Anal. Chem. 1996, 68, 3979-3986
A Modified Cross-Polarization Magic Angle Spinning 13C NMR Procedure for the Study of Humic Materials Robert L. Cook and Cooper H. Langford*
Department of Chemistry, University of Calgary, 2500 University Drive NW, Calgary, Alberta, Canada T2N 1N4 Raghav Yamdagni
Mass Spectrometry and NMR Laboratory, Department of Chemistry, University of Calgary, 2500 University Drive NW, Calgary, Alberta, Canada T2N 1N4 Caroline M. Preston
Pacific Forestry Centre, Forestry Canada, 506 West Burnside Road, Victoria, British Columbia, Canada V8Z 1M5
A new approach to the use of high-field instruments for cross-polarization (CP) magic angle spinning (MAS) 13C NMR for analysis of humic materials is described. This technique consists of using a high sample spinning rate and a ramp CP pulse sequence, which, among other advantages, addresses chemical shift anisotropy effects. The theoretical aspects of high spinning rates on line broadening for nonrigid solids are reviewed. Also, the ramp CP pulse sequence and its implications for humic materials are discussed. It is shown that the highest resolution and most informative spectra can be obtained via a high-field instrument if the sample is spun at a rate higher than 0.25 of the anisotropy of the chemical shift of the aromatic moieties and a ramp CP pulse sequence is used. This study raises concerns that previous methods may have underestimated the amount of aromatic carbons in humic materials. Because of the similarities between humic materials and coals, this work may also have implications in the CP-MAS 13C NMR approach to coals.
Humic substances, the major organic constituents of soils and important factors in natural water chemistry, pose a special problem for the analytical chemist. They are complex polymeric mixtures whose properties reflect their structural diversity, as well as the processes of aggregation, conformational change, and surface charge distribution. Attempts to fractionate humics into singular molecular components have met with little success, and the fractions obtained are still heterogeneous mixtures. Therefore, techniques that can directly interrogate intact samples are more suitable. As well, individual molecular components would not reveal properties emeging upon interaction. Probes that can reveal changes that occur in response to functional activity, such as cation binding and aggregation, are most useful; for example, we have recently discussed the use of synchronous fluorescence in this context.1 (1) Cook, R. L.; Langford, C. H. Anal. Chem. 1995, 67, 174-180. S0003-2700(96)00403-9 CCC: $12.00
© 1996 American Chemical Society
Carbon-13 nuclear magnetic resonance (NMR) spectroscopy has proved to be a powerful tool for the investigation of humic substances.2 In particular, the technique of cross-polarization with magic angle spinning (CP-MAS) for solid samples has seen wide application to both humic substances and coal,3 as it allows interrogation of intact samples. The quantitative reliability of the 13C CP-MAS technique for insoluble organic matter has long been of concern,3-6 and has been thoroughly researched for fossil fuels.7-9 There are at least three factors that affect the intensity of a particular signal. The first is variation in cross-polarization rates for different carbon types. The second is the loss of intensity and distortion of peak areas by spinning sidebands, especially in the aromatic and carboxyl regions. The third is that carbons close to a paramagnetic center may be undetectable. This is because unpaired electron spins reduce the T1F(1H) of the hydrogens so much that they lose magnetization too quickly to transfer it to a carbon. For samples low in carbon, the percentage of carbon actually observed is typically 50% or less.10-13 The first problem can be addressed by one of several approaches, including acquisition of spectra with varying contact times so that the true intensity distribution can be calculated and Bloch decay acquisition (without cross-polarization). The latter generally requires samples high in carbon and low in paramag(2) (a) Preston, C. M. Soil Sci. 1996, 161, 144-167. (b) Hatcher, P. G.; VanderHart, D. L.; Earl, W. L. Org. Geochem. 1980, 2, 87-92. (3) Wilson, M. A. NMR Techniques and Applications in Geochemistry and Soil Chemistry; Pergamon Press: Oxford, 1987 (and reference cited therein). (4) Fru ¨ nd, R.; Lu ¨ demann, H.-D. Sci. Total Environ. 1989, 81/82, 157-168. (5) Kinchesh, P.; Powlson, D. S.; Randall, E. W. Eur. J. Soil Sci. 1995, 46, 125-138. (6) Wershaw, R. L., Mikita, M. A., Eds. NMR of humic substances and coal. Techniques, problems and solutions; Lewis Publishers: Chelsea, MI, 1987. (7) Jurkiewicz, A.; Marciel, G. E. Anal. Chem. 1995, 67, 2188-2194. (8) Pan, V. H.; Maciel, G. E. Fuel 1993, 72, 451-468. (9) Snape, C. E.; Axelson, D. E.; Botto, R. E.; Delpeich, J. J.; Tekely, P.; Gerstien, B. C.; Pruski, M.; Maciel, G. E.; Wilson, M. A. Fuel 1989, 68, 547-560. (10) Preston, C. M.; Schnitzer, M. Soil Sci. Soc. Am. J. 1984, 48, 305-311. (11) Preston, C. M.; Newman, R. H. Geoderma 1995, 68, 229-241. (12) Skjemstad, J. O.; Clarke, P.; Taylor, J. A.; Oades, J. M.; Newman, R. H. Aust. J. Soil Res. 1994, 32, 1215-129. (13) Wilson, M. A.; Vassallo, A. M.; Perdue, E. M.; Reuter, J. M. Anal. Chem. 1987, 59, 551-558.
Analytical Chemistry, Vol. 68, No. 22, November 15, 1996 3979
netics and/or large (2.5 cm3)14 rotors. The third problem of low visibility has no easy solution, but thus far, there is no clear indication that the observed C differs substantially from that rendered invisible by proximity to paramagnetic centers, at least for humic substances. For soluble fractions such as fulvic and humic acids, cross-checks of solution- and solid-state spectra have shown reasonable agreement.15 If it is not possible to use any of these approaches, a contact time of 1 ms has generally been found to be appropriate for coals and humic substances, giving the best compromise between quantitative intensity distribution and CP intensity enhancement. For the second problem, some attempts have been made to use mechanical manipulation of the spinning,16 but these approaches are complex, not commercially available, and not suitable for all types of samples, due to the relatively long delays (0.5-5 s) involved. Pulse sequences to suppress sidebands also have their own problems with signal loss and intensity distortions. Faster spinning should be the solution, but it has long been known that high MAS rates can interfere with the cross-polarization process, again leading to loss of signal and distortion of relative intensities.17 For this reason, and because much of the broadening in coals is due to chemical shift dispersion which is not improved by higher fields, it has been recommended that the best CP-MAS results for coals (and humics) will be obtained with low-field instruments (2.3 T, ∼100 MHz for 1H) and slow spinning rates less than 5 kHz.9 In any event, speeds much above 5 kHz were not readily available until recently. Two other mechanisms could cause broadening in the spectra of nonrigid solids such humic substances: motional modulation of the CH coupling and motional modulation of the resonance frequency by the chemical shift anisotropy (CSA). The latter should make an increasing contribution to line widths at higher fields, especially for aromatic and carboxyl carbons. As outlined in the Theory section, higher MAS rates should alleviate the broadening due to both of these mechanisms. Recently, MAS probes have become commercially available that allow spinning rates up to 20 kHz, and this has been coupled with the proliferation of high-field CP-MAS instruments with their greatly enhanced sensitivity. Concurrent with this, modifications have been developed for the CP-MAS pulse sequence to alleviate the CP problems due to high-speed spinning.18-22 The most promising pulse sequence for large macromolecules such as humic substances is known as ramp (ramped-amplitude) CP.22 With these advances, it is appropriate to examine the effects of high fields and high MAS rates when used with ramp CP NMR for humic substances. In this paper, we apply these techniques to two well-characterized humic substances, the Laurentian fulvic and humic acids (LFA, LHA). (14) Zhang, M.; Maciel, G. E. Fuel 1990, 69, 557-563. (15) Schnitzer, M.; Preston, C. M. Soil Sci. Soc. Am. J. 1986, 50, 326-331. (16) (a) Sardashti, M.; Maciel, G. E. J. Magn. Reson. 1987, 72, 467-474. (b) Zeigler, R. C.; Wind, R. A.; Maciel, G. E. J. Magn. Reson. 1988, 79, 299306. (17) Pruski, M.; dela Rosa, L.; Gerstein, B. C. Energy Fuels 1990, 4, 160-165. (18) Barbara, T. M.; Williams, E. H. J. Magn. Reson. 1992, 99, 439-442. (19) Wu, X.; Zilm, K. W. J. Magn. Reson. Ser. A 1993, 104, 154-165. (20) Peersoen, O. B.; Wu, X. Kustanovich, I.; Smith, S. O. J. Magn. Reson. Ser. A. 1993, 104, 334-339. (21) Peersen, O. B.; Wu, X.; Smith, S. O. J. Magn. Reson. Ser. A 1994, 106, 127-131. (22) Metz, G.; Wu, X.; Smith, S. O. J. Magn. Reson. Ser. A 1994, 110, 219-227.
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Analytical Chemistry, Vol. 68, No. 22, November 15, 1996
THEORY Line Broadening. Line broadening in a solid can take place by a large number of mechanisms. Two are important for nonrigid solids and are discussed below. This discussion is abstracted from much more detailed works on the subject,23-27 to which the reader is referred for a full theoretical basis. Motional Modulation of the CH Coupling in a CP Experiment. Motional modulation can provide pathways for relaxation in a solid and thus cause line broadening. One such mechanism comes about by molecular motion at the frequency, ω1H, which corresponds to the decoupling field strength, B1H, where ω1H ) 2πν1H ) γHB1H. The ensuing transverse relaxation time, T2m, contributes (πT2m)-1 to the 13C line width. If the proton irradiation is applied exactly on resonance, the sample spinning frequency (rate), ωrot ) 2πνrot, is much smaller than ω1H, and the motions dominating T2m occur at frequencies much smaller than the carbon Larmor frequency, then the expression for the motional-induced relaxation time is
(T2m)-1 ) 0.5M2CHJm(ω1Η)
(1)
where M2CH is the carbon-proton Van Vleck second moment expressed in rad2/s2 and Jm(ω1H) is the spectral density of motion at ω1H given by Jm(ω) ) ∫g(τ) exp(iωτ) dτ. For motion that is isotropic with a single correlation time, τc, and an exponential autocorrelation function g(τ), Jm(ω1H) is given by
Jm(ω1H) ) 2τc(1 + ω1H2τc2)-1
(2)
Thus, in the slow motion regime, i.e., τc2 . (ω1H)-2, so that Jm(ω1H) ∼ 2(ω1H2τc), and hence T2m ∝ ω1H2. In the other limit, τc2 , (ω1H)-2, i.e., the fast motion regime, Jm(ω1H) ∼ 2τc, thus T2m is independent of ω1H. Motional Modulation of the Resonance Frequency via Chemical Shift Anisotropy. A loss of phase coherence in the transverse 13C magnetization resulting from motional modulation of the resonance frequency via the 13C chemical shift anisotropy (CSA) is the second mechanism for line broadening in 13C spectra resulting from molecular motion. When a sample is being spun at the magic angle, each 13C resonance undergoes periodic changes in resonance frequency so that all of the mean resonance values are the same for chemically equivalent carbons over a rotational period, Trot. Hence, at t ) nTrot, where n is a positive integer, there will be a macroscopic coherence in the transverse magnetization, and these periodic coherences are known as “spin echoes”. For the understanding of line broadening due to the modulation of the CSA, it is essential to recognize that the mean frequency averaged over the Trot is uniform. A specific phase error will be induced at the next echo if a molecule executes a reorientational jump at (23) Axelson, D. E. Solid State Nuclear Magnetic Resonance of Fossil Fuels Multiscience Publications Ltd.: Canadian Government Publishing Centre, Supply and Services Canada, 1985; pp 120-123. (24) VanderHart, D. L.; Earl, W. L.; Garroway, A. N. J. Magn. Reson. 1981, 44, 361-401. (25) Rothwell, W. P.; Waugh, J. S. J. Chem. Phys. 1981, 74, 2721-2732. (26) Suwelack, D.; Rothwell, W. P.; Waugh, J. S. J. Chem. Phys. 1980, 73, 25592569. (27) VanderHart, D. L.; Garroway, A. N. J. Chem. Phys. 1979, 71, 2773-2787.
some point during a Trot interval. It will depend on the geometries involved and the relative time of the jump within the interval. These jumps are simple changes in spatial orientation which leave the isotropic chemical shifts unaffected, rather than jumps between conformational minima which do produce changes in the isotropic chemical shifts. Because of averaging over a collection of spins, these individual phase errors appear as a decrease in the amplitude of the next echo, thus producing relaxation described by transverse relaxation time T2σ and line width ∆νσ ) (πT2σ)-1. The dephasing effects of a jump are not cumulative over subsequent intervals unless the mean frequency changes as it might in conformational jumping. A distinction is made between two limiting cases, for which a single jump usually causes either a large or a small random phase error at the time of the next echo, in order to estimate line width contributions attributable to these reorientational jumps. These two regimes are known as the “strong” and “weak” collision cases, respectively. A single event usually produces a large phase error in the strong collision case when the two following conditions prevail:
(3)
Figure 1. (a) SACP and (b) ramp CP pulse sequences used in this experiment.
where ∆σ is the root-mean-square instantaneous change in chemical shift due to the jump, and
In the intermediate region, where 21/2ωrotτc = 1, coherent spatial averaging (which permits line narrowing in the strong collision limit) and incoherent averaging (which allows line narrowing in the weak collision limit) destructively interfere with one another, resulting in broadened NMR lines for this middle case. The following two equations can be derived from the discussion above:
-ωrot , γB0∆σ
-τc . ωrot-1
(4)
where τc is the the mean time between jumps. When this is the case, T2σ, the transverse 13C relation time associated with the mechanism, is
T2σ = τc
(5)
for a diffusional model, (T2)-1 )
In the weak collision case, the T2σ must consist of weak events, and the relaxation corresponds to the diffusional loss of phase coherence. If one or both of the following conditions prevail:
-ωrot . γB0∆σ
(6)
or -1
-τc
. ωrot g γB0∆σ
(7)
then
(T2σ)-1 ) (gB0∆σ)2τc(1 + ωrot2τc2)-1
(8)
The above equation can be simplified in terms slow or fast motion of the molecule. In the case of slow molecular motion (fast sample spinning), τc2 . (ωrot)-2, and T2σ-1 is proportional to ωrot-2τc-1; whereas for fast motion, τc2 , (ωrot)-2, and T2σ is proportional to τc. Since it is normally desired to choose as fast a sample spinning frequency as is needed to prevent spinning sideband interference, it is usual for eq 8 to apply.
1 2 2 ω σ (1 + n2/3)[τc/(1 + 4ωrot2τc2) + 15 0 2τc/(1 + ωrot2τc2)] (9)
and for an anisotropic diffusive model, (T2)-1 )
1 2 2 2 ω σ n [τc/(1 + 4ωrot2τc2) + 45 0 2τc/(1 + ωrot2τc2)] (10)
Both of these equations suggest that, at a sample spinning frequency, νrot, of 0.25σ, there should be substantial line narrowing. Ramp CP-MAS. The standard single-amplitude CP pulse sequence (called SACP from here on), shown in Figure 1a, uses single-amplitude CP pulses set so that the Hartmann-Hahn match (HH) condition (ω1H ) γHB1H ) γXB1X ) ω1X) is satisfied for the duration of the contact time (the duration of the CP pulse). Alternatively, in ramp CP, only one spin-lock is maintained at a constant amplitude, while the other spin-lock condition is varied. This variation can be done in several different ways.20-22 The one used in this study has been called ramping.22 The pulse sequence of Figure 1b shows how this ramp is applied, in this case on the proton channel (as done in this study). It can be applied to the X (e.g., 13C) channel as well. The ramp does not discriminate between channels. To understand why this is so, one must first understand what the ramp is doing. Analytical Chemistry, Vol. 68, No. 22, November 15, 1996
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Figure 2. SACP and ramp CP pulse sequences in terms of a Hartman-Hahn profile for two hypothetical types of carbons (see text).
In the simplest terms, the ramp is causing one of the channels to be spin-locked slightly off the HH, except at the center of the ramp, in which case the spin-lock is exactly HHed to the nonramped channel. In other words, the ramp, when applied on the H channel causes ω1H < ω1X > ω1H , where at the middle of the ramp ω1H ) ω1X . Now if we changed channels on which we applied the same ramp, the amount that the spin-lock leads to Hartman-Hahn mismatch would be the same (no matter which of the channels the ramp was on), and thus it does not matter on which channel the ramp is applied: the final results will be the same. This method of ramping allows one to overcome the motional modulation of the CH coupling caused by spinning the sample at a high rate, by changing one of the spin-lock conditions to compensate for the high-speed spinning effect. In terms of the above discussion, the HH is disturbed by ωrot. This disturbance makes a narrow continuum into a series of peaks so that it becomes very burdensome to find, set, and maintain the HH on the maximum of one of these peaks (and it may not be realistically possible). This can be more clearly seen if one looks at Figure 2. Figure 2 shows three different cross-polarization pulses on the protons vs a Hartman-Hahn profile projected vertically rather than horizontally (as is customary). For the sake of argument, the dark line might be thought of as representing the carboxyl carbons of a fulvic acid, while the light line (or smaller amplitude) might be considered to represent the aromatic carbons. In this figure, an exact HH occurs only at the maximum of each peak labeled as 1, 0, -1, and -2, to indicate sidebands where 0 is the central band. The hypothetical sample is being spun at 10 kHz, and the central sideband is at 50 kHz; thus, the 1, -1, and -2 sidebands are at 60, 40, and 30 kHz, respectively, for the carboxyl carbons. A SACP pulse is represented by the bar labeled A, which in this case is centered on the central band, i.e., the perfect SACP experiment. But, if for some reason this CP pulse is not perfectly centered, then as the sideband peaks get narrower (higher sample spinning speed), the further off the HH the CP pulse will be. However, if the SACP pulse is replaced by a ramp CP pulse, as illustrated in Figure 2 by bars B and C, a whole sideband or more, as in the case of bar C, can be covered. This, in turn, means that, no matter how narrow the sideband gets, there will always be an exact HH during the ramp CP pulse. Thus, by ramping we are able to compensate for the effect of ωrot by varying ω1H or ωX. The broader the ramp, the flatter the Hartman-Hahn profile, at the expense of signal strength.22 3982
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However, it has been shown that a ramp CP experiment on the -1 sideband will give a stronger signal per unit time than a SACP experiment on the 0 sideband.22 Ramp CP also promises to give better-resolved spectra. To understand this, refer again to Figure 2. It can be seen that the SACP experiment will only pick up on one HH, in this case the carboxyl HH, due to the fact that this signal is stronger, so that when the HH is selected, it is this HH that is chosen. On the other hand, the ramp CP experiments cover both the HHs of the carboxyl and aromatic carbons, thus giving no preferential treatment to one type of carbons at the expense of other types of carbons. Thus, ramp CP experiments are more likely to give better-resolved spectra and a more accurate depiction of all the types of carbons in a sample and will overcome the motional modulation of the CH coupling as well. EXPERIMENTAL SECTION Material and Equipment. This study exploits both the Laurentian fulvic acid (LFA) and Laurentian humic acid (LHA) extracted from a forest podzol from the area controlled by Laval University (Quebec, Canada). The acids were then prepared and purified as described in refs 28 and 29 . Both LHA and LFA have been the subject of extensive studies, including elemental composition, acid-base and metal titration curves, emission fluorescence, FT-IR, 1H NMR, and MCD,30-33 as well as synchronous and time-resolved fluorescence of the LFA.1 The composition of LFA was 45.1% C, 4.1% H, 1.1% N, 49.7% O,
