Biomacromolecules 2005, 6, 678-683
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Identifying the Characteristic Secondary Ions of Lignin Polymer Using ToF-SIMS Kaori Saito,† Toshiyuki Kato,‡ Yukiko Tsuji,† and Kazuhiko Fukushima*,† Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan, and Technical Center, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan Received August 16, 2004; Revised Manuscript Received December 9, 2004
The chemical structure of lignin, a complex, irregular polymer of phenylpropane units that occurs in plant cell walls, was investigated using time-of-flight secondary ion mass spectrometry (ToF-SIMS). The positive ToF-SIMS spectra of lignin isolated from pine and beech wood showed prominent secondary ions possessing guaiacyl (at m/z 137 and 151) or syringyl (at m/z 167 and 181) rings, which are the basic building units of lignin polymer. This shows that ToF-SIMS is a useful tool for lignin structural analysis. The peaks at m/z 137 and 167 were assigned as the C6-C1 ion, and the peaks at m/z 151 and 181 may be double-component, the C6-C1 ion and the C6-C2 ion. We confirmed the characteristic guaiacyl ions using a synthetic lignin model compound, dehydrogenation polymer (DHP), which was formed by polymerizing of unlabeled and deuterium-labeled coniferyl alcohols. The formation mechanism of the main secondary ions was deduced by labeling specific positions of coniferyl alcohols with a stable isotope to study the relationship between chemical structure and secondary ion formation in ToF-SIMS. Introduction Lignin is one of the main constituents of plant cell walls and is the second most abundant biopolymer in nature after cellulose. A major problem in studying lignin is the difficulty in obtaining information on its native state, because lignin is located in various morphological regions of the cell wall and is highly cross-linked and intertwined with other cell wall constituents, such as hemicellulose and cellulose.1,2 Therefore, the isolation of lignin from wood is accompanied by structural changes, and it is impossible to extract all of the lignin from the cell wall. A form of isolated lignin that is widely used for structural studies is milled wood lignin (MWL), which is obtained by extracting milled wood with dioxane containing water. MWL represents less than 50% of total lignin content and still contains some carbohydrates.3 Lignin polymer is built from phenylpropane C6-C3 units, which have three main patterns based on different methoxyl substitutions on the aromatic rings: guaiacyl (G), syringy (S), and p-hydroxyphenyl (H) units, as shown in Figure 1. The G, S, and H units are highly relevant to the origin and evolution of plants. Most of the softwood (gymnosperm) lignins are generally G-lignins, hardwood (angiosperm) lignins are GS-lignins, and grasses (monocotyledonous angiosperms) contain additional H units.4 These phenylpropane C6-C3 units are connected via various C-C and C-O linkages to form the complex and irregular structure of lignin polymer. The structural model Sakakibara5 proposed for typical softwood lignin is shown in Figure 1, although it is * To whom correspondence should be addressed. Phone: +81-52-7894159. Fax: +81-52-789-4163. E-mail:
[email protected]. † Graduate School of Bioagricultural Sciences, Nagoya University. ‡ Technical Center, Nagoya University.
impossible to state the exact molecular mass or draw the molecular shape of lignin. The structural complexity of lignin polymer further limits the information that can be obtained using chemical degradation methods. Acidolytic degradation has been developed for the structural identification, based on selective cleavage of the aryl alkyl-ether (so-called “uncondensed”) bonds that are the most abundant linkages in lignin.6,7 Thioacidolysis is a well-established depolymerization method for estimating uncondensed units in lignin and has been extended to detect other units using Raney nickel desulfuration of the thioacidolysis dimeric products.8 Thioacidolysis and subsequent desulfuration characterize about 30-50% of MWL or dehydrogenation polymer (DHP).9,10 Analytical techniques that reflect the entire lignin sample without the drawback of structural change that is observed during chemical degradation are physical methods, such as nuclear magnetic resonance (NMR),11,12 infrared spectroscopy (IR),13,14 and Raman spectroscpopy.15,16 These methods provide precise data on functional groups, interunit linkage types, and their environmental information. Spectroscopic analysis requires sufficient resolution to obtain detailed structural information, and in some cases, samples must be prepared by removing compounds that interfere with the spectra. In NMR analysis, the isolated polysaccharide-free lignin fraction provides well-resolved spectra, although information about all of the lignin is not obtained. Recently, a high-resolution solution-state NMR study of fully dissolved ball-milled plant cell walls showed the entire lignin fraction in the cell wall.17 ToF-SIMS is a powerful technique that provides chemical information about the surface of a solid sample that does
10.1021/bm049521v CCC: $30.25 © 2005 American Chemical Society Published on Web 01/22/2005
Secondary Ions of Lignin Polymer
Biomacromolecules, Vol. 6, No. 2, 2005 679
Figure 1. Phenylpropane (H, G, S) units and the model of softwood lignin proposed by Sakakibara.5
not require any treatment. This analytical method uses a pulsed primary ion beam, such as Ga, Ar, or Cs, with an energy between 1 and 25 keV. When primary ion particles bombard a surface, secondary particles, such as atoms or molecular ions, are emitted from the surface, and ionized species are detected by time-of-flight mass spectrometry in positive and negative modes.18 Most of the secondary ions originate from the top two or three layers (10-20 Å) of the surface.19 The ToF-SIMS technique is now widely used in organic and inorganic analyses.20-22 Several ToF-SIMS studies of wood pulp that examined pulp fiber surface or properties of paper have been reported.23-26 The significant advantage of ToF-SIMS for analyzing chemical complexes is that it is capable of providing mass spectral imaging by the direct measurement of an untreated solid surface, where the location of all or selected secondary ions are encoded spatially on the sample surface. ToF-SIMS image analyses have been applied widely to visualize the chemical composition and distribution on the surface27,28 and such information is unobtainable using other analytical techniques, such as NMR. This chemical mapping will also allow us to determine the histological origin of biological samples and is expected to demonstrate heterogeneity of the chemical distribution in wood tissues, However, there has been few histological imaging studies.29-31 Furthermore, concerning the relationship between chemical structures and ToF-SIMS spectra, there are no established rules for spectral interpretation, unlike traditional mass spectrometry such as EI (electron impact ionization), making data interpretation more difficult.32 For analyzing lignin macromolecules, studies with other mass spectrometric techniques, matrix assisted laser desorption ionization (MALDI) and electronspray ionizaion (ESI), were reported. Several investigations showed that MALDIMS is a powerful tool providing a molar mass distribution of lignin molecules.33,34 For example, the birch MWL MALDI mass spectrum showed a molar mass distribution up to 16 000 Da with the center of gravity at around 2600 Da.33 Recently, in addition to the molecular weight determination, ESI-MS using tandem mass spectrometry revealed
the structure of the lignin oligomers and the information on fragmentation patterns.35-37 The ESI-MS analysis with Fourier transform ion cyclotron resonance (FT-ICR) configuration reported that the lignin degradation product obtained after thioacidolysis of spruce MWL was identified as a tetrameric structure that contained 5-O-4′, 5-5′, and 8-1′ linkages.37 This study applied the ToF-SIMS technique to lignins in the plant cell wall and obtained exact chemical information on the structure of lignin. Material and Methods Materials. Milled wood lignins (MWLs) from pine (Pinus thunbergii) and beech (Fagus crenata) and the lignincarbohydrate complex (LCC) from pine (Pinus thunbergii) were prepared from the extractive-free wood meal according to the conventional method.38,39 Coniferyl alcohol, coniferyl alcohol-[9,9-2H2,OC2H3], and coniferyl alcohol-[7-13C, 9,92 H2] were synthesized, as previously described.40-42 Dehydrogenation polymers (DHPs) were prepared from coniferyl alcohol using horseradish peroxidase (Wako, Co., Japan) and H2O2 according to the procedure previously described.43 ToF-SIMS Analysis. ToF-SIMS analysis was performed using a TRIFT III (ULVAC-PHI, Japan) spectrometer. Positive spectra were obtained using a 15 keV primary beam 69Ga+ liquid metal ion source at a current of 2 nA, with a pulse width of 18.0 ns (
