( 9 ) Kula R. J., Sawyer, D. T., Chan, S. I., €hey, C. M., J. Am. Chem. Soc. 85, 2930 (1963). (10) Legg, J. I., Cooke, D. W., Znorg. Chem. 4, 1,576 (1965). ( 1 1 ) Lindqvist, I., Acta Cryst. 3 , 159 (1950). (12) Lindqvist, I., Nova Acta Regiae SOC. Sci. Upsaliemis 15, 22 (1950). (13) Maricic, S., Smith, J. A. S., J. Chem.. SOC.1958, p. 886. (14) Pecsok, R. L., Sawyer, D. T., J. Am. Chem. SOC.78, 5496 (1956).
(15) Sasaki, Y., Lindqvist, I., Sillen, L. G., J. Znorg. Nucl. Chem., 9, 93 (1959). (16) Schwarsenbach, G., Meier, J., J . Inorg. Nucl. Chem. 8 , 302 (1958). (17) Sillen, L. G., Martell, A. E., “Stability Constants of Metal-Ion Complexes,” Special Publication No. 17, The Chemical Society, London, 1964. (18) Weakliem, H. A., Hoard, J. L., J . Am. Chem. SOC.81, 549 (1959). (19) Wiberg, K. B., Nist, B. J., “The Interpretation of NMR Spectra,” W.
Pyrid oxa I-GI yci nate Co mp lexes with Metal Ions
A. Benjamin, Inc., New York, 1962. (20) Wittick, J. J., Rechnits, G. A., ANAL.CHEM.37, 816 (1965).
RECEIVEDfor review July 6, 1965. Accepted May 4, 1966. This work was supported in part by the Research Committee of the Graduate School of the University of Wisconsin, with funds made available by the Wisconsin Alumni Research Foundation, and by a Grant (GP-4423) from the National Science Foundation.
Some Diva lent
D. 1. LEUSSING and NURJAHAN HUQ Department of Chemistry, Ohio State University, Columbus, Ohio
b Titrimetric-pH investigations of pyridoxal-glycine systems have revealed the formation and coexistence of stable mono- and bis-Schiff base complexes with the metal ions Ni(ll) and Zn(ll). The pyridoxal pyridine nitrogen in these species is readily protonated to form pyridinium complexes. The formation constants of the protonated and unprotonated complexes have been evaluated and the implications toward earlier spectrophotometric studies reported in the literature are discussed. Under the acidic conditions encountered in these titrimetric studies Cu(ll)-pyridoxal-glycine solutions appear to undergo reactions other than complex formation. N o complex formation was observed with Ca(ll).
binary metal ion complexes as well as the ternary Schiff base complexes, similarly absorbing species) attempts were made to arrange conditions so as to favor the formation of only the lowest Schiff base complex having a pyridoxalamino acid metal ion ratio of 1 : l : l . Bis-Schiff base complexes however, have been isolated as solids with the ions Mn(II), Fe(II), Fe(III), Ni(II), and Zn(I1) (I). Cu(II), on the other hand, yields only the 1 :1 : 1 solid ( I ) . A continuous variation study on pyridoxalalanine-Ni(I1) solutions shows the formation of a stable 2:2:1 complex An interesting but further complicating aspect of these pyridoxal systems is the protonation of the pyridine nitrogen in the metal ion complexes. For the reaction R
S
basemetal ion complexes undergo a variety of interesting reactions (11) some of which duplicate those observed in enzymic systems involving amino acids and the cofactor pyridoxal (IS). Pyridoxal activity appears to arise via Schiff base intermediates and because of this much interest has been directed toward the investigation of simpler model systems of Schiff bases with the hope of improving our understanding of the mechanism of enzyme activity (4,9, IS). Bearing upon the present investigation previous spectrophotometric studies have been concerned with the solution stabilities of pyridoxal-valine complexes with the metal ions Mg(II), Mn(II), Ni(II), Zn(I1) (3) and Cu(I1) (8, 3 ) . Other amino acids were also cursorily investigated (3). Because of the complicated mature of the aqueous solutions of these species (dissociation of the Schiff bases, formation of simple ANALYTICAL CHEMISTRY
solids Christensen ( I ) reports somewhat higher values for Ni (pyridoxal-valine)? pKI. = 7.3, pKZD= 8.1 but pK. = 5.6 for the 1:1:l Cu complex. The basicity of this nitrogen appears to lie between that of pyridine (pK. = 5.45) and the dipolar form of pyridoxal,
I
(4).
I
CHIFF
1388
432 IO
I
H+ R I
Davis, Roddy, and Metzler (3) report pK.valuesof 6.5-6.7 [M+’ = Zn+a, Ni+*, R = (CHJpCH-1, 5.6[M’* = CU+’, R = (CH$zCH-], 6.05(M+*=C~+’, R = H ) . From the direct acidimetric titration of solutions of the dissolved
H+ pK.=8.5 (IO). Spectrophotometric studies on solutions in the absence of metal ions have demonstrated the formation of the species PV-2, PVH-, and PVHZ (P-= pyridoxal, V-=valinate) and the weaker Schiff bases PG-2 and PGH(G- = glycinate) (6, 8). The proton most likely occupies a hydrogen bonded position between the imine nitrogen and the phenolic oxygen. Titrimetric pH studies have been shown to have particular value in determining the equilibrium properties of solutions of Schiff base complexes especially where the uncomplexed Schiff base is highly dissociated (7). A large number of data points are easily obtained and subsequently processed using a high speed digital computer. Earlier studies in these laboratories have shown that the purely aliphatic Schiff bases, at least, form in solution a series of complexes in which the extensive coexistence of both mono and bis forme is possible. Since this overlap ww not considered in the previous pyridoxal imine studies, the present investigation
I
was undertaken to determine whether such effects are important and, if S O , to evaluate more accurately the interactions involved.
t
A
/-
?COR
EXPERIMENTAL
Pyridoxal hydrochloride was obtained from both Sigma Chemical and NUtritional Biochemicals. Titration of weighed samples with standard NaOH to the first end point indicated purities of the order of 99.9% so these reagents were used without further purification. Glycine, hlatheson, Coleman and Bell (ammonia free), was recrystallized and dried in vacuo. Standard sodium glycinate solutions were prepared by adding carefully measured equivalents of standard XaOH to weighed amounts of glycine and diluting accurately to the desired volume. All experiments were performed in a medium of 0.50JI KC1 at 25.0' c. The titrations were performed under a blanket of nitrogen. RESULTS AND DISCUSSION
Titration of pyridoxal hydrochloride solutions (0.005hI) with standard NaOH yielded average values of pK1, equal to 4.25 and pKz. equal to 8.54. A total of 6 titration curves was obtained. Agreement was found to lie within 0.02 pK unit of the average values irrespective of the source of pyridoxal. Repeating these experiments using sodium glycinate as the titrant in place of sodium hydroxide yielded data from which the formation constant of PGHcould be calculated. The formation of this species was indicated by an increase in the apparent acidity of pyridoxal in the region between the first and second end points. The best least squares value of the constant K p G H , defined by the reaction
*
P-
+ G - + H + F! PGH-,
(1)
was obtained through the back calculation of theoretical titration curves using trial values of K p G H . For each theoretical titration curve the sum of the squares of residuals U = Xi (pHcolcd.i- pHobad.i)P was obtained. By assuming the curve U us. KpGHwas parabolic, the value of KpGHa t Urnin. was calculated (1%'). The procedure was repeated using the value of KpGH so found until convergence to the true minimum was assured. In this way a best value of K p G H equal to 1.37 X 1011 was obtained. The value of Urnin. corresponded to a standard deviation of a point equal to *0.01 p H unit (two curves, 36 points). Although this standard deviation indicates an excellent fit within the experimental error, it should be emphasized that the value of K p o H is somewhat sensitive to systematic errors, since by setting this constant equal to zero a theoretical curve is obtained for which the standard deviation is only *0.05 pH unit.
\ I
I
I
.I
.!?.
.3
302mp I .5
I .4
I .6
I
I
.?
-8
1
MNaGtot
Figure 1. Spectrophotometric study of the interaction between P- and G0.010M NaOH, 0.50M KCI, 25OC. 1.406 X lO-'MPSolid lines are computed using the rerulh reported in the text and ED,-'(, = 1 * 18 1 O+' cp,-mz = 5.61 X 10" KPGH = 19.0
x
In the region from the start of the titration up to the first end point, the curves obtained using NaG as a titrant were indistinguishable from those obtained using NaOH. This indicates no appreciable formation of PGHz under our conditions. The unprotonated Schiff base, PG+, was found to be formed in too alkaline a region for reliable pH measurements to be made. To obtain KPGresort was made to spectrophotometric methods. Solutions, 0.010M in NaOH, 0.5M KC1, were made up to contain a total of 1.406 millimolar pyridoxal and varying amounts of NaG. Spectra were obtained using 0.100-cm. cells. The results at the two wavelengths 302 mp (P- max) and 367 mp (PG-2 max) were analyzed to obtain the formation constant of P G + and its associated extinction coefficients. Preliminary values of these parameters were calculated using the slope-intercept method. The values were then refined by back-calculating theoretical absorbance-composition curves and comparing the theoretical to the observed curves. In performing this procedure a two-dimensional network of KPO-I and ePG- values was scanned to locate the approximate Urnin.: (U = Xi(Aic.led. - Aiobrd.)'. The region of Urnin,was rescanned using finer incre-
Table 1.
ments. In the final computations, increments of 0.25 KPO-2unit and either 0.01 X ePG-2 unit at 302 mp or 0.05 X l O + 3 units at 367 mp were used. "Best values" in the least squares sense were found to be KPG-2 = 19.5, epc-l = 5.65 X l O + J for the data 367 mp and Kpc = 18.5, epG-l = 2.39 X l O + 3 for the data at 302 mp. The observed relative standard deviations of a point are 2.8y0 and 1.5%, respectively. The calculated curves and observed points may be seen in Figure 1. Metal ion-pyridoxal complex formation constants were estimated in the usual way from the results obtained by titrating approximately 0.002M metal ion and (usually) 0.005-0.010M pyridoxal hydrochloride solutions with a standard solution of NaOH. Contrary to earlier results (3) in which no complexation was found, evidence for weak but detectable complexes was found with Ni(II), Cu(II), and Zn(I1). Complex formation was negligible with Ca(I1). The results are presented in Table I. The formation constants for the binary glycinato metal ion complexes were determined in earlier work (6,7). The Schiff base complexes were studied by essentially repeating the titrations described in the preceding section but using NaG as the titrant of
Formation Constants for Binary Complexes
pK,a
PKI, Log constant (this work)" This This Metal workn Literature worka Literature ion 81 BZ Bs Pyridoxal 4.25 4.20 (10) 8 . 5 4 8.66 (10) Ni+* 1.85 ... ... Cu+*
Glycine
. ..
2.46 ( 7 ) ~
. ..
3.51 2.32 Ni+2 5.66 C U + ~ 8.11 Zn+* 4.88
Zn+f
9.70 (7)"
0.5M KCl, 25' C.
VOL 38,
7.0 ..
10.51 14.43 9.01
NO. 10, SEPTEMBER 1966
.., ...
14.0
...
11.0
1389
The slow equilibration times and s u b out shape of the curves shows that more than one glycinate is complexed per sequent side reactions precluded conzinc(I1) and the strong dependence of ventional titrations. The Ni(I1) and their position on the pyridoxal conZn(I1) systems were studied in batchcentration suggests, also, that more wise experiments: each point on a than one pyridoxal is involved in the titration curve representing a single equilibria. experiment. The pH of the solutions Mass balance leads to the following was measured after 3 hours with Zn(I1) equations which include all the species and after 10 hours with Ni(I1). A total found earlier (7) plus protonated forms: of 72 points was run with Zn(I1) and 6 with Ni(I1). The Zn(I1) results are presented in Figure 2 along with the M t = Mt2 M P + MG+ theoretical curves (solid lines) calMG2 MGsMPG culated using the values of the constants reported below. MPGZMPzGz-' MPGH+ Unlike the pyridoxal-valinate-Zn(I1) MPGzH MP~GzHsystem investigated by Davis, Roddy MPzG2H2 (2) and Metzler (3) the pyridoxal-glycinateZn(I1) species investigated here show neither fluorescence nor photolability when exposed to direct sun light for short times. The Ca(I1) systems equilibrated rapidly and showed no indication of forming Schiff base complexes. INTERPRETATION OF THE DATA AND THE EVALUATION OF THE TERNARY G~ = H,G HG GCOMPLEX FORMATION CONSTANTS. Consideration of the results shown in MG+ 2MGz 3MG3Figure 2 suggests that bis as well 89 MPG 2MPGz2MP2Gz-' mono N - pyruvylideneglycinato comMPGH+ 2MPGzH 4plexes are readily formed: the drawn-
the metal ion pyridoxal solutions instead of NaOH. These solutions were found to be slow to equilibrate, the pH drifting to lower values after an abrupt pH increase which occurred after each addition of titrant. Two types of reaction appeared to take place. A first relatively rapid decrease in pH reached a metastable plateau in 3-4 hours with Zn(I1) and 8-10 hours with Ni(I1). However, after this first st,age the pH of the solutions continued to drift at a much slower rate until a week later, additional decreases of several tenths of a pH unit were observed. Because of possible complications arising from slow molecular rearrangements in these systems the readings at the first plateau were taken to calculate the stability of the Schiff base complexes. Davis, Roddy, and Metzler (3) also report spectrophotometric equilibration times of the order of 3 hours for the Zn(I1) complexes. The Cu(I1) complexes unfortunately did not show a well defined plateau. Apparently under these acidic conditions side reactions occur a t about the same rate as the complexes are formed. Instability of Cu(I1)-Schiff base complexes in acidic solutions has been reported elsewhere (3, 6).
I
02 Figure 2.
I
I
I
0.6
I
1.0
I
+ + +
I
I
I .4
1
1.8
1
1
1
22
I
I
1
1
2.6 3.0 m l . O.IM Sodium Glycinotc
'
3.4
1
1
3.8
Titrant: 0.1 006M NaG
1 2 3 4 5
fir
1M)
0,00970 0.01067 0.01067 0.01 067 0.01067
Pt
(MI
0.02023 0.01979 0,00999 0.01099 0.03296
Ht (MI 0.04136 0.041 49 0.02226 0.02425 0.03672
ANALYTICAL CHEMISTRY
1
4.2
+
+ +
1
+
1
4.6
+
1
+
1
5.0
- 0.50M KCI, 25" C.
Initial vdurne ml.
The solid lines are theoretical curves calculated using the results reported in the text
1390
+
1
+
+ +
+
Titration of pyridoxal hydrochloride, zinc chloride solutions with sodium glycinate
Curve No.
+
+
+
+
+ + +
1 1 .oo 10.00 10.00 10.00 10.00
2hlPzGzH-
+ 2iLIPzGzH2 + PGH- + PG+
+ + + + + +
Hj = 2HzP+ HI’ 2HzG+ HG MPGH+ MPGzH 1IPzGzH2XIP2GzHz PGHH+-OH-
+ +
(4)
+
ji \ 0 c
._ Y Z0.f
(5)
t
Invoking equilibrium allows the substitution of terms in M+2, P-, G - and H + for the concentrations of the various species. Equilibrium constants are defined for the generalized reaction,
M+z
+ iP- + j G -
(MP+) =
G JIP,G,
(6)
(P-)
(7)
PlO(M+’)
(,\IG+) = P i 0 (,\I+‘) (G-1, (I\IG)z = @ z ~ ( h l +(G-)’ ~)
,
52
#
#
,
,
56
54
,
,
58
I
I.
=-
,
/
,
6 22 ’ 6 6. 44
60
,
PH
.
66
,
.
68
72
77.0 0
77.6 6
77.k k
Til‘n’5
L\
0.8
(8)
0
c 0.
.-
e
u
I
LL
0. 0.
5.6
5.8
6.0
6.2
64
68
6.6
7.0
7,2
7.4
7.6
?.6
PH
Figure 3. Figure 2
Calculated distribution of Zn(ll) during some of the titrations shown in
In order to reduce the number of parameters which need to be evaluated, it was assumed that inductive effects across a metal ion are negligible so that the inherent basicity is the same for all of the pyridine nitrogen atoms in the complexed Schiff base pyridoxal units. while for Thus, K,‘ = K.” = KOMpoa, statistical reasons, K1,”’ = 2Koxp~n and Kz.,“’ = ‘/z KahfpGH. The four constants were then evaluated using the generalized approach described above for obtaining KpoH (12).
The results are presented in Table 11. The standard deviation of the Zn(I1) points shown in Figure 2 is *0.033 pH unit and that of the K(I1) points is k0.08 pH unit. In analyzing the Zn(I1) results it was found that the species MPGz- was formed to too slight an extent to be characterized reliably: the uncertainty in plz was greater than its absolute value. This species was therefore ignored in analyzing the Ni(I1) data where fewer points were available. Mention should be made of the con-
Table II. Interactions in Pyridoxal-Imine Systems
etc., where
This work
P-+GaPG-* H + P G + -aPGHH + PGHPGHz (Protonation of pyridine nitrogen, estimated from valinate results) H + PGHz PGHs+ (Protonation of carboxylate group, estimated) M + z + P - + GM+Z - $ M=PNi+Z G c u +z
++ +
1.28 10.86
...
Log constant Literature 1.01 (8) 10.4 (8) 5.88 (8)
... 10.30
2.3 (8) Bll
...
16.O’c‘g’, 15.4 (Sj 8.4-9.1 (5)
8.43 Knowing the total concentrations and the simple equilibrium constants given in Table I these relationships yield four combined mass balance equations in the eleven unknowns M+’, P-, G-, H.+, 6’11, 812, 822, Kat, K,”, Kl,”’ and Kz,,”’.
19.84 16,863 7.W
*
Zn + p Intrinsic value. See text. pK1 and pKz of M(P)Z(Val)zHz.
7.34.
Bzz
...
RMPGH 6 . 5 (3) 7.3,8.1* ( 8 ) 7.3, 8.1s ( 8 )
VOL. 38, NO. 10, SEPTEMBER 1966
1391
vention adopted here for expressing the values of the acid dissociation constants. The activity of the hydrogen ion as obtained from measurements with a glass electrode was used together with the concentration ratio of the base to acid form of the ligand, so that,
K,
= aH
(h)I(HA)
(22)
The values will differ from those of the true concentration constants by the activity coefficient of the hydrogen ion appropriate to the medium employed, and will differ from the thermodynamic constants by the activity coefficient ratio of the base to acid forms. It should be emphasized that as long as an appropriate correction is made for the free hydrogen ion concentration of the solution when this term is not negligible, no error in the complex formation constants results. The complex formation constants are true concentration constants. Any arbitrary pH scale may be used without influencing the values of the complex formation constants as long as the data are internally consistent. To make the pK, values more meaningful relative to others reported in the literature, we have standardized the Radiometer 25 SE pH meter used in these studies against XBS standard buffers. The relatively high values of Pz2 obtained with TU’i(I1) and Zn(I1) ions indicate that the bis imine complexes
tend to form simultaneously ivith the mono imines. Such behavior is not likely to occur with Cu(I1) (2, 3) owing to its small tendency to attain a coordination number greater than four. The distribution of the Zn(I1) species during the titrations represented by curves 2 and 5 of Figure 2 has been calculated and plotted in Figure 3. The extensive over-lap of higher and lower complexes is quite apparent. The most important complexes are the protonated imines and, in more alkaline solutions, ZnPzGz-*. The binary glycinato complexes are formed only to a minor extent and those of pyridoxal are negligible. While certainly the distribution with valine is expected to be different it should be pointed out that the high amino acid levels used in the earlier work (3) will favor, in the more alkaline solutions employed, the disproportionation, 2MPV
+ HV M(PV)Z-*
+ hIV+ + H +
Thus, the constants reported in (3) probably are subject to some error. The similar independently determined values of K.MPGR for X(I1) and Zn(I1) found in this work and by Christensen (1) demonstrate a striking insensitivity to the strength of the metal-imine bond. Since this is the case, then certainly the considerably smaller interligand effects transmitted via the metal ion must be negligible as
has been assumed. This insensitivity may result from a high ionic character in the metal ion-phenoxide ion bond. Our values for K a lie between ~ ~those~ reported by Christensen (1) and those reported by Davis, et al. ( 3 ) . However, even Christensen’s values may be considered only approximate since dissociation of the complexes to give lower species had not been taken into account. LITERATURE CITED
(1) Christensen, H. N., J. Am. Chem. Soc. 79, 4073 (1957). (2) Christensen, H. N., Ibid., 80, 2305
(1958); (3) Davis, L., noddy, F., hletzler, D. E., I b i d . , 83, 127 (1961). (4) Eichhorn, G . L., Dawes, J. W., Ibid., 76, 5663 (1954). (5) Heinert, I)., Alartell, A . E., Ibid., 85, 188 (1963). (6) Lenssing, D. L., Hanna, E. hl., Ibzd., 88, 696 (1966). (7) Leussirig, 1). L., Schultz, D. C., Ibzd., 86, 4846 (1964). (8) Jfetzler, D. E., Ibzd., 79, 485 (1957). (9) hletzler, 1). E., Ikawa, M., Snell, E. E., Ibzd., 76, 648 (1954). (10) Jletzler, D. E., Snell, E. E., Ibzd., 77, 2431 (1955). (11) Pfeiffer, P., Offerman, R., Werner, H., J . Prakl. Chern. 159, 313 (1941). (12) Sillen, L. G., Acla C h e n . S c a n d . 16, 195 (1962). (13) Snell, E. E., Fasella, P. Jl., Braunstein, A,, Ilossi-Fanelli,A., eds., “Chemical and Biological Aspects of Pyridoxal Catalysis,” Jlacmillan, Yew York, 1963. RECEIVEDfor review June 6, 1966. Accepted July 7, 1966. Work supported by the National Science Foundation (GP 1627).
Preparation, Stability, and Use of Preformed Solutions of Iodine Isocyanate SAMUEL ROSEN and DANIEL SWERN Fuel Research lnstitute and Department of Chemistry, Temple University, Philadelphia, Pa.
b
Solutions 0.4 to 0.5N in iodine isocyanate (INCO) were prepared at -30” C. in the dark in ether, tetrahydrofuran (THF), and glyme by reaction of an excess of pure silver cyanate with iodine. Rate of generation of INCO i s highest in glyme (30 minutes), next in THF (60 minutes), and slowest in ether (120 to 180 minutes). Decomposition of INCO at - 1 1 O C. is highest in glyme (75% decomposition in 24 hours) but considerably slower in THF and ether (15 to 25% decomposition in 2 4 hours). A differential iodometric analytical method has been developed for determining iodine and INCO in solution, thus permitting the generation, decomposition, and consumption of INCO in reactions to be accurately monitored. Preformed INCO sohtions react extremely rapidly with
1392
ANALYTICAL CHEMISTRY
many types of unsaturated compounds; the rate is at least ten times that observed in the conventional in situ reaction method. Care must be taken in storing and handling INCO solutions, as decomposing INCO solutions generate an unknown, colorless gas and slowly precipitate a mildly explosive white solid.
bamates, amines, ureas, and aziridines: AgCNO >C=C
?-?< I
N=C=O
I
ODIPI’E isocyanate
(INCO), a reactive pseudohalogen readily prepared by the reaction of silver cyanate with iodine in dry ether, is a useful reagent for forming carbon-nitrogen bonds from a wide variety of unsaturated compounds ( I , 2, 4-6). The addition reaction is a typical electrophilic reaction (d, 3,5),proceeding by stereospecific trans-addition of INCO to the double bond (6). The initial reaction products are vicinal iodoisocyanates, readily converted to car-
>c-c
c-c
C-C< ‘N’
I
H
>c-c
