A SPECTROPHOTOMETRIC DETERMINATION OF THE
DISSOCIATION CONSTANTS OF p-NITROPHENOL
AND PAPAVERINE
BY A. I. BIGGS
Dept. of Chemistry, University of Malaya and the Government Dept. of Chemistry,
Singapore
Received 26th Janunry, 1954
The ultra-violet absorption spectra of p-nitrophenol and papaverine have been studied
in the ultra-violet region. From measurements at different pH's, the dissociation constants
of these two compounds have been determined.
Edwards 1 has recently described the use of the ultra-violet spectrophotometer
to measure the dissociation constant of salicylamide. In a search for a method
of analysis of the insecticide Parathion, it was noticed that one of its decomposition
products, p-nitrophenol, had an ultra-violet spectrum of an unusually simple
nature, consisting of two bands, one of which was enhanced in alkaline solution
and the other in acid solution. In addition, it exhibited one of the clearest isobiestic points yet observed and from the absorption at a series of pH values it
was possible to evaluate the dissociation constant. In continuation of a study of
the analysis of alkaloidal mixtures by the spectrophotometer,2 it has been observed
that the alkaloid papaverine has an absorption spectrum much less complex
than those of the alkaloids in general, and again the absorption is so dependent
on the pH of the system that the dissociation constant of the base can be evaluated
with ease. In view of the interest which attaches to the work on salicylamide,
it is worth while reporting briefly on these further applications of the spectrophotometric method.
EXPERIMENTAL
Measurements were made on a Uvispek spectrophotometer with 10-mm cells in an
air-conditioned room at 25" f 1". Papaverine and p-nitrophenol were purified by
recrystallization and had melting points 147" and 114" C respectively.
The absorption spectrum of p-nitrophenol (3.6 x 10-5 mole/l.) was investigated over
the spectral range 280-440 mp in sixteen solutions buffered at pH values between 5 and 9.
The buffers were made from mixtures of (i) disodium hydrogen phosphate and citric acid,
(ii) potassium dihydrogen phosphate and sodium hydroxide and (iii) boric acid, potassium
chloride and caustic soda and the pH in each case was checked with a glass electrode.
The " blank " cell of the spectrophotometer was filled with the buffer solution without
p-nitrophenol. Thirteen of the absorption spectra are shown in fig. 1 as a graph of optical
density against wave-length. In acid solution there is maximum absorption at 317 mp,
6 = 9720 ; the absorption at this wave-length diminishes as the solution becomes more
alkaline and the extinction coefficient approaches a minimum value of E = 1390 in very
alkaline solution. As the solution becomes more alkaline the extinction coefficient at
407 mp, which is negligible at pH 5, is enhanced and becomes a pronounced maximum
in the absorption spectra at pH values above 7. There is also a remarkably clear isobiestic point at 350mp. This would seem to be a clear case where the maximum at
317 mp is to be attributed to the acid form ofp-nitrophenol and that at 407 mp to the ionized
salt-like form. On this assumption two independent estimates of the dissociation constant of p-nitrophenol can be made. At a wave-length of 407 mp in acid solution, there
800
801
A. I. BIGGS
is no background absorption due to the peak at 317 mp, i.e. the acid form is not absorbing
at all at this wave-length. At pH 10, cB = 18,330 at 407 mp. This is the extinction coefficient when the substance is completely in the salt-like form. If, in a solution of intermediate pH, c( is the fraction ofp-nitrophenol in the salt form, then E&s. = wB. Similarly
h
FIG. 1.-p-nitrophenol
in m
p
in water ; 3.6 x 10-5 mole/l.
the absorption at 317 mp can be ascribed mainly to the acid form but even at pH < 5
there is a small amount of background absorption due to the salt form at 407 mp, so that
cB at 317 mp = 1390. With allowance for this, a second estimate of the fraction present
as salt can be made. From each of the estimates a pK, value can be calculated (table 1).
The average pK, value is 6.99 compared with other determinations of 7.02,3 7 ~ 1 6 ~and
4
7.19.5
TABLE1.-DISSOCIATION
8
X
CONSTANT OF p-NITROPHENOL
10-3
PK,
a
PH
3170 8,
40708,
3170A
40708,
3170 8,
4070 8,
4.0
9-72
9-72
9-03
8.61
8.19
7.36
6.39
1.66
2-28
3-99
0.083
0.133
0.184
0-283
0.400
0-500
0.633
0.733
0.816
0.09 1
0.124
0.218
7.04
7.01
7.05
7.00
6-98
7.00
6.96
6-96
6.95
7-00
7-05
6.95
7-01
6.99
740
6.96
6-97
6.95
5.0
6.0
6-2
6.4
6.6
6.8
7.0
7.2
7.4
7.6
7.8
8.0
9.0
10.0
5.55
5.14
7.22
9.16
0.280
0.394
0.500
0.636
13.40
0.73 1
15.00
0.8 18
2.08
16-90
1.81
17.50
6.99
1-39
18.33
1-39
18.33
cc = fraction of nitrophenol in salt form ; average pK, = 6.99.
4.45
3-61
2.92
11.65
6-99
802
DISSOCIATION CONSTANTS
Similar spectra for papaverine
(1.475 x 10-5 mole/l.) in various
buffer solutions are shown in
fig. 2. Again the ionized form
exhibits absorption at a longer
wave-length (251 mp) than the
non-ionized (239 mp) and there
is an isobestic point at 245 mp.
Analyzing the results as was done
for p-nitrophenol, the pKb values
can be calculated (table 2). The
value now obtained, pKb = 7.60,
would indicate that the base is
considerably stronger than previous results, 8-05,6 and 8-07,7
would suggest. These experiments
indicate that the spectrophotometric technique may provide a
simple and effective method for
determining dissociation constants
of acids and bases which are
not amenable to the more usual
methods.
h in
FIG.2.-Papaverine
in water ; 1475 x 10-6 N.
TABLE
2.-r\ISSOCIATION
6
The author thanks Prof. R. A.
Robinson for his interest in this
work.
mp
CONSTANT OF PAPAVERINE
x 10-4
PKb
U
PH
2390 A
2510 A
2390 A
2510 A
2.0
3.0
4-0
5.0
5.6
5.8
6.0
62
6.4
6-6
6.8
7.0
7.4
8-0
11.0
12-0
3.36
3.36
3.39
3-48
3.86
3.93
4.30
461
4-86
5-22
5-46
5-66
6.03
6.27
6.43
5.90
5-90
5-83
5-63
5.19
4.91
4.61
4.15
3.71
3.30
2.77
2.5 1
0.838
0.8 15
0.695
9.594
0513
0.396
0.318
0.253
0.836
0.772
0.703
9.597
0495
0.40 1
0.279
0.219
6.44
CI
= fraction
2390 A
7.69
7.56
7.64
7.63
7.58
7-58
7.53
7-47
7.69
7.67
7.63
7.63
7.61
7.57
7-81
7.55
2.00
1.63
1.56
1.56
7-56
of alkaloid in form of cation ; average pKb = 7.60.
Edwards, Trans. Faraday SOC.,1953,49,234.
Biggs, J. Pharm. Pharmacol., 1952,4, 547 ; 1953, 5, 18.
Hantzsch, Ber., 1899, 32, 3066.
4 LundCn, J. Chim. Phys., 1907, 5 , 574.
5 Hollemann and Wilhelmy, Rec. trav. chim., 1902, 21, 432.
6 Kolthoff, Biochem. Z., 1925, 162,289.
7 Veley, J. Chem. SOC.,1909, 95, 758.
1
2
3
2510 8,
7.64