M.Sc., for their interest in this work and to the directors of J. Lyons and Company, Ltd., for permission t o publish. REFEREXCES (1) FLEXYER, HAMMET~P, A N D DINGWALL: J . Am. Chem. SOC.57, 2103 (1935). (2) MARCHLEWSHI AND WYROBEH: Bull. Acad. Polonaise 1929A, 93; 1934A, 22. (3) SPIERS. ~ N DWIBAUT:Rec. trav. chin]. 66, 573 (1937).
NICOTINIC ACID ULTRAVIOLET ABSORPTION SPECTRUM
.4ND
E. B. HUGHES, H. H. G . JELLINEK,
DISSOCI-4TION CONSTANTS
~ N I )(PIIRS.)
B. A . AMBROSE
The Labotatoiies, J . Lyons and Conipang, Ltd., London, W . 14, England
Rccciaed Janiiui g 26, 1948
The determination of dissociation constants of acids and bases from the change of absorption spectra with pH has been carried out by a number of workers. Most of the work has been done on indicators in the visible range of the spectrum (l),but the method is essentially the same for ultraviolet spectra (2). The absorption spectrum of nicotinic acid in aqueous solution has been measured by Huneclce (3), but the change of the spectrum with change of pH values has not so far been repoyted. In the present work the nicotinic acid spectra a t different pH values have been studied and the thermodynamic acid and base dissociation constants of nicotinic acid have been evaluated from the spectra. Figure 1 s h o w the absorption spectra of nicotinic acid a t the following pH values: 1.28 (0.1 N sulfuric acid), 3.20, 3.40, 3.70, 3.85, 4.08, 4.40, 4.65, 5.00, 5.35, 5.60, 13.0 (0.1 N sodium hydroxide). The intermediate pH values were obtained by means of an acetic acid-sodium acetate buffer. The ionic strength of the various buffer solutions was kept constant a t a value of 0.01 by addition of the requisite amounts of sodium chloride. The concentration of the nicotinic acid was of the order of 0.0004 M . The spectrum of nicotinic acid shows an absorption maximum a t 2615 A. The extinctions of the maximum decrease as the pH values of the solutions increase, The spectra fall into two the maximum remaining a t the wave length of 2615 groups, each having two isobestic points indicating two equilibria. The first two isobestic points lie a t wave lengths of 2495 A. and 2695 A. and are associated with pH values from 1.28 to 4.08. They belong to the dissociation equilibrium of nicotinic acid when acting as a base,
.x.
()cooH \N/
+ HzO
(JCOOH
H+
+ OH-
ULTRAVIOLET ABSORPTION SPECTRUM O F NICOTINIC ACID
415
60 x JOz
50
40
30 t
900
A
ZSOQ
2CQO
WAVELENGTH IN
A.
2150
FIG.1. Absorption spectra of nicotinic acid a t different pH values
The second group of isobestic points a t 2465 b. and 2710 b.,covering a range of pH values from 4.4 to 13.0, is associated with the acid equilibrium of nicotinic acid:
;nCOOH
IN)
+ H ~ Oe /\coo- + H30+
416
IC. B. HUGHES, H. H. CT. JELLINEK, AND E. A. AMBROSE
The isoelectric point must lie a t a p H value intermediate between those pH values connected with the two groups of isobestic points, that is, between pH 4.08 and 4.4. The validity of Beer's law was confirmed in acid solution (pH 1.28), alkaline solution (pH 13.0), and in an acetic acid-sodium acetate buffer (pH 4.25) over a range of concentrations of nicotinic acid from cu. 1.6 to 5 X moles per liter.
CONCENTRATION I N MOLES PER L I T R E FIG.2. Denionstration of Beer's law in buffer a t 4.25
Figure 2 shows the straight-line relationships between the extinction coefficients and concentrations of nicotinic acid in the buffered solution. CALCULATION O F DISSOCIATION CONSTANTS
Figyre 3 shows the molecular extinctions for the wave lengths 2615 A. and 2580 A. plotted against pH. The following relationship is valid for all pH values at a particular wave length, since Beer's Iaw is obeyed by nicotinic acid, and its cation and its anion,
where
E = is the extinction of the whole mixture expressed for 1 mole of nicotinic acid dissolved el = the molecular extinction of the anion
€2
I"COO-t
= the molecular extinction of the cation "COOH
I !
ULTR.kVIOLET ABSORPTION SPECTRUM O F NICOTINIC ACID
417
eR = the molecular extinction of the undissociated molecule of nico-
tinic acid aland a2 = the respective degrees of dissociation
If the product of the acid and base dissociation constants of an ampholyte has a value of then it has a maximum of 83 per cent undissociated molecules a t its isoelectric point; if the product is it will have a maximum of about 61 per cent undissociated material. Hence it will not be possible to determine €3 experimentally for a substance of which the product of the acid and
FIG. 3 . Plot of molecular extinction coefficient against pH. 0 , experimental points; X , pli, = 4.9, p R b = 10.4, i.e.p. = 4.25, ea (2615) = 4.45 X lo3, e3 (2580) = 4.03 X lo3. m, pli, = 4.48, P I i b = 10.45, i.e.p. = 4 . 2 , e 3 (2645) = 4 . 5 X lo3, € 3 (2580) = 4.06 X IO3.
base constants ( I < a ' & ) is above and e2, however, can be determined experimentally in strongly acid or alkaline solution, respectively, where nicotinic acid is dissociated conipletely into either cations or anions. In order to calculate the dissociation constants, suitable values for the acid constant (ICa)and for the isoelectric point, which is known from the spectra to lie between pH 4.08 and 4.40, are chosen. The base dissociation constant (&) is then given by the following relationship where pI'b and pIL are the negative logarithms of the respective dissociation
418
E. B. HUGHES, H. H. G. JELLINEK, AND B. A . AMUROSE
constants, pR,, is the negative logarithm of the dissociation constant of water (taken as 14.0), and pHi is the pH value a t the isoelectric point. a1 and a2 are given by the following equations:
and
a1 and a2 can be evaluated for various pH values, and the respective degrees oi dissociation a t a particular pH value-preferably the pH value a t the isoelectric point-can then be put into equation 1 and c3 can be evaluated. Taking this value for c3, values for a1 and a2 for the whole range of pH values can be inserted into equation 1, E values can be obtained, and the calculated curve may be compared with the experimental one. The procedure must then be repeated for different pairs of li, and isoelectric point values until the best fit (compare figure 3) for the experimectal points is obtained. This has been done for two = 2615 b.)and the values finally obtained wave lengths (XI = 2580 A. and are as follows: I . Wave length 2615 8.: K , ,= 10-4.90,isoelectric point = 4.25, Kb = 10-10.40 €1 = 55.0 X 102,~zo= 29.6 X 1 O 2 , € 3 == 44.5 X 10' I I . Wave length 8580 .4.: Same values as before except for €1 = 4G.0 X lo2,€2 = 26.3 X lo2,€3 = 40.4 X 10' A second method was employed which was developed by Vlbs and Gex ( 6 ) . Their equations, however, contain some fundamental errors.' 1 VlBs and Gex in their derivations make the fundamental mistake of considering separately the amount of undissociated substance for the acid and base constituents of the molecule, i.e., ai,a 2 , l - 011, and 1 - az. The sum of all the species present in the solution should, of course, be unity, which i t is not in the deductions made by Vies and Gex, since a1 a2 (1 - 011) (1 - 012) = 2. They, therefore, obtain an incorrect expression for t h e extinction, i.e., EA = ale1 ~ Z E Z (1 al)ea (1 -
+ +
+
+
+
+
-
Equation 1 given above is the correct one, Similarly, VlBs andGexuse the wrong equations for calculating a 1 and 012: namely, PH
=
P K ~
+
log=
pH
=
pKb
+
log-
a1
and
The correct equations are
1
- a2 a?
419
ULTRAVIOLET SBSORPTION SPECTRUM O F NICOTINIC ACID
These errors have been remedied in this worlz and the correct equations are given below. The method consists in plotting a function c$ against pH, whereby a curve with a pronounced minimum or maximum is obtained. The function 6 is given by the ratio of the experimentally found extinctions for two wave lengths a t a particular pH value, i.e. E = 1 (4) Ex 2 Equation 1 is then substituted for the Ex values in expression 4. Further, a1 and a2 are substituted into equation 4 by equations 3a and 3b restated in a slightly different form, a1
=
a2
=
1 l O ~ " ~ - ~ " ( l o ~ ~ i - 1) ~'
+ +1
and 1
+ 1) + 1
lOPH-P~6(10PH-PK.
where PIG
=
PICw - pz
