Anal. Chem. 1987, 59, 1659-1664
CID spectra of precursor ions of a wide mass range with a single set of calibration data and results in mass assignments within f0.3 u or better, sufficient for the reliable interpretation of the product ion spectra of compounds ranging in molecular weight to at least 3000 mass units. Registry No. I, 2507-24-6; 11, 107982-40-1.
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(16) Hass, J. R.; Green, B. N.; Bateman, R. H.; Boa, P. A. Presented at the
32nd Annual Conference on Mass Spectrometry and Allied Topics,
San Antonio, TX, 1984 pp 380-381. (17) Carr, S.A.; Roberts. 0. D.; Hemllng, M. E.; Klllmer, L. B.; Johnson, W.;
Mentzer, M. Presented at the 34th Annual Conference on Mass Spectrometry and Allied Topics, Cincinnnati, OH, 1986;pp 630-631. (18) Kammei, Y.; Itagaki, Y.; Kubota, E.; Kunihiro, H.; Ishlhara, M. Presented at the 33rd Annual Conference on Mass Spectrometry and Allied Topics, San Diego. CA, 1985;p 855. (19) Matsuda, H.; Matsuo, T.; Fujita, Y.; Wollnik, H. Mass Spectrom. (JaLITERATURE CITED oanl r - , 1978. . 24.. 19. (20) Biemann, K.; Martin, S. A.; Scoble, H. A.; Johnson, R. S.; Papayanno(1) McLafferty, F. W.; Bente, P. F., 111: Kornfekl, R. T.; Sal, S.4.; Howe, poulos, I . A.; Biller, J. E.; Costello, C. E. I n Mass Spectrometry in the I.J. Am. Chem. SOC. 1973, 9 5 , 2120-2129. Analysis of Large Molecules; McNeal, C. J., Ed.: Wiley: Chlchester, (2) Beynon, J. H.; Cooks, R. G. Res./Dev. 1971, 22, 26. England, 1986;pp 131-149. (3) Boyd, R. K.: Beynon, J. H. Org. Mass Spectrom. 1977, 12, 163-165. (21) Boyd, R. K.; Bott, P. A.; Harvan, D. J.; Hass, J. R. Int. J . Mass Spec(4) Millington, D. S.; Smith, J. A. Org. Mass Spectrom. 1977, 12, trom. Ion Proc. 1988, 6 9 , 251-263. 264-285. (22) Carr, s‘ A’9 communication. (5) Bruins, A. P.; Jennings, K. R.; Evans, S. Int. J. Mass Spectrom. Ion (23) Matsuo, T.; Matsuda, H.; Katakuse, I.; Shimonishi, Y.; Maruyama, Y.; Phys. 1978, 2 6 , 395-404. Higuchi, T.; Kubota, E. Anal. Chem. 1981, 53,416-421. (6) Morgan, R. P.: Porter, C. J.; Beynon, J. H. Org. Mass Spechom. (24) Friedli, F. Org. Mass Specfrom. 1984, 19, 183-189. 1977, 12. 735-738. (25) Haddon, W. F. Anal. Chem. 1979, 51. 983-988. (7) Neumann, G. M.; Derrick, P. J. Org. Mass Spectrom. 1984, 19, (26) Biemann, K. I n The Proceedings of the Sixth International Conference 165-170. on Methods in Protein Sequence Analysis; Walsh, K. A,. Ed.; Humana: (8) Bricker, D. L.: Russell, D. H. J. Am. Chem. Sac. 1988, 108, Clifton, NJ, 1986;in press. 6174-6179. Costello, B.9 M.1.T.q 1986, unpublished (9) Ast, T,; Bozorgzadeh, M, H,; Wlebers, J. L.; B ~J, H,; ~ ~ ~A, ~ ~ (27) ~ ~ , t c. E.; ~Singh, B. ~ N.; Domon, , work. G. Org. Mass Spectrom. 1979, 1 4 , 313-318. (28) K’ B’; Crow, F‘ w’; Gross, M. L. J . A m . Chem. SOc. (IO) Gross, M. L.; Russell, D. H. in Tandem Mass Spectrometry; McLaffer105,5487-5488. ty, F. W., Ed.: Wlley: New York, 1983;pp 255-270. (29) Hemling, M. E.; YU, R. K.; Sedgwick, R. D.; Rinehart, K. L. Biochemis(11) Gllliam, J. M,; occo~ow~z, J. Presented at fie 31st ~~~~l Conference try 1984, 23,5706-5713. On Mass and AlliedTopics, P 193. (30) Barber, M,; brdoli, R, S.; Sdgwick, R, D,; J, C, J , Chern. (12) Johnson, E. G.; Nier, A. 0. Phys. Rev. 1953, 9 1 , 10-17. Soc., Farm%)’ Trans. 1 1982. 78, 1291-1296. (131 Futrell. J. H.:Miller, C. D. Rev. Sci. Inshum. 1988. 37. 1521-1526. i14) Todd, P. J.: McGllvew. D. C.: Baldwln, M. A.; McLafferW, F. W. I n ’ Tandem Mass Speciomefry; McLafferty, F. W., Ed.; Wlley: New RECEIVED for review December 18, 1986. Accepted March 12, York, 1983;pp 271-286. 1987* This work was supported by the Institutes (15) Amster, I. J.; Baldwin, M. A,; Cheng, M. T.; Proctor, C. J.; McLafferty, F. W. J . Am. Chem. SOC.1983, 105, 1654-1655. of Health, Division of Research Resources (Grant RR00317). lgS39
Analysis of Molecular Orientational Order in Solid Samples by Nuclear Magnetic Resonance: Application to Lignin and Cellulose in Wood Galen R. Hatfield, Maziar Sardashti, and Gary E. Maciel*
Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523
A two-dlmenslonal Fourier transform solid-state nuclear magnetic resonance experlment designed to yleld anlsotropy Information can be used to probe molecular orientational order of speclflc components In a complex, Inhomogeneous (e.g., blologlcal) solld. Thls technlque Is applied to wood In order to determlne the extent of macroscoplc orientation and ordering of llgnln and cellulose. Model systems are also considered to demonstrate the ablllty of thls technlque to probe orlentatlonal order. Results for Eucalyptus polyanfhemus wood reveal that there Is no net molecular orlentatlonal order of llgnln or cellulose over a macroscoplc volume of about 0.9 cm3.
Molecular orientational order is an important physical property in many areas of science, including biological membranes, surface chemistry, and oriented polymers. In many cases,the physical properties (and possibly chemical behavior) of these systems are dominated by the degree of molecular orientational order present. In order to understand, use, and modify these chemical systems, it is important to have a detailed knowledge of the type and extent of order present. To date, most methods for readily determining orientational order can be applied only to relatively simple homogeneous
samples or else have required substantial experimental limitations. For example, in highly ordered and chemically homogeneous systems, techniques that rely on X-ray or neutron diffraction may be useful. However, with samples that are chemically inhomogeneous and/or with small degrees of molecular orientational order, the diffraction patterns are poorly defined and orientational information is difficult, if not impossible, to obtain. For less ordered systems, 2H NMR has proven to be an excellent technique for extracting orientational information, based on the anisotropic nature of the nuclear electric quadrupole interaction (1). However, this method requires isotopic labeling, which presents a severe experimental limitation, especially if the system of interest is a natural biopolymer. We report here a two-dimensional Fourier transform (2-D FT) NMR experiment that readily permits analysis of molecular orientational order at the molecular level in both homogeneous and complex inhomogeneous solids. During the past few years the scope of NMR applications has been broadened from liquid samples to include a range of solid samples that represent a wide variety of chemical systems, from polymers and resins to molecules adsorbed on catalytically active surfaces (2). The chemical shift observed in an NMR experiment is the spectroscopic parameter used most often for the elucidation of molecular structure. This
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parameter depends upon two factors. These are (1)the disposition of the nuclear spin with respect to its electronic environment (i.e,, the chemical structural environment of the nucleus) and (2) the orientation of the structural environment of the nucleus with respect to the applied external magnetic field, Bo. In a typical liquid-state NMR experiment, this orientation dependence is averaged out by the tumbling (Brownian) motion of molecules in a liquid, yielding a sharply defined “isotropic chemical shift” average. However, in the solid state, motion is far more restricted, and the chemical shift anisotropy (CSA) gives rise to broad CSA powder patterns. These patterns contain valuable information on the orientation dependence of the chemical shift, but they can constitute serious line-broadening effects in the spectra of complex samples. If one wishes to obtain high-resolution spectra in such cases, say for 13C,the sample is usually rotated rapidly at the so-called “magic-angle” (54.7’) to average out this orientation dependence (3). The result of either magicangle spinning (MAS) of a solid or natural Brownian motion for a liquid is one or more sharp resonance lines characteristic of the isotropic chemical shifts, which reflect the chemical environments of the nuclei under study. However, in such experiments information on the orientation dependence of the chemical shift is lost. For relatively simple systems, CSA patterns containing orientational information can be obtained on static samples or from the spinning sideband patterns in a MAS experiment (3). However, for complex organic systems such as wood, the CSA patterns from many 13C nuclei in a wide variety of structural environments are hopelessly overlapped in the spectrum of a static sample, and the spinning sideband patterns in a slow-spinning MAS experiment are usually obscured by overlaps, preventing information on either the chemical structures orientation from being readily discerned. An experiment recently developed in this laboratory ( 4 ) permits the observation of both isotropic chemical shift information and CSA patterns associated with each of the isotropic chemical shifts. In this 2-D FT NMR experiment, the angle between the direction of Bo and the spinning axis of the sample is flipped between 54.7’ and another angle (typically 90’) during a time between the evolution and detection periods of the experiment. It can be shown ( 5 ) that, for sample spinning at 90°, the CSA is manifested as an anisotropy pattern that is reversed along the frequency scale and collapsed to half the width of the static (nonspinning) case, but the “shape” of the CSA pattern is the same as for the static experiment. After the evolution period, the sample-spinning axis is flipped to 54.’i0, where the chemical shift manifests itself as its isotropic average for detection. Details of this experiment have been described elsewhere ( 4 , 6 )and will not be repeated here. In order to determine the ability of this technique to probe molecular orientational order in a complex, inhomogeneous solid, a series of experiments were carried out on Eucalyptus polyanthernus wood. Wood is an excellent choice for studying order in a complex, inhomogeneous solid. The molecular architecture of wood and the organization of the cell wall itself have been the subject of intense study (7). Despite substantial strides, many crucial questions remain to be answered. Wood consists of a wide variety of constituents, including lignin, cellulose, hemicelluloses, tannins, and so forth. In this paper we will directly address the molecular orientational order of the two major constituents in wood, lignin and cellulose. Lignin has been generally considered to be somewhat “gluelike” in nature and randomly dispersed around oriented cellulosic fibers (8). Compared to cellulose, which is homogeneous at the level of primary polymer structure, lignin is much more heterogeneous, having a variety of different
bonding patterns and therefore an inhomogeneous primary structure. A recent study gave evidence suggesting that lignin in woody tissue “is more highly organized at the molecular level than had heretofore been recognized” and proposed “that the orientation of the phenyl ring is most often preferentially in the plane of the cell wall” (9). To address this type of issue, we have applied the above-mentioned 2-D F T NMR experiment designed to yield anisotropy information ( 4 ) to examine the molecular orientation of lignin and cellulose in whole wood. In solid-state NMR the sample is studied in its “natural” and unaltered solid state. The solid-state 2-D FT CSA approach of the present study provides information on anisotropy patterns associated with each distinct isotropic chemical shift. Thus, one can probe the orientation of each atomic site or moiety in a macromolecular system and detect the presence of molecular orientational order. We report here definitive physical proof that these components are largely unoriented, in a macroscopic sense, in unaltered, whole wood.
EXPERIMENTAL SECTION All spectra shown in this paper were accumulated at a 13C resonance frequency of 25.3 MHz on a home-built spectrometer using a Nalorac 89-mm magnet and a Nicolet 1180/293B datasystem/pulse-programmer. 13Cmagnetization was generated by means of cross polarization (CP) from proton spins (IO). I3C CP/MAS (11,12) spectra of Eucalyptus polyanthemus wood were obtained with a CP contact time of 2 ms and a repetition time of 1 s. I3C CP/MAS spectra of homovanillic alcohol were obtained with a contact time of 5 ms and a repetition time of 1 s. Both the cellulose and sucrose spectra were obtained at a contact time of 2 ms, with repetition times of 2 and 5 s, respectively. For the 2-D spectra, the t , period was incremented in steps of 83 p s In all cases, samples were spun at 2.4 kHz. RESULTS AND DISCUSSION It is possible to obtain precise information on the orientations of molecules under study from a mathematical analysis of an observed chemical shift pattern (13). In the limit of a single crystal, with only one molecular orientation per unit cell, the chemical shift or CSA pattern consists of only discrete sharp lines, each reflecting a specific ”chemical environment” and a specific orientation. In the limit of a completely disordered powder or a completely amorphous solid, the CSA pattern consists of an infinite number of resonance lines representing an infinite number of orientations. This infinite set of resonance peaks overlap to form a broad CSA pattern, reflecting a specific “chemical environment” and the statistical probabilities of the various orientations. Thus, a highly ordered system gives rise to sharp, discrete resonance lines, while a random system results in a broad, so-called “powder pattern”. It is this difference which serves as the basis for studying the orientation of lignin and cellulose in wood through the use of this 2-D FT NMR experiment. The strategy used in this study was to compare CSA patterns of cellulose and lignin resonances obtained when sample spinners were loaded in the following four ways: (1) with the wood grain collinear with the spinner axis, (2) with the direction of the grain oriented at an angle of 45’ with respect to the spinner axis, (3) with the wood grain perpendicular to the spinner axis, and (4)with powdered wood. If a wood component is highly ordered, the corresponding CSA patterns should be markedly different for each of the four samples. If, on the other hand, the component (e.g., lignin) is randomly oriented, the CSA patterns corresponding to a specific isotropic chemical shift should be the same for each sample and will appear no different from the CSA pattern obtained for powdered wood. The oriented wood samples used in this study were intact wood plugs removed from a naturally dried section of a fallen EucalSptus polyanthemus tree, taken from the sapwood regic\n located just beneath the inner bark. The plugs were
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Flgure 2. 13C 2-D FT CSA patterns for the specified chemical shifts in the spectrum of Eucalyptus polyanthemus wood: (A) grain collinear; (B) 45’; (C) perpendicular to the spinning axis; (D) powdered wood.
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Figure 1. I3C CP/MAS NMR spectra: (A) sucrose, showing with arrows the peaks at 73 and 106 ppm used for the 2-D FT experiments; (B) celiulose powder (Whatman CC31), showing with arrows the peaks at 7 3 and 106 ppm used for the 2-D FT experiments; (C) Eucalyptus polyanthemus wood used in this study, showing with arrows the lignin peak at 152 pm and the cellulose peaks at 106 and 73 ppm used for the 2-D FT experiments; (D) homovanillic alcohol, showing with an arrow the 149 ppm peak used for the 2-D FT experiments. roughly 1.8cm in length and 0.8 cm in diameter and thus probe molecular orientational order over a 0.9 cm3 volume. The powdered wood sample was prepared by collecting sawdust taken from the same region of sapwood and grinding it in a ball mill. Care was taken to ensure that the milling was not so extensive as to destroy the molecular structural integrity of wood components. 13CCP f MAS spectra were taken (not shown) in order to ensure that the lignin and cellulose contributions to the spectra remain unaltered by ball milling. Shown in Figure 1C is the normal 13C CP/MAS NMR spectrum of the wood used in this study. Lignin in Eucalyptus polyanthemus, a hardwood, is a heterogeneous mixture of polymers consisting of syringylpropane (I) and guaiacylpropane (11) repeat units (14,as well as significant quantities
of p-hydroxyphenylpropane moieties. Among the aromatic carbons in these structures are those with methoxy groups attached. These carbons have been shown (15) to give rise to 13Cresonances at 152-154 ppm and this position is marked in structures 1-111 by an arrow. The I3CCSA patterns corresponding to these carbons in the oriented and powdered wood samples, as given by the 2-D FT experiment ( 4 ) ,are shown in Figure 2. It is immediately obvious that the CSA patterns for the samples in which the wood grain was oriented with respect to the spinner axis are virtually identical with that of the powdered wood. This clearly proves that lignin can not be highly ordered or oriented in whole wood over a 0.9 cm3 macroscopic volume, and a “preferred” in-plane orientation for the aromatic rings over the macroscopic volume of the sample can be ruled out. Slight differences between the patterns are almost certainly within signal-to-noise limits and should be neglected. Even in the case of a partially ordered system, the CSA patterns would differ markedly and this is demonstrated below. We believe that this represents the first concrete physical proof that demonstrates lignin to be orientationally disordered with respect to a macroscopic sample of whole wood. In order to illustrate the ability of this 2-D FT NMR technique to detect orientation and order, a set of experiments was carried out on homovanillic alcohol (111). Note that the CH20H
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aromatic moiety in this model compound closely resembles that of a guaiacylpropane unit (11). More significantly, it contains a methoxy-substituted aromatic carbon in a local environment that is essentially identical with those found in lignin. The 13CCP/MAS spectrum of this compound is given in Figure 1D. The resonance at 149 ppm corresponds to this methoxy-substituted aromatic carbon, and CSA patterns obtained by the 2-D FT experiment were taken from this isotropic chemical shift. The CSA pattern corresponding to
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Figure 3. 13C CSA patterns obtained for the 149 pprn isotropic chemical shift for homovanillic alcohol: (A) powdered sample; (B) single
crystal at one orientation relative to the spinning axis; (C) the same single crystal at another orientation relative to the spinning axis; (D) collection of crystalline intergrowths (see text). this peak for powdered homovanillic alcohol is given in Figure 3A. The similarity of this pattern to the CSA patterns obtained for the wood samples (Figure 2) reinforces our judgement regarding the suitability of this compound as a model for a methoxy-substituted aromatic carbon of the type found in lignin. Also, the similarity of the CSA pattern of powdered homovanillic alcohol with those obtained for wood again demonstrates that the molecular orientation of lignin is macroscopically random in these wood samples. Attempts to grow a single crystal of homovanillic alcohol suitably large enough for the 2-D FT experiment, although ultimately successful, proved to be difficult at first. Figure 3D shows the CSA pattern obtained for a large sample comprised of a collection of crystalline intergrowths. Note that even for this partly oriented system, the CSA pattern is substantially different from that of the random powder case. Figure 3B,C shows that CSA pattern obtained for a single crystal of homovanillic alcohol a t two different orientations in the spinner. As expected for a highly ordered system, the CSA patterns contain only sharp lines and are different for the two orientations. X-ray diffraction analysis revealed the presence of four molecules per unit cell, each with a significantly different orientation. These give rise to the four 13C NMR lines shown in Figure 3B. Figure 3C also contains four lines, two of which overlap, and Figure 3D appears to have four broad contributing components, which reflect the limited order present. In addition, Figure 3D demonstrates by analogy that, if the aromatic rings of lignin were preferentially oriented in the plane of the cell wall (or in any other plane), dramatic differences in the CSA patterns would be expected among the wood plots of Figure 2. As this is not the case, we can confidently conclude that lignin is not preferentially oriented in any way over the dimensions observed in this experiment. It should be noted that the “dips” seen at about 160 ppm in the 2-D FT CSA powder patterns for the 152 ppm resonances in Figure 2 and the 149 ppm resonance in Figure 3A have been assigned to cross-polarization dynamics. Variable contact-time experiments (data not shown) on an isolated lignin sample showed similar dips in the 2-D FT CSA powder patterns obtained with a CP contact time of 2 ms. The nature of these distortions changes with contact time.
Having determined from these experiments that the molecular orientation of lignin is random and exhibits no preferred macroscopic orientation throughout whole wood, we sought to employ this 2-D FT NMR technique to probe order in the cellulose in wood. Cellulose has long been known to be well organized within the cell ultrastructure (8). Shown in Figure 1B is the 13C CP/MAS spectrum of cellulose powder (Whatman CC31). The 13C CP/MAS NMR spectrum of cellulose has been extensively studied (16-19) and the details of its spectrum will not be repeated here. The sharp intense peak at 73 ppm, indicated with an arrow, is largely responsible for the intensity a t 73 ppm in the spectrum of Eucalyptus polyanthemus, shown in Figure 1C. From this isotropic chemical shift, CSA patterns for cellulose in wood were generated by the 2-D FT NMR technique. These patterns are given in Figure 2. It is immediately obvious that these patterns are very similar, suggesting that cellulose, like lignin, is largely disordered in a macroscopic sense in wood. Slight differences in line width and line shape for the 73 ppm cellulose patterns in Figure 2 may reflect a slight degree of gross molecular order, but such differences are too small to be highly significant a t the present level of experimental error. The peak at 73 ppm has been previously assigned to carbons 2, 3, and 5 of cellulose (16-19). In order to ensure that the CSA patterns from these three different carbons were not overlapping in such a way as to obscure the CSA pattern of interest, we also generated patterns from resonance corresponding to the isotropic shift at 106 ppm. This resonance has been assigned to carbon 1 of cellulose. These patterns are also given in Figure 2. Like those patterns obtained a t 73 ppm, these are all very similar to each other and actually show less deviation from each other than those shown in Figure 2 for the 73 ppm resonance. This reinforces the result that cellulose is largely disordered in a macroscopic sense in wood. At first glance, this seems disturbing in light of the unquestionable gross order present for cellulose in wood (8). However, there are several possible reasons for this result, which are discussed later in this paper. To demonstrate how order a t the molecular level would affect the CSA patterns of cellulose, we carried out a similar set of experiments on sucrose. Sucrose consists of one glucose-type unit and one fructose-type unit, the former of which contains carbons essentially identical in local molecular environment with those found in cellulose. For sucrose, like cellulose, we again chose the resonances at 73 and 106 ppm from which to generate the CSA patterns. These resonances are marked with arrows in the 13C CP/MAS spectrum of sucrose shown in Figure 1A. Figure 4 gives the CSA patterns for powdered (A) and single-crystal (B, C) sucrose taken from the isotropic shifts at 73 and 106 ppm. Note that the powder patterns (A) are relatively broad and very similar to those for cellulose in wood (Figure 2). However, a single crystal of sucrose, which is highly ordered at the molecular level, results in sharp lines, and the effect of an oriented sample is seen by comparing corresponding parts B and C of Figure 4. Note the large differences in the 73 ppm line shapes between parts B and C of Figure 4. These differences may reflect the number of different carbons attributed to the peak at 73 ppm, as discussed above. While the 106 ppm line shapes in Figure 4 are very similar, note that the resonance positions differ by about 10 ppm. These two illustrations demonstrate that if cellulose in wood were, in fact, highly oriented at the molecular level over the macroscopic volume of our experiments, we would expect to see large differences between the cellulose patterns given for wood in Figure 2. Relevant to this study is an examination of the CSA behavior of a regenerated cellulose sample that was believed to be highly oriented a t the molecular level. Results of this
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Figure 5. I3C 2-0 FT CSA patterns for two specified chemical shifts in the spectrum of cellulose: (A) powdered cellulose (Whatman CC31); (8)cellulose fibers oriented collinear with respect to the spinning axis; (C) cellulose fibers oriented perpendicular to the spinning axis.
examination are summarized in Figure 5, which give the patterns obtained in the 2-D FT CSA experiment. Long strands of the regenerated cellulose sample were cut and placed into the spinner collinear with the spinning axis (Figure 5B)and perpendicular to the spinning axis (Figure 5C). For comparison, Figure 5A shows the CSA patterns corresponding to the isotropic shifts of 73 and 106 ppm, for powdered cellulose (Whatman CC31). Note in Figure 5 the general decrease in the width of the 73 ppm peak in spectrum B relative to that in spectrum A and the significant changes in line shape observed upon sample rotation. Similar changes in line width are also observed for the 106 ppm peak in Figure 5 and a roughly 10 ppm shift occurs when the sample is rotated, as seen in parts B and C of Figure 5. These samples are clearly much more oriented over the macroscopic volume of these experiments than is the cellulose found in wood. Within the cell wall ultrastructure there exist several subdivisions, each different from the next both in chemical composition and in orientation of the structural (i.e., cellulose) elements (8). Models for this cell wall organization have been developed and summarized elsewhere (7,8,19-21) and will
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not be repeated in detail here. However, it is pertinent to point out that the cell wall is typically divided into the following subdivisions: the primary (P),or outermost wall; the secondary wall, usually divided into two regions (S1 and S2); and the tertiary (T), or innermost wall. Between the individual cells there is a thin layer called the middle lamella (ML), which “glues” the cells together to form the tissue (8). From electron microscopy studies (23),considerable detail is known about the orientation of cellulose in each of these subdivisions. The ML is in principle free of cellulose. In the primary wall (P),the cellulose fibrils are arranged in thin, crossing oblique layers. The orientation here is very gross in nature, with the outermost region of P containing less order than the inner region. In S1, the orientation of the fibrils is somewhat more ordered, running nearly perpendicular to the cylindrical axis of the cell wall, while the fibrils in S2 are more ordered and run nearly parallel with the cylindrical axis of the cell wall. In T, the cellulose is less ordered than for SI, but with the same type of fibril orientation. Thus, in a gross sense, cellulose is very ordered in wood at a microscopic level (23). The 2-D FT CSA NMR technique employed in this study addresses the issue of molecular orientation, as manifested over the entire macroscopic size of the sample, in the present case 0.9 cm3. This technique does not provide information on the degree of molecular orientational order over a much smaller volume, say a few to several molecular volumes. Thus, it may be possible, or even likely, that lignin or cellulose macromolecules are highly ordered with respect t o some preferred orientation over linear dimensions of, say, 100 8, in or near a specific fibril. And, in some other 106-A3(volume) region in another part of the sample the same kind of molecular orientational order may exist, yet if the orientational characteristics for each of these 106-A3regions are not highly correlated (mutually oriented in some preferred manner) among the different regions, then evidence for macroscopic molecular orientational order, as summed over a 0.9-cm3 sample in the specific 2-D FT NMR experiments reported here, will not be manifested in the results. Hence, it may be true that a high degree of molecular orientational order exists for cellulose and lignin within each subdivision of the cell wall ultrastructure; but if that is the case, then the results reported here show that the microscopic orientational characteristics of the various subdivisions must be sufficiently randomized over a 0.9 cm3 sample to yield a zero degree of molecular orientational order in the macroscopic NMR experiment. This situation would be analogous to the case of randomly oriented crystallites in a powder, in which the highly ordered structure within each crystallite is “averaged” by the random orientation of the crystallites with respect to each other. The present 2-D FT NMR technique a t its current level of natural-abundance sensitivity is not capable of examining the possible (likely?) existence of molecular orientational order over the individual ultrastructure components in Eucalyptus polyanthemus wood. A major improvement in the signalto-noise ratio of the NMR experiment would permit one to examine the macroscopic molecular orientational order over a much smaller sample size than the 0.9 cm3 of the present study. One way of working toward this goal is to enrich the 13C concentration in the wood sample by employing I3C-enriched C02for the growing of the wood. Such experiments have been initiated.
CONCLUSIONS The results of this paper demonstrate how the 2-D FT CSA technique allows one to study molecular orientational order of the various molecular components in a complex, inhomogeneous sample. The technique was applied to Eucalyptus polyanthemus wood, where it was determined that no net
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molecular orientational order of lignin in cellulose exists over a macroscopic volume of about 0.9 cm3. Work is currently under way to enable one to quantitate the type and degree of order present in similar systems.
(10) Pines, A.; Gibby, M. G.;Waugh, J. S.J. Chem. Phys. 1973, 59, 569. (11) Schaefer. J.; Stejskal, E. 0. J. Am. Chem. SOC. 1978, 98, 1031. (12) Schaefer, J.; Stejskal, E. 0. Topics in Carbon-13 NMR Spectroscopy; Levy, G. C.. Ed.; Wiley: New York, 1979. (13) Veeman, W. S. Prog. Nucl. Magn. Reson. Spectrosc. 1984, 16, 193. (14) Browning, 6. L. The Chemistry of Wood: Robert E. Krieger Publishing Co.: New York, 1975. (15) Hatfieid, G. R.; Maciel, G. E.; Erbatur, 0.; Erbatur, G. Anal. Chem. 1987, 59, 172. (16) Vanderhart, D. L.; Atalia, R. H. Macromolecules 1984, 17, 1465. (17) Atalla. R. H.; Vanderhart, D. L. Science 1984, 223, 283. (18) Dudley, R. L. J. Am. Chem. Soc. 1983, 105, 2469. (19) Maciei, G. E.; Kolodziejski, W. L.; Bertran, M. S.;Dale, B. E. Macromolecules 1982, 15. 686. (20) Cote, W. A. Cellular Ultrastructure of Woody Plants: Syracuse University Press: Syracuse, NY, 1965. (21) Frey-Wyssling, A. The Plant Cell Wall; Borntraeger: Berlin, 1976. (22) Koilmann, F. F. P., Cote, W. A. Principles of Wood Science an Technology; Springer-Verlag: New York, 1968; Vol. 1. (23) Cote, W. A. Wood Ultrastructure; University of Washington Press: Seattle, WA, 1967.
ACKNOWLEDGMENT The authors thank Joseph Reibenspies for the crystallographic work and Cyril Heitner, Pulp and Paper Research Institute of Canada, for providing the oriented cellulose samples. Registry No. Lignin, 9005-53-2; cellulose, 9004-34-6;sucrose, 57-50-1;homovanillic alcohol, 2380-78-1. LITERATURE CITED (1) (2) (3) (4) (5)
(6)
(7) (8)
(9)
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RECEIVED for review December 8, 1986. Accepted March 3, 1987. The authors gratefully acknowledge partial support of this research by a grant from the Colorado State University Experimental Station. The Nicolet R3m/E diffractometer and computer system a t Colorado State University were purchased with funds provided by NSF Grant CHE-8103011.
Use of the Microwave-Induced Nitrogen Discharge at Atmospheric Pressure as an Ion Source for Elemental Mass Spectrometry Daniel A. Wilson,' George H. Vickers, and Gary M. Hieftje* Department of Chemistry, Indiana University, Bloomington, Indiana 47405
A microwave-induced nitrogen dlscharge at atmospheric pressure (MINDAP) is used as the Ion source for elemental mass spectrometry (MS)and compared to the use of the inductively coupled plasma ( ICP). Optimiratlon studies are presented to Illustrate the dependence of slgnais on various instrumental parameters. Detectlon llmits determhed for five elements range from 3 to 22 ng/mL, somewhat higher than those determlned with an ICP and the same mass spectrometer system. The background mass spectrum from the MINDAP ISdominated by NO'; oxide and hydroxide ion ratios are higher than for ICP-MS. The linear dynamic range is similar to that In ICP-MS, but Interferences caused by concomitant elements are much worse in MINDAP-MS.
Since the introduction of inductively coupled plasma mass spectrometry (ICP-MS) ( I ) , the technique has shown a great deal of potential for elemental analysis. Recent reviews ( 2 , 3 ) have described the advantages of ICP-MS, including a relatively simple mass-spectral background, the ability to perform isotope-ratio determinations, and sub-nanogramper-milliliter detection limits for many elements. 'Present address: Alcoa Technical Center, Bldg C, Alcoa Center,
PA 15069.
Although the ICP has been proven to be an attractive ion source for elemental mass spectrometry, it is important to investigate other types of plasmas as ion sources to assess whether further improvement in analytical characteristics is possible. Possible improvements that are desired include better sensitivity, greater freedom from interferences, ease of analytical use, lower operating and instrumental costs, and better precision. One type of discharge which has been used as an ion source is a microwave-induced plasma (MIP). In fact, the earliest work reported using continuum plasma sampling, the type most commonly used in ICP-MS at present, employed an argon MIP (4). This early work was later carried further and compared with ICP-MS (5). Detection limits in MIP-MS were about an order of magnitude lower than those from ICP-MS. However, matrix interferences were much more severe in the MIP than in the ICP, with the ICP being reported as matrix-free (5). More recent papers (6, 7) on ICPMS have revealed that matrix interferences are observed in that method also. Work with a microwave plasma as the ion source for mass spectrometry continues (8). These recent studies coupled both argon and helium MIP ion sources with a mass spectrometer adapted from a commercial gas chromatograph-mass spectrometer system. An alternative and attractive plasma which has been'used for atomic emission studies is the microwave-induced nitrogen discharge at atmospheric pressure (MINDAP) (9, IO). This
0003-2700/87/0359-1664$01.50/0 0 1987 American Chemical Society
