J. Phys. Chem. 1980, 84, 3085-3090
3085
Studies on Spin-Trapped Radicals in y-Irradiated Aqueous Solutlons of Glycine and L-Alanine by Hlgh-Performance Liquid Chromatography and ESR Spectroscopy Fuimlo Moriya,* Kelsuke Maklno, Nobuhlro Suzukl, SouJlRokushlka, and Hlroyukl Hatano Department of Chem/stw,Facutty of Science, Kyoto Universtty, Kyoto, 606 Japan (Received: April 23, 1080; In Fim/ form: June 27, t980)
Aqueous solutions of glycine and L-alanine were y irradiated in the presence of a spin trap, 2-methyl-2nitrosopropane. Stable spin adducts produced in the y-irradiated solutions were analyzed by means of high-performanceliquid chromatography and ESR spectroscopy. The following four spin adducts were found and identified t-BuN(O.)CH&OO- (I)and t-BuN(0.)CH(NH2)C00-(II)from glycine; t-BuN(O*)CH(CH,)COO(111) and t-BuN(0.)CH2CH(NH3+)COO-(IV) from L-alanine. It was found that the ESR spectra of these spin adducts changed with pH through the acid dissociation equilibria of the carboxyl or amino groups. The pK, values for the dissociation have been determined to be 3.2 for the carboxyl group of spin adduct 111, and 8.9 for the amino group of IV.
Introductilon In order to understand the radiation-chemical process of aqueous solutions of proteins and enzymes, several workers have recently done numerous studies concerning such systems as amino acids and peptides.lg Radicals produced in proteins by active radiolytic species of water, e, .OH, He.,etc., are quite conceivably the primary species responsible for radiation effects on protein^.^^^ The radical intermediatlas produced by ionizing radiation in aqueous solutions of many amino acids have been observed by ESR spectroscopy during in situ radiolysis4+ or in frozen The optical absorption of the transient species of amino acids during pulse radiolysis has been reported.1° Recently, ithe spiin trapping method has been developed, by which short-lived radicals are converted into fairly stable nitroxide radicals (spin-trapped radicals or spin adducts) through reactions with spin traps such as nitroso or nitrone cc~mpounds.l~-~~ Structures for the short-lived radicals can be inferred from the ESR spectra of the spin adducts in irradiated solutions. The radical intermediates produced by y radiolysis or photochemical reactions in aqueous solutions of many amino acids, peptides, and proteins have been studied by the spin-trapping method with 2-methyl-2-nitrosopropane (MNP) as a spin t r a ~ . ~ J ” l ~ In most of these systems the ESR spectra obtained from irradiated solutions are complicated due to the overlap of signals of several sipin adducts. From such complicated ESR spectra it is not easy to investigate the structures of individual spin adducts by ordinary computer simulation only. Liquid chromatography has been recently combined with ESR spectroscopy for separation and detection of radical mixtures.20 This technique has been applied to separate and identify the spin adducts in y-irradiated aqueous solutions of 5’-UMP,21 5’-TMP, 5’-CMP,22DLmethionine? L-valine,u L-isoleucine, and L-leucine= with MNP as a spin trap. The application to y radiolysis of MNP itself in an aqueous solution has also been reported.% The reaction of MNP with a short-lived radical (Re) is represented by eq 1. From the ESIt spectroscopic point t-BuN=O + R- -* t-BuN(O.)R I-Bu&+(O-)R (1) of view each (atomiin a spin adduct is designated a,p, y, etc., according to the increasing number of bonds counted from the nitrogen atom in the nitroxyl g r o ~ p . ~ ’
0022-3654/80/2084-3085$01 .OO/O
In this study, the spin trapping method with MNP was applied to y-irradiated aqueous solutions of glycine and L-alanine. The spin adducts were separated by highperformance liquid chromatography and identified by ESR spectroscopy. The pH and temperature dependence of their ESR spectra were investigated. From these results, the spin adducts could be characterized.
Experimental Section Glycine was purchased from Wako Pure Chemical Industries, Osaka, and L-alanine was from Kyowa Hakko Industrial Co., Tokyo. MNP was synthesized and purified according to the method of Stowe11.28 Di-tert-butyl nitroxide was purchased from Eastman Kodak Co., New York. All other chemicals were of reagent grade. Aqueous solutions of MNP (5 mg/mL) were prepared in the dark by stirring for 1 h a t 45 0C.29 The concentration of glycine and L-alanine in the MNP solutions was 1.0 M. In air the sample solution was cooled by ice and irradiated with “Co y rays at a dose rate of 6.0 X lo6rd/h to a total dose of 3.0 X lo6 rd. Immediately after irradiation, 2 M phosphate buffer ([Na2HP04]/[NaH2P04])= 1/9) was added It0 the solution. Then 1.0 mL of the mixed solution was loaded into the cation-exchange column (IEX-21OSC of Toyo Soda Manufacturing Co., Tokyo, 0.95 cm i.d. and 60 cm long). The eluate was passed through UV and ESR detectors and collected in fraction tubes. The chromatographic system was illustrated in the previous paper.21 The high-performance liquid chromatograph was a Toyo Soda HLC-803. The UV detector was a JACSO UVIDEC 100 (10 mm light path), which was tuned to 240 nm. The ESR spectrometer was a JEOL PE-3X, which was operated with 100-kHz modulation frequency at X-band. For detection of the radicals during chromatography, the magnetic field was fixed at the positions indicated by the vertical arrows in Figures 1and 5. The magnetic-field modulation was applied at a high amplitude of 10 G to cover a wide range. Conditions for the chromatography were as follows: pressure, ca. 70 kg/cm2; flow rate, ca. 0.2 mL/min; temperature, ca. 25 O C ; eluant, 0.2 M sodium phosphate buffer ([Na+] N 0.33 M), pH 7.0. In order to detect intact amino acids we developed each fraction with ninhydrin reagent by boiling in a water bath. Ninhydrin positive fractions were analyzed with an amino acid analyzer (Hitachi KLA-3B). Hyperfine splitting constants (hfscs) were measured by using Mn2+ in MgO as a reference (splitting between the central two peaks, 86.9 f 0.1 G). The pH of each solution was read
0 1980 American Chemical Society
The Journal of Physical Chemistry, Vol. 84, No. 23, 1980
3086
Moriya et al.
Figure 3. ESR spectrum of the separated spln adduct obtained from the fractions giving peak A in Figure 2 at pH 7.0.
A
I
I
Figure 1. ESR spectrum of an aqueous solution of glycine with MNP observed just after y irradlation (at pH 6.0). During chromatography the magnetic field was fixed at the posltion indicated by the vertical arrow.
0
10
Elution
20
Volume
30 ( ml )
Figure 2. Chromatogram of the y-irradiated aqueous solution of glycine with MNP drawn by ESR detection. A 0.2 M sodium phosphate buffer, pH 7.0, was used as the eluant.
IIIII IIIII IIIII
in a Hitachi-Horiba pH meter M-5. All experiments were carried out in the dark.
Results and Discussion Glycine. The ESR spectrum of a y-irradiated aqueous glycine solution with MNP is shown in Figure 1. Most of the spin adducts which led to this spectrum were fairly stable for several hours at room temperature. This spectrum is more complicated due to overlapping signals of spin adducts from glycine and those from the self-trapping of MNP.26 Liquid chromatography was applied to the solution immediately after irradiation and the subsequent addition of 2 M phosphate buffer. Preliminary experiments by use of the present column have revealed that with neutral eluants negatively charged compounds are eluted much faster than neutral and positive compounds with complete separation, and that most of spin adducts produced by the self-trapping of MNP26are adsorbed on the column and eluted very slowly with diffusion and the ESR signal intensities of these broad chromatographicpeaks are so small that they are not always observable. The chromatogram by ESR detection is shown in Figure 2, where three peaks were observed. A typical ESR spectrum for the peak A fractions is depicted in Figure 3. The ESR spectrum obtained at pH 7.0 for the peak B fraction was distorted, and this spectrum changed reversibly depending on pH. Figure 4A shows the spectrum observed at pH 11.1, which can be analyzed as composed of two spin adducts. These spectra can be clearly distinguished from the characteristic patterns of the spin adducts by the self-trapping of MNP.= Peak C did not always appear on the lower side of the base line and sometimes appeared on the upper side, and no significant ESR signals were observed for the fractions of
Flgure 4. (A) ESR spectrum of the separated spin adduct obtained from the fractions giving peak B in Figure 2 at pH 11.1, (B) Computersimulated spectrum and stick diagrams for (A). Parameters used for spectrum simulation are specified in the text.
this peak. Intact glycine detected by using an amino acid analyzer eluted around peak C. Figure 3 shows the ESR spectrum obtained for the peak A fraction, which can be analyzed as a triple triplet. The primary triplet of 16.1-G splitting is due to the nitrogen atom in the nitroxyl group. The secondary triplet of 8.45-G splitting with an intensity ratio of 1:2:1 is assigned to two equivalent /3 hydrogens. It is well-known that, when hydrated electrons react with a-amino acids, reductive deamination is caused by the addition of e, - to the carboxyl groups.l~' The ESR signal pattern in h g u r e 3 was assigned to the deaminated glycine adduct, whose structure is given in I. In the neutral and alkaline solutions, the f-Bu-N-CH2-COO-
I
0.
I
spectral pattern of adduct I was pH independent. However, in the acidic pH range from ca. 2 to 4, reversible pH-dependent shifts of the signals were observed. At pH 1.4, the hfscs, uN and (2 H), are 15.8 and 8.6 G, respectively. This acid dissociation property is ascribed to the existence of either a nitroxyl group or a carboxyl group in adduct I. The spectral pattern (uN = 17.2 G) of the di-tert-butyl nitroxide aqueous solution was independent of the pH between 0.5 and 13.2, which indicates that a nitroxyl group is not protonated in this range since the
Spin-Trapped Adducts of Glycine and L-Alanine
I I
10 G
t Figure 5. ESR spectrium of an aqueous solution of L-alanine with MNP observed just after y irradiation (at pH 6.1). During chromatography the magnetic fleld was fixed at the position indicated by the vertical arrow.
protonation of the nitroxyl group should give an additional H hfsc of several gauss.30*31Consequently, the spectral changes of spin adduct I caused by changing pH, which did not derive new hyperfine splittings, were attributable to the protonation of the carboxyl group whose pKa value is around 3. Figure 4A shows the spectrum obtained at pH 11.1, adjusted by addition of 2 M NaOH to the peak B fraction. The spectrum is interpreted to consist of two spin adducts whose stick diagrams are added to the bottom of Figure 4. Major signals (upper sticks in Figure 4) can be analyzed as a triplet of five lines with an apparent intensity ratio of 1:1:2:1:1. The primary hfsc of a nitrogen atom is 15.9 G and the seconday splittings are assigned to a @ hydrogen and a 0 nitrogen = 3.3 G and aS:N = 1.5 G). Minor signals due to another adduct (lower stlcks in Figure 4) are consistent with those of Figure 3, and are attributed to a trace of spin adduct I. The simulation of the resulting spectrum, shown in Figure 4B,was computed with Ag = g, - gl = -0.00006 1(+0.lG), Lorentzian peak-to-peak line widths of 0.16and 0.5 G for the major (gl) and the minor (gz)signals, respectively. The simulated spectrum is consistent satisfactorily with the observed one. The structure of the spin adduct from the major signals was assigned as ishown in 11. Spin adduct I1 is derived from (iab.~
NH,-CH-COO-. f-Bu-N-O*
I
I1
a short-lived radical produced by hydrogen abstraction from the carbon adjacent to the carboxyl group of glycine by OH radicals. The hfscs of adduct I1 measured a t pH 6 in the present study were almost consistent with those obtained in the UV photolysis of a DZOsolution of glycine with DzOz at pD 6.2 by Rustgi et al. (uN = 14.25 G, aa.H = 2.35 G).15 1.85 G, and L-Alanine. Figure 5 shows the ElSR spectrum of a yirradiated aqueous solution of L-alanine with MNP. The spin adducts which1 led to this spectrum were stable for several hours at room temperature, The chromatogram by ESR detection is shown in Figure 6, where three peaks were observed. Typical ESR spectra for the fractions of peaks A and C are depicted in Figures 7A and 9A, respectively, which are characteristic patterns of the spin adducts from L-alanine. These signals are partly revealed in the ESR spectrum in Figure 5. No significant ESR signals were observed for the fractions of peak B and these fractions contained the remaining intact L-alanine.
The Journal of Physical Chemisfty, Vol. 84, No. 23, 1980 3087
~~
0
10
30
20
Elution
Volume
( ml
Flgure 6. Chromatogram of the y-irradiated aqueous solution of Lalanine with MNP drawn by ESR detection. A 0.2 M sodium phosphate buffer, pH 7.0, was used as the eluant.
A
B
I
I
I I
Flgure 7. ESR spectra of the separated spin adduct obtained from the fractions giving peak A in Figure 6. (A) and (B) were observed at pH 7.0 and 1.3, respectively.
Figure 7A shows the ESR spectrum for the fraction of peak A, which can be analyzed as a double triplet of four lines with an intensity ratio of 1:3:3:1. The primary hfsc of a nitrogen atom is 16.1 G, and the secondary splittings are assigned to a @ hydrogen and three equivalent y hydrogens (u6.H = 5.3 G and U,.H = 0.42 G). This ESR signal pattern was assigned to the deaminated a-alanine adduct, whose structure can be written as in 111. In the neutral f-Bu-N-CH-COO-
I 1CH,
0.
III and alkaline solutions, the spectral pattern of adduct I11 was pH independent. However, in the acidic pH range, reversible pH-dependent shifts of the signals were observed. Figure 7B shows the ESR spectrum of the protonated adduct I11 at pH 1.3 whose hfscs, uN, u&H, and uH., (3 H), are 15.9,2.7, and 0.35 G, respectively. The @-Hhfscs for adduct 111were plotted as a function of pH in Figure 8. In the acid dissociation equilibrium of the carboxyl group of adduct 111, if the interchange between the acid form (A) and the base form (B)is rapid, which is the case here, the observed hfsc, a, represents the weighted average of the two forms.32 For a given solution the acid dissociation constant can be calculated by eq 2, where U A and
ag are the hfscs for the acid and base forms, respectively, and f A and f B refer to the fraction of each form cfA + f B
3088
The Journal of Physical Chemistty, Vol. 84, No. 23, 7980 5.5
I-
Moriya et ai.
TABLE I: Hyperfine Splitting Constants (G) of Spin Adducts (t-BuN(0.)R) from Glycine and L-Alanine'
I
structure, R
-
PH
CH,COO- (I)b 4.0
t
CH(NH,)COO- (11)
a0-H
ah'
Glycine 7.0 16.1 1.4 15.8 11.1 15.9
8.45c 8.6c _ . 3.3
ap-N
5-H
1.5
L- Alanine
CH(CH,)COO(III)b . -.
2.51,
I
0
1
,
, 2
,
,
,
3
I
4
,
I
5
,
, 6
,
Il
.
7
PH Flgure 8. The effect of PH on the #-H hfsc for win adduct 111. Clrcles indlcate experimental results. Best fitted solid curve was calculated by use of eq 3.
7.0 16.1 1.3 15.9 7.0 16.4
5.3 0.42d 2.7 0.36 d CH,CH(NH,+)16.7, 0.45 c o o - (IVP 10.9 16.Se 15.4,e f 11.3e 10.5 16.6 14.1, 0.65 10.9 1.7 16.3 16.3, 0.5 13.7 ' Unless otherwise stated, hfscs were measured at room Two temperature. Shown as structures at pH 7.0. equivalent hydrogens. Three equivalent hydrogens. e Obtained by increasing the temperature of the sample at pH 7.0 to ca. 80 "C. f Expected 7-H hfs unresolved. .
I
r
groups of a double quartet whose inner pair is selectively broad. The stick diagram is added to the bottom of Figure 9A in which the minor doublets are neglected. The primary hfsc of a nitrogen atom is 16.4 G, and the secondary splittings are assigned to two nonequivalent P hydrogens and a y hydrogen (us.H = 16.7 G and 10.9 G, and ay.H = 0.45 G). Such a spectrum shown in Figure 9A is a typical pattern for a nitroxide radical which has an a methylene and an asymmetric /3 carbon in the molecule, that is, R'N(O.)CH,*CXYZ (X f Y # Z X). The origin of the characteristic four-line splitting pattern from the two /3 hydrogens in such a radical has been interpreted by assuming, for instance, interconvertion between two minimum energy conformers which have mutually different sets of dihedral angles between the plane of the HB-C, and C,-N bonds and the plane including the C,-N bond and the p orbital on nitrogen.39i34The spin adduct was assigned to structure IV derived from a short-lived radical which
+
Flgure 9, ESR spectra and stick diagram of the separated spin adduct obtained from the fractions giving peak C in Figure 0 (at pH 7.0): (A) room temperature; (6) ca. 80 OC.
= 1). From eq 2 the hfsc, a, is derived as a function of pH, as follows: aA
a=
t aB(lOPH- pKa) 1 + 1OPH - PKa
NH-*:FH-COO-
yl e I
f-Bu-N-O*
IV
(3)
where CIA = 2.7 G and UB = 5.3 G for adduct 111. The best fit with the experimental points was obtained by substituting pKa = 3.2 to eq 3 as shown by the solid line in Figure 8. Figure 9A shows the ESR spectrum for the fraction of peak C, which appears as a mixture of six narrow and two broad lines. Each of the former lines slightly splits into a doublet. It was observed that the two broad lines became narrow with increasing temperature and their peak heights approached those of the six narrow lines almost independent of temperature. Near 80 OC the ESR spectrum for this fraction, asterisked in Figure 9B, newly revealed four broad lines concealed behind the central four narrow lines with small shifts of the original lines. (The hfscs are given in Table I.) Therefore it was concluded that the ESR spectrum shown in Figure 9A consists of six narrow and six broad lines with equal intensities, that is, the spectrum is due to only one kind of spin adduct with line width alternation. The spectrum can be analyzed as three
is produced by hydrogen abstraction from the methyl group of L-alanine by OH radicals. Spin adduct IV exhibited reversible spectral changes accompanying pH variation of the fraction as shown in Figure 10. Figure 10A reveals the spectrum of negatively charged adduct IV having a deprotonated amino group (uN = 16.6 G, U.+H (2H) = 14.1 G and 10.9 G, and = 0.65 G). The six broad lines became narrow with increasing temperature as well as a t neutral pH. It further supports the assignment to structure IV that the peak heights of all lines became almost equal at 60 "C. The ESR signal pattern shown in Figure 10F, which is due to the zwitterion form, did not change in the pH range 3-8. The pH dependence in the alkaline range denotes that an ESR spectrum at the pH around pK, consists of two overlapping signal sets of the zwitterion form and the negatively charged form with their concentration ratio because the interchange between them is slow. A simulated ESR spectrum at the pH equal to the pKa of the amino group, shown in Figure 10D, was computed with Ag = g4- g3 = -0.00012 (+0.2 G), concentration ratio of 1:l for the two forms, and Lorentzian peak-to-peak
Spin-Trapped Adducts of Glycine and L-Alanine
A
B
C
The Journal of Physical Chemistv, Vol. 84, No. 23, 1980 3088
dicates that most of short-lived radicals previously reported in y-irradiated aqueous solutions of glycine and L-alanine1p4i5are spin trapped. Spin adducts I and I11 are derived from deaminated radicals produced by the addition of e,; to the carboxyl groups of glycine and L-alanine, respectively. The hfscs for adducts I and I11 are in good agreement with previously reported value^.'^^^^ Spin adducts I1 and IV are derived from radicals produced by hydrogen abstraction by OH radicals from the carbon adjacent to the carboxyl group of glycine and from the methyl group off L-alanine, respectively. In the case of L-alanine, spin adduct V corresponding to I1 was not found. NH,+-C(CH,I-COO-
f-Bu-N-0.
I
V
D
E
F
G
This is not necessarily in conflict with previous observations of the corresponding short-lived radical from a-alanine,5y36since it is conceivable that spin adduct V has decayed rapidly after y irradiation or during chromatography, and that the OH radical may react at the CH3 site to a considerable extent since in a neutral solution the CH site is highly deactivated by the adjacent NH3+ grou?. The ESR spectra of the four spin adducts change with pH through the acid dissociation equilibria of the carboxyl or amino groups in the vicinity of the nitroxyl groups. The pK, values for the dissociation have been determined to be 3.2 for the carboxyl group of spin adduct 111, and 8.9 for the amino group of IV.
Acknowledgment. The authors express their sincere gratitude to Dr. Yashige Kotake, Osaka University, Dr. Hitoshi Taniguchi, Yamaguchi University, and Dr. Fumiko Yamamoto-Murakami, Kyoto University, for helpful suggestions and valuable discussion. The authors are indebted to Toyo Soda Manufacturing Co., Ltd., Tokyo, for supplying a high-performance liquid chromatograph, HLC803, and a cation-exchange column, IEX-21OSC. References and Notes
Flgure I O . ESW spectra of spin adduct I V observed at room temperature: (A) pH 10.5; (B) pH 9.4; (C) pH 8.9; (D)simulated spectrum at the pH equal to the pK, of Its amlno group. Parameters used for spectrum simulation are specified in the text: (E) pH 8.5; (F) pH 5.9; (G) pH 1.7.
line widths of 0.45 and 1.5 G for the zwitterion form (g3) and of 0.45 and 0.6 G for the negatively charged form (g4), respectively. The good agreement of the observed spectrum shown in Figure 1OC with the simulated spectrum suggests that the pK, value for the amino group of adduct IV is about 8,9. In the acidic pH range ca. 0.8 to 3, reversible spectral changes were also observed with pH-dependent shifts of the signals. For example, at pH 1.7 (Figure 10G) ,the hfscs are U N = 16.3 G, u0-H (2 H) = 16.3 G , and 13.7 G, and u.~-H = 0.5 G. The spectral changes are due to the protonation of the carboxyl group of adduct IV. Conclusions Four kinds d spin adducts in y-irradiated aqueous solutions of glycine and L-alanine with MNP as a spin trap were separated and identified by high-performance liquid chromatography and ESR spectroscopy. Their structures and hfscs are summarized ;n Table-I This method in-
Garrison, W. M. Radlat. Res. Rev. 1972, 3 , 305, and references therein. Riesz, P.; Rustgi, S. Radlat. Phys. Chem. 1979, 13, 21, and references therein. Klapper, M. H.; Faraggi, M. Q. Rev. BiqDhys. In press, and references therein. Neta, P.; Fessenden, R. W. J. Phys. Chem. 1970, 74, 2263. Neta, P.; Fessenden, R. W. J. Phys. Chem. 1971, 75, 738. Neta, P.; Fessenden, R. W. J. Phys. Chem. 1971, 75, 2277. Sevilla, M. D. J . Phys. Chem. 1970, 74, 2096. Sevlila, M. D.; Brooks, V. L. J. Phys. Chem. 1973, 77, 2954. Sevlila, M. D.; DArcy, J. B.; Suyanarayana, D. J. Phys. Chem. 1978, 8 2 , 2589. Neta, P.; Simic, M.; Hayon, E. J. Phys. Chem. 1970, 74, 1214. Janzen, E. G. A m . Chem. Res. 1971, 4, 31, and referencestherein. Lagercrantz, C.J. Phys. Chem. 1971, 75, 3466, and references thereln. Terabe, S.; Konaka, R. J. Chem. Soc., Perkln Trans. 2 1972, 2163. Sargent, F. P.; Gardy, E. M. Can. J. Chem. 1974, 5 2 , 3645. Rustgi, S.; Joshl, A.; Moss, H.; Riesz, P. Int. J. Radlat. Bld. Relat. Sfud. Phys., Chem. Med. 1977, 3 1 , 415. Rustgi, S.; Joshi, A.; Rlesz, P.; Friedberg, F. Inf. J. Radlat. Bbl. Relat. Sfud. Phys., Chem. Med. 1977, 32, 533. Tanlguchi, H.; Hatano, H. Chem. Lett. 1974, 531; 1975, 9. Joshi. A.; RustgY, S.: Moss, H.; Rlesz, P. h t . J . Radlat. Bb/.Relat. Stud. Phys., Chem. Med. 1978, 33, 205. Rustgi, S.; Rlesz, P. Inf. J . Radiat. Bid. Relat. Stud. Phys., Chem. Med. 1978, 3 4 , 127, 301, 449. Rokushika, S.; Taniguchi, H.; Hatano, H. Anal. Lett. 1975, 8 , 205. Kominami, S.; Rokushika, S.; Hatano, H. Int. J. Radlat. Blol. Relat. Stud. Phys., Chem. Med. 1976, 30, 525. Kominaml, S.; Rokushika, S.; Hatano, H. Radlat. Res. 1977, 72, 89. Maklno, K.; Hatano, H. Chem. Lett. 1979, 119. Makino, K. J. Phys. Chem. 1979, 8 3 , 2520. Maklno, K.; Suzuki, N.; Moriya, F.; Rokushika, S.; Hatano, H. Anal. Lett. 1980, 13, 301. Makino, K. J. Phys. Chem. 1980, 84, 1016. Makino, K. J. Phys. Chem. 1960, 84, 1968.
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(26) Makino, K.; Suzuki, N.; Moriya, F.; Rokushika, S.; Hatano, H. Chem. Lett. 1979, 675. Makino, K. J. Phys. Chem. 1980, 84, 1012. (27) Kotake, Y.; Kuwata, K.; Janzen, E. G. J. Phys. Chem. 1979, 83, 3024. (28) Stowell, J. C. J . Ofg. Chem. 1971, 31, 3055. (29) Makino, K.; Suzuki, N.; Moriya, F.;Rokushika, S.;Hatano, H. Anal. Lett. 1980, 13,311. (30) Hogeveen, H.; Gersmann, H. R.; Praat, A. P. Recl. Tfav. Chim. Pays-Bas 1967, 86, 1063.
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Spectrophotometric Determination of N-Methylformamide Autoprotolysis Constant T. Oncescu, A . 4 . Oancea, Polltechnicai Institute of Bucharest, Instltute of Chemlstry, Depattment of Physical Chemistry, Bd. Republicii 13, 7003 1 Bucharest, Romania
and L. De Maeyer" Max-Planck-Institutfuer biophysikaiische Chemie, 0-3400 Qoettingen, Germany (Received:March 17, 1980)
The autoprotolysis constant of N-methylformamide obtained from spectrophotometric measurements on M2harmonizes well with p-nitrophenol and p-nitrophenoxide solutions is reported. The value K,, = 1.8 X the autoprotolysis constants of other related compounds.
The study of proton-transfer reactions between different alkyl amines and nitrophenols in N-methylformamide (NMF) which is in progress in our laboratory indicated that the solvent itself is involved in acid-base equilibria. This participation is not unexpected since the N-monoalkylated amide solvents are known to possess both basic and acidic properties,l as proved primarily by amide hydrochloride precipitation when hydrogen chloride was bubbled into solvent and by hydrogen evolution accompanied by potassium salt formation when metallic potassium is added to solvent. In our experiments the acid-base properties of NMF were revealed by analysis of electronic spectra of p-nitrophenol (PNP) and sodium p-nitrophenoxide (NaPNP) in NMF. Starting from either PNP or NaPNP solution, both phenol and phenoxide absorption bands were present in the spectrum. This behavior we associated with the presence of basic and acidic impurities, but after thorough purification it was found that PNP was sufficiently acidic to protonate and NaPNP sufficiently basic to deprotonate NMF, within a concentration range suitable for spectrophotometric measurements. As the acidic and basic impurities compete with the solvent in acid-base equilibria, special attention was paid to solvent purification. Experimental Section Solvent. NMF (Fluka purum) kept over molecular sieves of 4 A was treated with charcoal (Merck) and thoroughly stirred, filtered, refluxed over BaO until the vapor temperature remained constant (-318-320 K), and distilled under reduced pressure (
