Nuclear Magnetic Resonance Studies of Protonation of Polyamine and Aminocarboxylate Compounds in Aqueous Solution JAMES L. SUDMEIER and CHARLES N. REILLEY Department o f Chemistry, University of North Carolina, Chapel Hill, N. C.
b Application of nuclear magnetic resonance to the determination of protonation schemes of chelating agents of the polyamine and aminocarboxylate types is illustrated. Substituent shielding constants obtained from a study of methylenic proton chemical shifts of model compounds as a function of pH are employed to determine the distribution of protons among 'various sites on the ligand. These distributions, in conjunction with acidic dissociation constants measured in identical solution media, are useful in accounting for contributions of electrostatic and inductive effects to the average microscopic acid-base equilibria of these analytically important complexing agents.
C
INTEREST has been shown in the problem of determining protonation schemes of aminocarboxylate and polyamine chelating agents. Experimental methods most frequently employed include potentiometry (1, 15, 18, 19), calorimetry (5, 1 4 ) , ultraviolet spectrometry ( I S ) , viscometry ( 4 ) , and infrared spectrometry (3, 12, 1 7 ) . Several authors (3, 10) have shown the applicability of nuclear magnetic resonance spectrometry to this problem and have concluded that the first two protons added to the tetraanion (Y-4) of (ethylenedinitril0)tetraacetic acid (EDTA) attach solely to nitrogen atoms. Because of the great importance of the common aminocarboxylate and polyamine chelating agents as masking agents and as titrants for the determination of metal ions, a more complete understanding of their microqcopic acidbase behavior is necessary for securing optimum insight into their metalchelating properties. NSIR provides a powerful tool for determination of the activity of various protonation sites in such compounds. Therefore, N M R spectral studies of EDTA, ethyl ether diaminetetraacetic acid (EEDTA), ethylene glycol bis(p-aminoethyl ether)N,"-tetraacetic acid (EGT-I), N-hydroxyethylethylenediaminetriaceticacid (HEDTA), diethylenetriaminepentaacetic acid (DTPA), diethylenetriamine ONSIDERABLE
1698
ANALYTICAL CHEMISTRY
(dien), triethylenetetramine (trien), and tetraethylenepentamine (tetren) were undertaken. Spectra of trans-( 1,2-cyclohexylenedinitri1o)tetraacetic acid (CyDTA) and analogous compounds exhibit important and interesting peculiarities which will be discussed in the following paper (21). The use of N M R for determination of protonation schemes is based upon measurement of chemical shift values of nonlabile protons (protons attached to carbon atoms) as a function of pH. Acidic protons attached to nitrogen atoms and carboxylate groups are quite labile except a t low pH values; hence, their resonances are combined with that of water. However, nonlabile protons attached to carbon atoms adjacent to any functional group are sensitive to changes in the electronic environment caused by protonation, as shown by Grunwald, Loewenstein, Meiboom ( 7 ) , Loewenstein and Roberts (If), and others. As an aid to assignment of methylenic proton resonances and determination of protonation schemes, a table of shielding constants for some common substituents is derived. These substituent shielding constants should be useful in predicting proton magnetic resonance spectra of known compounds (particularly amino acids and peptides) and in identification of unknown compounds. The chemical shift data presented and interpreted in this paper should prove valuable in perfecting conditions for analytical determinations of mixtures of chelating agents by NRIR; saturation effects would, of course, need to be studied in any quantitative work. The data should also be useful for assignment of resonance peaks in WMR studies of metal chelates. EXPERIMENTAL
Proton magnetic resonance spectra were recorded using a Varian A-60 High Resolution Spectrometer. Chemical shift values were measured relative to t-butyl alcohol, a convenient internal standard, but. are reported relative to sodium 3-(trimethylsily1)-1,ropane sulfonate (available from Eastman; henceforth, abbreviated tms *), probably more universally acceptable as an in-
ternal standard in aqueous solution. The chemical shift value of the methyl resonance of t-butyl alcohol (1.29 p.p.m.) was independent of ionic strength and pH within experimental error. Sharp resonance peaks are measured with a precision better than j10.01 p.p.m., broad or highly split resonances with a precision better than 3Z0.03 p.p.m. All spectra were recorded a t the ambient temperature of the cell compartment, 32" f 2" C. Bulk susceptibility corrections were not made. The p H values were measured a t room temperature (26-27 ") using a Leeds & Northrup Model 7401 lineoperated pH meter equipped with Model 124138 microelectrode assembly. The meter was standardized before each set of measurements with two different National Bureau of Standards buffers. Because acidic pK, values were not available for the rather concentrated solutions used in these studies, the pK, value of each compound was determined using the same solution employed in chemical shift measurements. Semi-automatic recording of p H titration curves provided a rapid and accurate method of determining pK, values. A Sargent Recorder was connected to a variable span adjust (100ohm ten-turn Helipot) across the p H meter recorder terminals. Thus, with pH recorded continuously, the chart is advanced manually after each increment of titrant has been added and sufficient time allowed for attainment of equilibrium at that point. The titrant was standard 6 M C02-free potassium hydroxide (,J. T. Baker, AR). The concentration of solutions is generally -0.5M. Allthough sharp peaks, such as those of EDTA, are readily detected at concentrations as low as 0.01.11, the former concentration provides a good signal for more multiplesplit or broad resonance bands. Hecause potassium exhibits the least tendency to form alkali metal complexes, all solutions were prepared as potassium salts. Any chelon obtained as the sodium salt or neutrally charged acid was converted to the potassium form. 411 solutions were prepared using C0,free deionized water. Chemicals of the highest purity available were obtained and used without further purification. EDTA was obtained from the Fisher Scientific Co. HEDTX, EEDTA, EGTA, and DTPA were obtained from Geigy Chemical Co. Trien was used as the liquid
amine available from Union Carbide. Tetren pentahydrochloride was prepared from the hydrosulfate by precipitating the s'ulfate with slightly less than one equivalent of barium chloride per sulfate. Thus, a Jmall amount of urireacted tetren hydrosulfate was filtered along with the Bas04 and barium contamination was minimized. Tetren hydrosulfate was obtained by a procedure previously described (16). RESULTS AND DISCUSSION
When a compound contains two or more different types of functional groups capable of accepting protons, microscopic equilibria exist among the various forms which have all possible combinations of protonated and unprotonated sites. \Then one equivalent of acid is added to the fully basic form of such a compound, each site is protonated for a certain average fraction of time. Methylenic (and other nonlabile) protons are deshielded by protonation of a nearby baGc site and by an amount which depends upon the nature of the basic site, its proximity to the particular methylenic protonq, and the fraction of time protonated. This may be expressed as
where A8*c is t,he total calculated protonat'ion shift (change in chemical shift d u e caused by protonat,ion) of t'he i t h methylenic resonance (i = a, b, c,. . C,, is the protonation shift, constant of the i t h resonance for total protonation of the j t h basic site ( j = 1, 2, 3,. . .T), and f j is the average fraction of time during which the J'th site is protonated. Implicit in Equation 1 are two assumptions: first, that, the shielding contribution of a particular basic site is linearly related to the fraction of time protonated; second, that the contributions of protonating different, sites are perfectly additive. The former assumption, based upon t'he time-averaging effect of rapid exchange, is well substantiated by Grunwald, Loewenstein, and Meiboom (7'); the latter has been shown to apply reasonably well for niethylenic substitueiits by Shoolery and coworkers (6. 2 0 ) . I n order to dc,tcwnine \-dues off,, it is first necessary to determine C,, value:< for substituents most commonly found in chelating agrnts. For this purpose, methylenic I m t o n chemical shift:: were measured for a number of simlile model compounds in 1%-hichresonance peaks and ])rotonation shift contributions could unarn1)ipuoualy assip;ned. Model Compounds. Table I is a eoml)ilation of chemical 3hift values of model ronipoundq in aqueous solution a t various valuer: of T I , the numb o r of equivalents of acid added to 1 x 3
t h e fully unprotonated form. Only those chemical shift values which correspond to extremities of nonprotonation (all f, = 0) and complete protonation (allfi = 1) are reported in Table I. Chemical shift data of several compounds in Tables I and 111 have previously been reported ( 3 , 9 ) . Chapman, Lloyd, and Prince (3) have reported comparable data for ethylenediamine, N,iV, N', X' - tetramethylethylenediamine, glycine, S,.V-dimethylglycine, betaine, iminodiacetic acid, and nitrilotriacetic acid. Converting the results of Chapman et al. from T to 6 scales and setting tms at, f0.04 DS. tms* (subtracting from 10.04 l1.p.m.) yields values in excellent agreement with tho..qe reported in this paper for ethylenediamine, glycine, and betaine. Poor agreement is obtained for the remaining compounds, particularly for iminodiacetic acid. The data in Table I may be cast into simultaneous linear equations which, upon solution, yield the substituent shielding constant,s given in Table 11. These values are found to be internally consistent'to within =k0.03 p.p.m. in t8he limited number of cases in which comparison is possible. The normal chemical shift value of methylenic protons is arbitrarily set a t 1.25 p,p.m. us. tme*. The chemical shift value of a pair of methylenic protons is obtained by adding shielding constants of the two attached substituents to the normal value of 1.25 p.p.m. For example, the chemical shift value of the italicized met'hylenic protons in t,he formula
HJ-CH~--CH~-COOH
is equal to 1.80 0.35 1.25 = 3.40 p.p.m. us. tms*. Substituent Shielding Constants. Several trends may be noted in the substituent values in Table 11. Chemical shift increases as the distance from t h e functional group- is decreased, as shown b\- t h e first seven substituents. Protonation shifts also increase as t h e distance from t h e site of protonation is decreased. For example, protonation of a primary amine causes a chemical shift, of 0.05 p.p.m. when located two carbons away, 0.25 p.p.ni. when located one carbon away, and 0.45 13.p.m. !Then directly attached to the methylene group under observation. These trends are expected on the basis of electronegativity and inductive effects. Protonation shifts of amines increase in the order Io < 11" < 111' (0.45 < 0.55 < 0.75 p.12.m.). Hydroxyl and ether groups greatly deshield methylenic protons. These groups cause a greater chemical shift of adjacent niethylenic protons than quaternary nitrogen atoms. Thus, methylene groups att,ached to oxygen atoms are readily distinguished from
+
+
Table
I.
Chemical Shift Values of Model Compounds
(p.p.m. os. tnis*) n= n = 0 1 1,4-Butanediamine -CH*--S 2 65 -CHS1 48 1,3-Propanediamine -CHz--S 2 70 --CHZy 1 60 Ethylenediamine 2 67 Ethylamine -CH,i2 68 3 . 1 2 -CH; 1 10 1 . 3 2 Diethyl&ne -CH*2 64 3 . 1 5 -CH, 1 08 1 . 3 2 S,SCdTkethyle thylenediamine -CH*2 69 -CHI 2 37 Triethylamine -CHZ2 55 3 . 2 4 -CH, 1 05 1 . 3 2 .V,.Y-dimethylethylenediamine -C"z--SHz 2 75 -CH*--SRz 2 53 --CHI 2 27 A',.\, .Y',~V'-tetrarnethyle t hylenediamine -CH*2 53 -CH, 2.27 Tetraethylammonium bromide -CH*3.34 -CH, 1.32 Tetra-n-propylammonium bromide -CH,--S 3.24 -CHS1 75 - CH, 1.02 Ethanolamine -CH,-O 3 67 3 90 -CH,--S 2 78 3.24 E thyleneglyrol 3 73 E: thyleneglyrol monornethyl ether CH,--OH 3.79 CHl-OR 3.62 3.45 CH, Propionic acid -CH?2 23 2 45 -CH, 1 10 1 12 Suceinnic acid 2 47 Adipic acid 2 25 -CHp-CO -CH,T 1 58 Methylamine 2 34 2 65 Dimethylamine 2 34 2 78 Trimethylamine 2 23 2 97 Tetraniethylammonium chloride 3 27
n= 2 3.08 1.80 3.20
2.10 3.44
3.54 2 88
3.57 3.57 3.05
3 72 3 12
2 73
other types of methj lene g r o u p in that the resonance occurs a t lon field ytrengths and is not appreciabl> influenced by change in pH ~ a l u e ,the 0x1 gen atom being a very n eak base in w c h compound, Reionances of meth? 1ene g i o u p ~ attached directl? to piotonated nitrogen atoms ma! occur at w i i l a r l j lon field strengths but are shifted *harplj upfield upon wmo\ a1 of the proton The plotonation shift of methylene groups diicctl? attached to carbokylate VOL. 36, NO. 9 , AUGUST 1964
1699
groups is only 0.20 p.p.m., an important feature in distinguishing various protonation schemes. Application of Substituent Shielding Constants to Selected Test Compounds. The model compounds used
in obtaining the substituent shielding constants in Table I1 are generally symmetrical and monofunctional. Perfect additivity of two substituents X and Y , as pointed out by Jackman ( 8 ) , is possible only when X and Y exist in a constant average orientation to one another. Thus, the additive property of substituent shielding constants obtained from simple model compounds may tend to break down for compounds having different average orientations.
Table II. Methylenic Substituent Shielding Constants
(normal -CH2at 1 25 p -CH~-CH~-CH~--NHI+ -CHrCH2--?JHz -CHn-CH2--NH3 -CH2--NH2 --CH2--NHa+
p m. us. tms*) 0 00 0 05
10 15 40 (0 45)” -“, 35 --?;Ha+ 80 ( 1 85Ia -CHI 05 -CH*--NRH 0 15 (0 20)” -CHI--NRH2 + 0 40 (0 50)a --?;RH 1 30 1 85 i l 9OP 0.15 0.50 1.15 1.90 2.00 0.20 2.25 0.25 -CH,-OR 2.15 -OR 0.30 -CH,--C020.35 -CH,-COOH 0.90 -co* 1.10 -COOH a Modified for application t o polyamine +
0 0 0 1 1 0
\
Piperazine Triethylenediamine p-alanine -4HQ-N -CH*Glycine N-methyl glycine -CH2-CH3 Y,S-dimethvl glycine ” -CHZ-CH3 Betaine -CH, -CH3 Iminodiacetic acid ( K + salt) (?;a+ salt) Xitrilotriacetic acid
1700
CH2-COz-
(1)\S-CHz-CHz-N (2”) -02C-CH2
I
expected, deviations are found, but they are relatively small and appear to follow regular patterns. The diamines, for example, exhibit negative deviations ( 6 , ~ < 6,) whereas the amino acids exhibit positive deviations ( & e > 8,). Zwitterionic forms of amino acids exhibit the largest deviations and cationic forms the smallest. Increasing the extent of N-methyl group substitution does not lower the
Table 111.
n=O Found
deviations as much as increasing the extent of N-acetate group substitution. Since aminocarboxylate chelating agents are highly substituted with N-acetate groups, the latter observation is particularly helpful. As will be shown, agreement between calculated and observed chemical shift values in the chelating agents is usually very good. Furthermore, the systematic component of the deviations is largely eliminated by using only net differences-Le., calculating protonation shifts, As,., to fit only the observed net protonation shifts, A&. E D T A . Chemical shift values of methylenic protons in E D T A as a function of pH have been reported by Kula et al. (IO). Experimental work in this laboratory yielded results in close agreement. Kula et al. conclude that the first two equivalents of acid added to EDT.4 protonate nitrogen atoms and not carboxyl groups. It is, therefore, of interest to test this conclusion by application of protonation shift constants derived from Table 11.
(2) -02C-CHz
compounds.
Calcd.
Nonadditivity may be enhanced by long range shielding effects of magnetically anisotropic groups such as carboxylate. For this reason, the shielding constants were slightly modified for application to polyamine compounds (dien, trien, and tetren) and these modified values enclosed in parentheses in Table 11. Chain-stiffening effects in the fully protonated compounds may be responsible for appreciable deviations which occur using the first quoted values. Therefore, the best model compounds for determining protonation shift constants applicable to polyamine compounds are the polyamines themselves in the fully unprotonated (n = 0) and protonated ( n = 3‘) forms. In Table 111, the calculated and observed chemical shifts of a number of test compounds are listed. For all aminocarboxylate test compounds, calculated values are based on the assumption that nitrogen atoms are fully protonated before any protonation of carboxylate groups takes place. As
/
\CHr-CO2-
(2”’)
The EDT.4 tetraanion (YP4) contains six basic sites capable of accepting protons: two identical nitrogen atoms (labeled 1 and 1’) and four identical carboxylate groups (labeled 2 , 2‘, 2”; and 2”’). The primary object of this study is to determine the proton distribution a t various values of n by evaluating f i , fi’, fz, fz‘, f ~ ” and , f2”‘. Because of rapid exchange of ionizable protons
Chemical Shift Values of Selected Test Compounds (p.p.m. us. tms*) n = l ni-2
n = 3
Calcd.
Found
Dev.
Calcd.
Found
3.10 3.10
3.25 3.37
-0.15 -0.27
3.50 3.65
3.73 3.94
Dev. -0.23 -0.29
2.70 2.55
2.85 2.85
Dev. -0.15 -0.30
2.90 2.30 3.50
2.87 2.37 3.23
0.03 -0.07 0.27
3.35 2.55 3.95
3.25 2.62 3.62
0.10 -0.07 0.33
3.40 2.75 4.15
3.30 2.87 3.95
0.10 -0.12 0.20
3.55
3.18 2.35
0.27
4.00
3.69 2.80
0.31
4.20
4.00 2.87
0.20
3 30
3 05 2 30
0 25
4 05
3 77 2 98
0 28
4 25
4 12 3 05
0 13
4 15
3 97 3 32
0 18
4 35
4 30 3 39
0 05
3 45 3 45 3 30
3 22 3 22 3 25
0 23 0 23 0 05
4 00 4 00 4 05
3 72 3 72 3 87
0 28 0 28 0 18
4 10 4 10
3 95 3 94
0 15 0 16
ANALYTICAL CHEMISTRY
(2’)
/(1’)
Calcd.
Found
Dev.
4 20 4 20
4 13 4 13
o
07
0 07
with water, the extent of protonation a t all equivalent basic sites must be equal and thus the following may be written: fi
= f,’; fi =
f2’
=
I g”
= f2’”
(2)
The protonation shifts of acetate a and ethylenic b protons are (calculated using Table I1 and Equations 1 and 2 as follows : htSaC= (1.90 - 1.15)fi
-
1.15)fi -t (0.50 - 0.15)fi’ = 1 lOfi
(4)
Contributions from additional terms are assumed to be negligible. The total time spent on all sites by protons for TZ = 1 must be equal to unity, and more generally,
2 x,
a,f, = n
(5)
j=1
where a, is the multiplicity of the j t h basic site. This relationship makes possible elimination of one of the variables f l and fz. Because M , the number of equations (one for each proton resonance) is now greater than N - I , the number of unknowns, all values of f , are overdetermined in the case of EDT.1 and in all subsequent cases. Thus, any set of f l values leads to values of A 6 , c which itre not in perfect agreement 1% ith the corresponding observed Aak values. For reasons previously discussed, this lack of agreement arises essentially from errors caused by inadequate additivity of substituent shielding constants. In the absence of specific, quantitative knowledge of average rotamer populations on the shielding contributions of individual rotamer states, these errors are assumed to independent randoni and normally distributed and least squares methods were applied. The set of coefficients fi, j 2 , . .fN-l is chosen such as to satisfy the condition ~
~
I
M
(As,. - A6,)2 = 2=1
2 M
=: minimum
(6)
a=1
where d, is the deviation of the i t h methylenic proton resonance. Partial differentiation of Eqiiiation 6 with respect to f , , for all values of j leads to a set of N - 1 normal equations which yield a unique solution. .\ program was written in IT language for setting up and solving the normal equations on the Univac 1105 computer. Once the values of f l , M f2.
. .f%-land
Table IV.
+
(1.10 - 0.901f2 = 0.75,fi f 0 20f2 (3) A6bc = (1.90
squares methods. I n order to find the standard deviation of the variable eliminated using Equation 5 , an alternate variable was eliminated and the computation repeated. .-I measure of the overall. agreement obtained between calculated and ob-
d t 2 are known, then a=1
u,,, the standard deviation of f,, is computed by straightforward least
n = O
served data is given by the standard deviation U , where
The
3.25 3.30 2.80 2.65 3.67 3.55
3 95 4 02 3 60 3 37 3 87 3 89
EGTA ( a ) Obsd. Calcd. ( b ) Obsd. Calcd. ( c ) Obsd. Calcd. ( d ) Obsd. Calcd.
3.27 3.30 2.82 2.65 3.70 3.55 3.72 3.65
3 4 3 3 3 3 3 3
HEDTA (a)Obsd. Calcd. ( b ) Obsd. Calcd. ( b ‘ ) Obsd. Calcd. ( c ) Obsd. Calcd. ( d ) Obsd. Calcd. ( e ) Obsd. Calcd. DTPA ( a ) Obsd. Calcd. ( b ) Obsd. Calcd. ( c ) Obsd. Calcd. ( d ) Obsd. Calcd. Dien (a)Obsd. Calcd. ( b ) Obsd. Calcd. Trien (a)Obsd. Calcd. ( b ) Obsd. Calcd. ( e ) Obsd. Calcd. Tetren (a)Obsd. Calcd. ( b ) Obsd. Calcd. ( c ) Obsd. Calcd. ( d ) Obsd. Calcd.
magnetic
Chemical Shift Values of Chelons (p.p.m. us. tms*) n = l n = 2 n = 3 n = 4
EDTA ( a ) Obsd. Calcd. ( b ) Obsd. Calcd. EEDTA ( a ) Obsd. Calcd. ( b ) Obsd. Calcd. ( c ) Obsd. Calcd.
3.22 3.30 2.70 2.55
proton
3 3 3 3
67 69 25 13
3 4 3 3
resonance
n = 5
97 02 74 61
92 01 55 36 97 88 78 65
3.25 3.30 2.77 2.55 2.77 2.55 2.77 2.60 3.72 3.65 3.24 3.30
3 3 3 3 3 3 3 3 3 3 3 3
62 62 25 05 25 09 22 61 88 83 64 69
3 95 3 95 3 75 3 55 3 75 3 57 3 52 3 31 4 04 3 98 4 05 4 08
3.25 3.30 2.73 2.55 2.73 2.55 3.20 3.30
3 3 3 2 3 2 3 3
43 50 10 89 10 95 53 62
3 3 3 3 3 2 3 3
92 94 40 24 14 96 33 41
3.97 4.05 3.52 3.37 3.32 3.31 3.68 3.93
2.77 2.80 2.70 2.70
2 3 2 2
99 06 91 91
3 3 3 3
16 31 04 03
3.57 3.60 3.57 3.51
2.79 2.80 2.71 2.70 2.75 2.75
2 3 2 2 2 2
94 02 88 89 87 88
3 3 3 3 2 2
12 25 04 07 95 97
3.35 3.45 3.31 3 30 3 19 3.21
3.60 3.60 3 60 3 60 3 65 3 65
2.79 2.80 2.73 2.70 2.76 2.75 2.76 2.75
2 2 2 2 2 2 2 2
9i 98 89 87 89 88 89 89
3 3 2 2 2 2 2 3
07 18 98 97 95 94 98 02
3 3 3 3 3 3 3 3
3 3 3 3 3 3 3 3
15 32 03 05 08 06 20 28
45 52 41 41 33 32 29 30
VOL. 36, NO. 9, AUGUST 1964
3 3 3 3 3 3 3 3
55 60 62 60 69 66 69 65
1701
spectrum of E D T A 1 tetraanion (YF4) shows two sharp resonance peaks, one attributed to acetate (a)protons and containing twice the area of the other, attributed to ethylenic ( b ) protons. Table IV gives the observed chemical shift values of a and b protons us. tms* for n = 0, 1, and 2. Values calculated using substituent shielding constant. a t n = 0 agree well with this data, except for the small positive deviations of acetate protons and small negative deviations of ethylenic protons, which occur in test compounds. This agreement further supports assignment of a to acetate protons and b to ethylenic protons. Table V reports the calculated values of f l , u j 1 ,f2, u j l l and u for EDTri at n = 1 and 2 . [-sing these valueq of f i and f2, protonation shifts of a and b are calculated and added to calculated values a t n = 0, yielding the results Shown in Table IT' at n = 1 and 2. Except for small deviationq also present a t n = 0, which are deliberately held constant, agreement is very good. These results indicate that when two equivalents of acid are added t o the virtually comE D T h tetraanion (YP4)> plete protonation of nitrogen atoms takes place, in agreement with Kula et al. (10).
Table V.
EEDTA. Figure 1 shows the structural formula of EEDT.1, a typical proton resonance spectrum, and the chemical shifts of various methylenic resonances as a function of p H . T h e structural formula shows eight identical acetate protons (labeled a ) , four identical methylenic protons adjacent to nitrogen atoms (labeled b ) , and four identical methylenic protons adjacent to oxygen atoms (labeled c). Numbers 1 and 2 designate the amine and carhoxylat,e sites, respectively. Because a protons are not appreciably spin-coupled to other nuclei, they exhibit a sharp resonance peak. On the other hand, b and c prot'ons are appreciably spin-coupled to each other (and to no other nucleus) and exhibit a pair of triplets when A& is sufficiently large. The centers of t'he peak and two triplets are given in Table IV along with values calculated from substituent shielding constants. Excellent agreement is obtained a t n = 0, aiding in assignment of resonances. I3ecause of the small differences between pK3 and pK4 in E E D T A and EGT-A] chemical shift, values are not' reported for n = 1. These small values of ApK are very close to the statistical minimuq for dibasic acids (ApK = 0.6), indicating that two sites of equal
Per Cent Protonation at Various Basic Sites
Compound
fr
fl
f2
53 1 5 96 f 2
-2 f 3 2 1 1
f 6 5 1 30
96 f 11
2 f 5
fll 3
n = 2
94 f 6
3 1 3
f 6 4
HEDTA n = l n = 2
42 f 7 87 16
97 f 5
26 f 1 85 f 5 80 f 11
41 f 1 15 16 64 f 13
41 f 4 92 f 12 98 i 6
18 f 8 16 f 25 104 f 12
fll 9 1 5 9
36 f 6 76 f 10 99 i 7 100 f 1
14 f 6 24 f 10 51 f 7 100 f 1
f 5 2 f9 2 f 6 4 f l 0
29 f 1 60 i 11 99 f 18 104 f 6 99 f 5
13 f 1 11 f 11 9 f 19
EDTA n = l n = 2
f3
U
EEDTA n = 2
EGTA
DTPA n = l n = 2
n = 3 Dien n = l n = 2
n = 3
Trien n = l n = 2 n = 3 n = 4
Tetren n = l n = 2 n = 3 n = 4 n = 5
1702
52 f 6
67 f 6 101 f 5
ANALYTICAL CHEMISTRY
3 f 18 0 f 15
0 f 21 16 i 18
f5 8 It4 8
7 f 2 0 f 10 76 f 23
4 ' 28
f l 0 f 3 7 f13 0
...
1 39
16 f 1 40 f 15 84 f 25
58 f 8 100 f 7
f O (i f h 1
f13
i
f 4 3
1 38
basicity are protonated simult,aneously as n goes from 0 to 2, making n = 1 a rather arbitrary point. For increasing n from 0 to 2, ASa and ASb are larger than A&. The triplet exhibit'ing the larger prot,onation shift is properly assigned t.0 methylenic protons lying closer to the site undergoing protonation. Calculated values of f l and f 2 in Table V indicat'e that in E E D T . l protonation of both nitrogen atoms is virt'ually complete a t n = 2. EGTA. Figure 2 gives the structural formula of E G T 1 , a typical spectrum, and the chemical shift values of various methylenic resonances as a function of p H . Except for t h e presence of four additional d protons lying adjacent to oxygen atoms, t'he structure is identical to that of EEDT-A. Hence, the spectrum should be similar to that previously discussed except for the presence of an additional singlet, and this is found to be the case. h s shown in Table IV, observed values of the resonance centers are in good agreement with calculated chemical shift values for n = 0, lending support to the given assignments. For increasing n from 0 to 2, ASa and &b are larger than A&, and ASd is very small. This sequence agrees well with the expect,ed sensitivities of various met'hylene groups to changes in the electronic environment of basic sites. Calculated values of fl and f2 in Table V indicate that in the case of E G T h , as in previous cases, protonation of both nitrogen atoms is virtually complete a t n = 2. HEDTA. The struct'ural formula in Figure 3 shows four identical acetate protons (labeled a) adjacent' to nitrogen atom 1 and t'wo acetate protons (labeled e) adjacent to nitrogen atom 2 . The methylenic protons adjacent to nitrogen atom 1 (labeled b ) are not necessarily equivalent to the methylenic protons adjacent to nitrogen atom 2 (labeled b ' ) , although no chemical shift difference has been observed in this study. Nethylenic protons c (adjacent, to nit,rogen 2) and d (adjacent to the hydroxyl group) are spin-coupled and should exhibit a pair of triplets when AScd is sufficiently large. I t can further be predicted from substituent shielding constants that d protons are the most highly deshielded. The spectrum in Figure 3 shows a triplet at low field strength, two overlapping singlet,s, one having twice the intensity of the other, and a large peak midway between two smaller signals. Lowering pH reveals the latter to be a sharp singlet superimposed upon a trilllet of area equal to that of the triplet' at IOK fitld strtngth. This illustrates the usefu1nes.q of pH variation in interiiretation of S 3 I R spectra. A;; qhown in Table IT'] observed values of the resonance centers are in good agreement
12
PH IC .pK4’ 9
8
E
c I
4.0 40
3.5
S, p.p.m Figure 1 .
,
.
.
8,
3.0
vs. t ms”
,
Figure 2.
(
.
,
3.5 p.p.m. vs. tms”
,
I
,
,
L
3.0
Chemical shift of EGTA a t various pH values
Chemical shift of EEDTA a t various pH value
with calculated chemical shift values for n = 0, lending support to the given
assignments. During addition of the first equivalent of acid, resonances undergo protonation shifts of magnitude ASb = Alibi, > As, > A& > A& > ASd The e resonance crosses over the a resonance, emerging downfield of a. The c resonance undergoes a larger protonation shift from n = 0 to n = 1 than from n = 1 to n = 2. Although these facts suggest a tendency of protons 00 populate nitrogen 2 more extensively than nitrogen I , the fact that b arid 6’ exhibit a sharp singlet a t all p l l values indicates nearly equal protona.tion of nitrogen atoms. Calculated values in Table V reflect these observations in the fact that f2 is somewhat larger than fi at n = 1. However, the magnitudes of calculated standard deviations are such that no appreciable difference in basicities of the nitrogen atoms is indicated. It is of interest to compare this result with that of Nakamoto, hlorimoto, and Martell (f 2 ) who concluded from infrared qtudies that protonation of nitrogen 1 occurs to a somewhst greater extent
than protonation of nitrogen 2 at n = 1. At n = 2, protonation shifts of all resonances are nearly double their respective values for n = 1, with the exception of c . Calculated values of fi, f2, f3, and f4 in Table V indicate that nitrogen atoms are almost fully protonated a t n = 2, in agreement with Nakamoto et al. (12). DTPA. T h e structural formula of D T P A in Figure 4 shows eight acetate protons labeled a a n d two acetate protons labeled d. Because neither a nor d protons are appreciably spincoupled to a n y other nucleus, two sharp singlets containing relative areas of 4:1 are expected. The two types of ethylenic protons b and c are strongly spin-coupled and should give rise to .&Bz patterns when is sufficiently large. I n alkaline solution (n = 0), 6, and 6 d are nearly equal, giving rise to adjacent singlets, and bb and 6, are nearly equal, giving rise to a slightly broad peak. The vertical dotted lines in Figure 4,indicating peak half-width, are approximately equal to 6 b and 6,. However, as shown by the calculated values in Table TV, there is no means for
differentiation of the pair a t n = 0, and both 6* and 6, are reported as the peak center. Addition of one equivalent of acid (n = 1) lowers the solution p H value to a point which lies midway between the numerical values of pK4 and pK5. I n approaching this point, it is apparent that d protons are deshielded more rapidly than a protons, implying a higher population of protons on the middle nitrogen atom (site 2 ) than on either end nitrogen atom (site 1 ) . This insight is confirmed in the calculated values of f l ,f2, and f 3 in Table V. A rather large negative value of f4 is obtained unless f 4is set equal to zero, as is done in the present calculation. These results indicate that the middle nitrogen atoms are more strongly basic than the end nitrogen atoms. This result opposes that of Nakamoto et al. (12) who found end nitrogen atoms to be more strongly basic at n = 1. As addition of a second equivalent of acid (n = 2) takes place, d resonance undergoes a negative protonation shift, once again crosses over the a resonance, and moves upfield close to the previous value of at, n = 0. The spectrum in VOL. 36, NO. 9, AUGUST 1964
1703
Figure 3.
Chemical shift of HEDTA at various pH values
Figure 4 was recorded at a p H value immediately below the crossover point. Simultaneously, a and b resonances undergo large protonation shifts, and Agbc becomes large. The dzBz pattern thus formed becomes a pair of symmetrical bands whose centers of gravity are reported as approximate values of gb and 6,. Values of f l , j2,fa, and f4 in Table V indicate a predominance of protonation a t end nitrogen atoms, showing the effect of electrostatic repulsion. This distribution is in general agreement with that found by Kakamoto et al. (12). In going from pa = 2 to n = 3, d undergoes the greatest change in chemical shift, followed by c , b, and a in that order. Calculated values of fi,f ~ and , j 3 in Table V indicate that the middle nitrogen atom (site 2) and the middle carboxylate group (site 3) undergo the greatest increases in protonation during this step. A large negative value of j4is obtained in the general solution. The assumption that j 4 is equal to zero was made in the present calculation, and yields a smaller standard deviation than the assumption that f3 is equal to zero. The magnitude
1704
ANALYTICAL CHEMISTRY
Figure 4.
Chemical shift of DTPA at various pH values
of the standard deviation indicates relatively poor agreement between calculated and experimental data. This appears to be caused by rather large nonideality of d protons, whose environment is unique among compounds studied in this paper. It is interesting to note the generally good agreement of these results with that of Nakamoto et al. (12) who estimated values of fi = 100%) f2 = 25% f3 = 75%, andf4 = 0%. Dien. T h e structural formula of dien in Figure 5 shows two different types of methylenic protons: a, adjacent to end nitrogen atoms (site 1) and b, and adjacent to middle nitrvgen atoms (site 2). Because of spincoupling between a and b protons, mutual splitting occurs when becomes sufficiently large, as occurs when protons become unequally distributed among sites 1 and 2. At pH = 13, a rather broad single peak, whose center is indicated by the solid line in Figure 5 , is observed. The vertical dotted lines in Figure 5 , indicating peak half-width, are given as approximate chemical shift
values of a: and b, in order of increasing field strength. At pa = 1, the p H value of the solution lies numerically midway between pK4 and pKs. Calculated values of f i and fz, based upon the slightly modified polyamine protonation shift constants in Table 11, indicate a somewhat greater extent of protonation a t end nitrogen atoms, partly due to the presence of appreciable diprotonated species. At n = 2, mutual splitting of a and b occurs to maximum extent, producing a spectrum similar to that shown in Figure 5 . Approximate values of 6. and were obtained by taking centers of gravity on either side of the line bisecting the pattern. Calculated values of f l and f2 in Table V show almost complete protonation of end nitrogens and very slight protonation of middle nitrogens. I n this distribution originally proposed by Prue and Schwarzenbach ( I @ , the molecule should have minimum energy of electrostatic repulsion. At n = 3, 6, becomes equal to 6b,
1.
I
Figure 5.
*
.
3.5
{
I
3.0* 6, p,p,m. v s . tms
l
2.5
l
Chemical shift of dien a t various pH values
and the d z B z pattern collapses into a sharp singlet. Calculated values of f i andfz fall within standard deviation of loo%, showing a high degree of consistency in the polyamine protonation shift constants. The above protonation scheme is compatible with results of calorimetric studies of Ciampolini and Paoletti ( 5 ) . Trien. As ,ahown in Figure 6, trien is composed of three types of methylenic protons: 13, lying adjacent to end nitrogen atoms (site 1 ) ; b , lving between a and middle nitrogen atoms (site 2 ) ; a n d I:, lying between pairs of middle nitrogen atoms. I n general, a and b exhibit AzBz patterns similar to those of dien. Because of symmetry, c protons always give rise to sharp singlets. I n strongly alkaline solut'ion (n = 0), a slightly broad symmetrical peak is observed. The band center is given as 6, and the half-width points are given as 6, and 6a, in order of increasing field strengt,h. At n = I , the spectrum is composed of a singlet immediately upfield of a triplet-like .4zBz pattern (whose center is indicated by solid line). The latter is solved for approximate values of 6, and Sb (indicated b;y vertical dotted lines) by a procedure described above. Values of f i and fz in Table V show t'hat in trien, as in the preceding case, pro-
35 Figure 6.
3.0
6, p.p.m. vs. tms
2.5
Chemical shift of trien a t various pH values
tonation of end nitrogen atoms is somewhat preferred. This is caused, in part, by the presence of appreciable diprotonated species concurrent with the overlapping of equlibria represented by K a and K 4 . During addition of a second equivalent of acid, the AzBz pattern broadens and becomes shifted downfield from the singlet, resulting in a spectrum similar to that shown in Figure 6. The calculated distribution given in Table V is expected to have a low energy of electrostatic repulsion. This distribution is realized in a statistical arrangement of two protons on four nitrogen atoms, forbidding 1-2 combinations and slightly emphasizing 1-4 combinations. i i t n = 2 , all resonances undergo nearly equal protonation shifts. causing little change in appearance of spectra. Calculated values of f, and f z show virtually complete protonation of end nitrogen atoms and approximately 50y0 protonation of middle nitrogens. With this distribution, originally proposed by Schwarzenbach (18), the molecule
should have the lowest energy of electrostatic repulsion. Addition of a fourth equivalent of acid causes the resonances of a and b to coalesce, forming a sharp singlet. The c singlet then crosses over the latter singlet, emerging on the downfield side. I n Table V t,he consistency in polyamine protonation shift constants is exemplified by calculated values of f i = 100%. The above protonation scheme is consistent with the findings of Paoletti, Ciampolini, and Vacca (14) based on calorimetric data. Tetren. As shown in Figure 7 , tetren contains two sets of spincoupled protons: a, coupled with b; and c, coupled with d. I n strongly alkaline solution ( n = 0) a slight'ly broad singlet is observed. Half-width points are assigned t,o a and b in order of increasing field strength and t'he peak center to c and d. At ra = 1 the spectrum shows a broad triplet-like AzBz pat'tern downfield of a sharper resonance. Qualitatively, this indicates a preference for protonatio2 of end nitrogen atoms. Calculated values of VOL. 3 6 , NO. 9 , AUGUST 1964
1705
fi, fz, and f3 in Table V support this observation. In proceeding to the point at which n = 2 , all resonances undergo nearly equal protonation shifts, producing little change in splitting patterns. Proton population, as shown in Table V, is highest on end nitrogen atoms (site I ) and on middle nitrogen atoms (site 3), partly caused by the presence of appreciable triprotonated species. At n = 3, the spectrum is composed of two overlapping A2Bzpatterns similar in appearance to that shown in Figure 7 (recorded at crossover point p H = 6.04). Both patterns reach maximum width a t this point implying a high degree of protonation at sites 1 and 3. The calculated distribution in Table V shows almost complete protonation of sites 1 and 3 and almost no protonation of'site 2 . I n proceeding from n = 3 to n = 4, the more upfield ilzB2 pattern crosses over the other AzBz pattern. The former collapses into a singlet while the latter collapses briefly and becomes broad once again. A key assumption in regard to assignment of resonances a t all values of n is introduced at this point : because of electrostatic forces which favor protonation at the ends of the molecule, the AzBz pattern undergoing the greater protonation shift is attributed to the a and b pair. Values of fi, f 2 , and f3 in Table V reflect dramatic changes in proton distribution caused by electrostatic repulsions. Having high proton density on end nitrogen atoms (site I ) , less on second nitrogen atoms (site 2 ) , and still less on middle nitrogen atoms (site 3), the tetren molecule conforms to the distribution expected to have minimum energy. -1ddition of a fifth equivalent of acid caubes 6, and ad to coalesce and cross over 6, and 6*, which become slightly separated. Values of f,,f2, and f3 in Table V are close to loo%, showing the consistency of polyamine protonation shift constants.
pH: 6.04 I'
t
7
"
I
3.5 Figure 7.
1
LITERATURE CITED
(1) Carini, F. F., Martell, A. E., J . A m . Chem. SOC.75, 4810 (1953). (2) Chapman, D., J . Chem. SOC. 1955,
1766.
1706
ANALYTICAL CHEMISTRY
'
8
6, p.p.m. vs.
I
3.0 tms
'
811'
'
I
25
Chemical shift of tetren a t various pH values
(3) Chapman, D., Lloyd, D. R., Prince, R. H., Zbid., 1963, 3645. (4) Charles, R . G., J . Am. Chem. SOC.78, 3946 (1956). (5) Ciampolini, M., Paoletti, P., J . Phys. Chem. 65. 1224 (1961).
ACKNOWLEDGMENT
The authors are indebted to S. P. Bag for invaluable aid in obtaining certain experimental data.
'
85. 2930 fl"963). (11) 'Loewenstein; A., Roberts, J. D., Zbid., 82, 2705 (1960). (12) Sakamoto, Tu'., Morimoto, Y., Martell, A . E., Zbid., 85, 309 (1963). (13) Olson, D. C., Maraeruni, D. W., Zbid., 82; 5602 (1960). -
(14) Paoletti, P., Ciampolini, M., Vacca, A. , J . Phys. Chem. 67, 1605 (1963). (15) Prue, J., Schwarzenbach, G., Helv. Chim. Acta 33, 985 (1950). (16) Reilley, C. S . , Vavoulis, A,, ANAL. CHEM.31, 243 (1959). (17) Sawyer, D. T., Tackett, J. E., J . Am. Chem. SOC.85, 314 (1963). (18) Schwarzenbach, G., Helv. Chim. Acta 33, 974 (1950). (19) Schwarzenbach, G., Ackermann, H., Zbzd., 30, 1798 (1947). (20) Shoolery, 3. S . , Technical Information Bulletin 2, No. 3, Varian Associates, Palo Alto, Calif., 1959. (21) Sudmeier, J. L., Reilley, C. N., ANAL. CHEM.36, 1707 (1964). RECEIVED for review November 19, 1963. Accepted May 22, 1964. Research supported in part by Sational Institutes of Health Grant RG-8349. Presented at the Southeastern Regional ACS Meeting, November 15, 1963.
