Morphology of Dry Lignins and Size and Shape of Dissolved Kraft

kraft lignin, NaOH/NaOD 0.1 M, Rh = 1.0−2.2 nm (Mw = 1600−12100) ... Curan 100 kraft lignin (CKL) was from Lignotech Sweden AB, Wargön, Sweden. ...
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Langmuir 2004, 20, 9736-9744

Morphology of Dry Lignins and Size and Shape of Dissolved Kraft Lignin Particles by X-ray Scattering Ulla Vainio,*,† Natalia Maximova,‡ Bo Hortling,§ Janne Laine,‡ Per Stenius,‡ Liisa Kaarina Simola,⊥ Janis Gravitis,| and Ritva Serimaa*,† Division of X-ray Physics, Department of Physical Sciences, P.O. Box 64, FI-00014 University of Helsinki, Helsinki, Finland, Laboratory of Forest Products Chemistry, Helsinki University of Technology, P.O. Box 6300, FI-02015 HUT, Finland, KCL Science and Consulting, P.O. Box 70, FI-02151 Espoo, Finland, Division of Plant Physiology, Department of Biosciences, P.O. Box 56, FI-00014 University of Helsinki, Helsinki, Finland, and Latvian State Institute of Wood Chemistry, 27 Dzerbenes Street, Riga LV1006, Latvia Received June 28, 2004. In Final Form: August 18, 2004 Lignin is a highly branched polymer consisting of phenylpropane units, and it is one of the ingredients of the supporting matrix in plant cell walls. The morphology of several lignins extracted from plant cell walls using different methods was studied by small-angle and ultra-small-angle X-ray scattering. A powerlaw type intensity was observed for the dry lignins, but on the basis of the power-law exponent the fractal approach often applied to lignins is not fully justified. However, the intensity of kraft lignin did show a power law with surface fractal dimension Ds ) 2.7 ( 0.1. The specific surface area of the lignins ranged from about 0.5 to 60 m2/g with 20% relative accuracy. The radius of gyration was determined from smallangle X-ray scattering data for aqueous solutions of kraft lignin. The shape of the particles in NaCl and NaOH solutions was found to be elongated. The particles were about 1-3 nm thick, while the length (5-9 nm) depended on the solvent and on the lignin concentration. The size of these primary particles was approximately the same as the size of the pores in the fractal aggregates of the dry kraft lignin. Their size was determined to be about 3.5 nm.

1. Introduction Cellulose, lignin, and hemicelluloses are the main components of wood cell walls. Partly crystalline cellulose gives wood its strength and stiffness. However, without the matrix of amorphous polymers, lignin, and hemicelluloses, the cell walls would not stay intact.1 Tens of millions of tons of dissolved lignin are annually produced in the kraft pulping processes all over the world. Lignin is a biopolymer consisting of phenylpropane units with an oxygen atom in the p-position (as OH or O-C) and with no, one, or two methoxyl groups in the o-positions to this oxygen atom (p-hydroxyphenylpropane, guaiacylpropane, and syringylpropane). The phenylpropane units are attached to one another by a series of characteristic linkages (β-O-4, β-5, β-β, etc.). The polymer is branched, and cross-linking occurs.2 During pulping, lignin undergoes more or less drastic degradation reactions depending on the pulping conditions. The terms kraft lignin and alkali lignin refer to the soluble lignin degradation products in the spent liquor after pulping. Because of the other components in wood, it is difficult to examine the morphology of lignin in situ with X-rays (or any other method for that matter), and since lignin tends to form covalently bonded aggregates with carbohydrates (lignin-carbohydrate complexes) it is not easy * Corresponding authors. E-mail: [email protected], [email protected]. † Department of Physical Sciences, University of Helsinki. ‡ Helsinki University of Technology. § KCL Science and Consulting. ⊥ Department of Biosciences, University of Helsinki. | Latvian State Institute of Wood Chemistry. (1) Sjo¨stro¨m, E. Wood Chemistry: Fundamentals and Applications, 2nd ed.; Academic Press: San Diego, 1993. (2) Lignins: Occurrence, formation, structure and reactions; Sarkanen, K. V., Ludvig, C. H., Eds.; Wiley-Interscience: New York, 1971; p 55.

to separate the lignin from the cell walls. Milled wood lignin, which is considered to be close to native lignin, always contains minor carbohydrate material,1 and it represents only around 40% of the total lignin content in wood cell walls. One way of characterizing the morphology and aggregation of materials is to determine their fractal dimension. The fractal dimension of lignins has been studied previously with a variety of methods. Viscometry, sedimentation, and diffusion measurements in the solution state,3,4 the box counting method applied on a lignin surface,5 and simulations of the cell wall structure have brought up the idea about fractal lignin.6 Norgren et al.7,8 have successfully described the aggregation of kraft lignin fragments and determined the fractal dimensions of kraft lignin clusters using quasielastic light scattering. According to their study, aggregation of kraft lignin starts with the self-association of macromolecular kraft lignin into compact colloidal particles in solutions of simple electrolytes. These particles then associate to fractal aggregates ranging in size from about 100 nm to 1-2 µm, as deduced from cryogenic transmission electron microscopy photographs. The specific surface area, defined as the total surface area of the sample divided by the mass of the sample, on the other hand, gives a measure of the porosity of the (3) Kokorevics, A.; Gravitis, J.; Ozols-Kalnins, V. Khimiya Drevesiny (Wood Chemistry) 1989, 1, 3-24 (in Russian). (4) Gravitis, J. In Ligno-Cellulosics. Science, Technology, Development and Use; Kennedy, J. F., Phillips, G. O., Williams, P. A., Eds.; Ellis Horwood: New York, 1992; pp 613-627. (5) Radotic´, K.; Tasic´, M.; Jeremic´, M.; Budimlija, Z.; Simic´-Krstic´, J.; Polzovic´, A.; Bolzˇovic´, Z. Gen. Physiol. Biophys. 2000, 19, 171-180. (6) Jurasek, L. J. Pulp Paper Sci. 1996, 22, J376-J380. (7) Norgren, M.; Edlund, H.; Wågberg, L. Langmuir 2002, 18, 28592865. (8) Norgren, M.; Edlund, H.; Wågberg, L.; Lindstro¨m, B.; Annergren, G. Colloids Surf., A 2001, 194, 85-96.

10.1021/la048407v CCC: $27.50 © 2004 American Chemical Society Published on Web 10/02/2004

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Table 1. Previous Studies Concerning the Size and Shape of Lignin Particles in Solution lignin

solvent

size and shape assessment

analytical method

ref

maple lignin isolated by methanol-HCl and by NaOH-ethanol milled wood, dioxane, kraft lignins alkali lignin solubilized by a mild procedure

different organic solvents

3 × 16 × 100 au; elliptical particle; shape factor ) 7.5

viscosity, spreading, and trough techniques

16

spread on water

spreading and trough

13

sedimentation, viscosity

17

spruce Bjo¨rkman lignin dioxane lignin

pyridine aqueous NaOH, 0.2-4 N

film thickness ) 1.7 nm; area per kraft lignin molecule ) 2.1-2.4 nm2 Rg ) 44-170 nm; microgel particles surrounded by a layer of loosely coiling chains; hydrodynamically between a random coil and a rigid sphere Rh ) 2 nm for Mw ) 7150 effective Rh ) 2.2-2.3 nm; globular particles

18 19

pine dioxane lignin thyoglycolic acid lignin

diluted organic solvents: DMSO, DMF, dioxane, pyridine pyridine-DMS-H2O

viscosity, sedimentation intrinsic viscosity, potentiometric back-titration viscometry, photon correlation spectroscopy spin labeling and viscosity

organosolv lignin

aqueous solution, pH 10-3

kraft lignin

aqueous alkali 0.1 M

kraft lignin

NaOD 1.0 M, aqueous buffer

kraft lignin acetylated kraft lignin

NaOH/NaOD 0.1 M CHCl3 1.0 M

hardwood kraft lignin

DMSO, DMF, methyl-cellulose, pyridine

a

NaHCO3-NaOH buffer, pH 9.5

size ) 110-157 nm or 9-23 nm depending on solvent and Mw apparent Rh ) 0.97-2.09 nm and 0.78-2.09 nm, assuming Einstein spheres with strongly immobilized tight network core and a more loose surface region size ) 40 nm at pH 10 and 150 nm at pH 3; 70% of particles 2-50 nm; primary particles at nm-range; agglomerates, 65 nm expansion factor ) 2.5-3.7; expanded random coil conformation without effects of long chain branching Rh ) 2.05-2.28 nm based and 38 nm as agglomerate (D2O, pH 6.5) Rh ) 1.0-2.2 nm (Mw ) 1600-12100) Rh ) 0.5-1.31 nm; oblate ellipsoid with axial ratio e 18 size ) 2.4-2.7 nm or 120-350 nm depending on molecular weight

20 21

filtration, photon correlation spectroscopy

22

size exclusion chromatography, ultracentrifugation

23

self-diffusion, PGSE-NMRa

14

self-diffusion, PGSE-NMR self-diffusion, PGSE-NMR

15 14

photon correlation spectroscopy

24

Pulsed field gradient spin-echo NMR.

material. It illustrates the coarseness of the delignification process. It is thus interesting to compare the specific surface of lignins prepared with different methods. To our knowledge, the specific surface area of lignin has not been characterized before to this extent. For kraft lignin, nitrogen absorption experiments give a value of 0.77 m2/g for the specific surface area,9 while the specific surface area of cellulose and fibers seems to vary mostly between 0.1 and 2 m2/g. A higher value, 68.4 m2/g, has been obtained for swollen cellulose.10 Dry lignins are thought to be composed of subunits of somewhat irregular size and shape. It should be possible to obtain some estimate of the size and shape of the subunits by investigating lignin in the dissolved state. The characteristics of kraft lignin particles especially are of particular interest due to the large kraft pulping industry. Goring et al. were the first to propose an oblate shape of the lignosulfonate macromolecules obtained from pulping.11,12 On the basis of electron microscopy studies, they claimed that lignin particles in solution would be oblate and about 2 nm thick. The idea was inspired by a previous work in which the thickness of monolayers of lignins spread on aqueous solutions in a Langmuir trough was found to be about 1.7 nm.13 This previous study actually suggested that the shape of milled wood lignin particles is elongated. Earlier studies also indicate that the shape of kraft lignin particles depends on the solvent. Garver and Callaghan14 studied the diffusion of acetylated kraft lignin in 1.0 M CHCl3 solution by pulsed field gradient NMR (9) Sˇ c´iban, M.; Klasˇnja, M. Holz Roh Verkst 2004, 62, 69-73. (10) Belgacem, M. N. Cellulose Chem. Technol. 2000, 34, 357-383. (11) Goring, D. A. I.; Vuong, R.; Gancet, C.; Chanzy, H. J. Appl. Polym. Sci. 1979, 24, 931-936. (12) Favis, B. D.; Goring, D. A. I. J. Pulp Paper Sci. 1984, 10, J139J143. (13) Luner, P.; Kempf, U. Tappi J. 1970, 53, 2069-2076.

and found the particles to be oblate with an axial ratio of ≈18. They also determined the hydrodynamic radius to be 2.29 nm for kraft lignin (Mw ) 4500) in 1.0 M NaOD. Using the same method and assuming the shape of the macromolecule to be spherical, Norgren and Lindstro¨m15 found that the mass-weighted hydrodynamic radius of several lignin fractions (concentration, 1.0 wt %) ranged from 1.0 to 2.2 nm in 0.1 M NaOH/NaOD. The extensive literature reports on lignin’s structure and physicochemical and hydrodynamic properties show that it is rather difficult to get a general picture of the exact shape and the actual size of an isolated lignin particle. Table 1 summarizes the size and shape of the lignin particle, as found in previous studies. Information on the morphology of the dry and dissolved lignin and the making of a clear distinction between primary lignin particles and their agglomerates should shed light on the interfacial properties of lignin. We have studied lignins both in the solid state and in solution with small-angle X-ray scattering (SAXS) and ultra-small-angle X-ray scattering (USAXS). Most of the samples used in this study have gone through a rough delignification process in which they are heated to high temperatures, so they cannot be considered to be native in the sense that they could answer the questions of the morphology of lignin in the cell wall. However, they do give information on the effect of the treatments on the morphology of the native lignin. The lignins representing the native lignins are milled wood lignin and released suspension culture lignin,25 which has not experienced the chemical stress during isolation. (14) Garver, T. M.; Callaghan, P. T. Macromolecules 1991, 24, 420430. (15) Norgren, M.; Lindstro¨m, B. Holzforschung 2000, 54, 528-534. (16) Loughborough, D.; Stamm, A. J. Phys. Chem. 1936, 40, 11131133. (17) Gupta, P.; Goring, D. Can. J. Chem. 1960, 38, 270-279.

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Vainio et al.

Table 2. Key to Abbreviations of the Samples abbreviation

samplea

CKL AOL RSCL BJOb MWL SEL SOL

Curan 100 kraft lignin (softwood) Alcell organosolv lignin (softwood) Released suspension culture l. (spruce) Bjo¨rkman lignin (spruce), impurities milled wood lignin (pine) steam explosion lignin (birch) B. DIV soda lignin (straw)

a The plant species from which the lignins were obtained are mentioned in brackets. b BJO contained a small amount of crystalline impurities and was therefore not included in the study except for comparison. MWL and BJO are extracted from wood using a similar method.

Another aspect of our study concerns the size and shape of redissolved kraft lignin in NaOH and NaCl solutions. To promote better understanding of kraft lignin adsorption on fibers, we used the same lignin solutions as used earlier in absorption measurements.26 2. Experimental Section 2.1. Samples. 2.1.1. Dry Lignins. Lignin samples with their origin and abbreviations are listed in Table 2. The steam explosion lignin (SEL) was prepared using a laboratory steam explosion unit (The Latvian State Institute of Wood Chemistry, LSIWC). Curan 100 kraft lignin (CKL) was from Lignotech Sweden AB, Wargo¨n, Sweden. Alcell (AOL), which was prepared by organosolv pulping, and B. DIV soda lignin (SOL) were obtained from The International Lignin Institute, Lausanne, Switzerland. A sample of Bjo¨rkman lignin (BJO) from spruce was also received, but it contained a small amount of crystalline impurities (determined by wide-angle X-ray scattering) and is therefore included here only for reference. The milled wood lignin sample (MWL) (from KCL Science and Consulting, Espoo, Finland) was isolated from pine wood (Pinus sylvestris) using ball milling of wood powder and extraction with dioxane-water (9:1). Further purification of the MWL was conducted according to the Bjo¨rkman method,27 which was slightly modified by using an ultrasonic extraction step at 15 °C instead of the Soxhlet extraction. The yield after purification was 45%, calculated on the lignin in wood. The released suspension culture lignin sample (RSCL) from Norway spruce (Picea abies) was prepared at the Division of Plant Physiology at the Department of Biosciences (University of Helsinki, Finland). The lignin sample was separated by centrifugation from the liquid medium of RSCL suspension cultures. After rinsing with cold distilled water, the pellet was freeze-dried. Details on the procedure are given by Simola et al.28 (18) Alekseev, A.; Reznikov, V.; Bogomolov, B.; Sokolov, O. Khimiya Drevesiny (Wood Chemistry) 1971, 7, 31-36. (19) Chupka, E.; Obolenskaya, A.; Nikitin, V. Khimiya Drevesiny (Wood Chemistry) 1970, 5, 53-58. (20) Bogolitsyn, K.; Rjabeva, N.; Volkova, N. Behaviour of native lignins in organic solvents. In International Symposium of Wood and Pulping Chemistry, 8th, Helsinki, 1995; Association of Finnish Paper Engineers; Vol. 2. (21) To¨rma¨la¨, P.; Lindberg, J.; Lehtinen, S. Paperi Puu 1975, 57, 601-605. (22) Richter, W.; Zaenker, H.; Nitsche, H. Forschungszent. Rossendorf e. V., FZR 1998, 76-77. (23) Sarkanen, S.; Teller, D. C.; Abramowski, E.; McCarthy, J. L. Macromolecules 1982, 15, 1098-1104. (24) Maier, L.; Bogolytsin, K.; Ivanova, M. Zhurnal Prikladnoi Khimii (Sankt-Peterburg) 1997, 70, 487-489. (25) Lewis, N. G.; Davin, L. B.; Sarkanen, S. The nature and function of lignins. In Comprehensive natural products chemistry, 1st ed.; Barton, D., Nakanishi, K., Eds.; Elsevier Science Ltd.: Oxford, U.K., 1999; Vol. 3. (26) Maximova, N.; Osterberg, M. K. K.; Stenius, P. Cellulose 2001, 8, 113-125. (27) Bjo¨rkman, A. Sven. Papperstidn. 1956, 60, 477-485. (28) Simola, L. K.; Lemmetyinen, J.; Santanen, A. Physiol. Plant. 1992, 84, 374-379.

Table 3. Percentages of Functional Groups (of Dry Weight) in Lignin Samplesa sample

[OCH3] (%)

[OH]t (%)

[OH]p (%)

[OH]c (%)

CKL AOL SEL MWL

12.05 18.55 18.52 16.2

4.4 6.7 4.0

3.4 4.3 4.0

1.0 2.4

a

t stands for total, p for phenolic, and c for carboxylic.

Relative amounts of functional groups in the lignins are listed in Table 3. CKL, AOL, and SEL were analyzed at LSIWC with potentiometric high-frequency titration, and MWL was analyzed at KCL. From the methoxyl content, it is possible to deduce whether the lignin comes from softwoods or hardwoods. The hydroxyl content on the other hand is a rough measure of the purity of the lignin. 2.1.2. Dissolved Lignin. CKL was used in studies of solutions. Dry lignin (in H-form) is insoluble in water and can only be dissolved in alkaline solution. However, there is a hysteresis of lignin solubility; once it has been dissolved in alkali, that is, transferred into Na-form, the lignin solution remains stable at low pH levels upon the addition of acid. A stock lignin solution was prepared by dissolving 20 g of lignin powder in 1 L of 0.1 M NaOH. The solution was left to stabilize for at least 48 h to allow all the lignin to dissolve. The pH of the stock solution was 12.8. A set of alkaline solutions of lignin (20, 10, 5, 2, and 1 g/L) were prepared from the stock solution by dilution with 0.1 M NaOH. A set of neutral solutions was prepared by adjusting the pH to 7 by adding concentrated HCl and adding NaCl to obtain the desired ionic strength (0.1 and 0.025 M NaCl). A solution containing 10 g lignin/L at pH 7 in 0.1 M NaCl was further diluted with 0.1 M NaCl to 5, 2, and 1 g/L. A solution containing 2.5 g lignin/L at pH 7 was further diluted with 0.025 M NaCl to 2 g/L. 2.2. Determination of Molar Mass. The molar mass distributions of the lignins were determined by gel permeation chromatography at KCL,29 using a Superdex 75 prep grade gel in a 30 × 1 cm column, 0.5 M NaOH as the eluent, and monodisperse Na-polystyrene sulfonates (from American Polymer Standards Corp.) for calibration. The elution curves were monitored by a UV detector at 280 nm. Phenol (Riedel-de Haen, pa) was used as an external standard both in the calibration and in the measurements. The flow rate was 1 mL/min. 2.3. SAXS Measurements. A conventional sealed X-ray tube (Cu anode, point focus) was used. The beam was monochromatized with a nickel filter and a totally reflecting glass plate to obtain Cu KR radiation (λ ) 1.542 Å). The beam was reduced with slits to 0.1 mm in width and 0.5 mm in height on the sample surface. The sample powders and liquids were installed in 0.8 mm thick steel rings covered with Kapton film. The scattered X-rays travelled through a vacuum chamber to the 2D proportional gas detector, a HI-STAR Area detector. The position of the direct beam and the transmitted flux were obtained from the image of the beam through a copper beam stop, which was installed inside the vacuum chamber. With the sample-to-detector distances of 20 and 120 cm, the cross section of the beam on the detector was 0.6 × 0.9 and 1.4 × 2.8 mm2, respectively. Correspondingly, the range of the length of the scattering vector q was 0.05-0.8 and 0.014-0.18 Å-1 for these distances and the size of the beam on the detector was 0.012 Å-1 × 0.018 Å-1 and 0.006 Å-1 × 0.01 Å-1. The length of the scattering vector is defined here as q ) 4π sin θ/λ, where 2θ is the scattering angle and λ is the wavelength. The q values were determined using a silver behenate standard (Roche Chemicals Limited London), d ) 58.373 Å.30 The intensity curves obtained by integrating from the 2D pattern measured with different sample-to-detector distances were united at q ) 0.05 or 0.06 Å-1 depending on the scattering power of the sample. The detector response and the spatial distortion of the intensities were corrected by measuring the intensity of a uniform radiation (29) Hortling, B.; Turunen, E.; Kokkonen, P. In Handbook of Size Exclusion Chromatography Techniques, 2nd ed.; Wu, C.-S., Ed.; Chromatograhic Science Series, Vol. 91; Marcel Dekker: New York, 2003; Chapter 13. (30) Huang, T. C.; Toraya, H.; Blanton, T. N.; Wu, Y. J. Appl. Crystallogr. 1993, 26, 180-184.

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field from a 55Fe source and by using the SAXS (version 4.1.09) measuring program of Bruker AXS. Dissolved CKL samples were measured with 20 and 50 cm sample-to-detector distances. All results were confirmed by measuring at least two samples of the same solutions. The lowest concentration of lignin for which reliable results could be obtained was 5 g/L in 0.1 M NaCl. The wide-angle X-ray scattering (WAXS) intensities were measured with the SAXS setup using the perpendicular transmission geometry. The sample-to-detector distance was 6 cm, and the scattering vector was calibrated with silver behenate and silicon (111) reflection, d ) 3.1354 Å.31 The SAXS intensities were corrected before analysis for absorption, air scattering, and WAXS background of parabolic type,32 while WAXS intensities were corrected for absorption, air scattering background, geometrical distortion, and polarization.33 From the isothermal compressibility and electron density of water, the absolute intensity of water samples (measured thicknesses of 0.8, 1.6, and 2.4 mm) at small angles can be calculated to be 0.0166 cm-1 at T ) 298 K.34 This value was used to calibrate a secondary standard Lupolen sample (transmission, 0.404; thickness, 2.72 mm), which then had an intensity maximum at q ) 0.03 Å-1, the intensity being 5.5 cm-1. This value is consistent with other studies where point focus geometry was used.35 The Lupolen standard was used to set the intensity of each sample onto an absolute scale so that the specific surface of the solid material in the samples could be calculated. 2.4. USAXS Measurements. The experiments at HASYLAB were performed using the USAXS beam line BW4 with a proportional area detector at 4 and 13 m sample-to-detector distances. With the used energy of 8.979 keV, these distances correspond to the q ranges 0.008-0.1 and 0.0015-0.035 Å-1. The primary beam intensity was monitored at the position of the beam stop. The intensities of dry lignins were united at 0.01 or 0.02 Å-1, and the dissolved kraft lignin was only measured with 4 m distance. The diameter and thickness of the sample holder were 5 mm and 0.8 mm, respectively, for dry samples and 1 cm and 2 mm for solution samples. The lowest concentration of lignin for which reliable data was observed was 2 g/L for 0.025 M and 1 g/L for 0.1 M NaCl solutions. The q values were calibrated by measuring rat tail collagen (d ) 650 Å). 2.5. Electrophoretic Mobilities. The electrophoretic mobility of dissolved lignin samples was measured with a Coulter Delsa 440 (Doppler Electrophoretic Light Scatter Analyzer) instrument. Lignin solutions of different concentration (1-20 g/L), ionic strength (0.025 M NaCl, 0.1 M NaCl, and 0.1 M NaOH), and pH (12.8 and 7) were prepared in the same way as for SAXS/USAXS studies, but before measuring the electrophoretic mobility, the solutions were diluted 1/20 with water, to ensure that the conductivity was below 1 mS/cm. The measurements were conducted at a run time of 120 s, a voltage of 5 V, a frequency of 500 Hz, and at constant temperature (25 °C). 2.6. Data Analysis of SAXS and USAXS. The SAXS intensity curves of all dry samples followed the power law -R

I(q) ∝ q

(1)

This can be interpreted as arising from fractal structures within the sample if the characteristic length scale R of a fractal satisfies the condition Rq . 1.36 For surface fractals in three-dimensional space R ) 6 - Ds, in which Ds is the surface fractal dimension (31) Wyckoff, R. Crystal structures, 2nd ed.; John Wiley & Sons: New York, 1963; Vol. 1, p 26. (32) Balta´-Calleja, F. J.; Vonk, C. G. Polymer Science Library 8: X-ray scattering of synthetic polymers; Elsevier: Amsterdam, 1989. (33) Guinier, A. X-ray Diffraction: In Crystals, Imperfect Crystals, and Amorphous Bodies; Dover: New York, 1994. (34) Orthaber, D.; Bergman, A.; Glatter, O. J. Appl. Crystallogr. 2000, 33, 218-225. (35) Russell, T. P.; Lin, J. S.; Wignall, G. D. J. Appl. Crystallogr. 1988, 21, 629-638. (36) Schmidt, P. W. In Fractal Approach to Heterogeneous Chemistry; Avnir, D., Ed.; John Wiley & Sons: Chichester, 1989; Chapter 2.2.

describing the fractality of the surface. The fractal nature of a mass fractal continues across the whole object, while the surface fractal is limited to the surface regions. Thus, for surface fractals the mass fractal dimension Dm is 3, while Ds can vary between 2 and 3. The Porod law, R ) 4, is valid for the scattering of a compact particle with a smooth surface (Ds ) 2, Dm ) 3). A power law with R < 3 is caused by a mass fractal for which R ) Dm ) Ds < 3. Continuous charge density transitions can cause R to be larger than 4.37 For the determination of the specific surface area, we assume that lignin can be described as a porous material with pores of no particular shape. We denote the volume fraction of lignin by φ1 and that of pores by φ2 and correspondingly the electron densities by F1 and F2. In this case, the average electron density38 Fj is

Fj ) φ1F1 + φ2F2

(2)

It can be shown that the invariant Q ) ∫∞0 i(q)q2 dq, where the scattered intensity i(q) is of magnitude 1 for one electron, takes the form

Q ) 2π2(∆F)2φ1φ2V

(3)

V is now the total volume of the sample. When the system becomes dilute, so that φ2 can be put to 1, we get the same equation as for particle scattering only multiplied by the number of scatterers N, since then φ1 ) NV1/V, where V1 is the volume of a lignin particle. The same applies to a sparce network of pores, only then φ1 ) 1. The modified surface-to-volume ratio is given by

S πφ1φ2 ) limq4i(q) V Q qf∞

(4)

This equation is valid also for intensities in relative units. If the absolute intensity is known, this approach can be extended further. The normalized differential scattering cross section dΣ/ dΩ (unit cm-1) is in this case referred to as the absolute intensity. The intensities from different samples can be put on the same scale by dividing by the measurement time t, the (optical) thickness l, and transmission T of the sample.34,39 The optical thickness l of a sample is defined by

l ) -ln T/µl

(5)

and to determine it, the linear absorption coefficient µl must be known. The weight fractions of oxygen, carbon, and hydrogen in milled wood lignin have been reported to be wO ) 0.2968, wC ) 0.6366, and wH ) 0.0604.2 The density of lignin Fl is assumed to be 1.4 g/cm3,40 and thus the linear absorption coefficient of lignin is estimated to be

( (µF)

µl ) Fl wC

C

+ wO

(µF)

O

(µF) ) ) 8.83 cm

+ wH

-1

H

for Cu KR radiation and 6.04 cm-1 for 8.979 keV radiation.41 The transmission of the dry lignin samples varied from 1.02 (RSCL) to 2.10 (SOL) depending on the sample, while variation for the same sample in different measurements was about 0.1. However, this was not a problem, since the transmission was measured through the beamstop at the same time with the scattering experiment. (37) Schmidt, P. W. J. Appl. Crystallogr. 1991, 24, 414-435. (38) Porod, G. In Small-angle X-ray scattering; Glatter, O., Kratky, O., Eds.; Academic Press: London, 1982; Chapter 2. (39) Spalla, O.; Lyonnard, S.; Testard, F. J. Appl. Crystallogr. 2003, 36, 338-347. (40) Stamm, A. J.; Hansen, L. A. J. Phys. Chem. 1937, 41, 10071016. (41) International tables for crystallography; Wilson, A. J. C., Ed.; Kluwer Academic: Dordrecht, 1992; Vol. C, Chapter 4.2.

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Vainio et al. Table 4. Calculated Weight Average Mw and Number Average Mn Molar Masses of the Studied Lignins lignin

Mw (g/mol)

Mn (g/mol)

PDa

P1b

P2

P3

CKLc

5800 7120 2650 2530 5440 6820 10860

2760 3000 1850 1880 2770 3220 3830

2.1 2.4 1.4 1.3 2.0 2.2 2.8

94 88 87 94 96 96

62 25 25 56 63 68

24 0.5 1 14 20 42

CKL AOL RSCL MWL SEL SOL

a PD is the polydispersity index M /M . b P1, P2, and P3 are the w n estimated percentages of log M g 3.0, log M g 3.5, and log M g 4.0. c CKL lignin which was used in the study of lignin solutions. Other lignins were studied in the dry state only.

Figure 1. Beginning of the SAXS intensity curves of some of the dissolved kraft lignin samples. Curves A, B, and D are 1, 5, and 10 g/L of lignin in 0.1 M NaCl (pH 7). Curve C is 20 g/L in 0.1 M NaOH (pH 12.8). The solid line is the fitted Guinier law. By combining eqs 3 and 4 and dividing by φ1, thus taking into account the volume fraction of the solid only, the specific surface of the solid matrix (not the whole sample) is simply39

Sl )

N(q) K S 1 ) limq4 V φ1F 2π(∆F)2lF qf∞ T

(6)

where N(q)/T is the measured count rate divided by transmission and from which background scattering has been subtracted. The electron density difference between lignin and vacuum ∆F is 1.2591 × 1011 cm-2 using the aforementioned values, while F is the density of the solid matrix (lignin), 1.4 g/cm3. The area of the beam is hidden in the constant K by which the otherwise corrected intensity curve of Lupolen has to be multiplied in order to get it on the absolute scale. The SAXS intensities of the dissolved kraft lignins were observed to follow the Guinier law (see Figure 1) at small values of q

Figure 2. Smoothed WAXS patterns of the dry lignin samples. The curves are shifted vertically for better visualization. The curve labeled Avicel is the intensity of nearly amorphous cellulose. For Avicel and BJO, the intensities are from diffractometer measurements.

3.1. Molar Masses. The average molar masses, which are calculated relative to Na-polystyrene sulfonates, are given in Table 4. SOL had the highest molar mass, while the molar masses and polydispersities of AOL and RSCL were close to each other. While we measured by X-ray scattering Curan 100 kraft lignin in the dissolved state and in the dry state, the two samples are from different

batches and have different polydispersities when dissolved in the same way. The molar masses are calculated for the total elution curve, which means that especially for SOL the part eluting at the void volume (sharp peak) is not distributed according to molar mass and thus its value is too low. The estimate of lignin fractions of different size is also given in the table. The polyelectrolyte effect is assumed to be partly suppressed due to the high alkalinity and simultaneously high ionic strength of the eluent. However, some association between the lignin molecules in the eluent cannot be excluded. 3.2. Dry Lignins. The WAXS intensity curves of the dry lignin samples (Figure 2) show that the samples are mostly amorphous, although they might contain small amounts of impurities, most probably hemicelluloses. As a reference, an intensity curve of nearly amorphous cellulose measured using a diffractometer (in symmetrical transmission mode) is also presented in the figure. The amorphous cellulose was made from microcrystalline cellulose by ball milling for 60 h. Crystalline monoclinic (Iβ) cellulose gives intensive reflections at 2θ values of 14.6° (11 h 0), 16.6° (110), and 22.4° (200).45 The reflection 200 is the most intensive and should be clearly seen if there is any crystalline cellulose present. This reflection, however, was not visible in the WAXS curves of the lignin samples. Instead, all the samples gave intensity curves very similar to those measured by Ahtee et al.46 for kraft, dioxane, and milled wood lignin. The WAXS pattern of SOL, however, was different, since for our sample the amorphous peak is not as broad as in the previous work. The intensity curve of BJO in the figure is from a

(42) Svergun, D. I. J. Appl. Crystallogr. 1991, 24, 485-492. (43) Feigin, L. A.; Svergun, D. I. Structure Analysis by Small-Angle X-ray Scattering; Plenum Press: New York, 1987. (44) Svergun, D. I. Biophys. J. 1999, 76, 2879-2886.

(45) Sugiyama, J.; Vuong, R.; Chanzy, H. Macromolecules 1991, 24, 4178-4175. (46) Ahtee, M.; Hattula, T.; Mangs, J.; Paakkari, T. Paperi Puu 1983, 65, 475-480.

I(q) ) I(0) exp(-Rg2q2/3)

(7)

where Rg is the radius of gyration of the particles.33 The radius of gyration was determined by fitting a line to (q2, ln I) using the least-squares method with Matlab. On the other hand, Rg was also obtained using the program GNOM (version 4.4).42 The limit at which the law applies is Rg < 1.3/qmax.50 The distance distribution function p(r) was also calculated:

p(r) )

r2 (2π)3

∫ dq 4πq sinqrqrI(q) ∞

2

0

(8)

which represents the probability of finding two points in the particle separated by a distance r.38,43 The largest diameter of the particle can be obtained from the p(r) function. The program DAMMIN (version 4.3b)44 was used to restore the average shape of the lignin particles in solution. The program is based on building a scattering unit from dummy atoms. Simulated annealing is employed to find a configuration that fits the SAXS data.

3. Results and Discussion

Morphology of Lignins

Langmuir, Vol. 20, No. 22, 2004 9741

Figure 3. USAXS intensities of dry lignins at room temperature. The intensity of MWL has been extended with a SAXS curve. The intensities are corrected for absorption and background scattering. (A) RSCL (filled circles), BJO (open circles), MWL (line). (B) SOL (filled circles), CKL (line), SEL (open circles), and AOL (dash-dot). The dashed line is the q-4 power law in the same scale in both pictures. Every fourth point is drawn in the case of circles to avoid crowding of the points. Table 5. Surface Fractal Dimension, Ds, of the Lignins at Room Temperature Determined from SAXS and USAXS Data sample

Ra

region (Å-1)

Dsb

Slc (m2/g)

CKLd

4.0 3.3 4.1 4.0 4.1 4.1 4.1 4.0

0.03-0.2 0.009-0.03 0.003-0.15 0.02-0.2 0.01-0.25 0.02-0.25 0.005-0.2 0.01-0.25

2.0 2.7 2.0 2.0 2.0 2.0 2.0 2.0

2.4

AOL RSCL MWL BJO SEL SOL

0.5 60 34 35 1.3 3.6

a The accuracy of R is (0.1. b The mass fractal dimension D is m 3 for every sample. c Sl is the specific surface area of lignin from absolute intensity (see the text). The relative error of Sl is estimated to be about 20%. d For CKL, two power laws were observed.

symmetrical transmission mode measurement with a diffractometer. A small amount of crystalline impurities were seen in the WAXS pattern measured with HI-Star for the same sample. SAXS intensities of the lignins obeyed a power law, and the power-law exponents were determined by fitting a line with the least-squares method to the logarithms of intensity and q. The results of the fits are presented in Table 5. A power-law exponent indicating a fractal structure was obtained only for CKL. The lower limit of the power law was not observed, but the upper limit qmax was at 0.03 Å-1. The length scale of the smallest unit can therefore be approximated as 1/qmax.36 Thus for CKL the fractal region started from about 3.5 nm and continued onto larger sizes. For RSCL, AOL, MWL, SEL, and SOL, the exponent was about -4, indicating that the particles are compact aggregates with sharp phase boundaries. All these results were confirmed with USAXS (Figure 3). CKL was observed to be inhomogeneous, since four measurements gave the exponent -3.3 and one measurement gave -2.9.47 With other samples, this kind of inhomogeneity was not observed. (47) Vainio, U.; Serimaa, R.; Gravitis, J.; Hortling, B. Fractal dimensions of extracted lignins at elevated temperatures by smallangle X-ray scattering. In Proceedings of the 7th European workshop on lignocellulosics and pulp, Turku, 2002; A° bo Akademi.

The other samples were found to follow the Porod law which arises from the surfaces of pores in the lignin particles or from the surfaces of the particles themselves. In the case of RSCL, MWL, and the reference BJO sample, the power law did not continue throughout the whole range of the scattering vector at the lower q values, which indicates that the dimensions of the scatterers in RSCL, MWL, and BJO are smaller than in AOL, SOL, and SEL. Using eq 6, the specific surface area of the solid lignin in the samples, Sl, was calculated from SAXS intensity curves put on absolute scale. For the less modified lignins MWL, BJO, and RSCL, the Sl was found to be 34, 35, and 60 m2/g, respectively. The estimated relative error is about 20% due to the uncertainties in the determination of the absorption of the sample and the low accuracy of the absolute intensity. For the other samples, the specific surface area was much lower (Table 5). Even though the density used in the calculations was for MWL, the values for other samples can be considered almost as accurate as for MWL, since the content of impurities for example in CKL is estimated to be low48 and some of the errors cancel in the divisor (density vs linear absorption coefficient). The specific surface area Sl calculated directly from the absolute intensity curves can be compared with that obtained by extrapolating the beginning of the USAXS scattering curve according to the Guinier law in the case of MWL, BJO, and RSCL. In these cases, the invariant Q can be calculated. Equation 4 yields S/(φ1φ2V/F) ) 78, 60, and 110 m2/g, respectively. The fraction of air φ2 can be evaluated from the sample thickness compared to the optical thickness, giving φ2 ) 0.75, 0.83, and 0.79. With these values, the calculations using the invariant give Sl ) 58, 50, and 88 m2/g for MWL, BJO, and RSCL with 20% estimated relative error. The volume fractions may be somewhat inaccurate due to the difficulties in the accurate determination of the thickness of the samples. However, the values are in reasonable correlation with those obtained directly from the absolute intensities. Brunow et al.49 studied the soluble fractions of RSCL from Norway spruce with 1H and 13C NMR. They found (48) Marton, J. Tappi J. 1964, 47, 713. (49) Brunow, G.; Kilpela¨inen, I.; Lapierre, C.; Lundquist, K.; Simola, L. K.; Lemmetyinen, J. Phytochemistry 1993, 32, 845-850.

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Figure 4. Radius of gyration (eq 7) as a function of lignin concentration c: (0, 9) 0.1 M NaCl (pH 7); (4, 2) 0.1 M NaOH (pH 12.8); (1) 0.025 M NaCl (pH 7). Open and filled symbols indicate measurements from different sample series (open, with laboratory SAXS apparatus; filled, at HASYLAB BW4).

the chemical structure of RSCL to resemble closely that of MWL from spruce and claim that RSCL is a highly branched polymer. According to SAXS measurements, the power-law exponent -R of RSCL was equal to -4, which indicates a compact aggregate with a smooth surface. The SAXS curves of RSCL from spruce and MWL from pine are nearly identical (Figure 3), so the morphologies of the two most native lignins can be considered to be similar despite the differences in the WAXS curves. The specific surface areas of these lignins are very close to each other and much higher than the specific surface areas of other lignins. According to previous studies, the relative specific surface area decreased as a function of temperature for all lignins.47 The low specific surface area of AOL already at room temperature may explain why the heating of AOL resulted in a total loss of intensity already at 140 °C, while the decrease in Sl for the other lignins only began at that temperature. The specific surface area Sl is also a measure of the porosity. Due to the small values of Sl, we may qualitatively say that the samples are mostly compact. It may be interpreted that MWL, RSCL, and BJO have particles of the size scale 100 nm and they are smaller than particles in for example AOL, which has the largest particles among the studied samples based on the very small Sl. Another interpretation would be that there are pores of the size scale 100 nm in MWL, RSCL, and BJO. CKL had a small Sl, meaning that the basic units of the fractal, which were about 3.5 nm in size, must be pores. This conclusion is made since particles of that size would give a much larger Sl. Nitrogen absorption experiments9 give a smaller value of 0.77 m2/g for Sl, possibly because the technique cannot see closed pores or because the pores are only partially penetrated by the gas. It seems that the fractal CKL in the wet state observed by Norgren et al.7 collapses to a more compact conformation as the structure dries. 3.3. Dissolved Kraft Lignin. The radius of gyration Rg of the dissolved kraft lignin particles could be reasonably well determined, and it varied between 1.6 and 3.5 nm depending on the solvent and concentration (Figure 4). Due to the rather broad molar mass distribution of the dissolved kraft lignin, the Rg is only an average value. In 0.1 M NaOH, the Guinier law extended for a wide range of q values for every concentration studied, indicating that there exists a large amount of particles of about equal size, while with 0.025 and 0.1 M NaCl the polydispersity is seen more clearly as a slight increase in Rg when the Guinier law is fitted at smaller q values and therefore the particle shape determinations may represent only an average over a size range. The values in the figure are those obtained by Matlab. Comparison of the results of

Vainio et al.

Figure 5. Examples of “good” p(r) functions that were calculated by GNOM from the intensity curve of 20 g/L lignin in 0.1 M NaOH (pH 12.8) (solid line) and 5 g/L 0.1 M NaCl (pH 7) (dashed line). Examples are also shown of obtained lowresolution shapes of the particles calculated by DAMMIN. The particle is viewed from the side and the top for both samples.

GNOM and Matlab indicates that the accuracy of the determination is about 0.1 nm. The shapes of the p(r) functions (Figure 5) clearly indicate that the shapes of the particles must be elongated, since an oblate shape would give a more symmetric p(r) function.50 Table 6 compares the radius of gyration Rg with the maximum diameters gained from the p(r) functions as well as with some qualitative estimates of the thicknesses of the particles based on the low-resolution shapes obtained by DAMMIN. On the basis of the DAMMIN models, the lignin particles in the solutions are elongated and about 1 nm thick and 2.5 nm wide. The molar mass calculated from the dimensions of the DAMMIN models from results in 0.1 M NaOH agrees qualitatively with the Mw values obtained by gel permeation chromatography in 0.5 M NaOH. However, in the different solvent 0.1 M NaCl the molar mass is larger according to the DAMMIN models, which may be caused by association or lowered density of the particles. A lower density would cause the calculation to give a value that is too large for the molar mass of the DAMMIN model particles. It is interesting to consider how the radius of gyration and the size depend on the chemical environment and the charge of the particles. The electrophoretic mobility is proportional to the surface charge density of the particle and depends on the pH, ionic strength, viscosity, temperature, and dielectric constant of the suspending liquid. The mobilities of the dissolved kraft lignin (Table 6) agree with previous results on ζ-potential.51 The negative charge of lignin in alkaline solutions is predominantly due to dissociation of phenol hydroxyls (pKa of about 9.5).1 As a result, the lignin particle in alkali is more charged and more mobile than in neutral solution. The mobilities at pH 7 vary between 2.0 and 2.7 (µm s-1)/ (V cm-1). Increasing the ionic strength from 0.025 M NaCl to 0.1 M NaCl will compress the diffuse double layer around the lignin particle and will thus result in a decrease in the radius of gyration. For rodlike particles, the changes in mobility resulting from the combination of these two effects are difficult to predict. An increase of particle size has been reported to take place both upon increase and decrease of pH. The increase of particle size due to increase of pH was ascribed to polyelectrolyte swelling due to breaking of intramolecular hydrogen bonds and dissociation of ionizable functional (50) Svergun, D. I.; Koch, M. H. Rep. Prog. Phys. 2003, 66, 17351782. (51) Dong, D.; Fricke, A. L.; Moudgil, B. M. Tappi J. 1996, 79, 191197.

Morphology of Lignins

Langmuir, Vol. 20, No. 22, 2004 9743 Table 6. Characteristics of the Dissolved Kraft Lignin (CKL) Particles

solutiona (1) (2) (2) (2) (3)

c (g/L) 2 1 5 10 20

pH 7 7 7 7 12.8

Rg (nm)b 3.5 2.2 2.3 2.6 1.6

Rmaxc (nm) ndg nd 7.3 ( 0.3 8.7 ( 0.5 5.3 ( 0.3

td (nm)

wd (nm)

mobilitye [(µm s-1)/(V cm-1)]

Mf (103 g/mol)

nd nd 1.2 ( 0.2 1.4 ( 0.2 1.0 ( 0.2

nd nd 2.5 ( 0.5 3.0 ( 0.5 2.0 ( 0.5

-2.4 -2.0 -2.1 -2.7 -4.0

nd nd 19 ( 6 31 ( 9 9(4

a Solutions: (1) 0.025 M NaCl, (2) 0.1 M NaCl, (3) 0.1 M NaOH. b The radius of gyration (R ; accuracy, (0.1 nm). c R g max is the maximum diameter of the particles obtained from the p(r) functions. d Qualitative estimates for the thickness (t) and width (w) of the particle obtained from DAMMIN models. e The electrophoretic mobilities were measured for particle concentrations that were 1/20 of the original concentration. The error of mobility is estimated to be (0.25. Note that the mobility increases with decreasing particle size. f The molar mass M is calculated from the particle dimensions (approximating the particle as rectangular and using a density of 1.4 g/cm3). g nd means not determined.

groups (e.g., refs 19 and 14), while the association due to decrease of pH was ascribed to formation of intramolecular hydrogen bonds: when the carboxylic groups are protonized, intramolecular hydrogen bonds are formed in the loose surface region of the particle, and intermolecular hydrogen bonds are formed between the particles.52 The decreasing charge density of lignin when the pH is decreased also promotes the formation of associated complexes favorably.53 According to SAXS measurements, an increase (up to 50%) in the particle radius (implying about a 3-fold increase in volume) occurs when the pH decreases from 13 to 7 at a constant ionic strength of 0.1 M. Thus there is no indication of the formation of large associated complexes or aggregates. Norgren et al.7 reported that very high salt concentrations are needed for extensive self-aggregation to occur and that the resulting aggregates are fractal. On the other hand, according to ultrafiltration and light-scattering studies (at a wavelength of 632.8 nm) reported by Woerner and McCarthy54 large stable complexes of kraft lignin exist in neutral and weakly alkaline solutions. As alkalinity is increased, these complexes are broken to yield small molecules, and when alkalinity is decreased from pH 13.8 to 8.5 large stable associated complexes (Mw of up to 200 000) grow from smaller lignin molecules (Mw of 3500).54 Any associated complex would most probably give a power-law type SAXS intensity curve, but such intensity curves were not seen. This means either that there is no association when pH decreases from 12.8 to 7 in aqueous solutions of kraft lignin or that the size of the associated complexes must be larger than about 100 nm. It cannot be excluded that some of the increase of the particle size taking place as pH decreases is due to an occasional association of macromolecules. The intensity and the dimensions extracted from it would then be an average over such aggregates and single particles. The following picture emerges: Due to dissociations of phenolic hydroxyls lignin acquires negative charge in alkaline solution, which results in high electrophoretic mobility and strong electrostatic repulsion between the lignin particles. The repulsion prevents association of lignin particles in the solution, which is consistent with the small diameters of lignin particles measured by SAXS. When pH decreases, the negative charge on lignin is partly neutralized which leads to a decrease of the electrophoretic mobility of the lignin particle. However, the electrostatic repulsion between the lignin particles is still sufficient to prevent extensive association in neutral solution. (52) Lindstro¨m, T. Kolloid Z. Z. Polym. 1979, 257, 277-285. (53) Sarkanen, S.; Teller, D. C.; Stevens, C. R.; McCarthy, J. L. Macromolecules 1984, 17, 2588-2597. (54) Woerner, D.; McCarthy, J. Macromolecules 1988, 21, 21602166.

On the basis of the low-resolution shapes obtained by DAMMIN, the lignin particles consist of oblate and prolate parts which have associated into elongated structures. The shape remains irregular but elongated at different concentrations of lignin and in different aqueous solvents. This is in contrast to another study,14 where acetylated lignin in 1.0 M CHCl3 solution was found to be an oblate ellipsoid with an axial ratio of less than 18 using PFGNMR. The reason this previous study concluded the shape was so extremely oblate could be due to the principal difficulties with the solvent diffusion model. The authors of the paper themselves stated it is easier to interpret the solvent diffusion data from an oblate ellipsoid than from a prolate one.14 We found the thickness of the dissolved lignin particles to be about 2 nm, which is consistent with results reported by Goring et al.11 and Luner and Kempf.13 4. Conclusions According to USAXS measurements, the dry CKL is formed of fractal aggregates with surface fractal dimension of 2.7 ( 0.1, while the other studied lignins showed no fractal properties at length scales smaller than 200 nm. The dry CKL was concluded to have pores of size 3.5 nm approximately, and the pores are near or at the surface of the aggregates. SAXS studies indicate that when the CKL powder is redissolved the particles in the solution are about 1-3 nm thick “chains”. The length of the chains increases with increasing polymer concentration, and the width of the chain varies within the chain from about 10 to 40% of the chain length. Significant association of the particles was not observed when pH is lowered from 12.8 (0.1 M NaOH) to 7 by adding HCl. The width of the particles is about the same size as the pores in dry CKL. Imperfect packing of the particles could result in a structure like that, while perfect packing would leave no pores at all, when the particles are aligned in the same direction. The combination of these packings would give a low specific surface observed in the dry state measurements. The specific surface area of the dry lignins was found to vary with the delignification method. An interesting observation concerning softwood lignins was that the specific surface area of milled wood lignin of pine was close to that of released suspension culture lignin of spruce, which has not experienced the chemical stress during isolation. The morphologies are close to each other even though the polydispersities in the dissolved state and the impurity levels of the two lignins are different. The other extraction methods such as steam explosion, Alcell, soda, and kraft pulping lower the specific surface area. The plant species from which the lignin is extracted does not appear to affect the morphology of the aggregate as much. Acknowledgment. We thank ILI for supplying the AOL and SOL samples and Lignotech for the CKL samples.

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Langmuir, Vol. 20, No. 22, 2004

The help of Dr. Girt Zakis and Ms. Brigita Neiberte in analysis using potentiometric high-frequency titration is greatly appreciated. We thank Seppo Andersson, Lic. Phil., for supplying us with the data for the nearly amorphous cellulose and Mr. Pekka Pihkala for making the sample holders used at the USAXS beam line for the liquid samples. Ms. Sabine Cunis and Dr. Rainer Gehrke are thanked for their kind help at the beam line BW4,

Vainio et al.

HASYLAB, Hamburg, and the HASYLAB organization is gratefully acknowledged for giving us the opportunity to do the measurements. The graduate school of the University of Helsinki and the Academy of Finland (Project 1104837) are thanked for financial support. LA048407V