Anal. Chem. 1982, 5 4 , 615-621 (9) Desreux, J. F.; Merclny, E.; Loncln, M. F. Inorg. Chem. 1981, 20, 987-991. ( I O ) Everhart, Dnnnls S.; Evllla, Ronald F., University of New Orleans, unpublished work. (11) Pemberton, Jeanne E.; Buck, Rlchard P. S. fhys. Chem. 1981, 85, 248-262. (12) Cralg, Norman C.; Levln, Ira W. Appl. Spectrosc. 1979, 33, 475-476. (13) Gallagher, P. K. J. Chem. Phys. 1984, 4 1 , 3061-3069. (14) Sarasua, Martha M. Ph.D. Thesls, Unlverslty of North Carolina, 1980. (15) Kropp, John L.; Wlndsor, Maurice W. J. Chem. fhys. 1985, 4 2 , 1599- 1608. (16) Habenschuss, Anton; Speddlng, Frank H. J. Chem. Phys. 1980, 7 3 , 442-450. (17) Albertson, JBrgen; Eldhg. Inga Acta Crysfallogr.. Sect. 8 1977. 833, 1480-1469. (18) Hoard, J. L.; Lee, Byungkook; Llnd, M. D. J . Am. Chem. Sot. 1985, 87, 1612-1613. (19) Lee, Byungkook Ph.D. Thesis, Cornell Unlversity, 1967. (20) Nasslmbenl, Lulgl R.; Wrlght. M. Robert W.; van Nlekeirk, Jill C.; McCallum, IPamela PI. Acta Crysfallogr., Sect. 8 1979, 835, 1341- 1345. (21) . . Martln. Leslie L.: Jacobson. Robert A. Inom. Chem. 1972,. 1 1., 2785-2789. (22) Martln, Leslie L.; Jacobson, Robert A. Inorg. Chem. ‘1972, 11, 2789-2795. (23) Kostromlna, N. A.; Tananaeva, N. N. Russ. J. Inorg. Cham. 1971,
-
--.
16. 1256-1:!59.
(24) Geler, G.; Jargensen. C. K. Chem. Phys. Lett. 1971, 9 , 263-265. (25) Durham, D. A; Frost, (3. H.; Hart, F. A. J. Inorg. Nucl. Chom. 1969, 39, 633-838. (26) Sayre, Edward V.; Freed, Simon J. Chem. Phys. 1956, 2 4 , 1213-12 19.
615
(27) Albertsson, Jorgen Acta Chem. Scand. 1972, 26, 1023-1044. (28) Slnha, Shyama P. “Europium”; Sprlnger-Verlag: New York, 1967; p 116. (29) Rellley, Charles N.; Good, Bennle W.; Ailendoerfer, Robert D. Anal. Chem. 1978, 48, 1446-1458. (30) Sayre, Edward V.; Miller, Donald G.; Freed, Simon J . Chem. Phys. 1957, 26, 109-113. (31) Melo, S6rglo M.; Serra, Osvaldo A. “The Rare Earths In Modern Sclence and Technology”; McCarthy, Gregory J., Rhyne, James J., Silber, Herbert B., Eds.; Plenum Press: New York, 1980; Vol. 2. (32) Kuya, M. K.; Serra, Osvaldo A. J. Coord. Chem. 1980, 10, 13-18. (33) Galduk, M. [.; Zolin, V. F.; Galgerova, L. S. “The Luminescence Spectra of Europium” [In Russian]; Nauka: Moscow, 1974; p 166. (34) Judd, B. R. Mol. Phys. 1959, 2 , 407-414. (35) Bleaney, B. J. Magn. Reson. 1972, 8 . 91-100. (36) Golding, R. M.; Pyykko, P. Mol. Phys. 1973, 26, 1389-1396. (37) Evans, Dennis F.; Tucker, John N.; de Vlllardi, George C. J. Chem. SOC..Chem. Commun. 1975, 205-208. (38) Wentzel, T. J.; Sievers, R. E. Anal. Chem. 1961, 53, 393-399. (39) Elgavish, Gabriel A.;Reuben, Jacques J. Am. Chem. Soc. 1977, 99, 1762- 1785. (40) Perrln, Charles L. Org. Magn. Reson. 1981, 16, 11-13.
RECEIVED for review August 5,1981. Accepted December 18, 1981. We gratefully acknowledge research support from the National Science Foundation. The laser laboratory facility a t the University of North Carolina at Chapel Hill was established through Grant CHEM 77-14547 of the National Science Foundation.
Water- SioIubIe Paramagnellic ReIaxat ion Reagents f or Carbon- 13 Nuclear Magnetic Resonance Spectrometry Thomas J. We!nzel,I Martin E. Ashley, and Robert E. Sievers” Depatiment of Ctremlstry and Cooperative Institute for Research In Environmental Sciences, University of Colorado, Boulder, Colorado 80309
Studies have boen performed In whlch metal complexes of the llgands ethylenedlamlnetetraacetlc acld (H,EDYA), dlethylenetrlamlneipentaacetlc acld (H,DTPA), and trlethylenetetraamlnehexaacetlc acld (HelTHA) were utlllzed ais paramagnetic relaxatlon reagents for ‘‘C NMR spectrometry In aqueous solutions. The Fe( I I I ) and Cr( I I I ) compllexes of DTPA are especlally siilted for facllltatlng spin relsrxatlon. These complexes can be used to decrease the relaxatlon tlmes of carbon nuclel, resultlng In enhanced slgnal to nolse ratlos. They allso quench the nuclear Overhauser effect (NOE), allowing one to obtain quantltatlve 13C NMR spectra. Through the use of these reagents, pulse angles can be Increased and scan tlmes can be decreased, resultlng in the acqulsltlon of hlgher signal lntensltles in shorter limes. Examples of modell cornpounds wlth whlch these reagents are effectlve Include1 arginlne, ascorblc acld, nlcotlnamlcle, and 5,5-dImethylhydimtoIn.
Paramagnetic metal complexes can be added to solutions of organic compounds in order to decrease carbon-13 spinlattice relaxation times as well as suppress the nuclear Overhauser effect (PJOE)observed for protonated carbonri (1-4). The addition of these reagents to a sample causes changes to ‘Present addrens: D e p a r t m e n t of Chemistry, Bates College, Lewiston, ME 04240.
the relaxation times that render the collection of 13CNMR data faster and more efficiently. The signal intensities for carbons not attached to hydrogen are enhanced, pulse angles can be increased so that more signal is obtained per scan, and the delay time between pulses can be decreased, significantly reducing the total data acquisition time. Also, if the NOE is completely suppressed, quantitative integration of 13CNMR spectra may be performed. Paramagnetic relaxation reagents suitable for organic solvents have been studied quite extensively (5-13); however, only a few reports have appeared dealing with water-soluble relaxation reagents (64-19). Gansow et al. have described the use of gadolinium(II1) cryptand (14,15). Bose et al. studied the ethylenediamine complex of chromium, Cr(er),Cl3-3H2O, with a number of amino acids (16). Lettvin and Sherry studied the triethylenetetraaminehexaaceticacid (TTHA) complex of Gd(II1) as an aid in l8C NMR (17). Dechter and Levy have reported the utilization of Gd(TTHA),- and the Gd(II1) complex of diethylenetriaminepentaacetic acid, Gd(DTPA)2-, as relaxation reagents for 15NNMR in aqueous solutions (18), and Bearden et al. (19)reported an aqueous relaxation reagent formed in solution from Gd(N03)3.6H20and inositol. In this paper we compare the Gd(II1) complexes of EDTA, DTPA, and TTHA for their applicability to analytical 13C NMR spectrometry. Dechter and Levy have reported that the ‘ITHA complex interacts the least with nitrogen bases (18). In our studies which include compounds containing oxygen functionalities as well as nitrogen (ascorbic acid, arginine, nicotinamide, and 5,5-dimethylhydantoin), some interesting 0 1982 American Chemlcal Soclety
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contrasts were observed and the DTPA complex actually appears to be the best choice overall for 13CNMR spectra (20). In addition, we have studied the EDTA and DTPA complexes of Cr(III), Fe(III), Mn(II), and Co(II), and the DTPA complexes of Ni(II), Cu(II), and Mo(V) as aqueous paramagnetic relaxation reagents for 13C NMR spectrometry. These compounds have not previously been evaluated as relaxation reagents, and we have found the DTPA complexes of Cr(II1) and Fe(II1) to function as excellent relaxation reagents in aqueous solutions. For all of the compounds the 13C NMR spectra of which we have studied in the presence of the DTPA complexes of Cr(III), Fe(III), and Gd(III), significant enhancements of the nonprotonated carbon resonances were observed without the onset of unacceptable broadening. In addition, these reagents do not produce shifts in the resulting spectra. The DTPA ligand appears to encapsulate these metal ions quite effectively, producing stable, coordinatively saturated, complexes. The DTPA complexes of Cr(III), Fe(III), and Gd(II1) are stable and effective over the pH range of 1 to 12. The DTPA complex of Mn(I1) functioned quite well as a relaxation reagent for three of the four compounds studied. These complexes are easily synthesized from commercially available starting materials,
EXPERIMENTAL SECTION Reagents. All chemic& were used as received without further purification. Disodium ethylenediaminetetraacetic acid (Na2H2EDTA)was obtained from J. T. Baker Chemical Co,, Phillipsburg, NJ. Triethylenetetraaminehexaacetic acid (H6TI"A) was obtained from Pfaltz and Bauer, Stamford, CT, and L-ascorbic acid was obtained from Fischer Scientific Co., Fair Lawn, NJ. Nicotinamide was purchased from Chem Service, West Chester, PA. Diethylenetriarninepentaacetic acid (H,DTPA), L-(+)-arginine, and 5,5-dimethylhydantoin were obtained from Aldrich Chemical Co., Milwaukee, WI. Preparation of Metal Complexes. ( A ) Dihydrogen (Diethylenetriaminepentaacetato)gadolinium(ZII) Dihydrate,H2To a suspension of 5 g of Gd203in 60 mL [Gd(DTPA)].2H20. of water was added 11g of H,(DTPA). The resulting suspension was stirred and gently warmed. After about 10 h all the material had dissolved. After the solution cooled to room temperature, 150 mL of acetone was added and a gelatinous,white precipitate resulted. This was collected by filtration and dried in vacuo over p4°10.
Anal. Calcd for Hz[Gd(C14H18N3010)].2Hz0: C, 28.81; H, 4.14; N, 7.20. Found: C, 29.15; H, 4.02; N, 7.19. (E)Trihydrogen (Triethy1enetetraaminehexaacetato)gadolinium(ZZI)Pentahydrate,H,[Gd(TTHA)].5H20. This was synthesized by a procedure identical with that for H2[Gd(DTPA)]. Anal. Calcd for H3[Gd(C18HaN4012)]-5H20: C, 29.26; H, 5.04; N, 7.58. Found: C, 28.89; H, 4.47; N, 7.58. (C) Monohydrogen (Ethy1enediarninetetraacetato)gadolinium(IZI)Pentahydrate,H[Gd(EDTA)].BH,O. This was synthesized by a procedure identical with that for H2[Gd(DTPA)]*PH@. Anal. Calcd for H[Gd(Clo€IlzN2O8)]~5H2Q: C, 22.38; H, 4.31; N, 5.22. Found: C, 22.30; H, 4.36; N, 5.36. (0)Disodium (Ethylenediaminetetraacetato)manganese(IZ) Dihydrate Nu2[Mn(EDTA)].2H20. All the transition metal chelates were synthesized according to the procedure of Sawyer and Paulsen (21) except H,[Cu(DTPA)] and H,[MOO,(OW)~(DTPA)], which were synthesized by the procedure of Sievers and Bailar (22). In some cases, addition of the ethanol led to the formation of a thick oil. These oils were separated and placed in vacuo over P4OI0. In all cases, the material solidified within a few hours. For the chromium chelates, reactih times on the order of 24 h were needed and typically some insoluble material had to be removed by filtration prior to the addition of the ethanol. (E)Disodium (Diethylenetriaminepentaacetato)chromium(IZl) Hexahydrate, Na2[Cr(DTPA)].6H20. Anal. Calcd for Na2[Cr(C14H18N3010)]*6HzO:C, 28.29; W,5.09; N, 6.86. Found: C, 28.02; H, 4.80; N, 6.76.
(F)Disodium (Diethylenetriaminepentaacetato)iron(IZZ) Dihydrate,Naz[Fe(DTPA)].2H20. Anal. Calcd for Na2[Fe-
(C14H18N3010)]*2H20:C, 31:96; H, 4.21; N, 7.99. Found: C, 31.72; H, 4.35; N, 8.01.
(G)Trisodium (Diethy1enetriaminepentaacetato)manganese(II) Tetrahydrate,Na3[Mn(DTPA)].4Hz0. Anal. Calcd for Na3[Mn(C14H18N3010)].4H20: C, 28.78; H, 4.48; N, 7.19. Found: C, 29.01; H, 4.46; N, 7.13. NMR Measurements. Proton-decoupled carbon-13 NMR spectra were recorded at a frequency of 22.5 MHz with a JEOL FX-9OQ spectrometer at an ambient probe temperature of 40 "C. For the qualitative relaxation studies, the spectra were recorded over a 5000 Hz range defined by 8192 data points. The repetition rate was 0.83 s and 1024 scans were taken. This repetition rate is faster than what is normally used for 13C NMR; however, significant enhancements of resonances of carbons not attached to hydrogens would still be obtained at slower repetition times when the relaxation reagents are added. The relaxation reagents were added either as aliquots from stock solutions or weighed out as solids and added directly to the sample. It is important to use a glass or plastic syringe for transfer of the solutions of relaxation reagents because we have observed corrosion of metal surfaces in the presence of the metal complexes. Spin-lattice relaxation times (TJwere measured by either the inversion-recovery (Tl< 3 s) or saturation-recovery (23) (Tl > 3 s) techniques with a JEOL FX-9OQ spectrometer at a probe temperature of 40 "C. The longer Tlvalues were measured on degassed samples in sealed tubes. AU relaxation time spectra were recorded at 5000 Hz spectra widths defined by 16384 data points. RESULTS AND DISCUSSION In an effort to develop stable, coordinatively saturated, water-soluble, paramagnetic relaxation reagents that do not shift the resonances in the NMR spectra of the compound of interest, we have studied the complexes of Cr(III), Fe(III), Mn(II), and Co(I1) with EDTA and DTPA. The complexes of Gd(II1) with EDTA, DTPA, and TTHA were synthesized and evaluated, as were the Ni(II), Cu(II), and Mo(V) complexes with DTPA. Many of these particular metals were chosen because they have been employed with other complexing agents in organic solvents in previous relaxation studies (2). It was hoped that these particular ligands would encapsulate the metal, creating stable relaxation reagents that are less reactive than the aquated metal ions. From our studies, the DTPA complexes of Fe(III), Cr(III), and Gd(II1) appear especially well suited as relaxation reagents for NMR spectrometry. The compounds ascorbic acid, arginine, nicotinamide, and 5,5-dimethylhydantoin were chosen as model analytes since they encompass a number of functional groups that may be typically encountered in water-soluble compounds. Arginine was of particular interest because it contains a carboxylic acid functional group. The results obtained for the Cr(III), Fe(III), and Gd(II1) complexes with these compounds are summarized in Table I and are discussed in more detail below. For the four compounds we have studied, the Cr(III), Fe(111),and Gd(II1) complexes of DTPA functioned effectively as relaxation reagents. In these cases, significant enhancement of the carbon resonances of carbons not directly bound to protons, without the onset of severe broadening of any peaks in the spectrum, was observed. A positive result in Table I is not indicative of whether complete suppression of the NOE, and hence a quantitative 13CNMR spectrum of the analyte, was observed. The ability to use the complexes of DTPA in order to obtain quantitative 13C NMR data is discussed separately. For all the other metal complexes listed in Table I, there is a t least one analyte for which unsatisfactory results were obtained. In these cases, severe broadening of one or more of the carbon resonances was observed prior to adequate enhancement of the carbon resonances of carbons not bonded to hydrogen. Chromium(II1). Complexes of chromium(III), especially chromium(II1) acetylacetonate, are the paramagnetic relaxation reagents most widely used in organic solvents (5,11-13,
ANALYTICAL CHEMISTRY, VOL. 54, NO. 4, APRIL 1982 ~
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Table I. Comparative Relaxation Reagent DataU ascorbic argi- nicotin- 5,5-dirnethylrelaxation reagent acid nine amide hydantoin Cr( N0,);9H2O Na[Cr(EDTA)I Na,[Cr(DTPA)] MnSO;H,O Na,[Mn(EDTA)] Na,[Mn(DTPP!.)] Fe(NO3);9H,0 Na[Fe(EDTA)] Na,[Fe( DTPA.)] Gd(NO,);GH,O H[ Gd(EDTA)I H,[Gd(DTPA)I H,[Gd('I'THA)]
d
617
c
t
+
._ .-
+
__
-I-1._
t t t
t -
t
+
t
iti.
Key : t = worked effectively as a relaxation reagent for this compound. - = not satisfactory as a relaxation reagent for this compound. a
Flgure 1. Proton-decoupled 13C NMR spectra of 850 pmol of nicotinamide in 1 mL of D,O wkh (a) no relaxation reagent, 0.83 s repetition rate, (b) no relaxation reagent, 2.1 s repetition rate, (c) 10 pmol of Na,[Cr(DTPA)], 0.83 s repetition rate, (d) 25 pmol of Na,[Cr(DTPA)], 0.83 s repetition rate.
24,25). Only one aqueous relaxation reagent involving Cr(II1) has appeared in the literature (16), and problems are encountered with tlhis complex a t high pH values. In our studies we have compared the behavior of the complexes 1Ja2[Cr(DTPA)] and Na[Cr(EDTA)] with that of Cr(N03)3-9H20. The sodium salts of the DTPA and EDTA complexes are used only because of their enhanced solubility in water vs. that of the protonated forms. The effectiveness of these various compounds with ascorbic acid, nitocinamide, arginine, and 5,5-dimethylhydantoin is demonstrated in Table I. For all four model compounds, the Na2[Cr(DTPA)]clomplex was found to work satisfactorily as a relaxation reagent. Significant enhancement of the intensity of the resonances for carbons not bonded to hydrogen was observed without noticeable broadening. In Figure 1,the spectra obtained with and without Na2[Cr[DTPA)] added to a solution of nicotinamide in IlZOare shown. For 850 pmol of nicotinamide, up to 200 pmol of I\la2[Cr(DTPA)] was added without severe broadening of the carbon resonances. After addition of 100 pmol, however, no additional improvement in the intensity for the carbons not bonded to hydrogen or further Suppression of the NOE for the carbons bonded to hydrogen was observed. In Figure la, the spectrum without any relaxation reagent, the resonance for the carbonyl carbon is not detectablle and the substituted rimg carbon is quite low in intensity. The sane spectrum was alslo obtained at a typical 13C repetition rate of
Flgure 2. Protondecoupled I3C NMR spectra of 850 pmol of argiriine in 1 mL of D,O with (a) no relaxation reagent and (b) 10 pmol of Na,[Fe(DTPA)]. Repetition rate held constant from (a) to (b). See Experimental Section.
2.1 s and is shown in Figure lb. In this spectrum, the resonance for the substituted ring carbon is a little larger in intensity while the carbonyl resonance is barely observed above the base line. The results obtained after the addition of Na2[Cr(DTPA)],shown in Figure lc,d, are substantially improved. Significant enhancements of the two resonances for the carbons not bonded to hydrogen are observed. The four resonances for carbons bonded to hydrogen are reduced in intensity because of partial suppression of the NOE. These results are in contrast to those obtained for nicotinamide when Na[Cr(EDTA)J was added. In this case, unacceptable broadening was observed for carbons b, d, and e on addition of the reagent. Apparently, the chromium in Na[Cr(EDTA)] can bond to the ring nitrogen of nicotinamide and broadening is observed. The DTPA ligand appears to better encapsulate the metal producing a less reactive relaxation reagent. This is a general trend we have observed for all of the metal coinplexes we studied. The Na[Cr(EDTA)] was found to work satisfactorily for arginine, ascorbic acid, and 5,5-dimethylhydantoin, but the DTPA complex must be recommended as superior for general usage. Cr(N0J3.9H2O WM unsatisfactory as a spin relaxation agent for all the compounds studied. In the case of arginine, a precipitate formed. Apparently, the carboxyl group in arginine is too reactive with the aquated chromium(I1I) ion. Another problem with Cr(N03)3-9H20is that severe broadening of the deuterium lock signal occurs before significant enhancement of the resonances fox the carbons not bonded to a hydrogen has taken place. The EDTA and DTPA complexes also broaden the lock signal, however, not nearly so extensively as that observed witahhydrated chromium(II1). Iron(II1). Iron(III), with five unpaired electrons, is a more efficient ion than chromium(II1) as a relaxation reagent. In organic solvents, iron complexes have found only limited use because of the instability of F e ( a ~ a c()6~, 9 , 1 0 , 2 6 , 2 7 ) .The complexes Na2[Fe(DTPA)]and Na[Fe(EDTA)] are quite stable in water and the DTPA complex is an effective relaxation reagent. The results for the three iron compounds tested are shown in Table [. Once again, the DTPA complex produced excellent results with all four compounds studied. The Na[Fe(EDTA)Jresultad in broadening for carbons a, b, d, and e in nicotinamide and carbons c, d, e, and f i n ascorbic acid. Fe(N0&.9H2O did not work effectively as a relaxation reagent for any of the compounds studied, and with arginine a precipitate resulted. The spectrum of arginine is shown in Figure 2a. The two carbons not bonded to hydrogen exhibit resonances that are very low in intensity. Addition of Na2[Fe(DTPA)] leads to significant enhancement of both resonances. Also, with Na2[Fe(DTPA)],very little broadening of the deuterium lock signal is observed. The lock signal is much sharper than was found with Na2[Cr(DTPA)] and this allows for much better shimming of the magnet and reduces drift that will lead to
618
ANALYTICAL CHEMISTRY, VOL. 54, NO. 4, APRIL 1982 IC
Ib la b
I I
b o HOCHCH20H HO,,?>CH
I
Ill Flgure 4. Protondecoupled NMR spectra of 850 pmol of 5 3 dimethylhydantoin in 1 mL of D,O with (a) no relaxation reagent and (b) 15 pmol of Na,[Mn(DTPA)].
Figure 3. Protondecoupled NMR spectra of 850 pmol of ascorblc acid in 1 mL of D 2 0 with (a) no relaxatlon reagent and 4.5 ps pulse (40°), (b) no relaxation reagent and 9 ps pulse (80°), (c) 20 pmol of Na,[Fe(DTPA)] and 4.5 ps pulse, and (d) 20 pmol of Na,[Fe(DTPA)] and 9 ps pulse.
broadening in the spectrum. For this reason, the use of Naz[Fe(DTPA)] is usually preferable to Naz[Cr(DTPA)]. One advantage of adding relaxation reagents is that larger pulse angles can be used, with a resultant increase in the signal obtained for each scan. As an example, the spectrum of ascorbic acid obtained at two different pulse angles is shown in Figure 3a,b. At the larger angle, the intensity observed for the three carbon resonances arising from carbons not bonded to hydrogen is reduced because of their long relaxation times. The intensities of the three carbon resonances in which the carbon is bonded to hydrogen are greater since they have relatively fast relaxation times facilitated by the proximity of the hydrogen nucleus. In Figure 3c,d, the results obtained after the addition of Naz[Fe(DTPA)] are shown. The iron complex reduces the relaxation time of the carbons not bound directly to hydrogen, and now increasing the pulse angle leads to an enhanced signal for all six carbon resonances. We have also examined the spectra of 850 pmol of nicotinamide in 1 mL of DzO with increasing amounts of Naz[Fe(DTPA)]. Up to 200 pmol was added without any severe broadening. However, no additional beneficial effects were observed in the spectrum after 100 pmol of Naz[Fe(DTPA)] had been added. Manganese(I1). As sources of manganese(II), neither MnSO4.H20nor Naz[Mn(EDTA)]functioned effectively a5 relaxation reagents. In all cases, severe broadening resulted in the spectrum even when only small amounts of the manganese compounds were added. Na3[Mn(DTPA)],however, exhibited quite different results, especially for nicotinamide, 5,5-dimethylhydantoin,and arginine. With these compounds, enhancement of the resonances for the carbons with long relaxation times was observed without any significant broadening. Typically, for 850 pmol of an analyte, up to 35 pmol of Na3[Mn(DTPA)] was added without the occurrence of any noticeable broadening. The results obtained for 5,5dimethylhydantoin with Na,[Mn(DTPA)] are shown in Figure 4. The two carbonyl carbon resonances were not observed above the base line until the relaxation reagent was added. The resonance of the methyl-substituted carbon is also enhanced in intensity in the presence of Na,[Mn(DTPA)]. With 850 pmol of ascorbic acid in 1mL of DzO, only a small amount (3 pmol) of Na3[Mn(DTPA)] led to appreciable broadening of the resonances for carbons e and f, indicating that some type of complexation was occurring between ascorbic acid and the manganese(I1) in Na,[Mn(DTPA)]. Because of this, Na3[Mn(DTPA)]can only be used as a relaxation reagent for certain classes of compounds, and the iron and chromium complexes are preferable for general use. Gadolinium(II1). Gadolinium(II1) with its seven unpaired electrons is very effective in spin relaxation. Complexes of
Gd(II1) have found only limited use as relaxation reagents in organic solvents (lo,%) and have actually been studied more extensively in aqueous solutions (14,15,17,18). One serious drawback of most Gd(II1) chelates is that they can expand their coordination number by bonding with suitable donors (29). This bonding leads to severe broadening of the resonances of carbons close to the site of complexation. The suitability of the gadolinium complexes, H[Gd(EDTA)], Hz[Gd(DTPA)], H3[Gd(TTHA)], and Gd(N03),.6H20, as aqueous relaxation reagents in 13C NMR was studied. The results obtained with ascorbic acid, nicotinamide, arginine, and 5,5-dimethylhydantoin are summarized in Table I. The protonated forms of the complexes were studied since they are quite soluble in water. As with chromium(II1) and iron(III), the DTPA complex of gadolinium(II1) again appeared to be the least reactive of the three ligands. The H,[Gd(DTPA)] complex gave satisfactory results with all four model compounds studied. The crystal structures of several lanthanide EDTA complexes are known (30),and the EDTA ligand is too small to fully encapsulate the metal, leaving an open pocket in which donor groups can form a bond. This is especially evident with arginine, as carbons b, d, and f broaden with only a small amount of H[Gd(EDTA)] added to the solution. The carboxyl group would appear to bond quite strongly to the gadolinium in this complex. H[Gd(EDTA)] did function effectively as a relaxation reagent for the other three compounds studied. For all four compounds, addition of Gd(N03),.6Hz0 led to broadening of certain carbon resonances before adequate enhancement occurred. The TTHA ligand might be expected to create the least reactive gadolinium complex; however, the elongated nature of the ligand may actually prevent it from fully wrapping around the metal. Dechter and Levy, in studying 15NNMR spectra, typically added 10 times as much H,[Gd(TTHA)] as H,[Gd(DTPA)] (18). For ascorbic acid, nicotinamide, and 5,5-dimethylhydantoin we have observed essentially identical results in the 13C NMR spectra with either the DTPA or TTHA complex of Gd(III), and the onset of broadening took place at the same concentration levels. For arginine, the H2[Gd(DTPA)]functioned effectively as a relaxation reagent; however, addition of H,[Gd(TTHA)] resulted in a precipitate. Apparently, the carboxyl group in arginine reacts with gadolinium in H,[Gd(TTHA)]. Neither of these complexes is truly unreactive toward the compounds we have studied, which can be verified by comparing the proton NMR spectra of the compounds with the spectra obtained after adding H,[Pr(DTPA)] or H3[Pr(TTHA)]. In contrast with the behavior of Pr(II1) complexes in organic solvents, Pr(II1) complexes in aqueous solutions normally function as downfield shift reagents if a substrate bonds to the metal (31,32). We therefore compared the shifts in the proton NMR spectra of ascorbic acid, arginine, 5,5dimethylhydantoin, nicotinamide, pyridine, pyrollidine, and imidazole with equal concentrations of H,[Pr(DTPA)I or H,[Pr(TTHA)] added, The spectra of ascorbic acid and
ANALYTICAL CHEMISTRY, VOL. 54, NO. 4, APRIL 1982
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619
d
I
Protondecoupled NMR spectra of 850 pmol of nicotinamide in 1 mL of D20 with (a)no relaxation reagent and (b) 100 pmol of Na,[Fe(DTPA)]. Figure 6,
Flgure 5. Proton-decoupld 13C NMR spectra of 850 pmol of ascorbic , with (a) no relaxation reagent and (b) 3 pmol of acid in 1 m L of DO H,[Gd(DTPA)].
5,5-dimethylhydantoin were not shifted in the pre~enceof either Pr(IJ1) ctomplex. The NMR spectra of imidazole and arginine, which interestingly did not form a precipitate when H,[Pr(TTHA)] was added, were shifted by both reageints, with those in the presence of H,[Pr(TTHA)] slightly larger. The shifts observed for pyridine, nicotinamide, and pyrollidine were quite similar with both reagents. Flgure 7. Protoridecoupled (gated) NMR spectrum of 850 pmol In Figure 5, the spectra obtained with ascorbic acid after of ascorbic acld in 1 mL of D,O wlth 15 pmol of Na,[Cr(DTPA)]. adding H2[Gd(DTPA)] are shown. Enhancements are observed for all three carbons not bonded to hydrogen. Due to Table 11. Relaxation Times for Ascorbic Acid the efficiency of gadolinium(II1) as a relaxation reagent, for carbona,b amt, 850 pmol of a compound, only about 10 pmol of H2[Gdreagent pmol A B C D E F (DTPA)] could be added. By the addition of 15 pmol, a noticeable broadening was usually observed for the carbon none 0.8 1 . 3 1.3 108 34 124 resonances, although it was not too severe. Na,[Cr(DTPA)\ 1 5 0.5 0.6 0.5 0.8 0.8 0.9 While the H,[Gd(DTPA)] complex appears to be an efNa,[Fe(DTF'A)] 15 0.5 0.6 0.7 1.2 1.3 1.6 2 0.5 0.6 0.7 1.1 0.8 1.1 H,[Gd(DTF'A)] fective relaxation reagent for several compounds, neither the H,[Gd(TTHA)] 2 0.4 0.5 0.5 0.7 0.4 0.7 DTPA nor the TTHA complex of Gd(1II) is truly unreactive with respect to compounds containing an oxygen or nitrogen. a See Figure 3 for identification of carbons in ascorbic Relaxation times expressed in seconds. For this reason, the Cr(II1) and Fe(II1) complexes of DTPA acid. are recommended for general use since they are not us liable for carbons bonded to hydrogen. If the NOE is completely to bond to donor groups, and higher concentrations can be suppressed, quantitative integration of peaks in the 13CNMR added without running the risk of severe broadening in the spectrum can be perf'ormed. This application has been studied spectrum. rather extensively with the organic soluble relaxation reagents Cobalt(II), Nickel(II), Copper(II), and Molybdenum(33-37). Even if complete suppression of the NOE is not (V). The complexes Na2[Co(EDTA)] and Na,[Co(DTPA)] obtained, the faster relaxation times allow for spectra to be were compared with C O ( N O , ) ~ . ~ Hfor ~ Otheir effectiveness run much faster with the gated decoupling technique. as relaxation reagents. None of these three compounds, The application of relaxation reagents to quantitate peaks however, exhibited the desirable properties of a relaxation reagent. In a few cases with the EDTA or C O ( N O ~ ) ~ . ~ H ~ O in 13CNMR spectra must be done with a great deal of caution, complex, severe broadening was observed for one or more of however, and the limitations of this application have preuithe carbon resonances of the compound being studied after ously been discussed (37). In general, we have found that the addition of only a small m o u n t of the reagent. In most cases, gadolinium(II1) complexes are not suitable for this application. Their effectiveness as relaxation reagents typically leads to however, addition of the cobalt(I1) reagents did not produce signal enhancemient of the carbons not bonded to hydrogen. line broadening before quantitative spectra were obtained. The inefficiency of Co(I1) is not suprising because of its short The Na2[Fe(DTPA)]and Na2[Cr(DTPA)]complexes appear electron spin relaxation time (2). to be as effective in D 2 0 as Cr(acac), is in organic solvents. Very similar results were noted for Na3[Ni(DTPA)] and For example, in Figure 6, the spectrum obtained using 100 Na3[Mo202(0H),(DTPA)]. Addition of relatively large pmol of Na2[Fel(DTPA)]with 850 pmol of nicotinamide in 1 quantities (35 prnol of metal to 850 pmol of compound) promL of D20 is shown. This spectrum is essentially quantitative duced essentially no change in the 13C NMR spectrum by and was run under the same instrumental conditions as decomparison to the spectrum without the metal complex. By scribed in the Experimental Section. It is also worth noting contrast, in all cases with Na3[Cu(DTPA)],severe broadening that only minimal broadening was observed in this spectrum. of one or more carbon resonances resulted before adequate In cases in which the NOE is not completely quenched by enhancement of the resonances for carbons not bonded to the relaxation reagent, it is still possible to obtain quantitative hydrogen was obtained. 13C NMR spectra by performing the analysis with gated deQuantitative Spectra. The nuclear Overhauser effect coupling. Since the relaxation reagents significantly shorten observed for carbons bonded to hydrogen ordinarily makes the Tl values for the carbons not bonded to hydrogen, the quantitative integration of carbon-13 NMR peaks difficult. delay of 5T1 necessary between each pulse is drastically deA gated decoupling sequence can be used to eliminate the creased. As an example, the Tl values for ascorbic acid with NOE while still decoupling the protons; however, thie techand without the various relaxation reagents are shown in Table nique requires long delay times between pulses, and the 11. The relaxation times with the various reagents a t these process is quite time-consuming. Paramagnetic relaxation concentration levels are all less than 2 s, allowing a delay time reagents, in addition to shortening the relaxation times of of 10 s for the gated decoupling experiment. The spectrum carbons not bonded to hydrogen, will also suppress the NOE of ascorbic acid (850 pmol) with Na2[Cr(DTPA)] (15 pmol)
820
ANALYTICAL CHEMISTRY, VOL. 54, NO. 4, APRIL 1982
obtained after 400 pulses (totaltime of 1 h and 7 min) is shown in Figure 7. Integration of peaks in the spectrum is essentially quantitative (100:104:104:949997),in agreement with relative abundances of the respective nuclei. For ascorbic acid at the same concentration without any relaxation reagent added, a delay of about 600 s would be necessary, and 400 accumulations would require a total time of about 65 h. With identical conditions to those in Figure 7, quantitative spectra were obtained for arginine with Naz[Fe(DTPA)], and 5,5-dimethylhydantoin with Na,[Mn(DTPA)]. Similar quantitative spectra without any added relaxation reagent would require much longer analysis times. It is clearly demonstrated in these experiments that judicious utilization of certain relaxation reagents can greatly reduce the time (by a factor of 50 or more) necessary to acquire quantitative NMR data. pH Dependence. An important aspect of aqueous paramagnetic relaxation reagents is their stability at both acidic and basic pH values. The instability of certain complexes at basic pH has been a problem with some of the aqueous relaxation reagents previously reported (16), and the topic of pH has been ignored altogether or discussed only briefly in other reports (17-19). On the basis of previous work with the DTPA complexes of Fe(II1) (38)and the lanthanide(II1) ions (31, 32, 39), and our own results, the Fe(III), Cr(III), and Gd(II1) complexes of DTPA are altered as the pH is varied from 2 to 11; however, the changes in the complexes are minor and the ligand remains bound to the metal. In a study of Fe(II1) with DTPA in water, Vandegaer et al. (38) found that the following equilibria took place. H[Fe(DTPA)]-
OHOH% [Fe(DTPA)I2- ?
[Fe(DTPA)(OH)]3The protonated chelate exists in equilibrium with the (-2) chelate anion over the pH range of 2.5-4.5. The monohydroxo chelate is observed at pH values greater than seven. The important result of these studies, as far as aqueous relaxation reagents are concerned, is that the ligand remains bonded to the metal over the pH range from 2 to 11. In our studies, the pH of samples containing Naz[Fe(DTPA)] was adjusted to 1 and 12, and for neither extreme was precipitate (either HsDTPA at acidic pH, or Fe(OH)3at basic pH) noted within 24 h. A similar result was observed for Naz[Cr(DTPA)]. It appears then that the Fe(II1) and Cr(II1) complexes of DTPA remain stable over the pH range of 1-12 for at least a day and will function as effective paramagnetic relaxation reagents at these pH values. A similar set of equilibria has been observed for the lanthanide(II1) ions of DTPA and EDTA. A pK, value of 2.8 has been measured for the protonation of [Ln(DTPA)I2-(39), so at low pH values the ligand remains bound to the metal. No mention was made of the nature of these complexes at basic pH values. A detailed study of the pH dependence of Ln(II1) complexes of EDTA at both acidic and basic values has been performed by Elgavish and Reuben (31,32). They found that at pH values above 8 the hydroxo complex [Ln(EDTA)(OH)I2-was formed, and at pH values up to 12, the ligand is still bound to the metal with no decomposition observed. A similar result is expected for the DTPA complexes and we have observed no precipitate formation for at least 24 h when H,[Gd(DTPA)] is in aqueous solutions at pH values of 1 or 12. In fact, in the Elgavish and Reuben study, the shifts in the NMR spectrum of acetic acid were being measured and a t pH values less than 6, or greater than 10, the shifts were considerably reduced, indicating a reduction of interaction between acetic acid and the lanthanide. At values less than 6, the protonated acetic acid did not bond to the chelate. At values greater than 10, the acetate ion may not bond to the hydroxo complex. Elgavish and Reuben also pointed out the
importance of considering the changes to the analyte as the pH is varied. In some isolated cases, a pH change may render an analyte more suitable for partially or completely displacing the DTPA ligand from the metal coordination sphere, and broadening may result in the NMR spectrum of the analyte. In addition, as the pH is changed, the charge on both reagent and analyte may vary, producing a favorable situation for association of the two. Broadening in the NMR spectrum of the analyte may then be observed. Unfortunately, at this point there is no way to predict either of these types of behaviors beforehand. These results all show, however, that at pH values from 1to 12, the DTPA complexes of Cr(III), Fe(III), and Gd(II1) remain intact and should function effectively as paramagnetic relaxation reagents for a diverse group of analytes.
CONCLUSION The complexes Na2[Fe(DTPA)], Naz[Cr(DTPA)], and H,[Gd(DTPA)] have been found to function exceptionallywell as water-soluble paramagnetic relaxation reagents for 13C NMR spectrometry. The complexes of Fe(II1) and Cr(II1) are recommended over Gd(II1) for general usage on the basis that more of these can be added before broadening occurs. In addition, the Fe(II1) complex is considered the most preferable since it leads to less broadening of the deuterium lock signal than Cr(II1). In our studies, we have found that for 850 pmol of an analyte, 13CNMR spectra with a suitable signal to noise ratio for each carbon atom could be obtained at a pulse angle of 45O and a delay time of under 1 s after 10-35 pmol of Naz[Fe(DTPA)] or Naz[Cr(DTPA)]was added. These results allow one to considerably shorten the analysis time necessary for obtaining a I3C NMR spectrum, while increasing the certainty that all of the carbon resonances are observed. ACKNOWLEDGMENT We thank scientists at the Solar Energy Research Institute, Golden, CO, especially James Smart and Calvin Curtis, for providing time on the JEOL NMR spectrometer. The encouragement of William Harris of the National Science Foundation is gratefully acknowledged. LITERATURE CITED La Mar, G. N. J. Am. Chem. SOC. 1971, 9 3 , 1040. La Mar, G. N. Chem. Phys. Lett. 1971, IO, 230. Gansow, 0. A.; Burke, A. R.; Vernon, W. D. J. Am. Chem. SOC. 1972, 9 4 , 2550. Natusch, D. F. S . J. Am. Chem. SOC.1971, 9 3 , 2566. Gansow, 0. A,; Burke, A. R.; La Mar, G. N. J. Chem. SOC.,Chem. Commun. 1972, 456. Barcza, S.; Engstrom, N. J. Am. Chem. SOC. 1972, 9 4 , 1762. Henrlchs, P. M.; Gross, S. J. Magn. Reson. 1975, 17, 399. Holak. T. A,; Levy, G. C. J. Phys. Chem. 1978, 8 2 , 2595. Levy, G. C.; Komoroski, R. A. J. Am. Chem. SOC. 1974, 9 6 , 676. Levy, G. C.; Edlund, U.;Holloway, C. E. J. Magn. Reson. 1978, 2 4 , 375. Martin, L. L.; Chang, C. J.; Floss, H. G.; Mabe, J. A,; Hagaman, E. W.; Wenkert, E. J. Am. Chem. SOC. 1972, 9 4 , 8942. Tanabe, M.; Suzukl, K. T.; Jankowskl, W. C. Tetrahedron Left. 1973, 4723. Wenkert, E.; Bindra, J. S.;Chang, C. J.; Cochran, D. W.; Schell, F. M. Acc. Chem. Res. 1974, 7 , 46.Gansow. 0. A.; Kausar, A. R.; Triplett, K. M.; Weaver, M. J.: Yee, E. L. J. Am. Chem. SOC.1977. 9 9 , 7007. , E.; Roberts, J. Gansow. 0.A,: TrlDIett, K. M.; Peterson, T. T.; ~ o i t oR. D. Org. Magn. Reson. 1980, 13, 77. Bose, K. S.;Witherup, T. H.; Abbott, E. H. J. Magn. Reson. 1977, 27, 385
% l &n, J.; Sherry, A. D. J. Magn. Reson. 1977, 2 8 , 459. Dechter, J. J.; Levy, G. C. J. Magn. Reson. 1980, 39, 207. Bearden, W. H.; Cargln, V. M.; Roberson, W. R. Org. Magn. Reson. 1981, 15, 131. Wenzel, T. J.; Ashley, M. E.; Slevers, R. E. Abstracts, American Chemical Society, Meeting, Organic Divlsion, The Second Chemlcal Conaress of the North American Continent, Las Vegas, NV, Aug 1980; AbsGact ORGN 043. Sawyer, E. T.; Paulsen, P. J. J. Am. Chem. SOC. 1959, 81, 816. Slevers, R. E.; Ballar, J. C., Jr. Inorg. Chem. 1962, 1 , 174. Anderson, J. E.; Ullman, R. 1987, 71, 4133. Levy, G. C.; Edlund, U.; Hexem, J. G. J. Magn. Reson. 1975, 19, 259. Levy, G. C.; Cargloll, J. D.; Jullano, P. C.; Mitchell, T. D. J. Am. Chem. SOC. 1973, 95,3445.
621
Anal. Chem. 1982, 54, 621-624
(26) DiGioia, A. J.; Lichter, R. L. J . Magn. Reson. 1977, 2 7 , 431. (27) Levy, G. C.; Gargioli, J. D. J . Magn. Reson. 1973, IO, 231 (28) Levy, G. C.; Dechter, J. J.; Kowalewski, J. J . Am. Chem. Soc. 1978, 100, 2308. (29) Sievers, R. E., Ed. "Nuclear Magnetlc Resonance Shift Reagents"; ACademiic Press: New York, 1973. (30) Lind, U. D.; Hoard, J. L. J . Am. Chem. SOC. 1965, 8 7 , 1611. (31) Elgavlsh, G. A,; Reuben, J. J . Am. Chem. Soc. 1076, 98, 4755. (32) Elgavish, G. A,; Reuben, J. J . Am. Chem. Soc. 1077, 9 9 , 1762. (33) Farnell, L. F.; Randall, E. W.; White, A. I. J . Chem. Soc., Chem. Comniun. 1972, 1159. (34) Freeman, R.; Pachler, K. 0. R.; La Mar, G. N. J . Chem. Phys. 1971, 5 5 ,. 4586 -. - - -.
(35) Hawkes, G. E.; Herwig, K.; Roberts, J. D. J . Org. Chem. 1074, 39, 1017.
(36) Liebfrltz, D.; Roberts, J. D. J . Am. Chem. SOC.1973, 95, 4996. (37) Levy, G. C.; Edlund, U. J . Am. Chem. SOC. 1075, 95, 4996. (38) Vandegaer, J.; Chaberek, S.;Frost, A. E. J . Inorg. Nucl. Chern. 1959, I f , 210. (39) Harder, R.; Chaberek, S.J . Inorg. Nucl. Chem. 1959, 7f, 197.
RECEIVED for review July 17, 1981. Accepted December 7, 1981. The support of the National Science Foundation through Grant CHE 79-13022 is greatly appreciated. T.J.W. thanks John Frohliger and the Society of Analytical Chemists of Pittsburgh for a summer fellowship, awarded by the Analytical Division of the American Chemical Society.
Magnesium Nitrate as a Matrix Modifier in the Stabilized Temperature Platform Furnace Waiter SHavln," G. R. Carnrick, and D. C. Mannlng The Perkin-Elmer Corporation, Norwalk, Connecticut 06856
Magneslurn, as Mg(NO,),, has been added to solutions to be analyzed Ifor Mn and AI to permlt charrlng at hlgher tlemperature. The atomlzatlon Is delayed to a higher temperature whlch, with the platform technlque, permlts the tube to reach a stable temperature before atomlzatlon of Mn. We lbelleve that Mn, AI, and Mg are reduced to the oxlde In the solid phase, prior to vaporlzaiion of the oxide. The functlon of the Mg additlain Is to Imbed the analyte In a matrlx of Mg oxlde, delaying vaporlzetlon of the analyte untll the Mg oxide Is vaporlzed. With these condltlons, Interference effects on Mn and AI are greatly reduced. Prellmlnary data lndlcate tlhat thls matrix modlfler Is advantageous also for Cr, NI, and Co.
We recently described the determination of Mn in seawater (1). We found that there was less interference on Mn in the
seawater than in an equivalent concentration of NaC1. We could char a seawater sample at 1400 "C with little loss of Mn, while Mn in simple aqueous solutions was lost above 1200 "C. In studies of A1 (2), we found that adding a Mg salt, .MgC12, delayed the appearance of the Al peak in tlhe atomization step. Recently, Genc et al. (3) argued from activation energy measurements that Mn reached the atomic state after the Mn chloride, nitrate, or sulfate was converted to the oxide on the graphite surface. The oxide wa3 then vaporized and dissociated in the vapor state. Consistent with this model, they found that the appearance temperature for Mn was 11475 f 10 K indelpendently of whether the Mn was present as the chloride, nitrate, or sulfate. We speculated that the effect of the seawater on the Mn determination was due to Mg salts present in seawater. The alkaline earths were probably reduced to the oxide after hydrolysis, prior to vaporization. The Mn was trapped within the MgO crystals, delaying its volatilization. The bulk of the NaC1, on the other hand, was driven off at char temperatures near 1000 "C. This suggested that we might be able to improve the analytical conditions for metals that are atomized from their oxide in the furnace by adding a massive amount of a metal Eialt that is converted to a relatively refractory oxide. The experimental data shown here support our speculation. 0003-270~0/82/0354-0621$0 1.25/0
Table I. Instrument Parameters dry
char
atom
clean
cool
Manganese temp ("C) ramp (SI hold (s) int. gas flow (mL/min)
250 1
60 300
1400 10 70 300
2200a
2600
20
0 8 0
1 6
1
300
20 300
Aluminum temp ("C) ramp hold int. gas flow (mL/min)
250
1700
2400
2600
20
1 60
1
0
1
1
45 300
6
6 300
20 300
300
0
a Some of these experiments used an atomization temperature of 2300 "C, which we believe provided identical results.
EXPERIMENTAL SECTION The Perkin-Elmer Model 5000 atomic absorption spectrophotometer and HGA-500 furnace were used with the AS40 autosampler. The Data System 10 was used to record absorbance signal profiles. New pyrolytically coated graphite tubes (part no. B009-1504) and pyrolytic platforms (part no. 0290-2311) were used with 20-pL sample aliquots. The wavelength, slit width, and lamp conditions were taken from the Analytical Methods Book (4). The furnace programs are shown in Table I. Maximum power heating was used for the atomization step. The required char time varied depending on the matrix and the amount of dissolved solids. A shorter char time might be satisfactory for many applications. The purpose of the photodiode in maximum power heating is to indicate when the tube has reached a preset temperature. The maximum power available is applied to the furnace tube at the beginning of the atomization step until the photodiode viewing the emission of the tube indicates that the preset temperature has been reached. Then the voltage is reduced to a value which will maintain that temperature until the conclusion of the atomization step. Preliminary work indicated that the cutoff setting of the photodiode strongly influenced the shape of the thermal profile. The temperature conditions for the furnace were typically set (5) by programming a temperature on the keyboard which the pyrometer indicated provided the desired final 0 1982 Arnerlcan Chemical Society
