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The origin of lignin fluorescence
Journal of Molecular Structure 508 (1999) 19–27
The origin of lignin fluorescence Bo Albinsson a, Shiming Li b, Knut Lundquist b,*, Rolf Stomberg c a
Department of Physical Chemistry, Chalmers University of Technology, SE-41296 Go¨teborg, Sweden Department of Organic Chemistry, Chalmers University of Technology, SE-41296 Go¨teborg, Sweden c Department of Inorganic Chemistry, University of Go¨teborg, SE-41296 Go¨teborg, Sweden
b
Received 9 November 1998; accepted 27 November 1998
Abstract Spruce lignin exhibits fluorescence emission spectra that peaks at < 360 nm on excitation at wavelengths ranging from 240 to 320 nm. This can be explained by non-radiative energy transfer from lignin chromophores, that are excited in the wavelength range 240–320 nm, to an acceptor that emits fluorescent light at < 360 nm. Examinations of lignin samples and model compounds suggest that small amounts of phenylcoumarone structures in the lignin is a conceivable acceptor. Such structures and stilbene structures are formed from structural elements in lignin of the phenylcoumaran type on various treatments. The photophysical properties of models for phenylcoumarone structures [2-(3,4-dimethoxyphenyl)-7-methoxy-3-methylbenzo[b]furan, 2-(3,4-dimethoxyphenyl)-3-hydroxymethyl-7-methoxybenzo[b]furan] and stilbene structures (the E and Z forms of 2-hydroxy-3,3 0 ,4 0 -trimethoxystilbene) have been examined and are discussed on the basis of crystal structure determinations. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Lignin; Fluorescence; Phenylcoumarone; Stilbene; X-ray crystallography
1. Introduction The literature on the fluorescence properties of lignocellulosic materials has recently been reviewed [1]. Fluorescence spectroscopy can be used as a sensitive probe of the photochemistry of wood fibres and paper and for the analysis of lignin constituents in wastewaters from pulp mills. Emission spectra of dioxane–water (1 : 1) solutions of “milled wood lignin” from spruce (MWL) [2] exhibit a maximum at 358 nm on excitation at different wavelengths in the range 240–320 nm [3,4]. Removal of the carbonyl groups in the lignin by borohydride reduction did not change the position of the emission maximum but the intensity of the fluorescence increased * Corresponding author. E-mail address: [email protected] (K. Lundquist)
dramatically. To explain the observations it was proposed that energy is transferred in a radiationless way from different excited structural elements in the lignin to a certain type of structure, which acts as an “energy sink” and from which the fluorescent light is emitted. From experiments with lignin model compounds it could be concluded that structural elements of cinnamyl alcohol type (1) or phenylcoumarone type (2) are conceivable candidates for the “energy sink”. End groups of type 1 are known to be present in lignin while phenylcoumarone structures (2) have not been detected in untreated lignin. Acid treatment of lignin results in the conversion of structural elements in lignin of the phenylcoumaran type (3) into structures of type 2 and stilbene structures (4) (Fig. 1) [5]. Recent model compound studies suggest that milling leads to formation of phenylcoumarones of type 2 and stilbenes of type 4 from
0022-2860/99/$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S0022-286 0(98)00913-2
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B. Albinsson et al. / Journal of Molecular Structure 508 (1999) 19–27
Fig. 1. Reactions of lignin structures of the phenylcoumaran type (3) on acidolysis and oxidation.
phenylcoumarans of type 3 [6]. Biodegradation studies of lignin model compounds show that phenylcoumarans (3) can be oxidized to phenylcoumarones of type 5 [7]. As judged from the milling and biodegradation studies, it seems possible that phenylcoumarones (2, 5) are introduced in lignins by chemical transformations of phenylcoumaran structures (3) during the milling [2] involved in the preparation of
MWL’s, during the enzymic oxidation involved in the biosynthesis (or “ageing”) of lignins or during preparation/storage of MWL’s as a result of autoxidation. Acid-catalysed conversion of phenylcoumarans into phenylcoumarones (or stilbenes) (Fig. 1) is not likely to occur under the conditions prevailing in the plant or during the procedures applied to the isolation
B. Albinsson et al. / Journal of Molecular Structure 508 (1999) 19–27
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Table 1 Crystal and experimental data for 2-(3,4-dimethoxyphenyl)-7methoxy-3-methylbenzo[b]furan (9). T 293 K Crystal data Empirical formula Molecular weight (g mol 21) Crystal colour; habit Crystal dimensions (mm) Crystal system No. of reflections used for unit cell determination (2u range; 8) Lattice parameters:
Space group Z Dcalc (g cm 23) F(000) m (Cu Ka ) (mm 21); no correction Intensity measurements Diffractometer Radiation Scan type u range (8) Min. h,k,l / max. h,k,l No. of reflections measured
C18H18O4 298.32 colourless; rod fragment 0.45 × 0.35 × 0.26 Monoclinic 25 (79.3–79.9) ˚ a 14.916(1) A ˚ b 5.256(1) A ˚ c 19.032(1) A b 93.16(1)8 ˚3 V 1489.9(4) A P21/c (No. 14) 4 1.330 632 0.76 Syntex P21 ˚) Cu Ka (l 1.54178 A v –2u 2.0–62.5 0, 0, 2 21 / 17, 6, 21 Total: 2630 Unique: 2368 (Rint 0.023)
Average change in intensity of test reflections (%) 2 0.5 Corrections Lorentz-polarization Structure solution and refinement Structure solution Direct methods. Electron density difference maps Hydrogen atom treatment Refined xyzU Refinement Full-matrix least-squares P Function minimized w(uFou 2 uFcu) 2 2 2 Least-squares weights w 1/[s (Fo ) 1 (0.0514P) 2 1 0.3369P] P (Fo2 1 2Fc2)/3 Anomalous dispersion All non-hydrogen atoms Extinction coefficient (correction: 0.0153(8) SHELXL) No. of independent reflections used 2368 in the refinement No. of variables 272 Reflection / parameter ratio 8.71 R1/wR2/S (observed data) a 0.033/0.093/1.121 0.045/0.102/1.087 R1/wR2/S (all data) a Maximum shift/error in final 0.001 cycle Max./min. peak in final diff. map 0.155 / 2 0.123 ˚ 3) (e 2/A P Values of R1 , wR2 and S are defined as R1 u uFo u 2 P P 2 2 2 P 2 2 1=2 uFc uu= uFo u; wR2 { w Fo 2 Fc = w Fo } and S P { w Fo2 2 Fc2 2 = n 2 p}1=2 ; where n is the number of reflections and p is the total number of parameters refined. a
of the lignin. Fluorescence studies of MWL and model compounds presented in this paper show that the “energy sink” in MWL might be structural elements of the phenylcoumarone type (2 or 5). Fluorescence properties of lignin models representative of phenylcoumarone structures of type 2, compounds 6– 8, have been studied previously [3,4,8]. As far as comparisons are possible the results are in close agreement with those obtained in the studies of 9 reported in this paper. The photophysical properties of model compounds representative of stilbenes of type 4 are also examined in this paper. Such stilbene structures are formed from phenylcoumarans not only on acid treatments but also during milling [6] and on alkaline treatments ([9] and references cited therein). 2. Experimental 2.1. Materials Phenylcoumarones 9 [10] and 10 [11] and stilbenes 11 [5] and 12 [9] were prepared by methods described in the literature. Crystals of 9 suitable for X-ray analysis were obtained by crystallization from ethyl acetate (m.p. 1368C–1378C [10]). Spectrophotometric grade dioxane (Merck) and 2-methyltetrahydrofuran (Acros) were purified by distillation. All solutions
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Table 2 Atomic fractional coordinates and equivalent isotropic thermal PP ˚ 2) for 9. Ueq (/3) i j Uijai*aj*ai·aj parameters Ueq (A
were prepared immediately before examination and purged with argon prior to emission measurements.
Atom
x
y
z
Ueq
O1 O2 O3 O4 C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 C18
0.99351(9) 0.92232(8) 0.75026(7) 0.62393(9) 0.84341(10) 0.90742(11) 0.93198(11) 0.89305(11) 0.83029(11) 0.80560(11) 0.81666(10) 0.84182(11) 1.03616(15) 0.8882(2) 0.73466(11) 0.78871(11) 0.78353(13) 0.72491(14) 0.67016(13) 0.67377(11) 0.5633(2) 0.9082(2)
2 0.2037(3) 0.1351(3) 0.2675(2) 0.5502(3) 0.1062(3) 2 0.0622(3) 2 0.0492(3) 0.1351(3) 0.2992(4) 0.2852(3) 0.0932(3) 2 0.0488(3) 2 0.3909(4) 0.3281(5) 0.2348(3) 0.0440(3) 2 0.0139(4) 0.1265(4) 0.3166(4) 0.3736(3) 0.7021(5) 2 0.2600(4)
0.75790(7) 0.67434(6) 0.96826(5) 1.03831(6) 0.88057(8) 0.85512(9) 0.78654(8) 0.74078(8) 0.76539(9) 0.83462(9) 0.95325(8) 1.01110(8) 0.80187(12) 0.62744(11) 1.03802(8) 1.06701(8) 1.13868(9) 1.17633(9) 1.14612(9) 1.07518(8) 1.07529(12) 1.01963(12)
0.0621(4) 0.0589(4) 0.0458(3) 0.0626(4) 0.0399(4) 0.0450(4) 0.0440(4) 0.0436(4) 0.0465(4) 0.0451(4) 0.0412(4) 0.0428(4) 0.0600(5) 0.0671(6) 0.0434(4) 0.0444(4) 0.0546(5) 0.0578(5) 0.0552(5) 0.0472(4) 0.0619(5) 0.0592(5)
2.2. Determination of the crystal structure of 2-(3,4dimethoxyphenyl)-7-methoxy-3-methylbenzo[b]furan (9) Crystal data and conditions for the data collection, structure solution and refinement are given in Table 1. Programs used: SHELXS86 [12] (structure solution) and SHELXL93 [13] (structure refinement). Fractional atomic coordinates and equivalent isotropic thermal parameters for the nonhydrogen atoms are given in Table 2 and selected bond distances, bond angles and torsion angles in Table 3. Fig. 2 shows the molecule and the atomic labelling. 2.3. Absorption and fluorescence measurements The spectra were recorded at room temperature unless otherwise stated. Absorption spectra were measured with a CARY 4B UV/Vis spectrometer and corrected steady-state emission spectra were recorded on a SPEX Fluorolog t 2 spectrofluorimeter. Quantum yields of fluorescence (Ff) were determined relative to the quantum yield of an argon purged solution of 2,5-diphenyloxazole (PPO) in cyclohexane
Table 3 ˚ ), bond angles (8) and torsion angles (8) in 9 Selected bond lengths (A Distance O1–C3 O1–C9 O2–C4 O2–C10 Angle C11–O3–C7 C2–C1–C7 O1–C3–C2 O2–C4–C5 C8–C7–O3 C8–C7–C1 O3–C7–C1 C7–C8–C12 Torsion angles C9–O1–C3–C2 C10–O2–C4–C5 C2–C1–C7–C8 C6–C1–C7–O3 O3–C7–C8–C12
1.362(2) 1.419(2) 1.360(2) 1.426(3)
O3–C11 O3–C7 O4–C16 O4–C17
1.371(2) 1.390(2) 1.360(2) 1.422(2)
106.1(1) 121.5(2) 124.8(2) 125.2(2) 111.2(1) 135.3(2) 113.5(1) 105.8(2)
C7–C8–C18 C12–C8–C18 O3–C11–C12 O3–C11–C16 C12–C11–C16 C11–C12–C8 C13–C12–C8 O4–C16–C15
130.3(2) 124.0(2) 110.5(1) 125.3(2) 124.2(2) 106.4(1) 134.7(2) 126.9(2)
2 0.5(3) 3.0(3) 3.1(3) 2.0(2) 2 0.9(2)
O3–C7–C8–C18 C1–C7–C8–C18 C7–O3–C11–C12 O3–C11–C12–C8 C7–C8–C12–C11
178.4(2) 2 2.3(3) 2 0.5(2) 0.0(2) 0.5(2)
C1–C7 C7–C8 C8–C12 C8–C18
1.462(2) 1.365(2) 1.446(2) 1.490(3)
B. Albinsson et al. / Journal of Molecular Structure 508 (1999) 19–27
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order to avoid inner filter distortion. Low temperature measurements were performed in a temperature controlled liquid N2 cryostat (Oxford Instruments DN 1704 with temperature controller DTC2). The fluorescence lifetimes were measured by time correlated single photon counting using a nitrogen filled flash lamp (Oxford instruments) as the excitation source [16]. A diluted silica sol scattering solution was used as a reference. The sample and reference solutions were excited with linearly polarized light and the emission was detected through a polarizer set at the magic angle (558). The fluorescence decays were analyzed by convolution of the excitation pulse and multi-exponential fitting of the fluorescence decay using the Globals software [17]. This set up has an estimated time resolution of approximately 100 ps and it was controlled by measuring the lifetime of PPO (t found 1.45 ns, t litt 1.40 ns [18]). Fig. 2. A perspective drawing (Siemens. XP [14]) of phenylcoumarone 9 showing the atomic numbering. Small circles represent hydrogen atoms.
3. Results and discussion
(Ff 0.85) [15]. The measurements were performed on optically matched samples (the same absorbance at the wavelength of excitation) and the quantum yields are corrected for differences in refractive index. The fluorescence excitation spectra were measured on samples with maximum absorbance less than 0.1 in
Absorption and fluorescence spectra of phenylcoumarones 9 and 10 in dioxane solution are shown in Fig. 3. Spectroscopic data are collected in Table 4. The absorption spectra are unstructured and exhibit maxima at 306 nm. Both compounds show intense, structured fluorescence spectra with maxima at
3.1. Model compound studies
Fig. 3. Absorption and emission spectra of phenylcoumarones 9 (- - -) and 10 ( _____) in dioxane solution (cf. Table 4).
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Table 4 Photophysical data for lignin models 9–12 in dioxane solution (excepting l em for 12) Compound
l max(abs) (nm)
l max (M 21cm 21)
l max(em) (nm)
Ff
t f a (ns)
9 10 11 12
306 306 330 302
26 25 26 13
351 356 383 420 b
0.61 0.57 — —
1.25 1.27 — —
a b
400 000 500 200
Here l ex 313 nm, l em 360 nm. In 2-methyltetrahydrofuran glass at 80 K.
351 nm (9) and 356 nm (10). The vibrational structure gradually disappears when water is added to the dioxane solution. The fluorescence quantum yields are 0.61 and 0.57 for 9 and 10, respectively, in dioxane. The quantum yields are essentially unaffected by addition of water. The fluorescence lifetimes (t f) for excitation at 313 nm and detection at 360 nm are 1.25 ns and 1.27 ns for 9 and 10, respectively. The natural lifetime, t 0 t f/F f, is a measure of the transition probability for the emitting electronic transition and it is related to the transition probability of the corresponding absorbing electronic transition. A quantitative comparison between emission and absorption probabilities is provided by the Strickler–Berg relation [19]: Z 1 21 kf 2:88 × 1029 n2 kn~ 23 f l gl =gu 1 n~ d ln n~ t0
Here kf is the radiative rate constant, n is the refractive index of the solvent, knÄ f23l 2 1 is the reciprocal of the mean value of nÄ f23 in the fluorescence spectrum, (gl/ gu) 1 is the ratio of lower and upper state degeneracies, and 1 n~ is the molar absorptivity at wavenumber nÄ. From fluorescence quantum yield and lifetime measurements, the t 0 values were determined to be 2.0 ns (9) and 2.2 ns (10). These data compare fairly well with the values (<1.2 ns) determined by integrating the lowest energy absorption bands (260– 350 nm) and using the Strickler–Berg relation. It follows that the first electronic transition, S0 ! S1, dominates the lowest energy absorption band and that the same electronic states are involved in the absorption and emission of light. Nevertheless the appearance of the spectra (Fig. 3) suggests slight differences in geometry between the ground and excited states (the vibrational structure is different).
Fig. 4. Absorption and emission spectra of (E)-form (11) (- - -) and (Z)-form (12) ( _____) of 2-hydroxy-3,3 0 ,4 0 -trimethoxystilbene. All spectra were recorded in dioxane except for the emission spectrum of 12 which was recorded in 2-methyltetrahydrofuran (glass) at 80 K.
B. Albinsson et al. / Journal of Molecular Structure 508 (1999) 19–27
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Fig. 5. Emission spectra of untreated spruce lignin (-··-), NaBH4-reduced spruce lignin (- - -), phenylcoumarones 9 ( ____) and 10 (…) and stilbene 11 (-·-). All spectra were recorded in dioxane-water 9 : 1 and normalized to facilitate comparison.
The high absorbtivity and high fluorescence quantum yield of 9 and 10 suggest that the they adopt conformations with a planar p -system. Examination of 9 by X-ray crystallography showed that the benzofuran ring system is planar (the maximum deviation from the plane defined by the atoms in the ˚ ). The benzofuran benzofuran ring system is 0.021 A plane forms an angle of 3.02(9)8 with the ring plane C1–C6 (Fig. 2). Phenylcoumarone 10 adopts a similar conformation (the maximum deviation from the plane ˚ ; the defined by the benzofuran ring system is 0.017 A benzofuran plane forms an angle of 2.19(17)8 with the ring plane of the attached aromatic group); the crystal structure of 10 is reported in Ref. [11]. Absorption and fluorescence spectra of the (E)-stilbene 11 and the isomeric (Z)-stilbene 12 are shown in Fig. 4 (for spectral data, see Table 4). The spectral properties of the two stereoisomers differ dramatically. The conformations of 11 and 12 are of interest in connection with a discussion of the reasons for the spectral differences. The (Z)-isomer (12) adopts a non-planar conformation owing to steric hindrance (interactions between the aromatic rings). The angle between the aromatic ring planes in the crystalline form of 12 is 598 [9]. It is generally true that (Z)stilbenes adopts non-planar conformations [9,20]. The conformation of the crystalline form of the (E)isomer 11 is nearly planar (the angle between the aromatic rings planes is 148) [9]. It is probable that
the conformations in solution resemble those in the crystalline state. The conformations suggest that the p -conjugation is more pronounced in the nearly planar (E)-form than in the non-planar (Z)-form. This in turn provides an explanation for the fact that a change of the stereochemistry from (Z)-configuration to (E)-configuration is accompanied by a bathochromic shift of the absorption maximum (from 302 to 330 nm) and an increase of the absorptivity (Table 4). The (E)-stilbene 11 is a fairly strongly fluorescent compound. The emission spectrum (Fig. 4) has a maximum at 383 nm. The (Z)-stilbene 12 did not exhibit any intrinsic fluorescence (room temperature); the fluorescence observed emanated from the (E)isomer. This is caused by an efficient Z–E isomerization in the excited state (regarding the photochemical Z–E isomerization of stilbenes, see ref. [21]). However, (Z)-isomers of structural elements of type 4 in the lignin polymer may behave differently. It was therefore desirable to determine the fluorescence properties of 12. This was accomplished by recording the fluorescence spectrum of 12 in a 2-methyltetrahydrofuran glass at 80 K (Fig. 4). The spectrum was recorded using a non-illuminated sample and as small as possible excitation slits (0.05 mm) in order to minimize the interference of photochemically produced (E)-isomer. As it appears from the spectrum (Fig. 4), small amounts of the (E)-isomer have formed during the recording. The large Stokes shift
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B. Albinsson et al. / Journal of Molecular Structure 508 (1999) 19–27
Table 5 Spectroscopic data for lignin samples and lignin models dissolved in dioxane-water 9 : 1 Compound
l max(abs) (nm)
l max(em) a (nm)
9 10 11 12 MWL NaBH4-reduced MWL
306 310 330 305 280 280
351 357 385 — 364 360
a
Excitation at the respective absorption maxima.
(9000 cm 21) for the (Z)-isomer is presumably caused by a significant relaxation in the excited state prior to emission, i.e. the excited and ground state geometries are quite different. 3.2. Lignin studies Untreated and borohydride-reduced ‘milled wood lignin’ from spruce (MWL), dissolved in dioxane– water 9 : 1, exhibit similar emission band shapes (Fig. 5). The total intensity (quantum yield) increases dramatically on reduction [3]. In Fig. 5 the emission spectra of the lignin samples are compared with those of the phenylcoumarones 9 and 10 and the (E)-stilbene 11 (spectral data are given in Table 5). The phenylcoumarone spectra closely match the emission
from the lignin samples (the appearance of the stilbene spectrum clearly deviates from the lignin spectra). This points to the possibility that the lignin fluorescence is primarily emitted from structural elements in the lignin of the phenylcoumarone type (2,5). The origin of such structural elements is discussed in the introductory section of this paper. Phenylcoumarone structures of type 2 and 5 constitute, if present at all, only a very small fraction (,1%) of the structural elements in MWL. This is a serious objection to the hypothesis that lignin fluorescence is primarily emitted from structural elements of type 2,5. However, lignin chromophores with higher excitation energy than the phenylcoumarones (for examples, see Ref. [3]) can act as energy donors in non-radiative energy transfer to phenylcoumarone acceptors. The energy transfer efficiency depends on factors such as donor–acceptor distance and spectral overlap. The low concentration of phenylcoumarones (,1%) and, consequently, large donor–acceptor distance talks against their importance as acceptors/emitters. However, as judged from comparisons of spectra of borohydride-reduced MWL (Fig. 6), the lignin fluorescence may nevertheless be caused by energy transfer to phenylcoumarones and subsequent emission. Fig. 6 shows the excitation and absorption spectra of borohydride-reduced spruce lignin together with a presentation of the ratio between the fluorescence intensity and absorbance at different
Fig. 6. Absorption (- - -) and fluorescence excitation ( ____) spectra (l em 360 nm) of NaBH4-reduced spruce lignin. The relative fluorescence quantum yield (…) as a function of excitation wavelength is also shown.
B. Albinsson et al. / Journal of Molecular Structure 508 (1999) 19–27
wavelengths. This ratio is proportional to the quantum yield of fluorescence. It is clearly seen in Fig. 6 that the quantum yield is strongly dependent on the excitation wavelength and that it peaks around the wavelength where the phenylcoumarones have their absorbance maximum ( < 310 nm). The lignin spectra and the spectra of the phenylcoumarone models do not coincide completely (Fig. 5). This may be the result of the contribution of lignin chromophores other than phenylcoumarones to the lignin fluorescence or the fact that the phenylcoumarone models (9 and 10) differ structurally from the phenylcoumarone structures (2,5) in the lignin. It is in this context noteworthy that the emission from non-reduced MWL is comparatively strong in the normalized spectrum in the wavelengths range where lignin models carrying arylconjugated carbonyl groups emit fluorescence (370–465 nm [3]) (Fig. 5). References [1] J.A. Olmstead, D.G. Gray, J. Pulp Pap. Sci. 23 (1997) J571. [2] A. Bjo¨rkman, Sven. Papperstidn. 59 (1956) 477. [3] K. Lundquist, B. Josefsson, G. Nyquist, Holzforschung 32 (1978) 27. [4] K. Lundquist, I. Egyed, B. Josefsson, G. Nyquist, in: J.S. Gratzl, J. Nakano, R.P. Singh (Eds.), Proceedings of symposium on Chemistry of Delignification with Oxygen, Ozone and Peroxides. Raleigh, North Carolina, May 27-29, 1975, UNI Publishers, Tokyo, Japan, 1980; K. Lundquist, I. Egyed, B. Josefsson, G. Nyquist, Cellul. Chem. Technol. 15 (1981) 669.
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[5] S. Li, K. Lundquist, Holzforschung 53 (1999) (in press). [6] D.-Y. Lee, M. Matsuoka, M. Sumimoto, Holzforschung 44 (1990) 415. [7] T. Katayama, F. Nakatsubo, T. Higuchi, Mokuzai Gakkaishi 32 (1986) 535. [8] C. Noutary, P. Fornier de Violet, J. Vercauteren, A. Castellan, Res. Chem. Intermed. 21 (1995) 247. [9] R. Stomberg, S. Li, K. Lundquist, B. Albinsson, Acta Cryst. C54 (1998) 1929. [10] S. Li, T. Iliefski, K. Lundquist, A.F.A. Wallis, Phytochemistry 46 (1997) 929. [11] R. Stomberg, S. Li, K. Lundquist, A.F.A. Wallis, Z. Kristallogr. -NCS 212 (1997) 473. [12] G.M. Sheldrick. SHELXS86. Program for the Solution of Crystal Structures, University of Go¨ttingen, Germany, 1986. [13] G.M. Sheldrick. SHELXL93. Program for the Refinement of Crystal Structures, University of Go¨ttingen, Germany, 1993. [14] X.P. Siemens. Molecular Graphics Program. Version 5.03. Siemens Analytical Instruments Inc., Madison, Wisconsin, USA, 1994. [15] T. Takahashi, K. Kikuchi, H. Kokubun, J. Photochem. 14 (1980) 67. [16] J.E. Lo¨froth. Ph.D. Thesis, University of Go¨teborg, 1982. [17] J.M. Beechem, E. Gratton, W.W. Mantulin. Globals Unlimited, Revision 3, The Laboratory of Fluorescence Dynamics, University of Illinois at Urbana-Champaign, USA, 1992. [18] I.B. Berlman. Handbook of Fluorescence Spectra of Aromatic Molecules, New York, Academic Press, 1965. [19] S.J. Strickler, R.A. Berg, J. Chem. Phys. 37 (1962) 814. [20] H. Suzuki. Electronic Absorption Spectra and Geometry of Organic Molecules, Chapter 14, New York, Academic Press, 1967. [21] J. Saltiel, E.D. Megarity, J. Am. Chem. Soc. 94 (1972) 2742; J. Saltiel, A. Marinari, D.W.L. Chang, J.C. Mitchemer, E.D. Megarity, J. Am. Chem. Soc. 101 (1979) 2982.
The origin of lignin fluorescence Bo Albinsson a, Shiming Li b, Knut Lundquist b,*, Rolf Stomberg c a
Department of Physical Chemistry, Chalmers University of Technology, SE-41296 Go¨teborg, Sweden Department of Organic Chemistry, Chalmers University of Technology, SE-41296 Go¨teborg, Sweden c Department of Inorganic Chemistry, University of Go¨teborg, SE-41296 Go¨teborg, Sweden
b
Received 9 November 1998; accepted 27 November 1998
Abstract Spruce lignin exhibits fluorescence emission spectra that peaks at < 360 nm on excitation at wavelengths ranging from 240 to 320 nm. This can be explained by non-radiative energy transfer from lignin chromophores, that are excited in the wavelength range 240–320 nm, to an acceptor that emits fluorescent light at < 360 nm. Examinations of lignin samples and model compounds suggest that small amounts of phenylcoumarone structures in the lignin is a conceivable acceptor. Such structures and stilbene structures are formed from structural elements in lignin of the phenylcoumaran type on various treatments. The photophysical properties of models for phenylcoumarone structures [2-(3,4-dimethoxyphenyl)-7-methoxy-3-methylbenzo[b]furan, 2-(3,4-dimethoxyphenyl)-3-hydroxymethyl-7-methoxybenzo[b]furan] and stilbene structures (the E and Z forms of 2-hydroxy-3,3 0 ,4 0 -trimethoxystilbene) have been examined and are discussed on the basis of crystal structure determinations. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Lignin; Fluorescence; Phenylcoumarone; Stilbene; X-ray crystallography
1. Introduction The literature on the fluorescence properties of lignocellulosic materials has recently been reviewed [1]. Fluorescence spectroscopy can be used as a sensitive probe of the photochemistry of wood fibres and paper and for the analysis of lignin constituents in wastewaters from pulp mills. Emission spectra of dioxane–water (1 : 1) solutions of “milled wood lignin” from spruce (MWL) [2] exhibit a maximum at 358 nm on excitation at different wavelengths in the range 240–320 nm [3,4]. Removal of the carbonyl groups in the lignin by borohydride reduction did not change the position of the emission maximum but the intensity of the fluorescence increased * Corresponding author. E-mail address: [email protected] (K. Lundquist)
dramatically. To explain the observations it was proposed that energy is transferred in a radiationless way from different excited structural elements in the lignin to a certain type of structure, which acts as an “energy sink” and from which the fluorescent light is emitted. From experiments with lignin model compounds it could be concluded that structural elements of cinnamyl alcohol type (1) or phenylcoumarone type (2) are conceivable candidates for the “energy sink”. End groups of type 1 are known to be present in lignin while phenylcoumarone structures (2) have not been detected in untreated lignin. Acid treatment of lignin results in the conversion of structural elements in lignin of the phenylcoumaran type (3) into structures of type 2 and stilbene structures (4) (Fig. 1) [5]. Recent model compound studies suggest that milling leads to formation of phenylcoumarones of type 2 and stilbenes of type 4 from
0022-2860/99/$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S0022-286 0(98)00913-2
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B. Albinsson et al. / Journal of Molecular Structure 508 (1999) 19–27
Fig. 1. Reactions of lignin structures of the phenylcoumaran type (3) on acidolysis and oxidation.
phenylcoumarans of type 3 [6]. Biodegradation studies of lignin model compounds show that phenylcoumarans (3) can be oxidized to phenylcoumarones of type 5 [7]. As judged from the milling and biodegradation studies, it seems possible that phenylcoumarones (2, 5) are introduced in lignins by chemical transformations of phenylcoumaran structures (3) during the milling [2] involved in the preparation of
MWL’s, during the enzymic oxidation involved in the biosynthesis (or “ageing”) of lignins or during preparation/storage of MWL’s as a result of autoxidation. Acid-catalysed conversion of phenylcoumarans into phenylcoumarones (or stilbenes) (Fig. 1) is not likely to occur under the conditions prevailing in the plant or during the procedures applied to the isolation
B. Albinsson et al. / Journal of Molecular Structure 508 (1999) 19–27
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Table 1 Crystal and experimental data for 2-(3,4-dimethoxyphenyl)-7methoxy-3-methylbenzo[b]furan (9). T 293 K Crystal data Empirical formula Molecular weight (g mol 21) Crystal colour; habit Crystal dimensions (mm) Crystal system No. of reflections used for unit cell determination (2u range; 8) Lattice parameters:
Space group Z Dcalc (g cm 23) F(000) m (Cu Ka ) (mm 21); no correction Intensity measurements Diffractometer Radiation Scan type u range (8) Min. h,k,l / max. h,k,l No. of reflections measured
C18H18O4 298.32 colourless; rod fragment 0.45 × 0.35 × 0.26 Monoclinic 25 (79.3–79.9) ˚ a 14.916(1) A ˚ b 5.256(1) A ˚ c 19.032(1) A b 93.16(1)8 ˚3 V 1489.9(4) A P21/c (No. 14) 4 1.330 632 0.76 Syntex P21 ˚) Cu Ka (l 1.54178 A v –2u 2.0–62.5 0, 0, 2 21 / 17, 6, 21 Total: 2630 Unique: 2368 (Rint 0.023)
Average change in intensity of test reflections (%) 2 0.5 Corrections Lorentz-polarization Structure solution and refinement Structure solution Direct methods. Electron density difference maps Hydrogen atom treatment Refined xyzU Refinement Full-matrix least-squares P Function minimized w(uFou 2 uFcu) 2 2 2 Least-squares weights w 1/[s (Fo ) 1 (0.0514P) 2 1 0.3369P] P (Fo2 1 2Fc2)/3 Anomalous dispersion All non-hydrogen atoms Extinction coefficient (correction: 0.0153(8) SHELXL) No. of independent reflections used 2368 in the refinement No. of variables 272 Reflection / parameter ratio 8.71 R1/wR2/S (observed data) a 0.033/0.093/1.121 0.045/0.102/1.087 R1/wR2/S (all data) a Maximum shift/error in final 0.001 cycle Max./min. peak in final diff. map 0.155 / 2 0.123 ˚ 3) (e 2/A P Values of R1 , wR2 and S are defined as R1 u uFo u 2 P P 2 2 2 P 2 2 1=2 uFc uu= uFo u; wR2 { w Fo 2 Fc = w Fo } and S P { w Fo2 2 Fc2 2 = n 2 p}1=2 ; where n is the number of reflections and p is the total number of parameters refined. a
of the lignin. Fluorescence studies of MWL and model compounds presented in this paper show that the “energy sink” in MWL might be structural elements of the phenylcoumarone type (2 or 5). Fluorescence properties of lignin models representative of phenylcoumarone structures of type 2, compounds 6– 8, have been studied previously [3,4,8]. As far as comparisons are possible the results are in close agreement with those obtained in the studies of 9 reported in this paper. The photophysical properties of model compounds representative of stilbenes of type 4 are also examined in this paper. Such stilbene structures are formed from phenylcoumarans not only on acid treatments but also during milling [6] and on alkaline treatments ([9] and references cited therein). 2. Experimental 2.1. Materials Phenylcoumarones 9 [10] and 10 [11] and stilbenes 11 [5] and 12 [9] were prepared by methods described in the literature. Crystals of 9 suitable for X-ray analysis were obtained by crystallization from ethyl acetate (m.p. 1368C–1378C [10]). Spectrophotometric grade dioxane (Merck) and 2-methyltetrahydrofuran (Acros) were purified by distillation. All solutions
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B. Albinsson et al. / Journal of Molecular Structure 508 (1999) 19–27
Table 2 Atomic fractional coordinates and equivalent isotropic thermal PP ˚ 2) for 9. Ueq (/3) i j Uijai*aj*ai·aj parameters Ueq (A
were prepared immediately before examination and purged with argon prior to emission measurements.
Atom
x
y
z
Ueq
O1 O2 O3 O4 C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 C18
0.99351(9) 0.92232(8) 0.75026(7) 0.62393(9) 0.84341(10) 0.90742(11) 0.93198(11) 0.89305(11) 0.83029(11) 0.80560(11) 0.81666(10) 0.84182(11) 1.03616(15) 0.8882(2) 0.73466(11) 0.78871(11) 0.78353(13) 0.72491(14) 0.67016(13) 0.67377(11) 0.5633(2) 0.9082(2)
2 0.2037(3) 0.1351(3) 0.2675(2) 0.5502(3) 0.1062(3) 2 0.0622(3) 2 0.0492(3) 0.1351(3) 0.2992(4) 0.2852(3) 0.0932(3) 2 0.0488(3) 2 0.3909(4) 0.3281(5) 0.2348(3) 0.0440(3) 2 0.0139(4) 0.1265(4) 0.3166(4) 0.3736(3) 0.7021(5) 2 0.2600(4)
0.75790(7) 0.67434(6) 0.96826(5) 1.03831(6) 0.88057(8) 0.85512(9) 0.78654(8) 0.74078(8) 0.76539(9) 0.83462(9) 0.95325(8) 1.01110(8) 0.80187(12) 0.62744(11) 1.03802(8) 1.06701(8) 1.13868(9) 1.17633(9) 1.14612(9) 1.07518(8) 1.07529(12) 1.01963(12)
0.0621(4) 0.0589(4) 0.0458(3) 0.0626(4) 0.0399(4) 0.0450(4) 0.0440(4) 0.0436(4) 0.0465(4) 0.0451(4) 0.0412(4) 0.0428(4) 0.0600(5) 0.0671(6) 0.0434(4) 0.0444(4) 0.0546(5) 0.0578(5) 0.0552(5) 0.0472(4) 0.0619(5) 0.0592(5)
2.2. Determination of the crystal structure of 2-(3,4dimethoxyphenyl)-7-methoxy-3-methylbenzo[b]furan (9) Crystal data and conditions for the data collection, structure solution and refinement are given in Table 1. Programs used: SHELXS86 [12] (structure solution) and SHELXL93 [13] (structure refinement). Fractional atomic coordinates and equivalent isotropic thermal parameters for the nonhydrogen atoms are given in Table 2 and selected bond distances, bond angles and torsion angles in Table 3. Fig. 2 shows the molecule and the atomic labelling. 2.3. Absorption and fluorescence measurements The spectra were recorded at room temperature unless otherwise stated. Absorption spectra were measured with a CARY 4B UV/Vis spectrometer and corrected steady-state emission spectra were recorded on a SPEX Fluorolog t 2 spectrofluorimeter. Quantum yields of fluorescence (Ff) were determined relative to the quantum yield of an argon purged solution of 2,5-diphenyloxazole (PPO) in cyclohexane
Table 3 ˚ ), bond angles (8) and torsion angles (8) in 9 Selected bond lengths (A Distance O1–C3 O1–C9 O2–C4 O2–C10 Angle C11–O3–C7 C2–C1–C7 O1–C3–C2 O2–C4–C5 C8–C7–O3 C8–C7–C1 O3–C7–C1 C7–C8–C12 Torsion angles C9–O1–C3–C2 C10–O2–C4–C5 C2–C1–C7–C8 C6–C1–C7–O3 O3–C7–C8–C12
1.362(2) 1.419(2) 1.360(2) 1.426(3)
O3–C11 O3–C7 O4–C16 O4–C17
1.371(2) 1.390(2) 1.360(2) 1.422(2)
106.1(1) 121.5(2) 124.8(2) 125.2(2) 111.2(1) 135.3(2) 113.5(1) 105.8(2)
C7–C8–C18 C12–C8–C18 O3–C11–C12 O3–C11–C16 C12–C11–C16 C11–C12–C8 C13–C12–C8 O4–C16–C15
130.3(2) 124.0(2) 110.5(1) 125.3(2) 124.2(2) 106.4(1) 134.7(2) 126.9(2)
2 0.5(3) 3.0(3) 3.1(3) 2.0(2) 2 0.9(2)
O3–C7–C8–C18 C1–C7–C8–C18 C7–O3–C11–C12 O3–C11–C12–C8 C7–C8–C12–C11
178.4(2) 2 2.3(3) 2 0.5(2) 0.0(2) 0.5(2)
C1–C7 C7–C8 C8–C12 C8–C18
1.462(2) 1.365(2) 1.446(2) 1.490(3)
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order to avoid inner filter distortion. Low temperature measurements were performed in a temperature controlled liquid N2 cryostat (Oxford Instruments DN 1704 with temperature controller DTC2). The fluorescence lifetimes were measured by time correlated single photon counting using a nitrogen filled flash lamp (Oxford instruments) as the excitation source [16]. A diluted silica sol scattering solution was used as a reference. The sample and reference solutions were excited with linearly polarized light and the emission was detected through a polarizer set at the magic angle (558). The fluorescence decays were analyzed by convolution of the excitation pulse and multi-exponential fitting of the fluorescence decay using the Globals software [17]. This set up has an estimated time resolution of approximately 100 ps and it was controlled by measuring the lifetime of PPO (t found 1.45 ns, t litt 1.40 ns [18]). Fig. 2. A perspective drawing (Siemens. XP [14]) of phenylcoumarone 9 showing the atomic numbering. Small circles represent hydrogen atoms.
3. Results and discussion
(Ff 0.85) [15]. The measurements were performed on optically matched samples (the same absorbance at the wavelength of excitation) and the quantum yields are corrected for differences in refractive index. The fluorescence excitation spectra were measured on samples with maximum absorbance less than 0.1 in
Absorption and fluorescence spectra of phenylcoumarones 9 and 10 in dioxane solution are shown in Fig. 3. Spectroscopic data are collected in Table 4. The absorption spectra are unstructured and exhibit maxima at 306 nm. Both compounds show intense, structured fluorescence spectra with maxima at
3.1. Model compound studies
Fig. 3. Absorption and emission spectra of phenylcoumarones 9 (- - -) and 10 ( _____) in dioxane solution (cf. Table 4).
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B. Albinsson et al. / Journal of Molecular Structure 508 (1999) 19–27
Table 4 Photophysical data for lignin models 9–12 in dioxane solution (excepting l em for 12) Compound
l max(abs) (nm)
l max (M 21cm 21)
l max(em) (nm)
Ff
t f a (ns)
9 10 11 12
306 306 330 302
26 25 26 13
351 356 383 420 b
0.61 0.57 — —
1.25 1.27 — —
a b
400 000 500 200
Here l ex 313 nm, l em 360 nm. In 2-methyltetrahydrofuran glass at 80 K.
351 nm (9) and 356 nm (10). The vibrational structure gradually disappears when water is added to the dioxane solution. The fluorescence quantum yields are 0.61 and 0.57 for 9 and 10, respectively, in dioxane. The quantum yields are essentially unaffected by addition of water. The fluorescence lifetimes (t f) for excitation at 313 nm and detection at 360 nm are 1.25 ns and 1.27 ns for 9 and 10, respectively. The natural lifetime, t 0 t f/F f, is a measure of the transition probability for the emitting electronic transition and it is related to the transition probability of the corresponding absorbing electronic transition. A quantitative comparison between emission and absorption probabilities is provided by the Strickler–Berg relation [19]: Z 1 21 kf 2:88 × 1029 n2 kn~ 23 f l gl =gu 1 n~ d ln n~ t0
Here kf is the radiative rate constant, n is the refractive index of the solvent, knÄ f23l 2 1 is the reciprocal of the mean value of nÄ f23 in the fluorescence spectrum, (gl/ gu) 1 is the ratio of lower and upper state degeneracies, and 1 n~ is the molar absorptivity at wavenumber nÄ. From fluorescence quantum yield and lifetime measurements, the t 0 values were determined to be 2.0 ns (9) and 2.2 ns (10). These data compare fairly well with the values (<1.2 ns) determined by integrating the lowest energy absorption bands (260– 350 nm) and using the Strickler–Berg relation. It follows that the first electronic transition, S0 ! S1, dominates the lowest energy absorption band and that the same electronic states are involved in the absorption and emission of light. Nevertheless the appearance of the spectra (Fig. 3) suggests slight differences in geometry between the ground and excited states (the vibrational structure is different).
Fig. 4. Absorption and emission spectra of (E)-form (11) (- - -) and (Z)-form (12) ( _____) of 2-hydroxy-3,3 0 ,4 0 -trimethoxystilbene. All spectra were recorded in dioxane except for the emission spectrum of 12 which was recorded in 2-methyltetrahydrofuran (glass) at 80 K.
B. Albinsson et al. / Journal of Molecular Structure 508 (1999) 19–27
25
Fig. 5. Emission spectra of untreated spruce lignin (-··-), NaBH4-reduced spruce lignin (- - -), phenylcoumarones 9 ( ____) and 10 (…) and stilbene 11 (-·-). All spectra were recorded in dioxane-water 9 : 1 and normalized to facilitate comparison.
The high absorbtivity and high fluorescence quantum yield of 9 and 10 suggest that the they adopt conformations with a planar p -system. Examination of 9 by X-ray crystallography showed that the benzofuran ring system is planar (the maximum deviation from the plane defined by the atoms in the ˚ ). The benzofuran benzofuran ring system is 0.021 A plane forms an angle of 3.02(9)8 with the ring plane C1–C6 (Fig. 2). Phenylcoumarone 10 adopts a similar conformation (the maximum deviation from the plane ˚ ; the defined by the benzofuran ring system is 0.017 A benzofuran plane forms an angle of 2.19(17)8 with the ring plane of the attached aromatic group); the crystal structure of 10 is reported in Ref. [11]. Absorption and fluorescence spectra of the (E)-stilbene 11 and the isomeric (Z)-stilbene 12 are shown in Fig. 4 (for spectral data, see Table 4). The spectral properties of the two stereoisomers differ dramatically. The conformations of 11 and 12 are of interest in connection with a discussion of the reasons for the spectral differences. The (Z)-isomer (12) adopts a non-planar conformation owing to steric hindrance (interactions between the aromatic rings). The angle between the aromatic ring planes in the crystalline form of 12 is 598 [9]. It is generally true that (Z)stilbenes adopts non-planar conformations [9,20]. The conformation of the crystalline form of the (E)isomer 11 is nearly planar (the angle between the aromatic rings planes is 148) [9]. It is probable that
the conformations in solution resemble those in the crystalline state. The conformations suggest that the p -conjugation is more pronounced in the nearly planar (E)-form than in the non-planar (Z)-form. This in turn provides an explanation for the fact that a change of the stereochemistry from (Z)-configuration to (E)-configuration is accompanied by a bathochromic shift of the absorption maximum (from 302 to 330 nm) and an increase of the absorptivity (Table 4). The (E)-stilbene 11 is a fairly strongly fluorescent compound. The emission spectrum (Fig. 4) has a maximum at 383 nm. The (Z)-stilbene 12 did not exhibit any intrinsic fluorescence (room temperature); the fluorescence observed emanated from the (E)isomer. This is caused by an efficient Z–E isomerization in the excited state (regarding the photochemical Z–E isomerization of stilbenes, see ref. [21]). However, (Z)-isomers of structural elements of type 4 in the lignin polymer may behave differently. It was therefore desirable to determine the fluorescence properties of 12. This was accomplished by recording the fluorescence spectrum of 12 in a 2-methyltetrahydrofuran glass at 80 K (Fig. 4). The spectrum was recorded using a non-illuminated sample and as small as possible excitation slits (0.05 mm) in order to minimize the interference of photochemically produced (E)-isomer. As it appears from the spectrum (Fig. 4), small amounts of the (E)-isomer have formed during the recording. The large Stokes shift
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B. Albinsson et al. / Journal of Molecular Structure 508 (1999) 19–27
Table 5 Spectroscopic data for lignin samples and lignin models dissolved in dioxane-water 9 : 1 Compound
l max(abs) (nm)
l max(em) a (nm)
9 10 11 12 MWL NaBH4-reduced MWL
306 310 330 305 280 280
351 357 385 — 364 360
a
Excitation at the respective absorption maxima.
(9000 cm 21) for the (Z)-isomer is presumably caused by a significant relaxation in the excited state prior to emission, i.e. the excited and ground state geometries are quite different. 3.2. Lignin studies Untreated and borohydride-reduced ‘milled wood lignin’ from spruce (MWL), dissolved in dioxane– water 9 : 1, exhibit similar emission band shapes (Fig. 5). The total intensity (quantum yield) increases dramatically on reduction [3]. In Fig. 5 the emission spectra of the lignin samples are compared with those of the phenylcoumarones 9 and 10 and the (E)-stilbene 11 (spectral data are given in Table 5). The phenylcoumarone spectra closely match the emission
from the lignin samples (the appearance of the stilbene spectrum clearly deviates from the lignin spectra). This points to the possibility that the lignin fluorescence is primarily emitted from structural elements in the lignin of the phenylcoumarone type (2,5). The origin of such structural elements is discussed in the introductory section of this paper. Phenylcoumarone structures of type 2 and 5 constitute, if present at all, only a very small fraction (,1%) of the structural elements in MWL. This is a serious objection to the hypothesis that lignin fluorescence is primarily emitted from structural elements of type 2,5. However, lignin chromophores with higher excitation energy than the phenylcoumarones (for examples, see Ref. [3]) can act as energy donors in non-radiative energy transfer to phenylcoumarone acceptors. The energy transfer efficiency depends on factors such as donor–acceptor distance and spectral overlap. The low concentration of phenylcoumarones (,1%) and, consequently, large donor–acceptor distance talks against their importance as acceptors/emitters. However, as judged from comparisons of spectra of borohydride-reduced MWL (Fig. 6), the lignin fluorescence may nevertheless be caused by energy transfer to phenylcoumarones and subsequent emission. Fig. 6 shows the excitation and absorption spectra of borohydride-reduced spruce lignin together with a presentation of the ratio between the fluorescence intensity and absorbance at different
Fig. 6. Absorption (- - -) and fluorescence excitation ( ____) spectra (l em 360 nm) of NaBH4-reduced spruce lignin. The relative fluorescence quantum yield (…) as a function of excitation wavelength is also shown.
B. Albinsson et al. / Journal of Molecular Structure 508 (1999) 19–27
wavelengths. This ratio is proportional to the quantum yield of fluorescence. It is clearly seen in Fig. 6 that the quantum yield is strongly dependent on the excitation wavelength and that it peaks around the wavelength where the phenylcoumarones have their absorbance maximum ( < 310 nm). The lignin spectra and the spectra of the phenylcoumarone models do not coincide completely (Fig. 5). This may be the result of the contribution of lignin chromophores other than phenylcoumarones to the lignin fluorescence or the fact that the phenylcoumarone models (9 and 10) differ structurally from the phenylcoumarone structures (2,5) in the lignin. It is in this context noteworthy that the emission from non-reduced MWL is comparatively strong in the normalized spectrum in the wavelengths range where lignin models carrying arylconjugated carbonyl groups emit fluorescence (370–465 nm [3]) (Fig. 5). References [1] J.A. Olmstead, D.G. Gray, J. Pulp Pap. Sci. 23 (1997) J571. [2] A. Bjo¨rkman, Sven. Papperstidn. 59 (1956) 477. [3] K. Lundquist, B. Josefsson, G. Nyquist, Holzforschung 32 (1978) 27. [4] K. Lundquist, I. Egyed, B. Josefsson, G. Nyquist, in: J.S. Gratzl, J. Nakano, R.P. Singh (Eds.), Proceedings of symposium on Chemistry of Delignification with Oxygen, Ozone and Peroxides. Raleigh, North Carolina, May 27-29, 1975, UNI Publishers, Tokyo, Japan, 1980; K. Lundquist, I. Egyed, B. Josefsson, G. Nyquist, Cellul. Chem. Technol. 15 (1981) 669.
27
[5] S. Li, K. Lundquist, Holzforschung 53 (1999) (in press). [6] D.-Y. Lee, M. Matsuoka, M. Sumimoto, Holzforschung 44 (1990) 415. [7] T. Katayama, F. Nakatsubo, T. Higuchi, Mokuzai Gakkaishi 32 (1986) 535. [8] C. Noutary, P. Fornier de Violet, J. Vercauteren, A. Castellan, Res. Chem. Intermed. 21 (1995) 247. [9] R. Stomberg, S. Li, K. Lundquist, B. Albinsson, Acta Cryst. C54 (1998) 1929. [10] S. Li, T. Iliefski, K. Lundquist, A.F.A. Wallis, Phytochemistry 46 (1997) 929. [11] R. Stomberg, S. Li, K. Lundquist, A.F.A. Wallis, Z. Kristallogr. -NCS 212 (1997) 473. [12] G.M. Sheldrick. SHELXS86. Program for the Solution of Crystal Structures, University of Go¨ttingen, Germany, 1986. [13] G.M. Sheldrick. SHELXL93. Program for the Refinement of Crystal Structures, University of Go¨ttingen, Germany, 1993. [14] X.P. Siemens. Molecular Graphics Program. Version 5.03. Siemens Analytical Instruments Inc., Madison, Wisconsin, USA, 1994. [15] T. Takahashi, K. Kikuchi, H. Kokubun, J. Photochem. 14 (1980) 67. [16] J.E. Lo¨froth. Ph.D. Thesis, University of Go¨teborg, 1982. [17] J.M. Beechem, E. Gratton, W.W. Mantulin. Globals Unlimited, Revision 3, The Laboratory of Fluorescence Dynamics, University of Illinois at Urbana-Champaign, USA, 1992. [18] I.B. Berlman. Handbook of Fluorescence Spectra of Aromatic Molecules, New York, Academic Press, 1965. [19] S.J. Strickler, R.A. Berg, J. Chem. Phys. 37 (1962) 814. [20] H. Suzuki. Electronic Absorption Spectra and Geometry of Organic Molecules, Chapter 14, New York, Academic Press, 1967. [21] J. Saltiel, E.D. Megarity, J. Am. Chem. Soc. 94 (1972) 2742; J. Saltiel, A. Marinari, D.W.L. Chang, J.C. Mitchemer, E.D. Megarity, J. Am. Chem. Soc. 101 (1979) 2982.