4130
ROBERT F. STEINER AND EDWARD P. KIRBY
The Interaction of the Ground and Excited States of Indole Derivatives with Electron Scavengers1
by Robert F. Steiner and Edward P. Kirby Laboratory of Physical Biochemistry, Naval Medical Research Institute, National Naval Medical Center, Bethesda, Maryland (Received A p r i l 1, 1969)
20014
The quantum yields and fluorescentlifetimes of a series of indole derivatives are reduced in the presence of a wide range of substances which are efficient scavengers of hydrated electrons. Deactivation of the excited state of the indole ring, which is solvated in polar or polarizable solvents, requires a direct collisional interaction with the scavenger, One possible mechanism for quenching is by abstraction of an electron from the excited indole by the scavenger although other mechanisms cannot be excluded. At higher concentrations of added quencher, ground state complexes of the scavenger with the indole may be formed in some cases.
Introduction
The present paper is concerned with the intermolecular quenching of indole derivatives and with the I n earlier investigations by this and other laborabearing which this might have upon the intramolecular tories, the fluorescence properties of many indole and other nonradiative quenching processes which derivatives have been found to be dependent upon the terminate the excited state. We have been particularly nature of the chemical modification, as well as upon the interested in the possibility of quenching by electron temperature and solvent composition.2a-6 Under a transfer. particular set of conditions, the quantum yield and The latter interest has led us to examine the effects excited lifetime of fluorescence depend upon the comupon the fluorescence of indole derivatives of a series of petition between the emission of fluorescent radiation compounds known t o be active electron ~cavengers.~J and any radiationless processes which deactivate the The rates of combination with free solvated electrons, excited state. The possible factors which can conusually produced by pulse radiolysis of water, have been tribute to radiationless deactivation include intradetermined for over 300 substances of very diverse molecular quenching by a group in chemical attachment chemical nature and provide an index of their relative to the indole, intermolecular quenching by an external molecule or ion, electron ejection to the ~ o l v e n t , ~ efficiency in electron capture.* In brief, all the electron scavengers examined have been found t o be quenchers “tunneling” from the excited to the ground state, and of fluorescence and quenching by these agents generally intersystem crossing to the triplet state. occurs primarily by interaction with the excited state. If all of the processes which deactivate the excited singlet are first order with respect to the excited state Experimental Section
+
Q = quantum yield = kt/(kf Zki) (1) where kf and the set of ki are the rate constants for emission of fluorescence and for the various radiationless deactivation processes, respectively. The fluorescence lifetime, T , is given by T
=
l/(kf
+ Zki)
(2)
or (3) For a series of tryptophan derivatives formed by substitution on the a-amino or a-carboxyl group, the approximate constancy of Q / r 2 & and of the spectral distribution of fluorescencezb indicate that the basic characteristics of the indole excited state are not greatly changed by substitution of the non-indole portion and that the variation in fluorescence parameters arises primarily from the nonradiative processes. Q/T
The Journal of Physical Chemistry
= km
Methods. Measurements of relative fluorescence intensity and of the spectral distribution of fluorescence were made with an Aminco-Bowman spectrofluorometer, equipped with a spectral compensator to correct (1) From Bureau of Medicine and Surgery, Navy Department, Research task MR005.06-0005. The opinions in this paper are those of the authors and do not necessarily reflect the views of the Navy Department or the naval service at large. (2) (a) I. Weinryb and R. F. Steiner, Biochemistry, 7, 2488 (1968); (b) R. W. Cowgill, Arch. Biocham. Biophys., 100, 36 (1963). (3) R.W.Cowgill, Biochim. Biophys. Acta, 75,272 (1963). (4) R.W.Cowgill, ibid., 133,6 (1967). (5) M. S. Walker, T. W. Bednar, and R. Lumry, J. Chem. Phys., 47, 1020 (1967). (6) J. A. Gally and G. M. Edelman, Biochim. Biophys. Acta, 60,499 (1962). (7) L.I. Grossweiner and H. I. Joschek, Advances in Chemistry Series No. 50, American Chemical Society, Washington, D. C., 1965, p 279. (8) M. Anbar, Advances in Chemistry Series No. 60, American Chemical Society, Washington, D. C., 1965, p 55.
.INTERACTION OF INDOLE DERIVATIVES WITH ELECTRON SCAVENGERS for the wavelength dependence of the instrument sensitivity. Energy-corrected spectra were obtained.2” If no change in the shape or wavelength position of the emission band occurred, the effects of additives upon the fluorescence intensity were assessed by measurements a t the wavelength of maximum emission. The intensity for each level of additive was compared with that of a control solution containing no additive but otherwise identical. All experiments were run a t room temperature. A correction was applied for any attenuation by absorption of the incident or emitted beam, as described elsewhere.9 If the correction for attenuation was large (>20%), the extent of quenching was also measured for excitation a t longer wavelengths. Measurements of absorbance a t a single wavelength were normally made with a Gilford 2000 spectrophotometer. Difference spectra were measured with the Cary 14 spectrophotometer, using a set of fused tandem quartz cells. Excited lifetimes were determined using the TRW system (TRW Instruments, El Segundo, Calif).2a*10 Materials. Purified preparations of tryptophan (Trp), indole, acetyl tryptophan (AcTrp), acetyl tryptophan amide (AcTrp NH2),and tryptophan ethyl ester (TrpEt) were purchased from Sigma and from Cyclo. No differences in properties were observed between preparations from the two sources. All other preparations were reagent grade. Glass redistilled water was used for the preparation of all aqueous solutions. Solvents were Matheson Coleman and Bell “Spectroquality” reagents.
Results Table I summarizes the effects upon the fluorescence of acetyl tryptophan of the electron scavengers examined. All of the more than two dozen molecules and ions with electron-capturing ability which were tested were found to show significant quenching activity, although their efficiencies varied widely. Table I includes the corresponding values of the rate constants for electron capturee8 Quenching by Cations. Hydrogen and deuterium ions quench the fluorescence of acetyl tryptophan amide to an equivalent degree below pH 2 (Table I). Since this derivative contains no site which ionizes in the pH range 0-12, the observed quenching cannot be due to protonation of the ground state and must reflect an interaction of hydrogen or deuterium ion with the excited state of indole.l1,l2 It is pertinent that H3+0 is a very efficient electron scavenger.8 The metallic cations C U , ~ Pb,2+ + Cd,2+ and Mn2+ are effective electron scavengers and efficient quenchers of the fluorescence of tryptophan derivatives (Table I). I n contrast, Naf, K+, Mg,2+ NH,+, and Ba2+ (supplied as the chlorides) are inactive both as electron scavengers (8) and as quenchers at concentrations up to 1M .
4131
Table I: Quenching of Acetyltryptophan in Water Molarity for 60%
Reagent
H+
D+ cu2 + Na fumarate Acrylamide
NOa-
I08 Acetone Pb2+ Cysteamine Histidine Chloroform Thiourea Cysteine Cda+ Proline Mn2+ Glycylglycine Arginine Glycine HzPO4Lysine A1anin e @-Alanine
quenchinga
0.02 0.02 0.026 0.028 0.035 0.038 0.06 0.07 0.08 0.09 0.09 0.10 0.10 0.17 0.37 0.41 0.52 0.58 0.67 1.1 1.2 1.3 2 2.2
r/n for 50% quenchingb
0.49 0.46
kec X 10-10
2.3
8
3 0.8 2
8 13 13 8 8 13 8 8 15 13 8 15 8 15 8 18 15 15 8 15 15 15
3 0.8 0.6 4 2
0.52
0.46
0.48
Ref
0.7 2 0.3 0.9 1 0.002 0.008 0.025 0.015 0.0008 0.15 0.002 0.005 0.0004
a The solvent is 0.1 M KC1,O.Ol M KOAc, pH 5.0, except in the cases of Cu2+and Mn2+, for which the pH is 4.50, and that of Pbz+, for which the solvent is 0.01 M KOAc, pH 4.50. For Cu2+, H +, D +, and Pb2+, acetyl tryptophan amide replaced acetyl tryptophan. hex = 295 m r ; ht = 350 mp. * T h e ratio of the excited lifetime a t 50% quenching to the value in the absence of quencher. 0 The second-order rate constant for electron capture.
Quenching by Anions. The anions NOS-, IO3-, and fumarate are highly efficient as both electron scavengers* and quenchers, while HzP04- (but not HP0d2-) is moderately active in both respects (Table I). The following anions, which are unreactive with solvated electrons8%la exhibited no quenching activity when supplied as the sodium or potassium salt: CNS-, CH3COO-, HC 00-, s04’-, and citrate. (Acetate and formate were tested a t pH 7.0, where the unionized species are absent. The second-order rate constant for the quenching of acetyl tryptophan by NOS- (as computed from eq 4 below) was 5.2 X lo9,which approaches that predicted for a diffusion-controlled reaction (6.5 X lo9) of an ion of this size. As shown in Table I, the excited lifetime (9) R. F. Steiner, J. Roth, and J. Robbins, J . Biol. Chem., 241,560 (1966). (10) R. F. Chen, G. G. Vurek, and N. Alexander, Science, 156, 949 (1967). (11) A.White, Biochem. J.,71,217 (1959). (12) G. Weber, in “Light and Life,” W. McElroy and G. Glass, Ed., Johns Hopkins Press, Baltimore, Md., 1961. (13) E. J. Hart, J. K. Thomas, and 5. Gordon, Rudiut. Res. Suppl., 4, 74 (1964). Volume 78, Number 18 December 1980
4132
ROBERTF. STEINERAND EDWARD P. KIRBY
is reduced to approximately the same extent as the fluorescence intensity for 5001, quenching. Since interaction of NO$- with the ground state to form a nonfluorescent complex would not alter the lifetime, this implies that NO,- interacts directly with the excited state. This model was confirmed by the failure of any significant difference spectrum to appear for M) with 0.1 M mixtures of acetyl tryptophan (4X NaN03. Quenching by Xulfur-Containing Compounds. The sulfur-containing molecules carbon disulfide, thiourea, cysteine, and cysteamine combine with electrons at rates which approach those predicted for diffusioncontrolled p r o c e s s e ~ . All ~ ~ ~four ~ ~ are ~ also very efficient quenchers of the fluorescence of acetyl tryptophan (Table I). The reduction of fluorescence intensity by cysteine is accoriipanied by an equivalent fall in excited lifetime (Table I), suggesting that the quenching process involves primarily an interaction of cysteine with the excited state. However a barely perceptible difference M spectrum was developed for a mixture of 4 X acetyl tryptophan and 0.5 M cysteine, with a small positive peak a t 292 mp, whose magnitude was about 1% of the total absorbance at 280 mp. This raises the possibility of some minor degree of interaction of cysteine with the ground state of indole. The quenching by cysteine of four tryptophan derivatives was analyzed according to the Stern-Volmer equation1* 10 - =
I
1
+ Ksv[B] = 1 + le,r[B]
(4)
where I Oand I are the intensities of fluorescence in the absence and presence of quencher, respectively; KSVis a constant given by the slope of the line; lez is the second order rate constant for deactivation of the excited state; r is the excited lifetime in the absence of quencher; and [ B ]is the concentration of quencher. The variation of Io/I with cysteine concentration was linear for all four derivatives, with slopes decreasing in the order AcTrp > Trp, AcTrpn", > Trp > TrpEt (Table 11), which is that expected from the respective values of 7 (1). However the values of h,as comTable I1 : Rate Constants for Quenching of Tryptophan Derivatives by Cysteine Compound
TrP AcTrp AcTrpNHI TrpEt
Ksv
ka X 10-9
3.2 5.6 2.9 0.26
1.1 1.1 0.75 0.52
The solvent is 0.1 M KClO.01 M KOAc, pH 5.0. The values of kz are computed from eq 4. Estimated accuracy of ICz is
azo%.
T h e Journal of Physical Chemistry
puted from eq 4 were of a similar order of magnitude for all derivatives (Table 11). Quenching by Histidine. Histidine, cysteine, and cystine are the only amino acids whose rate constants for electron capture approach collisional values.8816 Shinitzky and coworkers have already reported the quenching of indole derivatives by imidazole-coctaining compounds, which they attributed to the formation of a nonfluorescent charge transfer complex by the ground state.16 Histidine was likewise found in this study to be an efficient quencher of the fluorescence of acetyl tryptophan (Figure 1 and Table I). However the parallel fall of excited lifetime with fluorescence intensity (Figure 1) indicates that deactivation of the excited state by interaction with histidine is a major factor in the quenching. The association constant for ground state complex formation by indole and histidine (-2) or by tryptophan and histidine (1.7) is too low to account for all of the q u e n ~ h i n g , ' ~predicting ,~~ that only 13-15% of the acetyl tryptophan is complexed a t the histidine level sufficient for 50% quenching. The pH profile of the quenching of acetyl tryptophan amide by histidine (Figure 1)parallels those for electron capture (Figure 2 of ref 15) and for the ionization of imidazole (pK S), suggesting that it is the protonated imidazole ring which is effective in both electron capture and in quenching. Quenchingby Other Amino Acids and Peptides. None of the remaining amino acids approach cysteine, cystine, or histidine in either electron capture efficiency or quenching effectiveness (Table I). Arginine is much more effective in both respects than glycine, alanine, lysine, or ,&alanine (Table I). However, proline, whose rate constant for electron capture is the same as that of lysine, is significantly more efficient as a quencher. This may be a consequence of ground state complex formation between proline and tryptophan derivatives. l7 Mixtures of acetyl tryptophan with proline, lysine, and arginine all gave small but significant difference spectra, which were qualitatively similar. Arginine gave the largest absorbance change, the magnitude of the small positive peak developed a t 293 mp being about 2% of the total absorbance a t 280 mp (Figure 2). Thus ground state complexing may make a finite contribution to the quenching by all three compounds. Formation of a peptide bond by glycine promotes both electron capture18 and fluorescence quenching (Table I). (Gly), is more effective than glycine. (14) 0.Stern and M. Volmer, Phys. Z., 20, 183 (1919), (16) R.Braams, Radiat. Res., 27,319 (1966). (16) M.Shinitzky, E.Katchalski, V. Grisano, and N. Sharon, Arch. Biochem. Biophys., 116,332 (1966). (17) S. C. K. Su and J. A. Shafer, J . Amer. Chem. Soc., 90, 3861 (1968). (18) R. Braams, Radiat. Res., 31,8 (1967).
INTERACTION OF INDOLE DERIVATIVES WITH ELECTRON SCAVENGERS
I
I.o
4133
i 5
0.02
AA 0
> W
I-
z -0.0 2
0.:
9
2 80
270
F
290
300
WAVE-LENGTH ( M p )
4w U
* Figure 2 . Difference spectrum developed by 3 X lob4M acetyl tryptophan plus 0.3 M arginine in 0.1 M KC1, 0.01 M KOAc, p H 5.0, with respect to the unmixed components.
I
0.I MOLARITY
I 0.2
L
0.3
HISTIDINE
Figure 1. Variation of relative fluorescence intensity ( 0 )and of fluorescence lifetime (A) for acetyl tryptophan (0.01 mg/ml) as a function of histidine concentration in 0.1 M KC1, 0.01 M KOAc, pH 5.0. ( 1 ) Inset. pI-1 variation of relative fluorescence intensity for acetyl tryptophan amide (0.009 mg/ml) in the presence of 0.19 M histidine in 0.1 M KC1.
Quenching by Uncharged Aliphatic Compounds. The chlorinated hydrocarbon chloroform, which combines with free electrons at a diffusion-controlled rate,s is also an efficient quencher of the fluorescence of tryptophan derivatives in water (Table I). The parallel decrease of fluorescence intensity and excited lifetime and the absence of any detectable difference spectrum (for 4 x low4M acetyl tryptophan plus 0.05 M chloroform) suggest that extensive ground state complexing is absent and that quenching occurs predominantly by interaction with the excited state. Similar statements can be made with respect to quenching by acrylamide (Table I), whose efficiency in electron capture has been attributed to its conjugation.8 Acetone is likewise effective both as an electron scavenger and as a quencher (Table I). The following compounds, which are inactive in electron capture, did not quench tryptophan derivatives a t concentrations up to 1 M : sucrose, ethanol, ethylene glycol, and urea. Solvent Eflects on Quenching of Indole Fluorescence. The quenching of indole and its derivatives by electron scavengers is not confined to aqueous solutions. The fluorescence of indole in dioxane is quenched by low
levels of carbon disulfide, chloroform, carbon tetrachloride, and acetone (Table 111), all of which react with solvated electrons in water a t collisional or nearcollisional rates.8 Acetic acid quenches at a somewhat reduced efficiency (Table 111). Table I11 : Quenching of Indole in Dioxane
a
Reagent
Molarity for 50% quenching
Acetone Carbon tetrachloride Carbon disulfide Chloroform Acetic acid
0.004 0.005 0.02 0.04 0.08
kea 10-10
0.6 2 2 2
0.02
Data of Hart, Thomas, and Gordon (1964).
Table IV compares the rate constants for quenching of indole by chloroform in a series of non-aqueous solvents, as computed from the Stern-Volmer equation using the limiting slopes a t low chloroform concentrations.l4 A significant degree of upward curvature was observed for chloroform concentrations above 0.1 M . The rate constant in methanol was an order of magnitude larger than in propylene glycol, which has a similar dielectric constant,, presumably as a consequence of the 60-fold higher viscosity of the latter solvent. The rate constant was highest in the nonpolar solvent cyclohexane, which is the only solvent of this series in which “exciplex” formation cannot occur.6
Discussion It is clear from the preceding results that the group of molecules and ions which have been found by pulse Volume 73, Number 12 December 1069
ROBERT F. STEINER AND EDWARD P. KIRBY
4134 radiolysis studies t o be efficient electron scavengers are also effective quenchers of the fluorescence of indole derivatives. All of the more than twenty members of this class which were studied were found to quench to some degree. (R. Lumry (private communication) has found that the scavenger NzO at a concentration of -0.008 M does not quench indole fluorescence.) In all the cases which were examined, the parallel decrease of the excited lifetime and the quantum yield suggests that radiationless deactivation of the excited state is a dominant mechanism. However, several of the amino acids, including arginine, histidine, proline, and cysteine, appear t o form complexes t o some extent with the ground state of tryptophan in water. This may also make a contribution to the observed intermolecular quenching by these molecules. It should be stressed that a particular molecule may quench by more than one mechanism; that its quenching efficiency may be influenced by several other factors (such as the nature of the substitution on the indole derivative, the size and charge density of the quencher, steric factors, etc.), and that quenching and solvated electron capture are different processes. Hence it is not to be expected that the quenching efficiencies should vary monotonically with the rate constants for electron capture. If the data of Table I are displayed graphically in a log-log plot, the computed correlation coefficient between the rate constants for electron capture and the molarities for 50 per cent quenching is 0.82 0.05. It is not unreasonable to suggest that the quenching of the fluorescence of indole derivatives by this series of compounds may result, a t least in part, from electron transfer from the excited state of indole to the acceptor molecule or ion. The evidence for an electron transfer mechanism for quenching is indirect and inferential, and cannot be regarded as conclusive. It may, however, serve as a convenient working hypothesis, pending more definitive information. It would be premature to attempt to elaborate the electron transfer hypothesis in any detail. Quenching might occur by (1) transfer of an electron by collisional contact of the quencher with an excited indole, (2) preliminary ejection of an electron from an excited indole to vicinal solvent molecules, combination with a scavenger being competitive with respect to recapture by the indole or escape into the free, solvated state, or (3) the formation of a transient complex of the chargetransfer type between excited indole and the quencher. (For case (3) the electron would not necessarily be retained by the quencher upon dissociation of the complex.) The exact nature of the most probable donor in any electron transfer process is uncertain. In a nonpolar solvent the donor would presumably be the excited indole ring itself, but in polar solvents a more specific transient complex is formed with the solvent of the
*
The Journal of Physical Chemistry
(iexciplex” type postulated by Walker, Bednar, and Lumry.5~~9 There are several instances where a molecule which acts as an intermolecular quencher of indole derivatives is also known to suppress fluorescence when in chemical combination. Cowgill has reported that a series of tryptophan derivatives containing a sulfhydryl or disulfide group in proximity t o the indole are all characterized by very low quantum yields of fluorescence.20 Similarly, Shinitzky and Goldman have found that a series of derivatives of the dipeptide HisTrp have low quantum yields (-0.02-0.03) when the imidazole group of histidine is protonated.21 The pH profile of quenching parallels the titration of histidine closely. Of the amino acids examined in the present study, only cysteine and histidine were highly
Table IV: Solvent Effects on Quenching of Indole Fluorescence by Chloroform
Solvent
r (nseo)
Ksv
kz X 10-0 for CHCl8
Water Methanol Propylene glycol Ethanol Isopropyl alcohol %-Butyl alcohol Dioxane Cyclohexane
4.1 3.5 4.4 3.8 4.0 3.7 4.5 5.1
23.2 32.3 3.9 29.8 26.1 23.5 35.0 68.5
5.7 9.2 0.9 7.8 6.5 6.4 7.8 12.8
The estimated accuracy of 7 is *0.3 nsec. Ksv was determined from Stern-Volmer plots as the slope of the line a t concentrations of CHC13 less than 0.1 M . The estimated acuracy of k2 is =t20%.
effective as intermolecular quenchers. (Cystine could not be examined because of its low solubility). It is of interest that the only amino acids which combine with electrons with very high efficiency (k, > 109) are cysteine, cystine, and histidine.15 I n these cases, there is a strong possibility that the inter- and intramolecular quenching processes occur by similar mechanisms, which may include electron transfer as a factor. It is worthy of mention in this connection that Dose has reported that ultraviolet irradiation of mixed solutions of tryptophan and cystine results in photodecomposition of tryptophan, accompanied by a reduction of cystine.22 The quenching of indole derivatives by hydrogen ions deserves special comment. A combination with indole in the ground state can here be ruled out for the (19) M. 8.Walker, T. W. Bednar, and R. Lumry, J . Chem. Phys., 45 3455 (1966). (20) R. W. Cowgill, Biochim. Biophys, Acta, 140, 37 (1967). (21) M. Shinitzky and R. Goldman, European J . Biochem., 3 , 139 (1967). (22) D. Dose, Photoclem. Photobiol., 7, 671 (1968).
4135
PROTON RESONANCE IN ALKALINITRATE MELTS range of hydrogen ion concentrations in which quenching occurs (pH 1-3). Two alternative mechanisms may be proposed. (a). Quenching might occur by attachment of a proton to the excited indole ring. This mechanism may suffer somewhat in plausibility from the failure of the existing molecular orbital calculation^^^ to indicate an obvious locus of excess negative charge on the excited indole ring. (b). In view of the high electron-capturing efficiency of hydrogen ion (8) it may be that the proton acts by abstracting an electron from the excited indole ring, thereby deactivating it. It has also been suggested that hydrogen ions may be the effective quenching agent for several tryptophan derivatives which contain a potential proton donor group,2a, 11,12 The question may also be raised as to whether the reduced quantum yield of zwitterionic tryptophan
(indole-3-alanine) in water, as compared with i n d ~ l e , ~ is related to the general intermolecular quenching caused by amino acids, including glycine and alanine, and whether the mechanism involves electron transfer, either directly to the protonated amino group or t o the hydrogen ion donated by this group. The two mechanisms are not, of course, mutually exclusive. The parallel increase of inter- and intramolecular quenching and of electron capture efficiency upon peptide bond formation is also suggestive.
Acknowledgment. The authors wish to acknowledge the able technical assistance of Mr. Theodore Lutins and Mr. Richard Kolinski. This work was partially supported by ONR Grant No. NR108-815. (23) P.-S. Song and W. E. Kurtin, Photochem.PhotobioZ., 9,175(1969)
Proton Resonance in Alkali Nitrate Melts' by Lieng Chen Siew and Benson R. Sundheim Department of Chemistry, N e w York University, N e w York, N e w York 10003 (Received A p r i l 3, 1969)
The viscosity, density, electrical conductivity, chemical shift, and proton relaxation times were determined for solutions of "03 in the LiNOs-KNOa-NaNO3 eutectic. Over the temperature range 140 to 220°, pronounced changes in many of these properties were noted a t a composition near mole fraction of HN03 equal to one-third. The data are interpreted as showing the presence of a more-than-two coordinated proton in this region and of the hydrogen dinitrate ion in dilute solutions.
Introduction Nuclear magnetic resonance measurements have been widely used in the study of chemical systems. Under favorable circumstances it is possible to obtain information about the nature of bonding, the identity and concentration of the various species in the system and of the lifetime of some of these species. Very little application of this technique has been made to the study of fused salt systems. Hafner and Nachtrieb2 first investigated the chemical shifts in fused thallium salts as a function of temperature and anions. There have also been rather extensive studies made on the structure of mineral acids, s ~ l f u r i c , ~ * ~ ~ and p e r ~ h l o r i c . ~Here ~ the various ways in which the proton can be bound were studied by coupling with other physical measurements such as infrared spectroscopy and electrical conductivity. I n connection with the studies of the structure of liquid nitric acid near room temperature, Happe and Whittakers examined the proton resonance spectrum
of nitric acid and of solutions of potassium nitrate in nitric acid. A systematic chemical shift dependent on concentration was observed and it was suggested that this is associated with a systematic change in structure. Studies on fused alkali nitrates have established that the cations are octahedrally surrounded by six oxygen atoms in a fairly regular pseudo-lattice.6 By comparison with the behavior of other molten salt systems, it seems not unlikely that nitric acid when dissolved in (1) Extracted in part from a dissertation by L. C. Siew submitted to the Graduate School of Arts and Science, New York University, in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Feb 1969. (2) S. Hafner and N. H. Nachtrieb, J . Chem. Phys., 40, 2891 (1964). (3) H. S.Gutowsky and A.Saika, ibid., 21, 1688 (1953). (4) (a) G.C. Hood and C. A. Reilly, ibid., 27, 1126 (1957); (b) G. C. Hood, 0.Redlich, and C. A. Reilly, ibid., 22,2067 (1954). ( 6 ) J. A.Happe and A. G. Whittaker, ibid., 30,417 (1959). (6) (a) D.Gruen, P. Graf, 8. Fried, and R. L. McBeth, Pure AppZ. Chem., 6,23 (1963); (b) K.E.Johnson and T. S. Piper, Discussions Faraday Soc., 32,32(1961).
Volume 73, Number 18 December 1969
