The use of spin counting for determining quantitation in solid state 13C NMR spectra of natural organic matter

Geoderma 96 Ž2000. 101–129

The use of spin counting for determining quantitation in solid state 13 C NMR spectra of natural organic matter 1. Model systems and the effects of paramagnetic impurities Ronald J. Smernik ) , J. Malcolm Oades Department of Soil and Water, Waite Agricultural Research Institute, The UniÕersity of Adelaide, Glen Osmond SA 5064, Australia Received 27 October 1998; received in revised form 4 November 1999; accepted 7 December 1999

Abstract The degree of quantitation achieved in the solid state 13 C nuclear magnetic resonance ŽNMR. spectra of a number of organic materials, an HF-treated soil, and a whole soil, was determined for both cross-polarisation ŽCP. and Bloch decay ŽBD. techniques using spin-counting experiments. The percentage of potential 13C NMR signal, which was actually observed Ž Cobs . was in the range 79–107% for the BD technique and in the range 29–103% for the CP technique. A number of materials, including cellulose, pectin, lignin, and palmitic acid gave quantitative spectra using both CP and BD. A second group, including chitin, collagen, and the HF-treated soil gave quantitative BD spectra, but significantly diminished CP spectra Ž Cobs-CP s 66–75%.. A third group including charcoal, a commercial humic acid, and the whole soil gave significantly diminished BD spectra Ž Cobs-BD s 79–87%. and severely diminished CP spectra Ž Cobs-CP s 29–35%.. Signal losses in the CP spectra were attributed to rapid relaxation rates Žshort T1r H. andror slow magnetisation build-up rates Žlong TCH .. The spin dynamics of the CP experiment were studied and a new method for correcting for differences in T1r H between the sample and the reference in CP spin-counting experiments was developed. Signal produced by the Kel-F rotor end-caps was significant for the BD spectra and a correction for the end-cap spectrum was

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Corresponding author. Fax: q61-8-8303-6511. E-mail address: [email protected] ŽR.J. Smernik..

0016-7061r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 1 6 - 7 0 6 1 Ž 0 0 . 0 0 0 0 6 - 9

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required prior to spin counting. The low 1 H content of the fluorinated Kel-F polymer ensured that the contribution of end-caps to the CP spectra was insignificant. The effects of paramagnetic cations on quantitation in solid state NMR spectra was investigated by doping model compounds with paramagnetic impurities. Three mechanisms, which bring about signal loss and operate on three different length scales, were identified. The magnitude of the signal loss brought about by a paramagnetic material was shown to be dependent on both the type of cation involved and on the intimacy of contact with the organic matrix. Chemically bound paramagnetic cations were shown to result in large signal losses in the CP spectrum, whereas paramagnetic salts in a physical mixture with an organic material affected both CP and BD spectra equally. q 2000 Elsevier Science B.V. All rights reserved. Keywords: spin counting; NMR quantitation; Bloch decay; cross-polarisation; paramagnetic; soil organic matter

1. Introduction Solid state 13 C nuclear magnetic resonance ŽNMR. spectroscopy is one of the most powerful techniques for determining the chemical structure of soil organic matter ŽSOM. ŽPreston, 1996, and references therein. and other complex organic substrates such as coal ŽSnape et al., 1989, and references therein. , lignocellulose ŽFrund 1989a. , and marine sediment organics Ž Hedges and ¨ and Ludemann, ¨ . Oades, 1997 . The main advantage of NMR spectroscopy is that it has the potential to enable quantitative determination of functional groups in a complex material because all equivalent nuclei Ž e.g., 13 C. potentially give rise to signals of equal intensity regardless of their chemical environment. However, solid state NMR spectra are not always quantitative. The NMR signal of some 13 C nuclei in SOM may be diminished or be rendered completely unobservable by a number of factors that interfere with the solid state NMR experiment, especially when the cross-polarisation Ž CP. technique is used. Foremost among these factors are the presence of paramagnetic centres, the remoteness of nearest 1 H nuclei, and high molecular mobility. The presence of paramagnetic centres can also interfere with quantitation in the more robust Bloch decay ŽBD. technique ŽBotto et al., 1987; Snape et al., 1989; Jurkiewicz and Maciel, 1995.. The issue of quantitation in solid state 13 C NMR spectroscopy of SOM has been discussed at length ŽVassallo et al., 1987; Preston et al., 1994; Skjemstad et al., 1994; Kinchesh et al., 1995b; Preston and Newman, 1995; Randall et al., 1995; Cook et al., 1996; Conte et al., 1997a,b; Schmidt et al., 1997. and has been reviewed recently by Kinchesh et al. Ž 1995a. . Quantitation in solid state 13 C NMR spectra of coals has also been investigated Ž Dudley and Fyfe, 1982; Sullivan and Maciel, 1982a,b; Packer et al., 1983; Hagaman et al., 1986; Botto et al., 1987; Vassallo et al., 1987; Snape et al., 1989; Jurkiewicz and Maciel, 1995..

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The term quantitation has been used in two different contexts throughout the literature, which will be differentiated here by the terms relative and absolute quantitation. These definitions differ slightly from those of Kinchesh et al. Ž1995a.. Relative quantitation will denote that the ratio of carbon types represented by different NMR resonances Že.g., aliphatic, aromatic, carbonyl. in the spectrum of a single sample is correct. Relative quantitation is achieved when an equal amount of signal is obtained from each 13 C nucleus in the sample. Absolute quantitation will denote that the signal intensities of different samples are proportional to the number of 13 C nuclei present. The term absolute quantitation will be used in two situations: Ži. where a sample and a reference material are present in the same rotor and, hence, their NMR spectra are being acquired simultaneously; and Ž ii. where NMR spectra of a sample and a reference material are acquired separately. Absolute quantitation is a more stringent requirement and hence relative quantitation is implicit for absolute quantitation. It is the concept of relative quantitation that is usually of more interest in studies of SOM and other complex organic materials, however, relative quantitation is difficult to measure. Comparing functional group ratios determined from solid state NMR spectra with those determined by other methods, including solution NMR Ž Frund 1989b; Conte et al., 1997a,b. or other ¨ and Ludemann, ¨ spectroscopic, pyrolysis or chemical methods, is of limited use because these other methods have their own quantitation problems. In contrast to relative quantitation, absolute quantitation is more easily determined by comparing the signal intensity of a sample with that of a standard containing a known amount of carbon. This process is termed spin counting. The results of spin-counting experiments are usually expressed as the fraction of expected NMR signal Ž based on the carbon content and mass of the sample. that is actually observed Ž Cobs .. Few spin-counting experiments have been reported on soils or soil extracts, and Cobs was alarmingly low in all cases. Wilson et al. Ž1987. reported Cobs in the range 30–62% for a series of humic and fulvic acids. Preston and Newman Ž 1995. and Preston et al. Ž 1994. reported Cobs in the range 7–38% for both HF-treated humic fractions and soil particle size fractions. Spin-counting experiments have been utilised to a much greater extent in solid state NMR studies of coals and related materials Ž Hagaman et al., 1986; Botto et al., 1987; Vassallo et al., 1987; Snape et al., 1989; Jurkiewicz and Maciel, 1995.. Values of Cobs vary greatly in the range 26–100%, depending on coal rank and maceral type. Most spin-counting experiments utilise the CP technique in which 1 H nuclei are magnetised by a radiofrequency pulse and the magnetisation is subsequently transferred from the 1 H population to 13 C nuclei. A number of spin-counting experiments have been performed using the BD technique, otherwise known as direct polarisation ŽDP. or single-pulse excitation Žor experiment. ŽSPE., in which 13 C nuclei are directly irradiated with a radiofrequency pulse. In studies in which both CP and BD techniques were

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used, Cobs was invariably greater for the BD technique than for the CP technique Ž Botto et al., 1987; Snape et al., 1989; Jurkiewicz and Maciel, 1995. . A lack of absolute quantitation may, but does not necessarily, affect relative quantitation. Functional group ratios derived from NMR spectra will be substantially in error if the ‘‘unobserved’’ carbon has a different functional group ratio to that of the observed carbon. However, if the loss of NMR signal intensity is equal across all functional groups, their relative ratios will not be changed, even though total signal is decreased. There are mechanisms by which either selective or non-selective signal loss can occur, and the relative impacts of these mechanisms need to be determined to assess the reliability of functional group ratios calculated from spectra where a substantial proportion of 13 C NMR signal is not accounted for in spin-counting experiments. The aims of this study were: Ži. to develop spin-counting methodology to best suit SOM samples; Žii. to carry out spin-counting experiments on a range of model compounds to determine which chemical structures are likely to be observed quantitatively and which are likely to be under-represented in solid state 13 C NMR spectra, and by how much; Žiii. to compare quantitation achieved using CP and BD techniques; and Živ. to investigate the effects of paramagnetic impurities on quantitation in solid state 13 C NMR spectra.

2. Materials and methods Cellulose Ž; 20 mm powder. , alkali lignin Ž henceforth referred to as ‘‘lignin’’. and humic acid, sodium salt Ž‘‘Aldrich HA’’. were used as supplied by Aldrich. Polygalacturonic acid, sodium salt Žfrom orange, minimum 85%, ‘‘sodium polygalacturonate, NaPGUA’’., chitin Žpractical grade, from crab shells. , chitosan Žfrom crab shells, minimum 85% deacetylated. , collagen Ž type I, insoluble, from bovine achilles tendon., and palmitic acid were used as supplied by Sigma. Glycine ŽAR grade. was used as supplied by Ajax Chemicals. Pectin was used as supplied by Nutritional Biochemicals CuCl 2 P 2H 2 O ŽAR grade. was used as supplied by BDH Laboratory Supplies. MnCl 2 P 4H 2 O and PrCl 3 P 6H 2 O were used as supplied by Merck. Charcoal was synthesised from red gum Ž Eucalyptus camaldulensis. wood chips using the method of J.O. Skjemstad and J.A. Taylor Ž personal communication.. A glass dish Ž170 mm diameter, 90 mm height. containing approximately 100 g of wood chips was covered with a watch glass and placed in a muffle furnace at room temperature. The furnace was heated slowly to 4508C and kept at this temperature for 1 h. The furnace was then turned off and allowed to cool

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to room temperature. The resultant charred material represented 36% of the mass of the starting material and contained 60% of the carbon of the starting material. The charcoal was ground Ž mortar and pestle. and sieved. The 53-mm– 0.2-mm fraction was used in this study. The soil used in this study ŽSS6. was collected from the surface horizon Ž0–5 cm. of a Hapludoll, great soil group — prairie soil, from Tallagalla, Queensland, Australia, and has been described in detail by Skjemstad et al. Ž 1994. . The soil was reported to contain 3.11% iron, and the major minerals present were kaolinite, illite, and quartz Ž Skjemstad et al., 1994. . A portion of the whole soil ŽSS6ws. was treated with HF to afford the de-ashed residue Ž SS6hf. , according to the procedure of Skjemstad et al. Ž1994. . A light Ž- 1 Mgmy3 . fraction ŽSS6lf. was isolated by flotation during HF treatment. The copper amended lignin sample was prepared by adding 0.5 g of lignin to 10 ml of a 0.5 M solution of copperŽII. chloride and shaking end-over-end for 1 h. The sample was centrifuged at 1000 = g for 20 min and the supernatant decanted off. Deionised water Ž 10 ml. was added, the sample was shaken for 2 min, then centrifuged at 1000= g for 20 min, and the supernatant discarded. This procedure of washing was repeated twice more before the residue was collected and freeze-dried. PraseodymiumŽIII. polygalacturonate Ž PrPGUA. was prepared by adding a solution of praseodymiumŽ III. chloride Ž0.299 g, 0.84 mmol. in 5 ml of deionised water to a solution of NaPGUA Ž0.5 g. in 5 ml of deionised water. A gelatinous, pale green precipitate formed immediately. The sample was centrifuged at 1000 = g for 20 min and the supernatant decanted off. Deionised water Ž10 ml. was added, the sample was shaken for 2 min, then centrifuged at 1000 = g for 20 min, and the supernatant discarded. This procedure of washing was repeated twice more before the residue was collected and freeze-dried. Carbon contents were determined by combustionrGCrMS. Finely ground samples were sealed in tin capsules, combusted, and the reaction products separated by GC to give a pulse of pure CO 2 for analysis of total C by the mass spectrometer ŽANCA-MS2020, Europa Scientific, Crewe, UK. . 2.1. NMR spectroscopy Solid state 13 C magic angle spinning Ž MAS. NMR spectra were obtained at a C frequency of 50.3 MHz on a Varian Unity 200 spectrometer. Samples were packed in a 7-mm-diameter cylindrical zirconia rotor with Kel-F end-caps and spun at 5000 " 100 Hz in a Doty Scientific MAS probe. Free induction decays ŽFIDs. were acquired with a sweep width of 40 kHz; 1216 data points were collected over an acquisition time of 15 ms. All spectra were zero filled to 8192 data points and processed with a 50-Hz Lorentzian line broadening and a 0.005-s Gaussian broadening. Chemical shifts were externally referenced to the methyl resonance of hexamethylbenzene at 17.36 ppm. 13

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Single contact time CP spectra were acquired using a standard CP pulse sequence Ž Wilson, 1987. . A 1-ms contact time and a 4-s recycle delay were used for all samples except cellulose Ž10-s recycle delay. and palmitic acid Ž 60-s recycle delay.. Recycle delays were chosen to avoid signal loss through saturation. Where T1 H had been measured ŽR.J. Smernik, unpublished results. , the recycle delay was set to ) 5 = T1 H. For other samples, the recycle delay was arrayed in preliminary experiments and a final recycle delay chosen for the spin-counting experiment for which the plateau of maximum signal had been reached. Between 1000 and 2000 transients were collected for each spectrum. BD spectra were acquired using a 6.2-ms Ž908. 13 C pulse. A recycle delay of 90 s was used for all samples except palmitic acid Ž 600 s. . These values were chosen to avoid signal losses through saturation and were based on preliminary experiments in which the recycle delay was arrayed. Between 900 and 1000 transients were collected for each sample except palmitic acid Ž 150 transients acquired.. The Kel-F rotor end-caps used give rise to a significant 13 C signal in the BD spectrum, which overlapped with the NMR signal of the samples. To correct for this, the end-cap spectrum was acquired separately Ž recycle delays 10 s, 7000 transients acquired. and was subtracted from each BD spectrum before integration. The CP 13 C NMR spectrum of the Kel-F end-caps is very weak due to the low 1 H content of the fluorinated polymer. The CP signal due to the end-caps equates to less than 1 mg of observable C. CP spectra were not corrected for end-cap signal because the contribution of the end-cap resonances is insignificant relative to the signal generated by the 13 C in these samples. It should be noted that for samples containing little carbon Ž - 10 mg C., signal derived from the Kel-F end-caps represents a significant proportion of the signal acquired for the sample and corrections should be made. Each spectrum Žboth CP and BD. was baseline corrected between y100 and 300 ppm, i.e., the intensity of the spectrum was set to zero at y100 and 300 ppm and the spectral baseline Ž zero intensity. was taken as a straight line between these two points. Each spectrum was then integrated between y10 and 300 ppm Žreferred to as total intensity. . T1r H was calculated from variable contact time Ž VCT. experiments Ž Wilson, 1987., which were acquired using an array of contact times between 10 ms and 12 ms. A recycle delay of 1–5 s was used for each sample, and between 400 and 4000 transients were collected. The FID corresponding to each contact time in the array was processed in the same way as for the single contact time CP spectra. T1r H was calculated as the negative reciprocal of the slope Žy1rm. of the line of best fit for the graph of lnŽtotal intensity. vs. contact time for the linear decay phase of the curve Žsee Eqs. 1–3 in Section 3. . Upper and lower bounds of the 95% confidence interval of T1r H were calculated from the standard error of the slope Ž s . as y1rŽ m q 2 s . and y1rŽ m y 2 s . , respectively.

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2.2. Spin-counting experiments Spin-counting methodology, including the calculation of Cobs values, is described below in Section 3. A brief discussion of sources and sizes of uncertainties in Cobs values follows. The main source of error was uncertainty in the integrated NMR intensities. Replicate measurements were carried out only for the glycine sample for which errors in total intensity of "5% for CP spectra and "10% for BD spectra were determined. The larger error for BD spectra was due to the lower signal to noise Ž SrN . ratio in these spectra. Since Cobs values are a function of two spectral intensities Žsample and standard., errors are compounded. For CP experiments, another source of error was uncertainty in T1r H, which is used in the calculation of Cobs-CP using Eq. 6. Overall, uncertainties were, therefore, estimated to be "10% in Cobs-CP and "15% in Cobs-BD.

3. Results and discussion 3.1. DeÕelopment of spin-counting methodology Spin-counting experiments involve comparing the 13 C NMR integrated signal intensity of a sample with that of a standard with a known mass and carbon Žspecifically 13 C. content. A variety of methods have been used in spin-counting experiments reported in the literature. These differ in the pulse sequence used ŽCP or BD., the corrections employed in accounting for differences in the spin dynamics of the sample and the standard in CP spectra, and in the type of standard used Žinternal or external.. 3.1.1. CP Õs. BD Most solid state NMR studies of SOM use a CP pulse sequence in which magnetisation is transferred from the abundant 1 H population to the 13 C nuclei. The advantage of CP over the DP or BD technique is improved sensitivity, mainly brought about by a shorter delay required between consecutive scans Žrecycle delay.. The length of the recycle delay is dictated by the rate of 1 H longitudinal relaxation Ž T1 H. for CP experiments that is generally much faster Žoften by an order of magnitude or more. than the rate of 13 C longitudinal relaxation Ž T1C., which dictates the recycle delay required for BD experiments. An increase in 13 C signal by, at most, a factor of g Hrg C f 4 Žwhere g H and g C are the gyromagnetic ratios of 1 H and 13 C nuclei, respectively. is also achieved using the CP technique. For many soil samples, the improved sensitivity offered by the CP technique is required to obtain a solid state 13 C NMR spectrum in a realistic timeframe.

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3.1.2. Accounting for differences in spin dynamics between sample and standard in CP spin-counting experiments Detailed accounts of the theory of solid state 13 C NMR can be found in Wilson Ž1987. and Kinchesh et al. Ž 1995a.. Aspects of CP, which affect quantitation and methods to correct for these effects, are discussed in detail below. The signal intensity of a CP spectrum is dependent on the contact time Ž t cp ., which is the period during which magnetisation is transferred between the 1 H and 13 C nuclei. CP spectra acquired with an array of different contact times ŽVCT experiments. show an initial increase in spectral intensity, since it takes a finite time for magnetisation to be transferred from the 1 H to the 13 C nuclei, followed by a decrease in signal intensity due to 1 H relaxation during the contact time. Both build-up and decay rates are usually modeled as being exponential, with rate constants 1rTCH and 1rT1r H, respectively. Signal intensity Ž I . is, thus, modeled as a function of contact time Ž t cp . by Eq. 1 ŽAlemany et al., 1983a.:

½

I s Cr 1 y Ž TCHrT1r H .

5 exp Žyt

cp rT1r H

. y exp Ž ytcprTCH .

,

Ž1.

which can be rearranged to give the alternate form Ž Wilson et al., 1987. ,

½

I s Cr 1 y Ž TCHrT1r H .

5 1 y exp Žya t

cp rTCH

.

exp Ž ytcprT1r H .

Ž2. where a s 1 y Ž TCHrT1r H. and C is proportional to the number of 13 C nuclei giving rise to the signal and is, thus, the important parameter for quantitation. If a contact time Ž t cp . can be chosen such that it is much larger than TCH and much smaller than T1r H Ži.e., TCH < t cp < T1r H., then I f C, i.e., the signal intensity depends only on the number of nuclei giving rise to the signal, and hence, quantitation is achieved. For most SOM samples, this condition is not met and the signal intensity is dependent on TCH andror T1r H for all values of t cp . The magnitude of the deviation of I from C is illustrated in Fig. 1 for T1r H s 5 ms Žtypical for HF-treated soils., and TCH s 0.05 Žtypical for protonated carbons. , 0.35 Žtypical for most non-protonated carbons. , and 1 ms Ž which may occur for carbons in structures containing very few protons, or for highly mobile phases.. The maximum value of IrC occurs at different contact times depending on the value of TCH . The value of the maximum also differs, being greatest for TCH s 0.05 ms Ž0.95., however, it should be noted that experimental build-up curves for protonated 13 C nuclei are not well described by simple exponential build-up Žsee below.. For the longer values of TCH of 0.35 and 1 ms, lower maximum values for IrC are observed Ž 0.82 and 0.67, respectively. . For many soil samples, T1r H is shorter than 5 ms Žsee, for example, Preston et al., 1984, 1994; Wilson et al., 1987. and the deviation of IrC from unity is larger.

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Fig. 1. Theoretical VCT magnetisation curves, according to Eq. 1, for different values of TCH . T1r H s 5 ms for all curves.

A number of methods have been used in an attempt to attain quantitative data from CP spectra, based on either minimising or correcting for errors brought about by the effects of T1r H and TCH on 13 C CP NMR signal intensities. These methods include: Ži. using a single arbitrary contact time Že.g., 1 ms. based on model compound studies ŽAlemany et al., 1983a,b. or similar samples ŽPreston and Newman, 1995; Preston et al., 1994. ; Žii. performing VCT experiments to determine the contact time at which signal intensity is maximised ŽConte et al., 1997a,b. ; and Žiii. performing VCT experiments and fitting signal intensities to Eqs. 1 or 2 ŽHagaman et al., 1986; Botto et al., 1987. , Eq. 3 Ž Vassallo et al., 1987, see below., or variants thereof Ž Wilson et al., 1987. . As illustrated in Fig. 1, methods based on measurement at a single contact time are subjected to large errors if samples or resonances are characterised by different values of TCH andror T1r H. However, methods utilising VCT experiments in order to measure TCH , T1r H and C are also subjected to errors, as shown below. For simple organic molecules that give rise to NMR spectra consisting of discrete, well-separated resonances, VCT signal intensities for each resonance can be fitted to Eq. 1 to obtain values for C, TCH , and T1r H. The results of the fitting procedure are shown in Fig. 2 for the 13 C CPrMAS NMR VCT spectra

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Fig. 2. Experimental VCT intensities and curves of best fit to Eq. 1 for carbonyl and methylene resonances of glycine. T1r H unrestricted in fit.

of glycine. A good fit is obtained for the carbonyl resonance, however, the fit is not as good for the methylene resonance. Difficulties in fitting 13 C signal intensities to Eq. 1 were encountered in other model compounds for all resonances for which there was rapid magnetisation build-up in VCT experiments, i.e., methylene Ž CH 2 . and methine Ž CH. carbons Ž data not shown. . Attempting to fit all VCT intensities using Eq. 1 led to a poor fit of both build-up and decay phases. A much better fit of the decay phase can be achieved by fitting only this section of the curve to a simple exponential decay ŽEq. 3.: I s I0 exp Ž ytcprT1r H . .

Ž3.

Eq. 3 is derived from Eq. 1 and the assumption that the term expŽytcprTCH . is negligible for longer values of t cp in the decay phase of the VCT curve. The constant I0 in Eq. 3, therefore, corresponds to the term Crw1 y Ž TCHrT1r H.x in Eq. 1. A full fit to the VCT curve is best achieved by varying TCH in Eq. 1 while keeping I0 and T1r H fixed to those values determined from the fit to Eq. 3. The result for glycine ŽFig. 3. is a good fit for the decay phase, while the fit to the build-up phase for methylene carbons is still poor. This indicates that magnetisation build-up for methylene Ž and methine. resonances does not follow simple exponential dynamics and the values of TCH determined are not truly exponential rate constants. They do, however, represent the best fit to the model dynamics of Eq. 1 and will be used as such in the following discussion.

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Fig. 3. Experimental VCT intensities and curves of best fit to Eq. 1 for carbonyl and methylene resonances of glycine. T1r H and I0 fixed at values determined by previous fit of decay phase Ž2–12 ms. to Eq. 3.

Non-exponential spin dynamics for methylene and methine carbons has been noted previously Ž Sullivan and Maciel, 1982a; Frund 1989a.. ¨ and Ludemann, ¨ The VCT fitting procedure was applied to selected resonances of model compounds to determine ‘‘best fit’’ TCH values ŽTable 1. . The fitting procedure was carried out only for resonances that represent identical or chemically similar environments and were well separated from other resonances. For example, even though the O-alkyl region of the NMR spectra of carbohydrates such as cellulose contained a number of distinct but overlapping resonances, each carbon is protonated, and hence, all are characterised by similar values of TCH . Therefore, they were treated as a single resonance in the VCT fitting procedure. On the other hand, the aromatic region of the NMR spectrum of lignin contains overlapping resonances for both non-protonated Ž O-aromatic, C-aromatic. and protonated ŽH-aromatic. carbons and, hence, the VCT fitting procedure could not be employed easily. T1r H relaxation rates did not differ greatly between resonances in the same model compound. This is to be expected due to the process of spin diffusion by which T1r H relaxation rates are rendered uniformly throughout a population of mutually coupled protons ŽWilson, 1987; Kinchesh et al., 1995a.. However, it should be noted that significantly different relaxation rates were observed for the O-alkyl and carbonyl resonances of pectin. TCH for the model compounds ranged from 0.30 Ž collagen. to 0.44 ms Žpectin. for non-protonated carbons and from 0.035 Žpalmitic acid, glycine. to 0.085 ms Žpectin. for methine and

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Table 1 Best fit values of T1r H and TCH for some model compounds. T1r H was determined first by a fit to Eq. 3. TCH was then determined with T1r H fixed at this value by a fit to Eq. 1 Sample Glycine Cellulose Pectin NaPGUA Chitin

Chitosan Collagen Palmitic acid

Resonance carbonyl methylene O-alkyl carbonyl O-alkyl carbonyl O-alkyl carbonyl O, N-alkyl methyl Ž N-acetyl. O, N-alkyl carbonyl alkyl methylene

T1r H Žms.

TCH Žms. .a

29 Ž26,32 28.0 Ž25.8, 30.6. 10.1 Ž9.4, 10.9. 3.4 Ž3.0, 3.8. 2.49 Ž2.42, 2.56. 3.8 Ž3.2, 4.6. 3.0 Ž2.7, 3.4. 6.3 Ž5.9, 6.8. 7.98 Ž6.78, 7.21. 6.82 Ž6.55, 7.11. 2.83 Ž2.55, 3.19. 3.8 Ž3.5, 4.3. 3.64 Ž3.52, 3.76. 52 Ž46, 60.

0.38 0.035 0.050 0.44 0.085 0.35 0.050 0.32 0.062 0.086 0.056 0.30 0.048 0.035

a

Values in parentheses represent lower and upper bounds, respectively, of the 95% confidence interval.

methylene resonances. This justifies the use of ‘‘typical’’ values for TCH of 0.05 ms for methylene and methine carbons and 0.35 ms for non-protonated carbons. TCH for the methyl Ž N-acetyl. resonance of chitin was 0.086 ms, just outside the range of TCH observed for other protonated carbons. For more chemically complex materials, such as SOM, where each resonance represents not just one but a range of chemical environments, fitting VCT intensities to Eq. 1 becomes even more difficult. The value of TCH may be different for nuclei in different chemical environments that have similar chemical shifts. In particular, protonated and non-protonated 13 C nuclei may have similar chemical shifts but will have vastly different magnetisation build-up rates. On the other hand, T1r H relaxation rates are often the same across the whole sample due to spin diffusion. The exception is where there are physically separated domains of different chemical structure within the sample Ž Preston and Newman, 1992, 1995; Smernik and Oades, 1999. . From the above discussion, it would appear advantageous to find a method for attaining quantitative data from CP spectra, which avoids fitting the build-up phase of the VCT experiment. One method, which avoids fitting the build-up section of the VCT experiment, is simply to fit only the decay phase of the VCT experiment to Eq. 3. However, this only yields T1r H and I0 , with I0 being a function of both T1r H and TCH . Assuming the CP dynamics of Eq. 1, I0 s Crw1 y Ž TCHrT1r H.x. In a number of studies, the factor of 1rw1 y Ž TCH rT1r H.x is ignored Žsee, for example, Vassallo et al., 1987. . This leads to an overestimation of non-protonated carbons by 7% when T1r H s 5 ms and by 14% when

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T1r H s 2.5 ms, assuming TCH for protonated and non-protonated carbons of 0.05 and 0.35 ms, respectively Ž Fig. 4. . The overestimation of non-protonated carbon comes about because the slow build-up rate of non-protonated carbon magnetisation means that less of its magnetisation is decaying at short contact times. Extrapolation of the decay curve to t cp s 0 overestimates both protonated and non-protonated carbon intensities, but more so for non-protonated carbons. This can be seen in the VCT curves for the methylene and carbonyl resonances for glycine ŽFigs. 2 and 3. where the intensity of the carbonyl resonance exceeds that of the methylene resonance at longer contact times. A better method for achieving quantitation between protonated and non-protonated carbons can be envisaged by considering the point in Fig. 1 where the TCH s 0.05 ms and TCH s 0.35 ms VCT curves cross, since this is the only value of t cp where relative quantitation of ‘‘typical’’ protonated and non-protonated carbons is achieved, i.e., the signal intensity is independent of the two different values of TCH . In terms of Eq. 2, we want to find a value of t cp for which Y, the product of terms involving TCH ŽEq. 4., is reasonably independent of TCH and T1r H Y s 1 y exp Ž ya t cprTCH . r 1 y Ž TCHrT1r H . .

Ž4.

Fig. 5 shows that for t cp s 1 ms, the contact time most often used in SOM NMR studies, Y deviates by no more than 6% from unity for either protonated Ž TCH s 0.05 ms. or non-protonated Ž TCH s 0.35 ms. carbons for T1r H ) 2.5 ms. This implies that a contact time of 1 ms results in good relative quantitation

Fig. 4. Plot of I0 r C vs. T1r H, according to Eq. 1, for different values of TCH .

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Fig. 5. Plot of Y vs. T1r H, according to Eq. 4, for different values of TCH .

under the above constraints. Also, under these constraints, Eq. 2 can be reasonably approximated by I Ž 1 ms . f Cexp Ž y1rT1r H .

Ž5.

C f I Ž 1 ms . rexp Ž y1rT1r H .

Ž6.

i.e., and, hence, C Ž which is directly proportional to the number of 13 C nuclei, which give rise to the resonance. can be closely estimated from the intensity of the 1 ms contact time spectrum and the measured value of T1r H. The performance of this approximation compares favorably with the approximation I0 s C Ži.e., ignoring the factor of 1rw1 y Ž TCHrT1r H.x., which was discussed above ŽFig. 4.. It must be noted that signal intensities of 13 C nuclei characterised by values of TCH considerably longer than 0.35 ms will be underestimated by Eq. 6 Ž e.g., TCH s 1 ms in Fig. 5.. This is most likely to occur for carbons far removed from the nearest proton or carbons in mobile structures whose rapid motion decreases the strength of 1 H– 13 C dipolar coupling. Significant errors will also occur when Eq. 6 is used for samples with T1r H - 2.5 ms. 3.1.3. Internal Õs. external standards for spin-counting experiments There are advantages and disadvantages to both internal and external standards. The advantage of an internal standard is that, since the calibration spectrum is run simultaneously with that of the sample, the acquisition parame-

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ters and conditions are identical. There are, however, a number of disadvantages. It is difficult to find a material whose 13 C NMR spectrum does not overlap with that of SOM samples, which contain resonances across a wide range of chemical shifts. Materials that have been used to overcome this problem include a methylsilicone polymer Ž d13 C s y2 ppm. Ž Sullivan and Maciel, 1982a,b. and a bicyclic ketone, which is 13 C labeled in the carbonyl position Ž d13 C s 217 ppm. Ž Jurkiewicz and Maciel, 1995. . Alternatively, procedures may be used to separate standard and sample signals, but these complicate measurement and are inevitably a source of error. Internal standards used in this way include hexamethylbenzene Ž Botto et al., 1987. and glycine Ž Hagaman et al., 1986; Vassallo et al., 1987; Wilson et al., 1987. . Another problem with internal standards is that NMR sensitivity is not constant throughout the whole sample, as shown by the following experiment. Glycine was placed in a rotor along with two ceramic spacers, which each occupy one third of the rotor volume and give rise to no 13 C NMR signal. Three spectra were acquired with the glycine either in the top, middle or bottom third of the rotor. The total intensities were 20%, 48%, and 31%, respectively, of the signal of a full rotor of glycine Ž containing three times as much glycine. . This shows that NMR sensitivity is greater in the middle of the rotor than at the ends. Radial sensitivity was not tested but may be expected to show similar non-uniformity, although, since the radial dimension is much smaller than the axial dimension, differences should be smaller. Owing to the spatial non-uniformity of NMR sensitivity, an internal standard needs to be uniformly distributed throughout the sample Ž Hagaman et al., 1986; Botto et al., 1987.. This introduces problems of potential reaction between standard and sample ŽVassallo et al., 1987; Wilson et al., 1987. and renders both sample and standard unrecoverable. The use of an external standard avoids signal overlap and the problems associated with having to mix standard and sample. However, problems do arise with running the sample and standard at different times and, hence, under potentially different spectrometer conditions. Factors, which may affect NMR sensitivity for different samples, include probe tuning, magic angle spin rate, the Hartman–Hahn matching condition, and differences in the magnetic properties Že.g., magnetic susceptibility, concentration of paramagnetic or ferromagnetic materials. between standard and sample. The effects of probe tuning, magic angle spin rate, and setting of the Hartman–Hahn matching condition on NMR sensitivity of a sample of glycine were tested. The effects of magnetic properties are discussed in Section 3.3. Probe tuning was shown not to significantly affect signal intensity for either the 1 H or 13 C channels, even when tuning was moved deliberately a long way from optimal settings. Magic angle spin rate was also shown not to affect the signal intensity at spin rates as low as 4 kHz, which is significantly outside the 5 kHz " 100 Hz rate usually employed. Deliberately setting 1 H power levels away

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from the Hartman–Hahn matching condition resulted in a decrease in signal intensity, especially for the carbonyl resonance, but the decrease was negligible in the range of settings in which the Hartman–Hahn matching condition drifts on the timescale of weeks to months. Therefore, these three factors are unlikely to cause problems when using an external reference for spin-counting experiments. However, since these factors may be instrument specific, the sensitivity of a spectrometer to variations in probe tuning, spinning speed, and Hartman– Hahn match should be checked before performing spin-counting experiments. In order to determine whether there was significant drift in NMR sensitivity over time, the NMR signal intensity of a sample of glycine was measured several times over the period of 2 months. Integrated intensities varied by less than 5% over this period. This suggested that NMR sensitivity is stable enough over time so as not to introduce significant errors in spin-counting experiments where an external standard is used. In order to minimise errors when performing spin-counting experiments using an external standard, it is suggested that the following procedures be followed. Ži. Calibrate the Hartman–Hahn match no more than 1 week before acquiring spectra for spin counting. Žii. Acquire the spectrum of the standard immediately before Ž or after. acquiring the spectrum of the sample. Where there are a number of samples, re-acquiring a spectrum of the standard on a weekly basis should suffice. Žiii. Use exactly the same parameters for the standard and sample spectra, with the possible exception of the recycle delay which should be set to at least five times T1 H for the most slowly relaxing component in the sample wSOM samples may contain components with different T1 H values ŽPreston and Newman, 1992, 1995; Smernik and Oades, 1999.x. Živ. T1r H also needs to be measured in order to calculate the NMR observable carbon content using Eq. 6, but VCT experiments need not be run at the same time as the 1 ms contact time spectrum. 3.2. Results of spin-counting experiments Spin-counting experiments were performed on model compounds using both CP and BD techniques. Glycine was used as an external reference because it is pure, stable, has a short T1 H Ž 120 ms., and a long T1r H Ž26 ms. . Model compounds were chosen, which either occur in SOM, or contain functional groups that occur in SOM. The model compounds used included cellulose, hemicelluloses Žpectin, NaPGUA. , biopolymers of amino sugars Ž chitin, chitosan., a protein Ž collagen., lignin, a fatty acid Ž palmitic acid. , and charcoal. Spin-counting experiments were also performed on a whole soil Ž SS6ws. as well as the light Ž- 1 Mgmy3 . fraction ŽSS6lf. and HF-treated material Ž SS6hf. derived from that soil, and, also, a commercial humic acid Ž Aldrich HA. . Being derived from lignite, Aldrich HA is not a representative of soil humic acids

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ŽMalcolm and MacCarthy, 1986. , but is included in this study as an example of a heterogeneous material for which NMR observability is vastly different for its different components. Spectra for each sample were acquired under near identical conditions. The only NMR acquisition parameter that varied was the recycle delay, which was set at 4 s for each CP spectrum except those of cellulose Ž 10 s. and palmitic acid Ž60 s.. The recycle delay for each BD spectrum was 90 s, except for palmitic acid Ž600 s.. These delays were used to ensure complete relaxation between transient spectra and, hence, avoid signal loss through saturation. Each spectrum was integrated between y10 and 300 ppm so that signal from all resonances, including first-order spinning sidebands, was measured. For each spectrum, the integrated intensity Ž I . was divided by the mass of sample in the rotor to give I ) ŽEq. 7., which is proportional to both the percentage carbon content Ž %C. of the sample and the NMR observability of the 13 C nuclei in the sample Ž Cobs . ŽEq. 8.. I ) s Irm

Ž7. Ž8.

I s k = %C = Cobs . The constant Ž k . was determined by assuming 100% observability of the glycine NMR signal, i.e., CobsŽglycine. s 100%. The results of spin-counting experiments are presented in Table 2. CP observabilities were determined both before Ž uncorrected Cobs-CP. and after Ž Cobs-CP. correcting for the effects of T1r H using Eq. 6. T1r H was determined )

Table 2 Results of spin-counting experiments on model compounds Sample

%C

Uncorrected Cobs-CP Ž%.

T1r H

Cobs-CP Ž%.

Cobs-BD Ž%.

Glycine Cellulose Pectin NaPGUA Chitin Chitosan Collagen Lignin Aldrich HA Palmitic acid Charcoal SS6hf SS6lf SS6ws

30.74 41.50 34.64 31.04 41.35 39.56 49.55 59.46 39.33 73.56 72.29 41.75 42.45 7.20

100 96 69 73 67 76 58 77 27 93 24 63 54 27

26 Ž23, 31. a 10.0 Ž9.0, 11.1. 2.80 Ž2.72, 2.89. 3.23 Ž3.07, 3.42. 6.44 Ž6.34, 6.54. 2.90 Ž2.73, 3.08. 3.9 Ž3.3, 4.8. 6.64 Ž6.37, 6.98. 3.72 Ž3.66, 3.78. 50 Ž43, 60. 4.0 Ž3.6, 4.5. 4.85 Ž4.79, 4.91. 4.24 Ž4.07, 4.43. 4.56 Ž4.36, 4.78.

100 102 95 96 75 103 72 87 35 92 29 75 66 33

100 90 92 91 100 105 93 89 79 90 87 99 107 85

a

Values in parentheses represent lower and upper bounds, respectively, of the 95% confidence interval.

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from VCT experiments using total signal intensities, i.e., y10–300 ppm. The value of uncorrected Cobs-CP ranged between 24% and 96%. Quantitation Ž Cobs f 100%. was only achieved for compounds with long T1r H, i.e., cellulose Ž T1r H s 10 ms. and palmitic acid Ž T1r H s 50 ms.. In contrast, quantitation was achieved for a number of other compounds when corrections for differences in T1r H values were applied. Cobs-CP for pectin Ž95%., NaPGUA Ž96%., and chitosan Ž103%. indicated that quantitation was achieved. The corresponding uncorrected Cobs-CP values were 69%, 73%, and 76%, respectively. Observabilities for the BD spin-counting experiments Ž Cobs-BD. were generally higher than for the corresponding CP experiments and ranged between 79% and 107%. The model compounds in Table 2 fall into three broad categories based on values of Cobs-CP and Cobs-BD. Ži. Materials that gave quantitative signals with both methods Ž Cobs-CP and Cobs-BD ) 87%.. These materials were cellulose, pectin, NaPGUA, chitosan, lignin, and palmitic acid. Žii. Materials that gave quantitative BD signals but moderately diminished CP signals Ž Cobs-CP in the range 66–75%. . These materials were chitin, collagen, SS6hf, and SS6lf. Žiii. Materials that gave slightly diminished BD signalsŽ Cobs-BD in the range 79–87%. and greatly diminished CP signals Ž Cobs-CP in the range 29–35%.. These materials were Aldrich HA, charcoal, and SS6ws. Differences in observabilities between CP and BD spectra were brought about by interference with the CP mechanism. The signal unobserved or under-represented in the CP spectrum was due to 13 C nuclei characterised by long TCH values andror short T1r H values. Long TCH values are due to weak 1 H– 13 C dipolar coupling, which can be caused by large distances from 13 C nuclei to their nearest 1 H neighbour or by high molecular mobility. Short T1r H values are usually brought about by paramagnetic centres, but may also be due to mobility. The effects of paramagnetic centres are discussed in detail in Section 3.3. In Figs. 6 and 7, CP and BD spectra are compared for the samples for which there were significantly different CP and BD observabilities. The vertical scales of the spectra have been set such that equal values of Cobs-CP and Cobs-BD would result in equal total integrals, i.e., the CP and BD spectra are directly comparable for each sample. The BD spectrum of chitin Ž Fig. 6aX . contains additional very broad resonances not evident in the corresponding CP spectrum Ž Fig. 6a. . This broadness may be indicative of rapidly relaxing nuclei Žshort T1C.. If this is the case, then T1r H is also likely to be shortened by the same mechanism, which may explain the absence of the broad signal in the CP spectrum. The situation for collagen was quite different. The BD spectrum of collagen ŽFig. 6bX . contains two additional very sharp resonances not observed in the CP

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Fig. 6. Comparison of CP and BD spectra for samples that gave quantitative BD spectra and significantly diminished CP spectra Ž Cobs in the range 66–75%.. The vertical scales of each pair of spectra have been adjusted to allow quantitative comparison between CP and BD spectra. Ža. CP spectrum of chitin; ŽaX . BD spectrum of chitin; Žb. CP spectrum of collagen; ŽbX . BD spectrum of collagen; Žc. CP spectrum of SS6hf; ŽcX . BD spectrum of SS6hf; Žd. CP spectrum of SS6lf; ŽdX . BD spectrum of SS6lf.

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Fig. 7. Comparison of CP and BD spectra for samples which gave significantly diminished BD spectra Ž Cobs-BD in the range 79–87%. and severely diminished CP spectra Ž Cobs-CP in the range 29–35%.. The vertical scales of each pair of spectra have been adjusted to allow quantitative comparison between CP and BD spectra. Ža. CP spectrum of Aldrich HA; ŽaX . BD spectrum of Aldrich HA; Žb. CP spectrum of charcoal; ŽbX . BD spectrum of charcoal; Žc. CP spectrum of SS6ws; ŽcX . BD spectrum of SS6ws.

spectrum ŽFig. 6b. , at 30.4 and 129.9 ppm. The absence of these resonances from the CP spectrum was unlikely to be due to rapid T1r H relaxation, but may have been due to very high mobility. Saturation caused by slowed T1 H relaxation can be ruled out as a possible mechanism, since the sharp signals were still not evident in the CP spectrum when the recycle delay was increased from 4 to 60 s. The resonance at 30.4 ppm is indicative of a lipid impurity. The CP spectrum of SS6hf Ž Fig. 6c. contains less intense resonances in the carbonyl and aromatic regions than the corresponding BD spectrum ŽFig. 6cX .. The signal missing from the CP spectrum may have been due to 13 C nuclei in

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domains with a high concentration of paramagnetic centres, either inorganic or organic. A similar situation is evident for SS6lf Ž Fig. 6d and dX . , but in this case, signal loss appeared to be less specific to the carbonyl and aromatic regions. A comparison of the CP and BD spectra for Aldrich HA Ž Fig. 7a and aX . shows that most of the signal missing from the CP spectrum is aromatic and carbonyl. This may be due to short T1r H values for nuclei close to organic or inorganic paramagnetic centres andror long TCH values for 13 C nuclei in condensed aromatic regions, which contain few protons. Differences in the relative intensities of the CP and BD spectra of charcoal Ž Fig. 7b and bX . are likely to be due to similar effects as described for Aldrich HA. All resonances in the CP spectrum of SS6ws ŽFig. 7c. are less intense than those in the corresponding BD spectrum ŽFig. 7cX . , although resonances in the carbonyl and aromatic regions are, again, the most diminished. Signal losses in the CP spectrum of this sample were most likely due to decreases in T1r H brought about by paramagnetic iron. 3.3. Effects of paramagnetic materials on quantitation The presence of paramagnetic impurities is one of the main causes of signal loss in solid state 13 C NMR spectra of soils ŽOades et al., 1987; Vassallo et al., 1987; Arshad et al., 1988; Preston et al., 1989; Skjemstad et al., 1994; Schmidt et al., 1997.. The effects of paramagnetic materials on quantitation in solid state 13 C NMR spectra were investigated by doping model compounds with paramagnetic impurities. The presence of paramagnetic centres affects solid state NMR spectra via three mechanisms. These mechanisms act on three different length scales, long Ž throughout the whole sample. , medium Ž within coupled spin systems., and short Žwithin a few bonds.. Long-range effects of paramagnetic materials on NMR signal intensities are brought about by decreases in field homogeneity. This tends to both broaden and decrease the intensity of resonances. Signal loss via this mechanism is equal for all resonances in the spectrum and hence relative quantitation is not affected. Fig. 8 compares the 13 C CP spectrum of cellulose with that of a mixture of cellulose and MnCl 2 P 4H 2 O Ž3.3% Mn by mass.. The vertical scales of the spectra have been adjusted to account for the differences in the mass of cellulose used in the analysis. The presence of MnCl 2 P 4H 2 O decreased the observability Žsignalrmass. of cellulose to 70% of that for neat cellulose, and caused signal broadening. Similarly, cellulose observability in the BD spectrum was decreased to 67% Ž Table 3.. In the above experiment, the MnCl 2 P 4H 2 O was present as sand sized crystals mixed evenly throughout the sample. The same sample Žcellulose plus MnCl 2 P 4H 2 O. was ground Žmortar and pestle. to test whether the particle size of the paramagnetic salt affected the observability of the cellulose NMR signal.

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Fig. 8. Effect of a paramagnetic salt, added as a physical mixture, on the CP spectrum of cellulose. Ža. Neat cellulose; Žb. celluloseqMnCl 2P4H 2 O Ž3.3% Mn by mass.. The vertical scales of the spectra have been adjusted to allow direct quantitative comparison between the spectra.

After grinding, cellulose observability was 67% for the CP spectrum and 69% for the BD spectrum Žwith respect to unamended cellulose. Ž Table 3. . These observabilities are not significantly different to those determined for the unground sample, indicating that there was no effect of the size of paramagnetic salt particles on cellulose observability. Increasing the paramagnetic content to 8.1% Mn resulted in further broadening and a further decrease in cellulose observability to 40% ŽCP. and 37% ŽBD.. The decrease in cellulose observability was found to be also dependent on the paramagnetic cation used. Cellulose observability was 98% in the presence of 9.9% Cu Žby mass. in the form of CuCl 2 P 2H 2 O ŽTable 3.. No broadening of cellulose resonances was observed, confirming that the presence of copper had no significant effect. Preston et al. Ž 1989. observed similar non-selective signal loss and broadening on the addition of an iron-rich mineral fraction to an isolated soil humin. Medium-range effects of paramagnetic centres on NMR quantitation are due to decreases in T1r H. The range of influence is dependent on the rate and extent Table 3 Relative observability a of cellulose 13C NMR signal in physical mixtures of cellulose and paramagnetic salts Žn.d., not determined. Sample

Relative observability ŽCP.

Relative observability ŽBD.

Cellulose CelluloseqMnCl 2P4H 2 O Žunground, 3.3% Mn by mass. CelluloseqMnCl 2P4H 2 O Žground, 3.3% Mn by mass. CelluloseqMnCl 2P4H 2 O Žunground, 8.1% Mn by mass. CelluloseqCuCl 2P2H 2 O Žunground, 9.8% Cu by mass.

100% 70% 67% 40% 98%

100% 67% 69% 37% n.d.

a

Observability Žsignal intensityrmass. relative to neat cellulose sample.

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of proton spin diffusion, but is at least of the order of 3–10 nm Ž Zumbulyadis, 1983; Newman and Condron, 1995. . The loss of signal brought about by decreases in T1r H can be compensated for by using Eq. 6 to calculate NMR observable carbon contents. However, very high concentrations of paramagnetic centres can decrease T1r H below the threshold value for which Eq. 6 is applicable Ž ; 2.5 ms.. BD spectra are not affected by changes in T1r H. The medium-range effect of paramagnetic centres on NMR quantitation was demonstrated by the effect that bound Cu2q ions had on the observability of lignin 13 C NMR resonances. A sample of lignin was amended with Cu2q by shaking with 0.5 M CuCl 2 . After removing excess free salt, the lignin sample retained some Cu2q ions. T1r H for the Cu2q amended sample was 4.02 ms which was considerably shorter than the value of 6.64 ms observed for unamended lignin. Preston et al. Ž 1989. reported that T1r H was negatively correlated with copper content in organic soils from a natural cupriferous bog. Pfeffer et al. Ž1984. also observed that T1r H decreased on the addition of Fe 3q to model sludges. In both cases, T1r H was most affected for O-alkyl resonances. The observability Žsignalrmass. of the BD spectrum for the Cu2q amended lignin sample was not diminished in relation to that of the unamended sample. However, the observability Ž corrected for differences in bulk T1r H. of the CP spectrum of the Cu2q amended sample was decreased by 24% in relation to that of the unamended sample. Since there was no loss in BD observability, the loss in CP observability must have been due to the decreases in T1r H for some nuclei over and above the decrease observed in bulk T1r H. It would appear that spin diffusion through the Cu2q amended lignin sample did not result in completely homogeneous T1r H relaxation rates, and that for around 24% of the 13 C nuclei, presumably in the near vicinity of bound cations, T1r H was reduced to an extent that they gave rise to virtually no signal in the CP spectrum. The signal loss was not selective, with all resonances equally diminished, and was not accompanied by spectral broadening ŽFig. 9. . Preston et al. Ž 1989. observed signal loss when FeCl 3 was added as a solution to an isolated humin. The signal loss was selective, with alkyl resonances least affected. Vassallo et al. Ž 1987. reported large and non-selective signal loss on the addition of a solution of FeCl 3 to lignite. Short-range effects of paramagnetic centres only operate on 13 C nuclei within a few bond’s distance of a paramagnetic centre. These nuclei are often rendered unobservable through extreme broadening andror shifting ŽAime et al., 1996. . Broadening may be due to extremely rapid relaxation rates or be the result of paramagnetic shifting of coupled proton resonances outside the 1 H chemical shift range which can be decoupled efficiently ŽBrough et al., 1993; Aime et al., 1996.. Short-range effects of paramagnetic centres are difficult to quantify since they are usually accompanied by medium-range effects on T1r H. Paramagnetic lanthanide ions, such as Pr 3q, are less efficient at reducing T1r H, but still cause

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Fig. 9. Effect of bound Cu2q cations on the CP spectrum of lignin. Ža. Neat lignin; Žb. Cu2q-amended lignin. The vertical scales of the spectra have been adjusted to allow direct quantitative comparison between the spectra.

short-range shifting and broadening of 13 C resonances. Short-range effects were observed for the Pr 3q salt of polygalacturonic acid ŽPrPGUA. , which was formed by the reaction of NaPGUA with PrCl 3. The observability of polygalacturonate 13 C resonances for the Pr 3q salt was decreased by 14% for BD and by 48% for CP, relative to that of the sodium salt. The presence of the Pr 3q ion also caused extreme broadening of the polygalacturonate 13 C resonances as shown by the comparison of the 13 C NMR spectra of the Naq and Pr 3q salts Ž Fig. 10.. The difference in observability between the

Fig. 10. Effect of bound Pr 3q cations on the CP spectrum of polygalacturonate. Ža. NaPGUA; Žb. PrPGUA. Note: these are not quantitatively comparable.

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Fig. 11. Effect of PrPGUA, added as a physical mixture, on the CP spectrum of cellulose. Ža. Neat cellulose; Žb. celluloseqPrPGUA. The vertical scales of the spectra have been adjusted to allow direct quantitative comparison between the spectra.

CP and BD spectra can be ascribed to decreases in T1r H, i.e., the medium-range effect Žbulk T1r H was 3.0 ms for NaPGUA and 2.7 ms for PrPGUA. . The 14% decrease in observability in the BD spectrum of the Pr 3q salt is due to either short- or long-range effects. To distinguish between these possibilities required the use of an internal standard. The CP spectrum of a physical mixture of cellulose and PrPGUA was acquired. The total signal intensity was the same Ž 97%. as would be expected if spectra were acquired separately. Thus, the signal of cellulose was not decreased by the presence of PrPGUA Žas it was by MnCl 2 P 4H 2 O., proving that the long-range mechanism was not causing significant signal losses. The 14% loss in observability in the PrPGUA BD spectrum Ž relative to that of NaPGUA. could, thus, be ascribed to the short-range mechanism. No broadening was apparent in the cellulose 13 C resonances in the CP spectrum of the celluloserPrPGUA physical mixture ŽFig. 11., in contrast to the significant broadening of cellulose 13 C resonances in the CP spectrum of the celluloserMnCl 2 P 2H 2 O physical mixture ŽFig. 8. .

4. Summary Spin-counting experiments were performed on a number of model compounds, using both CP and BD techniques. CP spin-counting experiments were complicated by the need to account for differences in T1r H between samples and

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the reference material. A new method was developed to correct for these differences. This method does not rely on the measurement of magnetisation build-up rates, which were shown to be poorly described by a single exponential rate constant. In the BD experiment, signal from the Kel-F rotor end-caps was shown to be significant for all samples and, therefore, the end-cap spectrum was subtracted from BD spectra of the samples prior to spin counting. The CP signal of the end-caps was shown to be minor due to the low 1 H content of the fluorinated polymer and no correction was required. For both CP and BD techniques, signal from first order spinning side bands was included in the spin-counting procedure by integrating the spectra between y10 and 300 ppm. The observability of 13 C NMR signal for the model compounds ranged between 79% and 107% in BD spin-counting experiments. This indicated that the BD NMR spectra substantially reflected the quantitative distribution of functional groups. The observability of 13 C NMR signal for the model compounds ranged between 29% and 103% in CP spin-counting experiments. A number of the model compounds gave quantitative CP spectra Ž Cobs-CP ) 87%.. A second group of materials suffered substantially decreased observability Ž Cobs-CP in the range 66–75%. and a third group suffered severely decreased observability Ž Cobs-CP in the range 29–35%. . This third group also suffered minor decreases in observability in the BD experiment Ž Cobs-BD in the range 79–87%.. Comparison of BD and CP spectra for the latter two groups showed that signal loss was selective, affecting some functional groups more than others. Chemical moieties prone to under-representation in CP NMR spectra included the aromatics in charcoal; carboxyl and aromatic groups in the whole soil, HF-treated soil, soil litter fraction, and commercial humic acid; and an apparent lipid impurity in collagen. The reduced observability of these moieties was attributed to the locally low values of T1r H for nuclei in the vicinity of organic or inorganic paramagnetics, andror to high values of TCH due to the remoteness of adjacent protons or high molecular mobility. The effects of paramagnetic materials on quantitation were examined by doping model compounds with paramagnetic impurities. Three mechanisms were shown to contribute to signal loss. These mechanisms were differentiated by the length scale on which they operated. The addition of MnCl 2 P 4H 2 O in a physical mixture with cellulose was shown to decrease the observability of all cellulose 13 C nuclei in both CP and BD experiments. Conversely, the addition of CuCl 2 P 2H 2 O in a physical mixture with cellulose was shown not to decrease the observability of cellulose 13 C nuclei in the CP experiment. Small quantities of bound Cu2q cations significantly decreased CP observability of lignin 13 C nuclei, but did not effect BD observability. Large quantities of bound Pr 3q cations decreased observability of polygalacturonate 13 C nuclei in both CP and BD spectra, but did not effect the observability of cellulose 13 C resonances when added as a physical mixture.

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5. Concluding remarks The quantitative reliability of solid state 13 C NMR spectra of SOM has been a topic of active debate over recent years. This paper describes spin-counting experiments using both CP and BD techniques, which allow the degree of quantitation in an NMR spectrum to be determined. These experiments require little additional NMR time and are relatively easy to carry out. Where high values of Cobs can be quoted, quantitation is assured, and questions regarding the reliability of functional group ratios determined from the NMR spectrum can be dismissed. Thus, spin-counting experiments on all, or at least a selection of, spectra in a solid state NMR study would add credence to conclusions based on these spectra. Low values of Cobs-CP alert the researcher to samples for which a large portion of the 13 C nuclei are unobserved or under-represented. Such samples may require the use of the more time-consuming, but more quantitatively accurate BD technique. This paper also identifies chemical structures likely to be under-represented in CP spectra. These include highly aromatic structures which are likely to have low protonation and hence high TCH values, and are also likely to stabilise organic free radicals and hence have low T1r H values; carboxylate groups in the presence of paramagnetic inorganic cations; and structures with high mobilities such as lipids near their melting point. On the other hand, structures such as cellulose, hemicelluloses, lignin, and lipids, which are not highly mobile, were observed quantitatively. Finally, this study shows the effect of paramagnetic cations on solid state 13 C NMR spectra depends on whether they are bound to the organic or exist as a separate phase. The magnitude and mechanism of signal loss also depends on the type of paramagnetic cation present.

Acknowledgements This work was funded by an Australian Research Council Ž ARC. grant.

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