PHEKOL TESTS. IV* A Study of the Velocity of Indophenol Formation. 2 , 6-Dibromobenzenoneindophenol. BY H. D. GIBBS
Introduction In the third paper of this series SE test for phenols was described. The test is based upon the reaction between a quinonechloroimide and a phenol forming an indophenol, the salt of which gives an intense blue solution. A special spectrophotometric study was made of the reaction between 2 , 6-dibromoquinonechloroiniide and phenol. On studying this reaction upon a quantitative basis enormous changes in the rate of the formation of the blue color were found to be dependent upon the pH of the solutions. Preliminary experiments failed to reveal any definite correlation or definite mechanism underlying the reactions producing the indophenol. Therefore, this systematic study was undertaken. It has been found that a very definite and simple relation exists. This type of indophenol formation is usually represented by the equation’ RT O=-, /==\-r;Cl + D O H 2NaOH = Br 2,6-dibromoquinonephenol chloroimide Br O = c > = N - a - O K a KaC1+ zHzO Br 2 , 6-dibromobenzenoneindophenol While this equation correctly expresses the proportions of substances entering into the reaction, which might be considered of the third order, it does not represent the true underlying mechanism of the indophenol production. It has been found that the reaction can be explained by assuming that the phenolate and the quinonechloroimide react in the sense of the equation
+
+
* Senior Chemist, Hygienic Laboratory. Published by permission of the SurgeonGeneral, United States Public Health Service. The question of nomenclature will be fully discussed in a later paper entitled STUDIES ON OXIDATION-REDUCTION. XII. PREPAR4TION O F INDOPHENOLS WHICH ARE ESEFUL AS OXIDATION-REDUCTION INDICATORS.
I054
H. D. GIBBS
There is some evidence of the formation of the intermediate compound
0=
Br - = N . O(-) Br
~
which, in alkaline solution, rearranges to the blue salt of the indophenol. It seems most probable that this rearrangement is very rapid and is not the governing factor in the speed of indophenol formation. Since, other things being equal, the speed of the reaction as expressed above is dependent upon thc concentration of the phenolate, the effect of pH upon the speed of the reaction is clear, and in buffered solutions in the alkaline ranges in the presence of a great excess of phenol, the measurements of the velocity of the reaction, calculated according to the first order, should give a constant, which they do. The reaction is, therefore, under these conditions, pseudomonomolecular. When the concentrations of the imide and of the phenol are more nearly equal, the velocity of the reaction, calculated according to the second order, gives a constant agreement with the values obtained in the former case. Theaveragesof ~Svaluesobtainedexperimentally fix thisvalue at 1.68X IO*. I t is interesting to note that technique has been developed by means of which the reaction can be followed and the velocity measured, with a fair degree of accuracy, at concentration so minute, that, heretofore, they have been below the limits of many of the available qualitative tests for phenol. Fair results have been obtained with I part of phenol in z million. Some measurements of an unsatisfactory nature, were obtained with I part phenol in 20 million. I n the early experimental work great difficulty was experienced in obtaining consistent results and it was soon discovered that the temperature coefficient of the reaction is considerable. This necessitated the construction of reaction vessels in which the temperature could be accurately controlled. It was also found that the various buffer solutions employed should be standardized to a fair degree of accuracy under uniform conditions. The standardizations were made electrometrically for the same dilutions employed in the reacting mixtures. I have Mr. W.L. Hall to thank for the electrometric standardizations. Mechanism of the Reaction The reaction between z , 6-dibromoquinonechloroimide and phenol in alkaline solutions may be represented as taking place in a number of ways. Since we have no evidence that indophenols form in acid solution (if they do form the rate is so slow that there is no evidence of the production of the red acid form), the first thought is to formulate a reaction of the third order as occurring between a = imide, b = phenol and c = hydroxyl. In buffered solutions the concentration of the hydroxyl ion, “c”, is constant and the velocity equation becomes
PHENOL TESTS
105s
dx _ _- kc(a-x) (b-x) dt
I.
which integrated gives I
k =
2.
tc (a-b) (b-c)
b(a-x) In a(b-x)
No constant values for “k” are obtained by the use of this conception. R e are evidently dealing with a complex reacting solution, the first stage being the formation of the phenolate ion, which reacts with the imide according to the equation
imide
phenolate
indophenol ion
This reaction appears to be of the second order. When it is carried on in buffered solutions the Concentration of the phenolate ion is determined by the pH of the buffer from the equation 4,
a
pH = pKa -t log(1
phenolate in which a is the ratio total phenol
-
).
,
The concentration of the phenolate ion is then the concentration of the total phenol times a. In reaction “3” if “a” equals the concentration of the imide, “b” the original concentration of the phenol, “ba” the concentration of the phenolate and “(b-x) cy” the concentration of the phenolate ion at any time, “t”, the velocity equation becomes
which integrated gives 6.
or 7.
kz
=
2In b(a-x) ~
t a (a-b)
a(b-x)
2.3 0 2 6 b(a - x) k? = _____ log ___ t a (a-b) a(b-x)
When the concentration of the phenol is very large in respect to the imide and when pH is high, the concentration of the phenolate ion becomes a constant for all practical purposes and the equation reduces to the form of a monomolecular reaction, the velocity constant of which is 8.
k = i l n L tb a (a-x)
1056
H. D. GIBBS
or 9.
k =
2
3026
a (a-x)
. I log __
w tb
While velocity equations 6, 7, 8 and 9 do not contain the OH- concentration directly, it is evident that the concentration of phenolate ion, is dependent, not only upon the concentration of the total phenol, but upon this factor also.
Experimental a. The Apparatus. All of the spectrophotometric measurements were made by means of a Keuffel and Esser color analyzer with the solutions held in I O cm tubes. During the early part of the work it was not realized that
maintenance of a uniform temperature of the solutions during the observation of the course of the reaction was necessary and, while approximately zoo was maintained, the heat from the apparatus gradually raised the temperature of the reacting vessels and good series of constants were difficult t o obtain. As soon as it was found that the temperature coefficient of the reaction was considerable, a special casing for the spectroscopic tubes was devised, whereby they were surrounded by a stream of water fed from a large supply, thermostatically controlled a t 20'. See Fig. I . All measuring flasks and pipettes were standardized by the C . S. Bureau of Standards. b. The manipulation. The phenol solutions were made from a carefully purified sample of phenol. It was constant boiling and colorless. Liter quantities of a number of concentrations varying from IO-* to IO-^ molar were made by dissolving in water. The 2,6-dibromoquinonechloroimide was made as described in the third paper of this series and approximately concentrated solutions were made by shaking with water a t 20' and filtering until clear. The water employed was practically ammonia free and no solutions over five hours old were employed.
PHENOL TESTS
I057
The solutions were standardized by the spectrophotometric method of measuring the indophenol formation as described in the third paper of this series. The buffering solutions employed were borate solutions of the Clark and Lubs series, and for the most accurate work were standardized electrometrically at the dilutions employed. Portions of 5 cc of the various phenol solutions were run into a j c cc measuring flask and then about 40 cc of the desired buffering solutions added. A measured volume, usually I to 2 cc, of the 2,6-dibromoquinonechloroimide solution was added, and the flask filled to the mark with the buffer. The spectroscope tube was filled with this solution, put in place in the spectrophotometer, and readings of the transmittancy taken at intervals of time measured by a stop watch. All readings were taken at IO,,, the peak of the absorption band for this indophenol. See third paper of this series. The dissociation constant of 2, 6-dibromobenzenoneindophenol (pKa = 5.7) is such that at the pH values used experimentally there was complete dissociation and, thercfore, full color. See Cohen, Gibbs and Clark (1924). c. The calculaticms. The concentration of the sodium phenolate was calculated from the Ka value for phenol at 18" of 1.3 X 10-l~.J. Walker (1900.) The percentages of sodium phenolate in the various buffer solutions employed are given in Table I. Many of the buffered solutions enumerated were employed only in the preliminary work, the data for which is not recorded here since the determinations of the pH of the buffers was not considered sufficiently accurate.
TABLE I Per cent of phenolate in various buffered phenol solutions salt PH = log -5 log Ka acid Ka for phenol = 1.3 X 10-l~at 18' pK. = 9.89
+
pH = 9.89 PH
8.4 8.5 8.6 8.733 8.884 9.0 9.129 9.2
acid salt
30.90 24.55
19.50 14.35 10.14 7.76 5.768 4.90
Per cent N a salt
axroo
3.14 3.91 4.88 4.5'
8.92 11.42 14.77 '6.95
+ log
a I -a
PH
9.28 9.3 9.4 9.5 9.528 9.6 9.917 10.0
scid
Per cent
salt
ax100
4,074
3.89 3.09 2.46 2.301
1.95 0.93;6 0.78
Ka salt
19.71 20.45 24.45 28.90 30.29 33.90 51.62
56.18
H. D. GIBBS
1058
The amount of indophenol formed at any given time was found from the reading of the per cent transmittancy of the solution in the spectrophotometer, the value -log T = I being that of an indophenol solution of a concentration of j X IO+. See third paper of this series. The reaction velocity constant, “k”, was calculated from the equation of a reaction of the first order a k = 2.3026 __ log __ 9. tba a-x where t = time in minutes, b = molar concentration of phenol, OL = phenolate a t the pH of the experiment, a = molar concentration of the 2, 6-dibromoquinonechloroimide, and x = amount of phenol or sodium phenolate, or 2, 6-dibromoquinonechloroimide reacting in time, “t”, which is also equivalent to the amount of indophenol formed. I n two cases (see Tables XI11 and XVIII) where the concentrations of the phenol and of the imide were nearly equal the velocity constant was also calculated from equation b(a-x) 2 3026 kz = L log 7. tda-b) aib-x) for a reaction of the second order. The agreement in the constants obtained in the various experiments indicate that the above described conceptions are correct. d. Results. The final series of the reaction velocity experiments are recorded in Tables 11-XXTi inclusive. The conditions of the 24 experiments and the velocity constants obtained are summarized in Table XXVI. TABLE I1 Reaction velocity of indophenol formation a = 2,6-dibromoquinonechloroimide3.24 X IO+ molar b = total phenol 103 molar Buffer solution = pH 8.733 t = time in minutes T = transmittancy x = indophenol formed in “t” a a - _I Ratios: phenol - 309 ’ Iia phenolate 2 0
.
~
X
T
-log T
I
0.70
0.155
3 4
I1
.44 .37 .31 .28 .26 .24
I7
.22j
t
5
6 8
,356 ,432 ’ 509 ,553 ’585 ,620 .648
times
106
0.78 1.78 2.16 2 55 2.77 2 93 3.IO 3.24 ’
a-x times
10-
2.46 I . 46 1.08 .69 ‘47 .3I ‘
14
0.00
Average
li
4.23 X io3 4.08 4.24 4.75 4.944.5’ 4.39
PHENOL TESTS
TABLE 111 Reaction velocity of indophenol formation a = 2,6-dibromoquinonechloroimide3.24 X IO+ molar b = total phenol = IO+ molar Buffer solution = pH 8.733 t = time in minutes T = transmittancy x = indophenol formed in 9” a a Ratios: -phenol - 30.9 ’ Na phenolate _f_
X
t
-1ogT
times
I
0.055
2
.097 . I31 .260 ,387 .432 .469
0.275 .485 ,655 I ,300 1.935
3 6 9 I2
I5
104
- _I 2
a-x times 10-3
2.965
13.7 X
2,755
12.2
2 ’ 585
11.5 13.1 ‘5.5 14.I 13.z
I ,940 I. 305
2.160
1.080
2.345
0.895
Average TABLE
k
13.3 X
108
103
IV
Reaction velocity of indophenol formation a = ~,6-dibromoquinonechloroimide 6.48 X IO+ molar b = Total phenol = IO+ molar Buffer solution = pH 8.733 t = time in minutes T = transmittancy x = indophenol formed in “t” Ratios:
a -2. a --I - 15.4’Na phenolate
phenol
I
a-x
X
T
t 2
0.63
3 4
.SO
5
.35
6 8
‘29
IO
,44
.22 .18
-log T 0.201
.301 ,357 .456 ’ 538 ,658 ’745
times
105
1.005 1.505
1.785
times
10-
5.475 4.975 4.695
2.280
4.200
2.690 3.290 3.725
3.790 3 . 190 2.755
Average
k 12.9
x
10s
13.5 13.2 13.3 I3 ’ 7 13.6 13.1
-13.3 X 1o3
1060
H . D. GIBBS
TABLE V Reaction velocity of indophenol formation a = 2,6-dibromoquinonechloroimide = 3.24 X IO+ molar b = Total phenol = IO+ molar Buffer solution = pH 8.733 t = time in minutes T = transmittancy x = indophenol formed in “t” Ratios:
a I -_ 3.09 ’ S a phenolate 0.2
2= _ f _ .
phenol
t
--log
T
3
0.022
5
,046
8 I3 18
.07I
X
times
0.110
,230 ,355 ,485 ,655 ,840 ,935
,097 ,131 ,168 ,187
22
28
105
a-x iimes los
3.130 3.010 2.885 2,755
k
17.7 X 22.5 22.2
2.585
24.I 19.2
2.400
21.0
2.305
18.7
-4verage
103
20.7 X
103
TABLE VI l{eaction velocity of indophenol formation ~,6-dibromoquinonechloroimide = 3.73 X IO-^ molar b = Total phenol = IO-^ molar Buffer solution = pH 9.129 t = time in minutes T = transmittancy x = indophenol formed in “t” I a -- _ Ratios: --.%- . phenol 270 ’ N a phenolate 40 3,
=
X
t
T
-log T
2
0.38 .26
’
585
3
.20
’
699
4
. I98
’
703
1
0.420
times
105
2 . IO0
2.925 3.495 3.515
a-x times IO^
1.630 ,805 ‘235 , 2 1j
Average
k
5.61 X IO^ 5 . ‘7 6.24 4.82
5.46 X
108
1061
PHENOL TESTS
TABLE VI1 Reaction velocity of indophenol formation a = 2,6-dibromoquinonechloroimide = 5.23 X IO+ molar b = Total phenol = IO-^ molar Buffer solution = pH 9.129 t = time in minutes T = transmittancy x = indophenol formed in “t” a - I . a Ratios: -- phenol 191’Na phenolate
- -I 28 a-x
X
t
T
-log T
times ioJ
I
0.260
2
.I25
2.925 4.515
3
.090
0.585 .903 I . 046
times
k 5 . 5 5 x Ioa 6.73
104
2.305
.715
. 000
5.230
6.14 X IO^
Average
TABLE VI11 Reaction velocity of indophenol formation
a = 2,6-dibron1oquinonechloroimide = 3.73 x
molar
10-6
b = Total phenol = IO+ molar Buffer solution = pH 9.129 t = time in minutes T = transmittancy x = indophenol formed in “t” a - I. a Ratios: - - phenol 27’Na phenolate X
t
T
-log T
times IO’
2
0.54 .44 .38 .34
0.278
1.39 1.78
3 4
5
.356 ,420 ,468
2 . IO
2.34
- -I 4 8-X
times
100
2.34 1.95 1.63 1.39
Average
k 15.78X 14.64 14.01 16.80
15.31 X
IO*
IO*
Note: Comparison tube contained buffer plus the same amount of 2,6dibromoquinonechloroimide.
1062
H. D. GIBBS
TABLE IX Reaction velocity of indophenol formation a = z,6-dibromoquinonechloroimide= 5.23 X IO^ molar b = Total phenol = IO-^ molar Buffer Solution = pH 9.129 t = time in minutes T = transmittancy x = indophenol formed in “t” Ratios:
a - I. a - -I -phenol 19’Na phenolate 2.9
~
T
-log T
0.46 .32
0,337 ‘495
‘24
,620
.18 j
,733 ’ 796 ,824
.16 ’
15
a -x
X
times
IO?
1.67 2.48 3.10 3.67 3.98 4.12
times
108
k
1.11
13.05 x 14.51 15.20 16.38 16.15 14.99
Average
I j . 05
3.56 2.75 2.13 I , 56 1.25
X
IOs
IO*
TABLE X Reaction velocity of indophenol formation a = z,6-dibromoquinonechloroimide= 5.23 X IO-^ molar b = Total phenol = IO-^ molar Buffer solution = pH 9.129 t = time in minutes T = transmittancy x = indophenol formed in “t” Ratios:
a a I . - -I - phenol 1.9’ Na phenolate 0.29 a -x
X
t
4.5 5 6 7
T 0.72 .70
-log T 0.I43
8
.61 .S6
‘I55 ,180 ,215 , 2 52
9
‘51
,292
.66
times
106
0.72 ‘78
times
10’
k
22.27 x
.90
4.51 4.45 4.33
21.30
1.08
4.I 5
22.39
1.26
3.97 3.77
23.32 24.63 -22.63 X loa
1.46
Average
I d
21.85
1063
PHENOL TESTS
TABLE XI Reaction velocity of indophenol formation a = 2,6-dibromoquinonechloroimide= 3.73 X IO-^ molar b = Total phenol = IO-^ molar Buffer solution = pH 9.129 t = time in minutes T = Transmittancy x = indophenol formed in “t” a a Ratios: - = L . phenol 2.7’ N a phenolate t
I1
T
0.86 .81 ’ 74 .61
-log T
0.066 ,092
. I31 ,215
times
0.4 a -x
X
3 4 6
- -I
IO’
0.33 .46 .66 1.08
times roe
3 ’ 40 3.27
3.07 2.65
Average
k 20.89 X 22.29 21.38
IO’
21.05
--
21 .40 X IO’
TABLE XI1 Reaction velocity of indophenol formation a = 2,6-dibromoquinonechloroimide = 7.46 X IO+ molar b = Total phenol = IO+ molar Buffer solution = pH 9.129 t = time in minutes T = transmittancy x = indophenol formed in “t” a 1 . a - -I Ratios: - - phenol 1 . 3 4 ’ Na phenolate 0.2 a-x
X
t
T
-log T
2
0.78
0.108
3
‘74
. I31
4
.68 .s3 ‘46 .42
,168 ,276 ‘337
6 8 IO
,377
times
IOO
0.54 .66 .84 I .38 1.69 1.89
times IO‘
6.92 6.80 6.62 6.08 5.77 5.57
Average
k (25.41)x 20.89 20.18 23.07 21.73 19.77 21.13 X
IOa
10)
1064
H. D. GIBBS
TABLE XI11 Reaction velocity of indophenol formation. a = phenol = IO-^ molar b = ~,6-dibromoquinonechloroimide= 7.46 X IO-^ molar Buffer solution = pH 9.129 t = time in minutes T = transmittancy x = indophenol formation in “t” b 1 . b - -I Ratios: - - phenol 1.34’Na phenolate 0.2 2.3026 b(a - x) log ___ from same - b), a(b - x)
calculated according to formula kz =
ta(a
data as table XII. X
t 2
3
4 6 8 IO
T
-log T
0.78 .74 .68 .53 .46 .42
0.108
times
. I31 ,168 ,276 ,337 ,377
105
b-x times 1 0 5
0.54 .62 .84 1.38 1.69 1.89
6.92 6.84 6.62 6.08
5.77 5.57
a-x times
IOO
9.46 9.38 9.16 8.62 8.31
kz
(26.10)X
IO
20.05
8.1 1
21.04 24.86 23.87 21.99
Average
22.36 X IO^
TABLE XIV Reaction velocity of indophenol formation a = z,6-dibromoquinonechloroimide = 3.73 X IO-^ molar b = Total phenol = 5 X IO-^ molar Buffer solution = pH 9.129 t = time in minutes T = transmittancy x = indophenol formed in “t” a I . a - -I Ratios: - = phenol 13.4’ Na phenolate 2 X
t 2
3 4
T 0.69 ’ 59
-log T
x 106
0.161 ,229
0.81
a-x
x 106
k
r.30
2.92 2.43
16.95X 19.34
.SO
.3OI
I . 52
2.21
17.71
5
,42
,377
1.89
1.84
19.I 4
8
.36
.444
2.22
1.51
15.31
Average
I 7.69 X
IO’
IO^
1065
PHEWOL T E S T S
TABLE XV Reaction velocity of indophenol formation a = ~,6-dibromoquinonechloroimide = 4 X IO+ molar b = Total phenol = IO-^ molar Buffer solution = pH 9.528 t = time in minutes T = transmittancy x = indophenol formed in “t” a - I. a Ratios: -- phenol 25o’Na phenolate
- -I 76 a-x
X
T
-log T
xI
0.28
0.553
2.77
. I9
.72 I
.I7
.770
3.62 3.85
x 108
d
k 103
.I 5
3.92 X 3.88 3.61
Average
3.80 X
103
1.23
.38
TABLE XVI Reaction velocity of indophenol formation
a = 2,6-dibromoquinonechloroimide= 4 X b = Total phenol = 104 molar
1 0 4molar
Buffer solution = pH 9.528
t = time in minutes T = transmittancy x = indophenol formation in “t” Ratios:
a = 5.
phenol
2s’ Na
a phenolate X
I E -
7.6 a -x
T
-log T
XI@
x 108
0.32
0.495 .602 ,670 .708
2.475
1,525
x
IO’
3.010
.990 ,650 ,460
14.28
Average
15.15 X
IO)
.25
.2 I4
,196
3.350 3.540
k
15.92 15.38 15.00
1066
H. D. GIBBS
TABLEXVII Reaction velocity of indophenol formation a = ~,6-dibromoquinonechloroimide= 4 x 10-6 molar b = Total phenol = 10-5 molar Buffer solution = pH 9.528 t = time in minutes T = transmittancy x = indophenol formed in “t” a - I . a Ratios: _ _ - phenol 2.j ’ E a phenolate T
-log T
times io5
0.72
0 .I43
0.72
5 6 7 8 9
. I87
.65 .61 .56 .53
.94
times
106
k 16.38X l o a
3.28 3.06
17.70
,215
I.08
2.92
17.30
. 25 2
1.26 I .38 1.42
2 ’ 74
17.82
2.62 2.58
17.50
,276
,284
4 2
0.76 a-x
X
t 4
- -I
Average
16.07
17,I I
x
103
TABLE XVIII Reaction velocity of indophenol formation. a = phenol = IO-^ molar b = ~,6-dibromoquinonechloroimide= 4 X IO-^ molar Buffer solution = pH 9.528 t = time in minutes T = transmittancy x = indophenol formed in “t” b - I . b _ _I Ratios: - - phenol 2.5’Na phenolate 0.76 k l calculated according to formal kz
=
2.3026 b(a - x) log ___ from same ta(a - b) a(b - x)
data as table I 7. X
t
4 5 6
7 8 9 I2
T 0.72
-log T 0.I43
times
101
0.72
.65 .61 .56
. I87
’
53
,276
.94 1.08 1.26 1.38
‘
52
,284
I!42
.so
.301
1.51
,215 ,252
b -x times IO’
a-x times IO’
3.28 3.06 2.92 2.74
9.28 9.06
2.62
2.58 2.49
8.92
8.74 8.62 8.58 8.49
Average
ki
(16.98)IO X’ 18.60 18.36 19.12 18.87 17.43 (14.21) 18.47x
103
1067
PHENOL TESTS
TABLE XIX Reaction velocity of indophenol formation a = ~,6-dibromoquinonechloroimide= 3.73 X
IO^ molar b = Total phenol = IO-* molar Buffer solution = pH 9.917 t = time in minutes T = transmittancy x = indophenol formed in “t” a - 1 , a Ratios: - - phenol 268’ Na phenolate T
.225 .205
-log T
0.620 .648 .688
138 a-x
X
0.24
- -I
times
IO’
3.10 3.24 3.44
times IO)
0.63 .49 ‘29
Average
k 1 . 6 8 X IO^ 1.31 1.00
1.33 X
103
TABLE XX Reaction velocity of indophenol formation a = ~,6-dibromoquinonechloroimide= 3.2 X IO+ molar
b = Total phenol = IO-a molar Buffer solution = pH 9.917 t = time in minutes T = Transmittancy x = indophenol formed in “t” Ratios:
2 3 -
phenol
a
I . 313’Na phenolate
X
T
-log T
times
106
- -I 161 a-x times
0.38
0.420
2.10
.25
,602
3.01
‘
.24
,620
3.10
10’
k
I . IO
1.04
I9
1.82
.IO
1.73
Average
X xo8
1.53 X
IO)
1068
H. D. GIBES
TABLEX X I Reaction velocity of indophenol formation a = ~,6-dibromoquinonechloroimide= 3.73 X IO-^ molar b = Total phenol = 104 molar Buffer solution = pH 9.917 t = time in minutes T = Transmittancy x = indophenol formed in “t” - I. a I - _ Ratios: -- phenol 27’Xa phenolate 13.8 X
t 1.5 2
3
T 0.41 .33 .3Q
-log T
0.387 .482 ’ 523
times
101
1.935 2.410 2.615
a-x times 1 0 6 1 ’ 795
1.320 I . I15
Average
k 9.45 X 10.04 7.80
9 . IO X
103
10s
TABLE XXII Reaction velocity of indophenol formation a = ~,6-dibromoquinonechloroimide = 3.20 X IO-^ molar b = Total phenol = 104 molar Buffer solution = pH 9.917 t = time in minutes T = transmittancy x = indophenol formed in “t” a - I. a - -I Ratios: -- phenol 31’ Ka phenolate 16 a -x
X
T 0.48 .32 .29
-log T
0.319 * 495 .538
times
106
1.60 2.48 2.69
times
106
1.60 .72
.SI
k 13.43 14.45 11.86
x
Average 13.25 X TABLE XXIII Reaction velocity of indophenol formation a = ~,6-dibrom0quin0nechloroimide= 3.20 X IO+ molar b = Total phenol = 5 X IO-^ molar Buffer solution = pH 9.917 t = time in minutes T = transmittancy x = phenol formed in “t” a a I . = IRatios: -phenol 16’ Na phenolate 8 times IO^
a-x times IO^
1.08 1.69 2.16
2.12 1.51 I .04
X
T 0.61 .46 .37
-log T 0.215
,337 ,432
Average
k 15.97 X 14.51 14.52 15.00 X
10’
103
103
10)
1069
PHENOL TESTS
TABLE XXIV Reaction velocity of indophenol formation a = 2,6-dibromoquinonechloroimide = 3.73 X IO+ molar b = Total phenol 5 5 X IO-^ Buffer solution = pH 9.917 t = time in minutes T = transmittancy x = indophenol formed in “t”
a a - -I Ratios: - = phenol 13 ’ N a phenolate 7 t
1.5 2
3 4
a -x
X
T
--log T
0.54
0.268
1.34
2.39
11.45
.44 .38
,356 ,420
1.78 2 .I O
1.95 1.63
.36
’
444
2.22
1.5’
12.57 10.69 8.76
times
105
times io6
Average
k
x
IO3
10.87 X
IO*
TABLE XXV Reaction velocity of indophenol formation a = z,6-dibr0m0quin0nechloroimide = 3.73 X IO-^ molar b = Total phenol = IO-^ molar Buffer solution = pH 9.917 t = time in minutes T = transmittancy x = indophenol formed in “t” a 1 . - -I Ratios: _a_ - phenol 2.7’Ka phenolate 1.38 a-x
X t
T 0.72
.64 .56 ,s2
-log T 0 .I43
. I94 .252
,284
times
106
times
106
0.72
3.01
.97 1.26 1.42
2.76 2.47 2.31
Average
k 20.29 X
IO*
19.45 19.96 (15. so) 19.90
x
IO*
H. D, GIBBS
1070
a.c
jP
2
X
N
10
iow
3
w
O
h
y e "
-
+
4
*
3
3
3
3
r
r
-
1
-
3
OD m
m
h h
r-0 0 .x. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N
m i& a m mw 3 3 W
h i
H
.............. -
-
e
-
c
e
r
e
**
ar-mm-t
Y
Y
2
B
3 &
......
..................
........
"-e3
w
-
3
3
3
3
e
3
3
c
3
-
-
-
.............. 3
c
e
3
N N N
3
3
N
3
3
xxxx xxxxxxxxx c c - 3 vivivivi
. . . .
\owwo
h h h h h h h h v i h h h h h h h
N
hm
w wwwmmw W 3 3 3 viw,-
e d b b * e * b m
. . . . . . . . .
3
3
3
H
H
H
3
3
. . . . . . .
h
v i v i v i v i N
N v i
xxxxxxx ? r,
m 0 0 " r " r
. . h . N. N. h. h.
C-N
mmmmmmm
X vi
x m m x N N "
1ovivivi
. . . .
ac\c.c.
PHESOL TESTS
1071
Discussion I t is to be noted from a study of Table XXT’I that experiments are recorded and reaction velocities measured in solutions when the phenol concentration varied from 10-3 to 5 >( IO-^ molar, the sodium phenolate varied from 5.162X varied 104 to 6.j1 X IO-? molar and the ~,6-dibromoquinoncchloroimide from 7.46 X IO-^ to 3.20 X IO-^ molar. The ratios of these substances varied in the various experiments recorded from the highest values of 3 I 3 parts of phenol to I part of z ,6-dibromoquinonechloroimide 161 parts of sodium phenolate to I part of z,6-dibromoquinonechloroiniide to the lowest values of I .34 parts of phenol to I part z,6-dibromoquinonechloroimide 0.2 parts of sodium phenolate to I part z,6-dibr0m0quin0nechloroimide Eighteen of the values give fairly satisfactory agreement in the velocity constant, “k”. The average of these 18 values is 1.68 x 104. I n these 18 experiments the ratios of the phenol and sodium phenolate to the z ,6-dibromoquinonechloroimide vaned from the highest value of 3 1 parts of phenol to I part of imide 16 parts of sodium phenolate to I part of imide to the lowest value of 1.34parts of phenol to I part of imide 0.2 part of sodium phenolate to I part of imide. When the concentration of sodium phenolate is greater than 16 times that of the imide the reaction is so rapid that the measurements are very uncertain. The constant seems to fall off rapidly under these conditions. It, therefore, seems reasonable to exclude the six values for “k” given in parentheses in Table XXVI, in obtaining the average value of 1.63 x 104. The accuracy of this value is dependent upon many experimental factors which were controlled as carefully as possible and also, basically, upon the Ka value for phenol which was determined by J. Walker in 1900to be 1.3 x 1 0 - l ~ at 18’. Stenstrom and Goldsmith (1926)have recently determined the dissociation consta?t of phenol by measuring the ultraviolet absorption at wave length 2825X at pH values varying from 4.5 to 13. The desired values of pH nere obtained by the addition of sodium hydroxide solution and were determined colorimetrically. The temperature during the exposures varied between 18’and 24‘. They reported two values I 38 X ~o-’Oand1.21 X IO-IO the average of which is 1.295 X 10-l~.This work does not afford any grounds for the correction of the values of Walker employed in my work. The actual activity coefficient of the phenolate ion in the buffers employed in this experimental work are not known and it is interesting to note that a change of Ka from 1.3 X 1 0 - l ~ to 2.3 X 10-l~may, in some cases, reduce the reaction velocity constant by so much as 50 per cent. It is, therefore, believed that an error arising from this source may be greater than that produced by any factor entering into my experiments. Since it has been my purpose to elucidate the underlying mechanism of the
1072
H. D. GIBBS
reactions involved in the indophenol formation, rather than determine a reaction velocity constant with great accuracy, for practical purposes it has not seemed necessary to redetermine the K a value of phenol. It has been found that 2,6-dibromoquinonechloroimidedecomposes in alkaline solution giving rise to an increased coloration. This is visible at 61omp, the wave length of the peak of the absorption band of the indophenol, and therefore, any of the imide which decomposes and escapee reaction with the phenol in the indophenol reaction will introduce an error into the readings. However, the amount of this decomposition during the indophenol reaction is very small, much less than the amount of decomposition in the alkaline buffers when phenol is absent. Any correction to be introduced for this cause seems to be negligible, and overshadowed by other errors of experimentation. A few experiments were performed employing an equal concentration of the imide and the same buffer solution in the comparison tube as employed in the reaction vessel. That is to say that the reaction tube and the comparison tube were equal in every respect, except that the former contained phenol while in the latter it was absent. The reaction velocity experiment recorded in Table VI11 was performed in this manner. This, and other similar experiments, showed that the error from the decomposition of the ~,6-dibromoquinonechloroimidewas very slight and indicated that water, or buffer solution, in the comparison tube was to be preferred. A study of the decomposition of z ,6-dibromoquinonechloroimide in various buffers mas undertaken. The Reaction of 2, 6-dibromoquinonechloroimide in Alkaline Buffers Br KCl, The coloration of ~,6-dibromoquinonechloroimide, 07 = Br in alkaline solutions can be followed by means of the spectrophotometer. Since the rate of reaction is accelerated by light, the readings were made as quickly as possible with the light passing through the solution for the shortest interval of time. The reactions give a light red solution which has very little absorption in the red. The greatest absorption is the blue. The absorption of the solution was investigated at various concentrations in the visible region of the spectrum, at which range it shows no particular evidence of the development of bands but only a gradual increase in absorption from the red t o the blue. See Fig. 2 . The rate of change was first followed by reading the absorption at 610mp for the reason that this wave length is the peak of the absorption band of the corresponding indophenol and it was, therefore, hoped to obtain results useful in the correction of the indophenol reaction velocity. The rate of change was also followed by measurements made at jzomp, a region nearer the maximum absorption. The z,6-dibronioquinonechloroi1nidesolutions employed were standardized with phenol by measurements of the indophenol
’ \-.> I=.====\
PHENOL TESTS
I073
2.0
1.9 18
1.7
1.6 1.5
14
1.3 12 1.1 1.0 0.9
0.8 0.7
T 0 . 6
0.5 a4 0.3 0.2
0.1 470 490 510 530 550 570 590 610 630 650 670 670700my& FIG 2 A Absorption curve of 2, 6-~bromoquinoneox1me 4 2 5 X IO-‘ molar. Only slight devlations occur in thw curve from pH 8 to pH IO. B. Absorption curve of an alkaline solution of 2, 6-dibromoquinonechloroimide after the reaction had roceeded for 8 minutes a t pH 9 13. I 37 x IO+ molar The position of thls curve IS ramelby prolonged action or increase in pH C. Same as “B” except pH 9.53 All values of T read in IO cm tubes Ordmates = -106 T.
I074
H. D. GIBBS
formation as previously described. They were then measured into alkaline buffer solutions and the color formation observed spectrophotometrically. Since neither the pure color compound nor its absorption spectrum are known, the maximum absorption obtained at a standard wave length, 61oand Szomp, was taken as complete transformation of the imide and the intermediate values for “x”, the amount of imide transformed in time “t” Calculated therefrom. The rate of change was found to vary greatly with the pH and measurements were, therefore, made at values varying from pH 8.j to 10.0. Constant values were obtained in each experiment when calculated according to a reaction of the first order. When these values are divided by the concentration of the hydroxyl ion all of the experiments at varying pH values are brought into agreement, the average of seven experiments at pH 8.5, 9.0, 9.13, 9.j, 9.53 and I O being 6.13 X 103. I t is therefore evident that the reaction under consideration is pseudo monomolecular and the equation IO.
a k = l l n tc a-x
where t = time in minutes, c = hydroxyl concentration of the buffers, a = 2,6-&bromoquinonechloroimide and x = amount transformed in “t”, is a true representation of the conditions. The hydroxyl ion concentration has been calculated from the pH value by means of the expression pH = log
11.
2
H+
employing the value of 14 for hk,. Since the dilution and other experimental factors not accurately determined may effect this value it seemed trivial to correct Kw for temperature. The seven reaction velocity experiments are recorded in Tables XXVIIXXXIII, inclusive, and a summary of the data is given in Table XXXIV. The values of kl and kt recorded in these seven tables are obtained from the formulae 2.3026 a 12. ki = log t a-x ~
13.
kz
2.3026
= -log
tc
a a-x
While the constants obtained leave much to be desired in agreement and accuracy, the effect of pH is definitely shown and magnitude of the constant is indicated. The tendency of the value of kl and kp to increase with time in some of the experiments is attributed to the accelerating effect of light, since in these experiments the irradiation was excessive due to the fact that readings were taken at several wave lengths.
I075
PHENOL TESTS
2, 6-Dibromoquinoneoxime (2, 6-dibromonitrosophenol) The reaction between 2,6-dibromoquinonechloroimide and alkalies is indicated by the reaction velocity experiments to be Br Br C1 O = /\-NCl O H O = '='-N.OH \=/Br A C z ,6-dibromoquinoneoxime being the principal reaction product. I n order to obtain some evidence upon the nature of the reaction, pure z ,6-dibromoquinoneoxime (nitrosophenol) was prepared by brominating pnitrosophenol as described by 0. Fischer and E. Hepp (1888). A saturated aqueous solution at 20' of this preparation was found to be 8.5 X IO^ molar as calculated from the determination of the nitrogen. One hundred cc of solution gave 0.00119 g nitrogen. This solubility compares with 1.88 x IO-^ molar for the unbrominated derivative. (See second paper of this series.) The compound was found to vary in the color of its solutions from colorless a t pH 3 to a yellow green in 0 . 2 ?; sodium hydroxide. The dissociation constant of this preparation, determined by the method of Salm (1906), is 4.6 in terms of pKa. This compares with 6.4 for the unbrominated compound. (See second paper of this series.) The solutions employed were: for the acid solution I cc of the saturated a,6-dibromonitrosophenol solution plus I O cc of buffer pH 3 ; for the alkaline solution I cc of z,6-dibromonitrosophenol solution plus I O cc of 0.2Nsodium hydroxide. Superpositions of these were matched against 2 cc of solution plus 9 cc of buffer pH 4.6 and 11 cc of water. The adsorption spectra of solutions of z,6-dibr0m0quin0ne0xime were measured in aqueous solution and at pH 8, 9 and IO, and very slight differences were observed under these conditions. Five cc of the saturated solution were mixed with 5 cc of the buffer solution. The typical curve is plotted in Fig. 2. It is quite flat from 700mp to soomp showing very little absorption in this region but rises rapidly showing almost complete absorption in the blue. There is some evidence of the formation of an incipient absorption band heading about 600mp which seems to be the most evident at pH 8.5 although not very marked under any of the conditions employed. 2,6-Dibromoquinoneoxime is quite stable in aqueous solution. In a buffered solution at pH 8.5 no change in the absorption spectrum was noted on 24 hours standing at room temperature. This is in striking contrast to the unbrominated bxime. Discussion From the previously described data, it is considered proved that 2,6dibromoquinonechloroimide reacts in alkaline solution, the velocity of the reaction showing it to be pseudo monomolecular the speed being determined by the concentration of the hydroxyl ion. The absorption curve of the resulting solution bears a sufficient similarity to that of 2,6-dibromoquinoneoxime,
+
+ -
b=/-
1076
H. D. GIBBS
see FIG.2 , to suggest that while the latter is not the only product it may be the first and most important stage of the reaction, other condensations later taking place to a more or less limited extent. The possibility of correcting the reaction velocity constant for the indophenol formation from any data obtained from a study of the decomposition of 2,6-dibromoquinonechloroimidealone in alkaline solutions seems to be rather remote and perhaps immaterial. If the velocity constant for the complete decomposition of the ~,6-dibromoquinonechloroimidewere employed it would reduce the velocity constant for the indophenol formation by only one-third. The decomposition of the z,6-dibromoquinonechloroimide is evidentally practically negligible when phenol is present to unite with it and the indophenol reaction proceeds. This is shown by the excellent results that are obtained by the standardization of the imide solutions by the indophenol formation at various pH ranges when a great excess of phenol is present. When the reacting quantities of ~,6-dibromoquinonechloroimide and phenol are nearly equal in molarity another state of affairs exists, and under these conditions, there is no doubt but that the decomposition of the imide is a material factor and that the maximum indophenol formation, as measured either by the amount of phenol present or the amount of imide present, is never reached. This can be attributed to the reaction of the z,6-dibromoquinonechloroimide with the alkali present. It is, therefore, evident that to obtain good results in the quantitative determination of phenol a considerable excess of the z,6-dibromoquinonechloroimide must be present and, vice versa, in standardizing 2,6-dibromoquinonechloroimide solutions a large excess of phenol must be present. In the latter case the maximum indophenol formation will take place before the amount of imide decomposed by the alkali becomes material.
Summary The reaction between 2,6-dibromoquinonechloroimideand phenol, producing 2,6-dibromobenzenoneindophenolJhas been quantitatively studied at various pH ranges in the alkaline region and the reaction velocity measured spectrophotometrically. This reaction has been found to take place in the alkaline region between 2,6-dibromoquinonechloroimide and the sodium phenolate and in buffered solutions with a large excess of phenol the reaction is pseudo monomolecular and is expressed by the equation 2.3026 tba
k = __ log
-.a a-x
When the concentrations of the phenol and the imide are nearly equal, the same value for the constant is obtained by calcu1ations:according to a reaction of the second order z 3026 b(a- x) kz = A log ta (a-b) a(b- x) The average value of k is 1.63 X io4 a t
20'.
PHENOL TESTS
I077
The production of this indophenol, giving a blue color in alkaline solutions, has been found to be a very delicate testafor phenol and the reaction velocity has been measured at phenol concentrations below the limit of many of the heretofore known phenol tests. The reaction has a considerable temperature coefficient and therefore the studies were carried on in specially constructed apparatus to maintain this factor constant. ~,6-Dibr0in0quin0nechloroimidedecomposes in alkaline solution and a study of the rate of this decomposition in various alkaline buffers shows that the reaction is pseudo monomolecular, the rate varying greatly with the hydroxyl ion concentration, and can be presented by the equation 2.3026 a ki = -log tc a-x in which t = time in minutes, c = OH, a = 2,6-dibromoquinonechloroimide and x the amount reacting in time “t”. The average of seven reaction velocity experiments indicates the value of kl to be 6.13 X io3. I t is indicated that 2,6-dibromoquinoneoxime is the primary product of the reaction but that other colored compounds are formed. 2,6-Dibromoquinoneoxime has been prepared and studied. The solubility in water at 20’ is 8.5 X IO-^ molar, and the pKa value is 4.6. The absorption spectra of ~,6-dibromoquinoneoxime, and of alkaline solutions of 2,6-dibromoquinonechloroimidearc charted. X
TABLE XXT’II Reaction velocity of z,6-Dibromoquinonechloroimide in alkaline buffer solutions. a = z,6-dibromoquinonechloroimide= 1.8 X IO-^ molar c = OH- at pH 8.5 t = time in minutes; x = transformed in “ t ” ; T = transmittancy The maximum absorption or final value of T = 0.31 Spectrophotometric readings at 610 mp; IO cm tubes Temperature 20’ Y
t 2
4
6 9 I2 15 21
27
T 0.97 .9I
-log T
times
0.013
0.046
,041
105
,145
a-x times 1 0 5
kl
1,754
(0.013)
1.65j I. 5 7 0
.86
.06j
,230
,815 ‘78
,087
1,492
t
02I
k2 (4.1 X
,023
7.2
. 0 2I
6.6
. I08
,308 ,382
1.418
,020
.I 2 5
6.3
.75
’
443
1.357
.68 .66
,167
’
590
1.210
,019 (.017)
,180
‘637
1.163
:s. 3)
Average
-0.02 I
103)
6.6
6.0
-
6.5 X
103
1078
H. D. GIBBS
It is shown that solutions of phenol and solutions of 2,6-dibromoquinonechloroimide can be accurately standardized by means of measurements of this indophenol formation. The experimental results are recorded in 34 tables. Two figures and a bibliography are included. TABLE XXVIII Reaction velocity of 2,6-Dibromoquinonechloroimide in alkaline buffer solutions. a = 2,6-dibromoquinonechloroimide= 1.8 X IO-^ molar c = OH- a t pH 9.0 t = time in minutes; x = “a” transformed in “t”; T = transmittancy The maximum absorption or final value of T = 0.31 Spectrophotometric readings at 610 mp; I O cm tubes Temperature 20’ X
t 2
4 6 9 I3
a-X
T 0.86 .76
ki
ki
0.065
0.230
1.570
(0.0684)
. I19
.42I
1.379
,0628
.70
’155 ,
.548 ,686 ,916
1.252
.64
,0605 ,0533 ,0547
(6.8) X 6.3 6.1 5.3
‘55
-log T
I94 .260
times
105
times
106
1.114 0.884 Average
0.0578
IO’
5.5 5.8 X
103
TABLE XXIX Reaction velocity of ~,6-dibromoquinonechloroimidein alkaline buffer solutions. a = 2,6-dibromoquinonechloroimide = 1.8 X IO+ molar c = OH- at pH 9.5 t = time in minutes; x = “a” transformed in ‘it”; T = transmittancy The maximum absorption or final value of T = 0.3 I Spectrophotometric readings a t 610 m p ; I O cm tubes Temperature 20’ X
t
T
-log T
2
0.66 .56 .46 .38 .3I
0.180
4 6 9 I2
.252
,337 .4zo .so9
times
IO$
0.637 .891 1.192 1.486 -
a-x times rob
ki
163 0,909
0.218
,608
,181 ’ I94
I.
,314
Average
,
I71
o 191
kn
6.9 X
10%~
5.4 5.8
6. I 6 o X 1oa
PHEFOL TESTS
T.4BLE
‘079
Xxx
Reaction velocity of 2,6-dibromoquinonechloroimidein alkaline buffer solutions. a = ~,6-dibromoquinonechloroimide= 1.8 X IO-^ molar c = OH- at pH 10.0 t = time in minutes; x = “a” transformed in “t”; T = transmittancy The maximum absorption or final value of T = 0.3 I Spectrophotometric readings at 610 mp; I O cm tubes Temperature 20’ X
t
T
2
0.j8
1 6 I2
43 .35 ’
.31
-log T
0,237 ,367 ,456 ,509
times
105
0.83 1.30 1.61 -
a --x times
104
ki
9.7
0.31
j.0
.32
.37
1.9 -
-
Average
0,33
kz
3.1 X
103
3.2 3.7
3.3 X
103
TABLE XXSI Reaction velocity of 2 ,6-dibromoquinonechloroimide in alkaline buffer solutions. a = ~,6-dibromoquinonechloroimide= 1.37X IO-: molar c = OH- a t pH 9.129 t = time in minutes; x = “a” transformed in “ t ” ; T = transmittancy The maximum absorption or final value of T = 0.2 j Spectrophotometric readings a t 610mp. IO cm tubes Temperature 20’ X
t
T
-log T
times
106
a--x times 1 0 5
0.097 ,181
0.22
1.15
0.41
4
0.80 .66 .62
.208
0.47
0.96 0.90
6
‘52
,284
7.5
.42
8
.39
,377 ,409
0.65 0.86 0.93
2
3.5
0.72
0.j I
0.41
Average
0.104
7.75 x103
1080
H. D. GIBBS
TABLE XXXII Reaction velocity of ~,6-dibromoquinonechloroimide in alkaline buffer solutions. a = ~,6-dibromoquinonechloroimide = 1.37 X IO-^ molar c = OH- at pH 9.129 t = time in minutes; x = “a” transformed in “t”; T = transmittanc:. The maximum absorption or final value of T = 0.05 Spectrophotometric readings at 5 2 0 mp. I O cm tubes Temperature 20’ a -x times 1 0 5
ki
1.12
(0,088)
.32
1.05
.089
48
.89 .86
,095 ‘093
X
t
T
2.33 3 4.5 5 6.33 7
0.58 ’ 50
8.33
‘14
.35 .33 .24 .2I
-log T
times
0.237
105
0.25
.301
,456 ,482 .620 .678 ,854
’
‘51
kZ
(6.50)X IO^ 6.56 7.08 6.90
.65
.72
. IO1
7.52
.7’ ,90
.66
,104)
(7.73)
.47
-
Average
.09j
7.01 X
103
TABLE XXXIII Reaction velocity of ~,6-dibromoquinonechloroimidein alkaline buffer solutions. a = 2,6dibromoquinonechloroimide = 1.37 X IO-^ molar a = OH- a t pH 9.528 = 3.39 X 10-~ molar t = time in minutes; x = “a” transformed in “t”; T = transmittancy The maximum absorption or final value of T = 0.05 Spectrophoometric readings at 5 2 0 mp. I O cm tubes Temperature 20’ a-x
X
t 2
3.25 4 5.33
T
-log T
times
105
0.35
0.456
0.48
.21
,678 .796 ,936
.71
6
.16 ,116 .094
1.027
7.33
.08
1.097
.84 .99 ~
times
105
0.89 .66
ki
kz
0.916
6.37 X
.225
6.63 6.99 7 ’ 09
53
,237
.38
,240
’
..?verage
0 . 2 29
6.77 X
103
103
PHENOL TESTS
I081
TABLE XXXIV Summary of reaction velocity data of 2,6-dibromoquinonechloroimidein alkaline buffer solutions from Tables XXVII-XXXIII, inclusive. Table
pH
KO.
27
28 29 30 31 32 33
Readings a t wave length
8.j 9.0 9.5
610
10.0
610
9.129
9.129 9.528
610 610
biolaritv of 2,6-dbromoquinonechloroimide
1.8 X IO-^ 1.8
1
-2.3026 log a k 2 =2.3026 -log -~t a-x tc
6.5 X 5.8 6.0 3.3 7.75
0.021
,058
.I91 .33
j20
1.8 1.8 1.37 1.37
.09s
7.01
j20
1.37
I229
6.77
610
,104
Average
6.2
X
a-x
103
103
BIBLIOGRAPHY B. Cohen, H . D. Gibbs, and W. M. Clark. Studies on oxidation-reduction. VI. A preliminary study of indo henols: (A) a dibromo substitution products of phenol indophenol; (B) substitutezindophenols of the ortho type; (C) miscellaneous, U. S. Public Health Re orts, Reprint No. 915,804 (1924). 0. Fischer unXE. Hepp. Ueber Dibromnitrosophenol. Ber., 21, 674 (1888). H. D. Gibbs. Phenol tests. I. A classification of the tests and a review of the literature. Chem. Reviews, 3, 291 (1926). H. D. Gibbs. Phenol tests. 11. Nitrous acid tests. The blillon and similar tests. Suectrophotometric investigations. J. Biol. Chem. 71,445 (1927 H.D. Gibbs. Phenol tests. 111. The indophenoi test. J. Biol. Chem., 72,649 (1927). E. Salm. Studien iiber Indikatoren. Z. physik. Chem.. 57,4 1 7 (1906). E. Salm. Die Verwendung der Indikatoren in der hlassanalyse. .Z..physik. Chem., 57, 485 (1907). W.Stenstrom and S . Goldsmith. Determinalion of the dissociation constants of phenol and the hvdroxyl group of tyrosine by mean of absorption measurements in the ultraviolet. J: Phvs. Chem., 30, 1683 1926). J. Walker. Vbe "die Beziehung zwischen den Dissociationskonstanten schwacher Sauren und der Hydrolyse ihrer Alkalisalze. 2. physik. Chem., 32,137(1900). J. Walker and W. Cormack. The dissociation constants of very weak acids. 3. Chem. SOC.,77,5 (1900).
.
