Spectmchimica
Acta, Vol. 26A, pp.
1479to1489.Per&wmm
Preaa 1969. Printed in Northern Ireland
The spectra of chloranilic acid and its complexes with amino acids M. A. SLIFKIN and B. M. SMITH Department
of Pure and Applied Physics, The University,
Salford
and
R. H. WALMSLEY Department
of Science, College of Technology (Received 16 Aw~t
and Design, Blackburn,
Lanes
1968)
Abstr&---The ultra-violet, visible and infra-red spectrum of chloranilic acid haa been determined. In mixtures of chloranilic acid and amino acids it is shown that 1: 1 complexes are formed in solution and 1: 2 complexes in the solid state. A study of the i&a-red spectra of amino acids, their hydrochloride8 and their complexes with chloranilic acid show that hydrogen bonded ionic species are formed. Data is given of equilibrium constants and enthalpies of dissociation of the complexes in solution.
DURING studies of some interactions of chloranil (I) in aqueous solutions [I],it was observed that chloranil gradually changed into trichlorohydroxyquinone (II) and chloranilic acid (III)
&;+&~H+O$$lH II 0 (I)
II 0
II 0
(II)
(III)
It has been shown [2-41 that amino acids and other amines form 1: 1 complexes with chloranil and trichlorohydroxyquinone. A study has now been made of the visible, ultra-violet and i&a-red spectra of chloranilic acid in water and KBr discs and of its interaction products with amino acids in the same media. EXPERIMENTAL Ultra-violet and visible spectra were obtained on a Unicam SP7OO recording spectrophotometer thermostatted to “C. Stoppered fused silica cuvettes of 10 mm path length were used. All i&a-red spectra were obtained with a Unicam SP200 recording spectrophotometer. Amino acid hydrochlorides were prepared by [l] M. A. SLIBKIN,R. A. SUMNER and J. G. IIE~~~~~~~,S~ectrochint.Acta SA, [2] M. A. SLIFEIN and J. G. HEATHCOTE, Spctrochim. Actu 23A, 2893 (1967). [3] J. B. BIRKS and M. A. SLIFKIN,Nature Lord 197, 42 (1963). [4] M. A. SLIFKIN,Spectrochim. Actu 20, 1643 (1964). 1479
1761 (1967).
1480
M. A. SIXXIN, B. M. SMITE and R. H. WALXSLEY
down solution of amino acid in hydrochloric acid solution. Chemicals were supplied by Messrs. British Drug Houses Ltd.
evaporating
RESULTS
AND DISCUSSION
1. Ultra-violet and visible spectra
The spectrum of 1O-4 M chloranilic acid in water consists of a major peak at 30.2 kK* with an absorbance of 2.16 and a very weak band at ~18 BK with an absorbance of 0.003. The pH of a 1O-4 M solution is 3.8. Heating a solution of chloranilic acid causes no change in absorbance over the range 20-80°C even when examined differentially, i.e., by using a 20°C solution as the reference and the 80°C solution as the sample. Decreasing the pH of the solution with de&normal HCI causes the major peak to shift to higher frequency while decreasing in absorbance and the minor band to increase its absorbance without changing its position. At pH l-5 the major peak has an absorbance of 1.76 and lies at 32-6 kK whereas the minor peak has increased its absorbance to 0.055. This pH 15 spectrum is very similar indeed to the spectrum of old chloranil in water or trichlorohydroxyquinone [Z]. Increasing the pH above 3-8 by adding de&normal caustic soda causes no shift in the 32.6 kK peak but a small increase in absorbance and a steady decrease in the absorbance of the minor band. These effects are fully reversible. Some of these spectra are illustrated in Fig. 1. It has been shown [5] that the sodium salt of chloranilic acid has a similar spectrum to that of the acid but the minor band has a much weaker absorbance. SCHWAEZENBAUE and SUTER [6J have studied the reduction potentials of chloranjfic acid solutions
as a function of pH. According to them chloranilic acid exists in three forms, the neutral form (yellow)? which exists at extremely low pH, the mononegative ion (dark violet) which is most stable at pH 2 and the dinegative ion (pale violet) stable at high pH. Consequently the spectrum of chloranilic acid is the sum of the three forms. The neutral form predominating at very low pH, the mononegative ion around pH 2 and the dinegative ion at higher pH’s. The change in spectrum of chloranilic acid in going down to pH 2 can be explained simply as due to the conversion of dinegative ion (pale violet) to mononegative ion (dark violet). The reason why the spectrum at pH 2 is very similar to that of trichlorohydroxyquinone is simply because trichlorohydroxyquinone can only form a mononegative ion which one would expect to be very similar to chlora~~c acid in spectral appearance. The effect of adding amino acids for 1O-4 M chloranilio acid solutions has been observed in both unbuffered and buffered aqueous solution. The use of water as a solvent suffers from the disadvantage that the addition of different concentration of
amino acid changes the pH. Thus solution of 1O-4 M chloran~ic acid and 10-a M neutral c+amino acid have pH in the region of 6-Oas compared to 3-8 for chloranilic * The value of 23.3kK given in OrganicElectronic Spectral Data Vol. 1. Interscience (1960) is a misreading of the data of E. H. TYEER, Anal. Chem. 20, 76 (1948) tmd is in fact an absorption minimum, not the maximum. t Golden is perhaps a better description. [a]
I.S. MUSTAFIN,L.O.~KATVEEV~II~E.A.KASHKOVSKAYA, Nauk. S.S.S.R. Inst. &x&him, Anal. Khim. 11, 87 (1960). [6] G. S~~~~~~c~andH.Su~~,~e~v. Che~.Acta2~,%17
Trudy. Komkii.Amal.Khim. (1941).
The spectra of chloranilic acid and its complexes with amino acids
1481
acid &lone. However the complic&ing effects of buffers are avoided and the range of pH in the mixtures is not too large (less than 0.5). The addition of amino acids* to chloranilic acid in water, show very rapid changes in the difference spectra (i.e., between chloranilic acid and chloranilic a&d plus
Frequency,
kK
Fig. 1. Absorption spectra of lOA chloranik acid in 1 cm path Iength. (a) at pH 1.5 (b) atpH3.G-----: (c) at pH 10.5- x - x -.
amino acid). There appears a large positive peak at about 29.5 kK and a negative peak at 342 kK. These peaks increase with increasing amino acid concentration. Furthermore isosbestic points sre to be seen in these spectra, illustrated in Fig. 2. BENESI-HILDEBRAND [7] plots for these spectra show that they arise from 1: 1 complexes. Eq~b~urn constants Ke for these complexes have been evaluated from these plots and are presented in Table 1. The effect of temperature on these difference spectra have been investigated. In the case of glycine and proline, increasing heat increases the positive peaks in the difference spectra, the converse is true with tryptophan. Plots of In H, vs. increase temperature have been made and using the relation In R = - AH”/RT + const. (from the Gibbs-Helmholtz equation) enthalpies * Glyoine, tryptophsn and proline (more proper& an imiuo a&d). [7] H. A. BENESI and J. H.
HILDEBRAND,
J. Am. Ohem. Sot. 71, 2703 (1949).
1482
M. A. i&x=x.
B. M. SMITE and R. H.
WALMSLEY
of dissociation MO of these complexes have been evaluated snd sre also listed in Table 1. Surprisingly the enthalpies of dissociation are positive (except for tryptophan) which is in very marked contrast to the behaviour of other amino acid complexes [2]. It should be noticed that the & values for the tryptophan chloranilic acid complexes sre very much larger thsn for the other amino acids. The effect on the spectral peak positions of changing the pH of these solutions over the range pH 4-10 with dilute acid or all&i is very slight.
35
Frequency, Fig.
2.
36
25
kK
Difference spectra. 1Oa M chloranilicwid and 10m4M chloranilicacid + glycine in 1 cm path length. 10-l M glycine. (&I ___(bf - - - - - - 2 x 100~ M glycine.
The interaction between these same amino scids and chloranilic acid has also been examined in buffered aqueous solution. Some typical spectra are shown in Fig. 3. Because of the high absorbance of the buffers themselves above 30 kK, only the spectra,below 30 kK are meaningful, the spectra in this region appear to ba very similar to those in unbuffered solution, namely, the intensification of absorbance in the region of 30 kK with a slight low frequency shift. Attempts were made to measure & values for the three amino acid complexes with c~or~n~o acid at pH ‘7. The values found were very low, and in view of the probable inhibiting effect of the buffers no attempt has been made to evaluate K, values at different temperature and pH’s nor to obtain AHo values. Values of K, in pH 7 buffer are given in Table 1. The overall effect of adding amino acids to aqueous chlor~nilic acid is to intensify
The spectra of chloranilicacid and its complexes with amino acids
1483
Table 1. Data on ohloranilioacid complexes 1. Proline
2.6 x lo-* M to 10-’ M with lo-’
(a) In water. K,*
At 37.6 kK 69 f 7 at 13%
100 f 10 st 32’C
M ohloranilio mid
134 f 28 at 40%iDC
240 f 30 at 4s&w
AH” = 7.4 -& 1.2 kcal mole &.t at 37.6 kK = 1640 f 120
(b) In pH 7 buffer At 28.4 kK K,+
= 0.93 f
0.16 at 26°C
7. Glycine 2 x lo-* M to 10-l M with 1OV M chlormilio acid (a) In water et 37.6 kK 147 f 30 xc* 78% 8 69 f 8 84f 13 at 14% at 46OC at 24% at 32°C
131 f 36 at 60°C
AHO = 2.2 f 0.8 Eat at 37.6 kK = 1600 f 200
(b) In pH 7 buffer at 27.6 kK. K,*
= O-13 f
0.006 at 26’K
3. Tryptophan 1.4 X 1OW M to 6 X lo-* M with 11F4 M chloranilia said (a) In water at 30 kK 360 f 60 K,* 840 f 90 680 f 80 430 f 60 at 60% et 26’C at 40% at 62’C (b) In pH 7 buffer at 26.7 kK k,* = 67 f 16 et 26’=K
AH” =
-4.7 f 0.6 kmls/mole .?.t at 37.6 kK = 1400 f 160
l Equilibrium aomtmts in I./mole. t Molecular extinction coeffiaient of oomplex.
the spectrum of chloranilic acid with a slight shift to lower frequencies, the i&ens& cation being proportional to the amino acid concentration. These interactions appear to be quite different to those observed between amino acids and chloranil or trichlorohydroxyquinone [2-41. In that work it was shown that the addition of amino acids causes large shifts to lower frequencies of the principal absorption bands of chloranil or trichlorohydroxyquinone. Furthermore the band shifts were pH dependent and took a very long time to develop (9 hr at pH 9 and 3 days at pH 4). Finally the interaction was exothermic whereas in the work reported herein the reaction appears to be endothermic (tryptophan being an exception). The chsnges in spectrum on adding amino acids for chloranilic acid cannot be explained simply as due to the increase in pH of the solutions causing conversion of mononegative ion to dinegative ion as the effects are also observed in buffered solutions. Furthermore our own studies of the effect of pH on the chloranilic acid absorption band at ~30 kK show that the effect of adding the amino acids is to cause an increase of absorbance several times that caused by the change in pH alone. Furthermore it is shown later that a definite reaction product is found between amino acids and chloranilic acid. The pH effect probably explains the rather large errors found in the values of K, and A.EZ”in Table 1. 2. Infra-red
spectra
Infra-red spectra in KBr discs have been measured of the amino acids, their hydrochlorides, chloranilic acid and the reaction product between chloranilic acid and the amino acids. The interaction products were prepared by evaporating down under reduced pressure in a rotary evaporator, solutions of amino acid and chloranilic acid in a molar
1484
M. A.
SLIFKIN, B. M.
SMITH and R. H. WALMSLIEY
80-
90.
.
loo--1
I
40
I
30
Frequency.
20
kK
Fig. 3. Difference spectra. 3.82 x 10v4 M chloranilic acid and 3.82 chloranilio acid plus proline in pH 7 buffer in 1 cm length. 2.48 M proline. -_---0.62 M proline. x - 0.41 M proline. -x-
x lOA M
ratio of 2: 1, it being found that this ratio gave the clearest spectra. An analysis of the precipitate coming down from saturated solutions of amino acid and chloranilic acid also showed the molar ratio to be 2 : 1. The spectrum of an amino acid (which can be taken as illustrative of [all the amino acids) is shown in Fig. 4. The a-amino acids which exist as zwitterions H,+N RCOO- in the solid state possess bands at 2130 cm-l arising from the COO
of
The spectra of chlorailio a&d and its complexes with amino acids
1486
100
60 i
01
5GQO
1
’
4000
I
3000
2ood'
I600 Wavenumber,
1600
1400
1200
1000
600
cm-’
Fig. 4. I&a-red spectrum of proline in KBr disc. 0.5 mg proline in 150 mg KBr.
I
so00
I
4000
3000
2oOd'
1600 Wavenumber,
1600 cm-’
1400
I
I
I200
I
I
1000
I
I
t,
600
Fig. 6. k&a-red spectrum of proline hydrochloridein KBr disc. 1 mg proline hydrochloridein 150 mg KBr.
The amino acid hydrochloride of which a typical spectrum is shown in Fig. 5 show the following differences to the amino acids. The carboxylic groups vibration are completely different. The C--O stretch occurs at 1710 cm-l for glycine and corresponds to the unionised carboxyl group. Other peaks associated with the unionised carboxyl group are seen at 853, 1200 end 1412 cm-l, for glycine HCl; at 860, 1195 and 1725 cm-l for tryptophan HCl; and 900, 1100, 1200, 1245, 1485 and 1725 for proline HCl. The NH,+ group for the hydrochlorides are very similar to those of the parent amino acid. Proline and its hydrochloride exhibits an NH,+ band at 2900 11
M. A.
1486
SLIFKIN, B.
M. SMITE and R. H. WALMSLEY
Table 2. Principal spectral absorption bands cm-l Glycine HCl 2896 1710 1676 1478 1412 1200 > 863
Glycine
N-H and O-H stretch, broad peak. C=O stretch of union&d carboxyl group NH,+ antisym. deformation. NH, gym. deformation coupling of O-H bending vib. and C--o+ stretch. -OH out of plane deformation.
1595 COO- antisym. stretch 1415 COO- sym. stretch 1609 NH,+ antisym. deformation 1526 NH,+ sym. deformation 1134 NH,+ rock frequency 2100 NH,+ bend 2940 N-H stretch broad and 2560 C-H stretc hI overlapping.
Tryptophan HCI 3310 2980 2900 1726 1680 1620 1200
Tryptophan
N-H stretch NH,+ stretch overlapping OH stretch > C =O stretoh of unionised carboxyl group NH$ antisym. deformation NH,+ sym. deformation OH in plane deformation.
3330 N-H stretch 2980 NH,+ stretch 2100 NH.+ bend 1666 CO& antisym. stretch 1690 NH,+ antisym. deformation 1684 NH,+ gym. deformation 1418 COO- sym. stretch 1100
Proline HCI
3
1725 1583 1486 1 1420 1246 lzoo}
N
-
H, C-H,
Proline
and OH stretch (if present)
C =O stretch of unionised carboxyl group NH,+ deformationmasked in free amino acid by COO- stretch double peak
I
I
NH,+
assignments based on observed changes in speotrmn when hydrochloride formed.
E3 1620 1405
N H and C-H &r&oh, broad and overlapping COO- antisym. stretch COO- sym. stretch.
tentatively assigned aa coupled OH bend and CO stretch
Several of the assignments for Proline and Proline HCl must be considered as tentative since there are very few NH,+ amino acids available for comparison. A weak absorption around 2100 cm-l assigned to NH, + by LEIPER and LIPPINCOT[3] is seen to be present in the spectra of Glyoine and Tryptophan but absent from that of Proline and also absent in all the spectra of the hydrochlorides.
cm-l instead of the NH,+ band of the a-amino acids. A list of these various bands with assignments is given in Table 2. The spectrum of chloranilic acid is illustrated in Fig. 6. Of interest are two carAn OH stretching vibration occurs at 3160 boxy1 bands at 1628 and 1635 cm-l. cm-l indicative of intramolecular hydrogen bonding [ 121. In saturated solution in deuterium oxide the -OH stretch occurs at 3340 cm-l indicative of polymeric intermolecular hydrogen bonding [12]. A peak also occurs at 1270 cm-l which we assign to -OH bend. The reaction product arising from glycine and tryptophan shows peaks characteristic of the NH,+ and COOH group in the hydrochloride, that from proline of NH,+ and COOH. Other bands of the amino acid are retained in the reaction products. stretching and bending vibration of chloranilic acid disappears in the The -OH reaction product. Two peaks at 760 and 700 cm-l seen in chloranilic acid are absent All other bands of chloranilic acid are retained in the reaction in the complex. products. A typical spectrum is illustrated in Fig. 7. [12] L. J. BELLAMY, The Infa-Red
Spectra of Complex Molecule.
Methuen
(1964).
1467
The spectra of ohloranilic acid and its oomplexes with amino aoids
of 5000
1
!
1
4000
f
I
3000
I\\1
t
zood’
9
L
is00 Wovenumber.
e 1600
I
t
L
1400
k
!
1200
I
f
IO00
,
8,
800
cm-’
Fig. 6. Infrared spectrum of chloranilic acid in KBr disc. 0.5 mg ehloranilic acid in 150 mg KBr.
100 -
i 0
60-
e I-
40-
20 -
I
4000
1
,
1
4000
3000
zoob
I
I800 Wovenumber,
Fig. 7. Infrared
/
I
1600
I
s
I400
s
x
I200
1
t
1
to00
L
*
BOO
cm-’
spectrum of proline chloranilio acid complex in KBr disc.
1 mg proline-chlorenilic
rtoid complex (molar ratio 2 : 1) in 150 mg KBr.
One other interesting point is that chloranilic acid is red in solid form but gives purple solutions in water as mentioned earlier. The solid reaction products formed with the amino acids are purple. So that we can say that in the reaction product chlorsnilic acid is present in the ionised form. This and the above evidence leads to the conclusion that the reaction produot consists of associated ions (IV) which may
1488
M. A. SLIFKIN,B. M. SLXITEand
R.
H. WAL~IZY
well be hydrogen-bonded, thus
0
0
II
II
*HOOCRNH +.;yJ;***Ha+NR 0 I,-,“’
2NH,+RCOO- + OH cl1
3
..
II 0
IV
* +. H,NRC!OOH HOOCRNH, - - - HO
Similar hydrogen bonded ion species have been postulated by SWTH [13] as stable structures occurring during the interaction of weak acids and weak bases. In addition other authors [14, 151in theoretical consideration of complexes using the charge transfer theory of hydrogen bonding also suggest such structures may occur also as reson&n~ehybrids ~ont~buting to some structure rather than as actual structures, thus OX- - * - H+Y t+ OXH * * *Y. In water at low pH we have shown that s, 1: 1 complex exists. Furthermore one must assume that the complexes will dissociate to form free ions pair thus: (I) NH*+ RCOO- + 0-XOH
+ O-X0-
- - . H,+NRCOOH + NH,+RCOOH
+ o-xoor (2) NH,RCOOH + 0-XOH
+ O-X0-.
- - H,+NRCOOH + NH[,+RCOOH
+ o-xoIn view of the rapidity of the ~~r~otion, the first mech~n~m is much the more probable. Slight evidence for the existence of the hydrogen bonded ion is given by the red shift of the electronic spectrum which is very characteristic of hydrogen bonding [IS] although this could well be due to a solvent perturbation effect. The formation of a 1: 1 complex in solution is not inconsistent with a 2 : 1 complex in the solid state. The formation of a 1: 1 complex in solution by negatively charging [13] J. W. SMITE,J.C~E~~..Z%ys.125 (1964). [I41 S. NAQAKIJRA, J.Chim. Phys. 217 (1984). [15] J. W. SBIITEI,Sci. Prog. 52, 97 (1964). [16] S. SUZUKI and H. BABA, J. Chem. Phys. 88, 349 (1963).
The spectra of chloranilicacid and its complexes with amino acids
1489
the chlorsnilic acid moiety will effectively prevent a second positively charged amino group from forming a second hydrogen bond. On condensation to the solid state the second amino acid will be forced into close proximity to the complex and bonding will occur. The increase in absorbance on heating in solution can be explained by the hydrogen bond being weaker than the bond binding the donated proton to the amino group, This bond must be a charge transfer bond due to the lone pair electronsofthe nitrogen in the amino group. Heating of the hydrogen bonded ion-pair will cause dissociation into the free ions. Thus the whole reaction will go to the right causing an increase of the visible spectrum associated with the chloranilic ion. The anomalous results with tryptophan might be due to the conjugated indole structure in tryptophan which could form an additional bond to the chloranilic acid by T-V charge transfer. It is interesting that the solid tryptophan chloranilic acid complex is a darker purple than the other complexes. This might be an indication of chargetransfer of a rr-electron of the indole ring in tryptophan to a n-orbital in the conjugated ring of the chloranilic acid. Charge transfer bonding usually has little effect on the infra-red spectrum of either the donor or acceptor and therefore one wouldn’t expect to find any difference between the i.r. spectrum of the tryptophan and the others although charge-transfer interaction does often give rise to marked visible colour changes [17]. It has been demonstrated that complexing takes place with chloranilic acid which is very different in form to amino acid complexes with chloranil or trichlorohydroxyquinone [2, 31. Yet the mechanism is similar in both cases, it is the lone-pair electrons on the nitrogen in the amino group which promotes the bonding; in the first case by donation to a n-orbital in the conjugated ring of chloranil to form the charge transfer complex and in the second to the proton of the chloranilic acid to form the hydrogen bonded ion. Several authors [14, 181 have treated the hydrogen bond in the same way as MULLIKEN treated the charge transfer complex [ 191and have shown that MULIJKEN’S theory can be equally applied to these types of hydrogen bonded complexes as it can to n-r charge transfer complexes. It is clear that the results previously reported for chloranil and trichlorohydroxyquinone [l, 21 cannot in any way arise from the presence of chloranilic acid in those solutions. Acknowkdgements-The SP700 spectrophotometer was obtained with a Medical Research Council Grant to M. A. S. [17] G. BRIEQLEB, Elektronen-Donator-Accepto+KompZexe. Springer-Verlag (1961). [18] R. G. PURANIKand V. KUMAR, Proc. Irad. Acad. Sci. 58, 29 (1963). [19] R. S. MN, J. Am. Chem. Sot. 74, 811 (1952).