V O L U M E 2 4 , NO. 6, J U N E 1 9 5 2
969 critical and difficult to reproduce. Because of this, the equipment would be unsatisfactory for quantitative determinations. This equipment cannot be used in conjunction with a continuously recording device without major changes in the design. Although the work described in this paper was conducted with a Thermocap relay, other commercially available instruments ( 3 ) would undoubtedly perform equally well or better; certainly an instrument specifically designed as a dielectric indicator would perform more satisfactorily. I t is hoped that this work will stimulate further investigations along these lines.
+ TIME IN HOURS
)OO
Figure 13. Meter Reading as Function of Time with Benzene Flowing through Cell A
(1)
c2)
thnt it is time t o change receivers, If desired, a bell or buzzer can be actuated by this relay, or a valve controlling the effluent can be closed. Although the instrument was satisfactorily stable over a clay’s run, the reproducibility from day t o day was less than desired. This is primarily because the position of the leads can never be completely reproduced and the condenser D adjustment is very
(3) (4)
(5)
LITERATURE CITED
Cassidy, H. G., “Adsorption and Chromxctography,”Kew ’iork, Interscience Publishers, 1951. Remick, 8.E., “Electronic Interpretations of Organic Chernistry,” New York, John Wiley & Sons, 1949. Sargent, E. H., and Co., Chicago, “Chemical Oscillometry.” 1951. TroitskiI, G., Biokhimiyu, 5, 375-80 (1940). Zechmeister, L., “Progress in Chromatography, 1938-1947,” New York, John W l e y & Sons, 1950.
RECEIVED for review October 1, 1951. Accepted March 15, 1952. Presented before t h e All-Day Chemical Conference sponsored b y t h e Chicago S e c t i o n , A M E R I c . ~CHEMICAL ~ SOCIETY,November 23, 1951.
Manometric Determination of Dissociation Constants of Phenols OSCAR G4WRON, M.4RJORIE DUGGAN, AND CHESTER J. GRELECKI D e p a r t m e n t of C h e m i s t r y , Duqccesne Cnit‘ersity, Pittsburgh, Pa. In connection with an investigation of the effects of substituents on the enzymatic hydrolysis of phenyl acetate, ~ K values A of the various phenol moieties at 38’ C. and in 8Yo dioxane were needed. These values were determined in a simple and fairly precise fashion by manometric measurement in the Warburg apparatus of the carbon dioxide evolved from a bicarbonatecarbon dioxide buffer on the addition of small amounts of the phenol in question. The method should be applicable to the determination of pK.4 values of weak acids and bases in the __ ~ K range A of approximately 6 to 11.
I
N CONNECTION with an investigation on the effects of substituents on the enzyme-ratalyaed hydrolysis of phenyl acetate, K A values of the various phenols were needed for application of a correction factor to rates of hydrolysis measured by rarbon dioxide evolution in the Warburg apparatus. Although the literature contains many referencrs to dissociation constants of substituted phenols. the reaction conditions, 870 by volume of dioxane and 38” C., employed in the above study, necessitated separate measurement of dissociation constants. This communication describes a simple manometric method for determining dissociation constants, applied to substituted phenols. These determinations were carried out in the FVarburg apparatus by measuring the amount of carbon d i o d e evolved from a bicarbonate-carbon dioxide buffer system on the addition of relatively small amounts of the phenol in question. The equilibrium for this reaction may be represented as
HA
+ HCO3-
.4-
+ HpO + CO?
(1)
H here H-4 and A - represent, respectively, undissociated phenol and phenolate ion. The carbon dioxide evolved is, therefore, equal to the amount of phenolate ion formed and the pIi of the phenol may be calculated from the relationship
where concentrations are elpressed in molee per liter and the subscripts o and e are initial m d evolved, respectively. The pK may also be calculated in a somewhat less evact fashion (a difference of several hundredths of a pK unit) by disregarding the evolved carbon diovide in the second term of the above vxpression, as it is small in comparison to the initial bicarbonate and rarbon dioxide concentrations. The measurements reported hetein were made a t pH 7.64 a t 38” and 7.Fj6 a t 25’ and arr limited a t these pH’s by cwnsiderations of phenol ionization, phenol solubility, and precision (average deviation from mean of duplicate manometer readings is 1.0 mm.) to phenols of appioximate p& range 6 to 10. This range ran lie somewhat evtencted bv ram! ing out the measurements a t lower or higher pH. No attempt was made in this investigation to vary the ionic strength of the medium, SO that the dissociation constants reported are apparent one-. WATER1 4 LS
Dioxane was purified ( 1 1 ) and freed from peroxides by treatment n i t h stannous chloride, followed by distillation over sodium. Solutions requiring dioxane a ere made up daily. Bicarbonate solutions were prepared from dry, c P sodium bicarbonate. The phenols used were either purchased or synthesized and purified until physical constants agreed with those in the literature.
970 Table I.
ANALYTICAL CHEMISTRY Measured Dissociation Constants and Literature Values Compound
Phenol o-Nitrophenol m-Nitrophenol o-Chlorophenol m-Chlorophenol p-Chlorophenol o-Bromophenol p-Bromophenol o-Cresol m-Cresol p-Cresol Catechol ~ D K I ) Resorcinoi’(pK1) Hydroquinone (pKi) o-Methoxyphenol m-hlethoxyphenol p-hlethoxyphenol o-Hydroxybenzaldehyde p-Hydroxybenzaldehyde
25’0
38’b
9.95 7.33 8.33 8.34 8.94 9.29 8.40
9.85 7.20 8.14
c.
-Log K Literaturec
c.
IT
f
9.95 7.23 8.35 8.50 9.02 9.38 8.42 9.25 10.20 10.01 10.17 9.12 9.15 9.91 9.93
8.86 9.14 8.22 9.07 10.06 9.76 9.88 9.14 9.14 9.73 9.58 9.33 9.75 10.16 7.95 7.52 7.66
n water. * I8% b y volume, dioxane.
a
.4t 25‘ except where otherwise noted.
‘
PROCEDURE
Measurements. All measurements were carried out in a rotary Warburg apparatus employing conventional flasks of approximately 15-ml. capacity and manometer fluid of density 1.033. Into the main compartment of the flasks were pipetted 2.20 ml. of distilled water and 0.30 ml. of 0.2500 iZI sodium bicarbonate solution. Into the side arm 0.50 ml. of the various phenol solutions was pipetted. The phenol solutions contained up to several tenths of a gram of phenol per 50 ml., the amounts varying with the acidity of the phenol. For the measurements in dioxane, 8% by volume dioxane solutions were used. After a 10-minute gassing period with 5% carbon dioxide-95% nitrogen and a 20-minute equilibration period a t the required temperature, pressures were equalized and flask contents mixed. Pressure readings were then taken a t suitable intervals until equilibrium was established, usually 60 to 90 minutes after mixing. (The presence of carbonic anhydrase would enable equilibrium to be reached more rapidly.) Blank runs (minus phenol j were included for correction purposes. All rune were made in duplicate. The pH’s of 0.0250 M bicarbonate in equilibrium with a partial pressure of 5% carbon dioxide at 25” and 38” are, respectively, 7.56 and 7.64. The presence of 8% dioxane had no effect on the pH as determined with a glass electrode. Calculations. Conversion of pressure changes due to evolved carbon dioxide to moles per liter, in both the gas and liquid phases, and calculation of the increase in concentration (liquid phase) of the carbon dioxide were carried out by the usual methods (IO). Values of pK’coz, the thermodynamic dissociation constant for the first ionization of carbonic acid, are those of Shedlovsky and MacInnes (9).
L
I
5
I
lo
I
15 / . lMolts
I
2 Cot $Olved
I
23
I
;Io
I
35
Figure 1. Relationship between ApK and Carbon Dioxide Evolved at Different Levels of Added Phenol A 10 pmoles. 0 20 pmolefi. X 30 pmoles. 0 40 p m o l e s .
changes in bicarbonate and carbon dioxide concentrations have been neglected. Figure 1 represents a plot of ApK us. carbon dioxide for several levels of phenol concentration. From the curves it is a parent that ApK is a minimum at CO, = 1/2 added phenol and t f a t the minimal ApK decreases with increasing amounts of added phenol; a t 10 micromoles of phenol it is 0.017 and at 40 micromoles of phenol, 0.004 pK unit. If 6 micromoles of carbon dioxide evolved is accepted as a convenient upper level for the manometers, working at added phenol levels greater than 30 micromoles reduces the ApK to 0.01 and less. I n this investigation measurements were made at 10 to 20 micromoles of added phenol and carbon dioxide evolutions of 1 to 3 micromoles. The error in duplicate determinations is, therefore, of the magnitude of 0.015 to 0.045 pK unit and the average deviation from the mean is of the magnitude of 0.008 to 0.023 pK unit. LITERATURE C I T E D
S. K. K., J . Indian Inst. Sci., 21A, 417 (1938). (2) Ibid., 23A, 99 (1941). (3) Boyd, D. R., J . Chem. SOC.,1915, 1538. (4) Fletcher, W.H., J . Am. Chem. SOC.,68,2726 (1946). (1) Abichandani, C. T., and Jatkar,
RESULTS
Table I lists the dissociation constants obtained in this investigation for the various phenols in 8% dioxane a t 38” and at an ionic strength of 0.025. For comparison purposes a number of constants determined in water at 25” and a t an ionic strength of 0.025 are included. When the latter are compared with carefully determined thermodynamic dissociation constants (6j agreement is seen to be fair, the average difference, not taking into account the sign of the difference, for these seven examples being 0.07 pK unit. With one exception (0-nitrophenol j the pK’s determined at p = 0.025 are lower than the quoted thermodynamic constants and it is possible these differences would be smaller if comparisons a t the same ionic strength were made. An estimate of the errors involved in the manometric method may be obtained as follows: For a difference of 0.1 micromole (2.24 mm., flask constant = 1) between duplicate manometer readings, - , it can be s h o m that
0.lA = 2.3X(A - X )
(3)
where pK is the differencein the pK, A is the micromoles of added phenol, X is the micromoles of carbon dioxide evolved, and
(5) Hantzsch, A.,Ber., 32, 3066 (1899). (6) Judson, C. &I., and Kilpatrick, SI., J . Am. Chem. Soc., 71, 3110 (1949). (7) Lunden, H., 2. physik. Chem., 70,249 (1910). ( 8 ) Pauly, H., Schubel, K., and Lockemann, D., Ann., 383, 308 (1911). (9) Shedlovsky, T., and MacInnes, D. A., J . Am. Chem. SOC.,57, 1683 (1935).
(IO) Umbreit, W. W., Burris, R. H., and Staufler, J. F., “Manometric Techniques and Tissue Metabolism,” Chap. I. 111, Minneapolis, Minn., Burgess Publishing Co., 1949. (11) Vogel, A,, “Practical Organic Chemistry,” p. 175, New Tork, Longmans, Green & Co., 1948. RECEIVEDfor review January 25, 1952. Accepted April 18, 1952. Presented in part before t h e Division of Biological Chemistry a t the 118th Meeting of the AMERICAN CHEXICALSOCIETY, Chicago, Ill. rlbstracted in part from the master’s thesis submitted b y Marjorie Duggan to Duquesne University. Work supported in part by a Frederick Gardner Cottrell Grant of t h e Research Gorp.
