BASICITY OF AMINO ACIDSIN D20
Nov., 1960
1653
THE BASICITY OF AMINO ACIDS IN DzO1 BY H.H. HYMAN,ARLENEKAGANOVE AND J. J. KATZ Argonne National Laboratory, Argonne, Illinois Receiucd April 14, 1060
The basicity of a number of amino acids in DzO has been measured by a spectrophotometric method. acids, the apparent ionization constant is approximately 0.5 p K unit higher in DzO than in H20.
One by-product of the nuclear energy program has been the availability of heavy water in essentially unlimited quantities a t moderate cost. This has stimulated a good deal of research, particularly in biological systems where there are important qualitative differences between ordinary and heavy water.2 The striking differences which show up in these biological experiments are believed to be the cumulative effects of many quantitative differences which may be unraveled in vitro. The general problem of acid-base interaction is of major importance in studying biological systems. The changes in equilibrium constants that occur when deuterium ion transfer is substituted for proton transfer may be measured in relatively simple experiments and seem to be important in understanding more complicated phenomena. A simple case of interest is the ionization constant of an amino acid. The acid ionization is usually representated as an equilibrium involving a cation and a dipolar ion.
For simple amino
strength measurement in both H 2 0and D20,it was decided to investigate a few amino acid systems by an indicator method. Experimental
The experimental procedure employed followed the classical procedure described by Sorensen.7 The pH of an amino acid-mineral acid mixture is measured by means of an indicator. The concentration of hydrogen ions corresponding to the indicator color is assumed to be the concentration of strong acid which would produce the same indicator spectrum. The difference between the stoichiometric concentration of strong acid in the system and the measured concentration of hydrogen ion is the concentration of amino acid in the cationic form. I n the H 2 0 system, pH is probably best defined as -log CHIo+ - log y * , where y* is the activity coefficient appropriate to the solution. The value of the activity coefficient may be either calculated, usually from a modified DebyeHuckel equation using a reasonable estimate of ionic size, or measured for similar solutions. For measurements made a t a total ionic strength of 0.11 M , -log T* % 0.10 and this value is used for all solutions discussed in this paper.* All the solutions studied were prepared by mixing stock solutions of amino acid and perchloric acid in sodium perchlorate. The sodium perchlorate concentration was 0.1 M and the perchloric acid or amino acid concentration +HaNRCOOHa0 +H3NRCOOH HzO 0.01 M , so that for all mixtures, the amino acid plus perWhile data have been tabulated for many amino chloric acid concentration totaled 0.01 M and the ionic acids in H20,3measurementsin DzOare very limited. strengthO.11 M . A number of indicators were investigated. For the pH Schwarzenbach, Epprecht and Erlenmeyer4stud- region of interest, thymol blue WM found most satisfactory ied the ionization constants of a number of weak and the data reported were obtained with this indicator. acids in DzO, and included glycine (H~NCHZ- Eastman Kodak thymol blue indicator was used without COOH). They used a deuterium gas electrode, a purification. pH measurements were also made using a conventional light-water saturated calomel electrode, and ob- glass electrode in both HzO and D20 systems. I n D10 tained 2.752 for the pK of glycine in 99.6y0 DzO as systems, glass electrodes were conditioned by immersion compared with 2.350 for an analogous measure- for periods up to one week in D20 buffer solutions with no change in the observed potentials. I n general, the glass ment in H2O. determinations of pH agreed with the indicator The ionization of weak acids in DzO was dis- electrode values within experimental uncertainties. cussed bv Sch~arzenbach~ and Rule and LaMers The sodium perchlorate and perchloric acid were reagent who objehed to liquid junctions in the cells chosen grade chemicals. The amino acids were the best readily by Schwarzenbach. There appears to have been no available commercial grades. All were used without addipurification. The D20 was redistilled water availstudy of amino acid basicity constants since larger tional able in this Laboratory containing more than 99.6 atom % quantities of D20became available, and macro tech- deuterium. All spectrophotometric measurements reniques could be employed. IVhile electrode meas- ported were made using a PerkiniElmer Spectracord in urements have been most frequently employed a temperature controlled room a t 25
+
+
+
.
for determination of basicity constants, indicator methods using modern spectrophotometry are capable of comparable precision. While there are uncertainties in the interpretation of any acid (1) Based on work performed under the auspices of the U. S. Atomic Energy Commission. (2) J. J. Kstz, H. L. Crespi, A. J. Finkel, R. J. Hasterlik, J. F. Thomson, W. Lester, Jr., W. Chorney, N. Scully, R. L. Shaffer and Sung Hwang Sun, Proceedings of Second International Conference on Peaceful Usea of Atomic Energy, Vol. 25, p. 173, 1958; J. J. Katz, H. L. Crespi. R. J. Hasterlik. J. F. Thomson and A. J. Finkel, J . Nat. Cancer Inst., 18, 641 (Mar. 1957). (3) E. J. Cohn and J. T. Edsall, "Proteins, Amino Acids and Peptides," A. C . 9. Monograph Series No. 90, Reinhold Publ. Corp., New York. N . Y.. 1943. (4) G. Schwaraenbach, A. Epprecht and H. Erlenmeyer, Helu. ehim. acta, 19, 1292 (1936). (5) G.Schwsrsenbach. 2. Eleklrochem.. 44,46 (1938). (6) C. K.Rule and V. K. LaiVer, J . Am, Chem. Soc., 60,1974 (1938).
0bservations The apparent pH measurements for a series of mixtures of glycine and perchloric acid in a constant ionic strength solution containing sodium perchlorate are given for both H2O and D20 systems in Table I. As mentioned above, these data were obtained by spectrophotometric observations using thymol blue indicator. Thymol blue has an absorption maximum at 560 mp in acid solution with a molar absorption (A,) of 4.04 x 104 and an absorption maximum a t 440 mp in basic solutions with A, = 1.51 X lo4. At 440 mp, the A, is about 6 X los in acid solution, (7) 8. P. L. Sorensen, 2. Biochem., 21, 131 (1909). (8) Symposium on pH Measurement, ASTM Special Technical Publication No. 190, 1956.
H. H. HYMAS,A. I~AGAXOVE ASD J. J. KATZ
1654
while the A , for absorption at 560 mp is well under lo3 in basic solution. The extinction coefficients are the same in H2O and D2O within the precision of the measurements. For a given solution, the fraction of indicator found in the acid form based on the molar absorption at 560 mp yields the most accurate measure of the pH. APP.4RENT
Ratio G/A
pH
OF
TABLE I GLYCINE-PERCHLORIC ACID MIXTURE IN 0.1 M NaC104
Concn. (mole/l.) Glyc. Acid
0
PD
Apparent pH D20
Ha0
in Dz0
0.01
2.10 1.70 2.105 2.28 1.90 2.30 .006 2.46 2.16 2.56 .005 2.33 2.73 2.57 2.49 2.89 .004 2.70 2.81 3.21 .0028 3.00 ,002 3.07 3.47 3.16 .01 5.6 5.6 6.0 a In D2O the pD of a 0.01 M DC10, solution is assumed to equal the p19 of a 0.01 M He104 solution in HzO. 0
,25 .667 1.00 1.667 2.57 4.0
.002 .004 ,005 .006 ,0072 ,008
.008
For indicator measurements pH Ei pK - log CIH+/CI. If the pH of a 0.1 M NaC104-0.01 M HC104 solution in HzO is taken as 2.10, the pK for thymol blue is 1.65 and the apparent pH of a 0.1 M NaC104-0.01 M DCl04 solution in D20 is 1.70. The apparent pH of a perchloric acid solution in D20 is 0.4 unit less than a similar solution in H20. As shown in Table I, this difference decreases as the amino acid replaces perchloric acid. For a nlvcine-sodium Derchlorate solution. with no added o u perchlcric acid,*the apparent pH is the same in HzO and D2O. In solutions containing only dilute perchloric acid and sodium perchlorate, the concentration of H30+ in H 2 0 solutions cannot differ substantially from the concentration of D30+ in D20. In both cases the equilibrium must favor essentially complete hydrogen ion transfer to the solvent from perchloric acid. Therefore, in the pH region where this indicator is useful, the pK of thymol blue is 0.4 unit higher in D 2 0than in H20; ie., 2.05. The pD of a D 2 0 solution in the acid strength region where thymol blue is useful may be taken therefore as 2.05 - log CIH+/CI. In fact, the glass electrode gives a quantitatively similar measurement. A number of other indicators show a comparable though not always identical difference between the apparent pH when comparing H2O and D 2 0 solutions containing an equal concentration of strong acid.g To calculate the ionization constant in D20, therefore, the pD of 0.4. any solution is taken as the apparent pH Sorensen6 used an ionization constant for the amino acids defined by the equation
Vol. 64
constant for the amino acid by a term depending on the ratio of the activity coefficients of the dipolar and cationic forms of the amino acid. In H 2 0 the difference between ~ K and A the ~ K Adetermined ' by the Sorensen method appears to be about 0.1 unit at 0.1 M and the difference between this correction term in H 2 0 and DzO must be very small indeed. Therefore no attempt was made to obtain the thermodynamic ionization constant as might be done by measurement at lower concentrations and extrapolation to infinite dilution. Table I1 shows the pD of equimolar mixtures of five amino acids with perchloric acid in D 2 0 and the ~ K A values ' for these acids. The ~ K A ' sare calculated by the method described above from measurements at a number of acid/amino acid ratios as shown in Table I for glycine. TABLE I1 IONIZATION OF SOME AMINOACIDSIN H20 AND D20 AT 25" pHa
pDb
~KA'c
Ad
Amino acid HzO D20 H20 DzO Glycine 2.57 2.73 2.24 2.78 0.54 Alanine 2.57 2.73 2.24 2.73 .49 Phgnylalanine 2.53 2.65 2.02 2.56 .54 Threonine 2.55 2.66 2.10 2.59 .49 Glutamic acid 2.52 2.68 1.96 2.59 .63 a Concn. amino acid 0.05 M = G; Concn. HC104 0.05 M = A; Concn. NaC104 0.1 M; Thymol blue indicator pK = 1.65. bConcn. amino acid 0.05 M; Concn. DCIOn 0.05 M ; Concn. NaCI04 0.1 M; Thymol blue indicator pK = 2.05. c p K ~ '= pH - log ( C / ( A - H + ) -1); H + = 1/ antilog (pH - 0.10). A = ~ K inA D2O - ~ K Ain' H20.
~
+
~ K A =' p H - log ((?/(A- H + ) - 1)
For the experiments reported here C is the concn. of amino acid (moles/l.) A is the concn. of added perchloric acid (moles/l.) H + i s the concn. of Hg0+ defined as l/antilog (pH
- 0.101
It is recognized that this ionization constant will differ from the true thermodynamic ionization (9) E. Hagfeldt and J. Bigeleisen, J. Am, Chem, Soc., 81, 15 (1960).
Conclusions A number of indicators commonly used for pH measurements, as well as the glass electrode, will show a lower apparent pH in D20 solutions containing a given D 3 0 + content than in H 2 0 solutions containing the equivalent H30+ concentration. This has been previously reported by Fischer and Potterlo and with improved accuracy by Hart." Quantitatively, the glass electrode and thymol blue indicator can be used in D20 in the pD region near 2 by adding 0.40 to the apparent pH to determine the pD. Hogfeldt and Bigeleisenghave extended the use of indicators in D2O to the Hammett type used in very strong acids, i.e., neutral molecules which are relatively weak proton acceptors. While they note a slight trend in favor of increasing A p K with decreasing acid strength of the protonated species, as predicted by Lewis and Schutz12and Halpern,13the effect is very much less than Rule and LaR4er6 believed. Hogfeldt and Bigeleiseng conclude that the type of acid is more important than the acid strength in so far as the difference of ionization constants between H2O and D2O is concerned. For simple amino acids, the apparent ionization constant is approximately 0.5 pK unit higher in (10) R. B. Fischer and R. A. Potter, MDDC-715, Sept. 1945. (11) R. G. Hart, C R E 423, Natl. Res. Council of Canada, June 1949. (12) G. N. Lewis and P. W. Schutz, J . An. Chem. Soc., 56, 1913 (1934). (13) 0.Halpern, J. Chem. Phya., 3, 456 (1935).
Nov., 1960
SPECTRA OF COMPLEXES BETWEEN KETONES AND CALCIUM ~IOSTMORILLONITE
D,O than in HzO. Glutamic acid has a second ionization and the slightly greater difference between p K in HzO and DzO may be due to a contribution from this second ionization. The difference of 0.5 pK unit is equal to the difference in pK for the ionization of acetic acid in light and A the acetic heavy water. Although the ~ K for acid ionization is 4.7 rather than -2, the proton transfer mechanism is the same for each of the amino acids as for the carboxylic acid. This is in agreement with the conclusion mentioned above that the most important single criterion in determining the difference in pK in HzO and DzO
1655
systems is the nature of the group to which the proton is attached. Unfortunately, this A p K is so close to 0.5 for all equilibria where valid measurements exist, that a systematic study of the differences which can be attributed to the specific bonds affected seems remote. When deuterium is substituted for hydrogen in an acid base interaction, the change in equilibrium constant measures a difference in the relative binding power of two bases for hydrogen and deuterium. There appears to be no simple way to determine how much of this difference between the two bases should be attributed to either base.
ISFRARED STUDIES OF SOME COMPLEXES BETWEEN KETONES AND CALCIUM M09TMORILLONITE. CLAY-ORGANIC STUDIES. PART IIIl BY
LOWELL G. TENSMEYER,~ REINHARD
w.HOFFMANN
AND
G. lv. BRINDLEY
Contribution N o . 59-87 f r o m the College of Mineral Industries, Dept. of Ceramic Technology, The Pennsylvania State University, University Park, Pennsylvania Received April 20,1960
Infrared spectra of one- and two-layer complexes of 2,5-hexanedione and 2,5,8-nonanetrione with calcium montmorillonite have been obtained by differential techniques. The samples were prepared by evaporation of clay suspensions with and without ketones on AgC1-windows and glass plates. Infrared absorption spectra and X-ray diffraction patterns were taken of the resulting films. Infrared spectra were obtained also of solutions of the ketones of CC1, and CS2, of liquid 2,5-hexanedione and of solid 2,5,S-nonanetrione1 the latter presented to the beam by several techni ues. The following vibrational assignments are made for the unadsorbed ketones: 3005 cm.-' CHI stretching a to C=8; 2959 cm.-l CH2 asymmetric stretching; 2912 cm.-l CH2 symmetric stretching; 1724 cm.-' C=O stretching; 1412 cm.-l CH2 deformation CY to C=O; 1401 cm.-l CH2deformation coupled to C-C; 1365 cm.-l CH3 deformation LY to C=O; 1360 cm.-l CH2 and CH, deformation a to C=O. Upon adsorption, significant changes occur in the carbonyl stretching frequency and the methyl and methylene deformation frequencies. Spectra of the one-layer complexes of 2,5-hexanedione and 2,5,&nonanetrione are quite similar to that of solid 2,5,&nonanetrione, whereas the two-layer complexes show less similarity. These data are interpreted in terms of a highly ordered one-layer complex and a decrease in order upon introduction of a second layer.
1. Introduction This investigation forms part of a program for studying the adsorption of organic molecules on clay minerals. A previous paper' gave results for the adsorption of neutral organic molecules from aqueous solutions on calcium montmorillonite. With a view to obtaining more detailed information on the state of the adsorbed molecules this study has been made of the infrared absorption spectra of two organic-clay complexes, chosen from among the organic materials examined in the previous work. The compounds selected were 2,Ei-hexanedione and 2,5,8-nonanetrione. Structurally similar to each other, they are adsorbed to differing extents on montmorillonite from aqueous solutions. The most interesting parts of their spectra, the C=O and C-H vibrations, have frequencies not obscured by the vibrations of the montmorillonite lattice. The interaction between clay mineral surface and adsorbed organic molecules would be expected to produce frequency and intensity changes in the spectrum of the adsorbed molecule. Intensity changes may also arise from different orientations (1) Part 11. R. W. Hoffmann and G . W. Brindley, ".4dsorption of Non-ionic Aliphatic Molecules from Aqueous Solutiona on Montrnorillonite," snbmitted to Geochzm. Cosmochzm Acta. (2) Temporary Research Fellow a t the Pennsylpania State Univerm t ) during Summer 1959, now a t Llnde Company, Indianapolis, Ind.
of the organic molecules on the clay surface and from different orientations of the clay particles with respect to the infrared beam. To interpret the spectra of the adsorbed molecules profitably and also to detect unadsorbed ketone in a clay-organic complex, one must have available the frequencies, frequency assignments and the extinction coefficients for the unadsorbed compound, at least for the spectral regions not obscured by infrared absorption of the clay. Strictly considered, only the frequencies of a gas at low pressure can be considered unperturbed, especially for molecules containing highly polar bonds. The spectrum of a dilute solution of a compound in a non-polar solvent approaches that of the gas, and dilute solution spectra are used as references in the present work, as well as the spectra of liquid 2,5-hexanedione and of solid 2,5,8-nonanetrione. 2. Experimental I. Materials.-( a) 2,BHexanedione was obtained from Aldrich Chemicals Co. and was redistilled before use; b.p. 82.2-82.7' (17 mm.). (b) 2,5,8-Nonanetrione was prepared in this laboratory as white plate-like and prismatic crystals, m.p. 55'. (5Methylfurfury1)-acetone was prepared according to Alder and Schmidt.3 The reaction was by no means spontaneous (3) K. Alder and
(1943).
C. H. Schmidt, Ber. deut. chem. Ges., 16B, 183
