- Email: [email protected]
Probing the lignin nanomechanical properties and lignin–lignin interactions using the atomic force microscopy
19 October 2001
Chemical Physics Letters 347 (2001) 41±45
www.elsevier.com/locate/cplett
Probing the lignin nanomechanical properties and lignin±lignin interactions using the atomic force microscopy Miodrag Micic a,*, Ivan Benitez b,f, Melanie Ruano b,f, Melissa Mavers c,f, Milorad Jeremic d, Ksenija Radotic e, Vincent Moy f, Roger M. Leblanc a,* a
Deptartment of Chemistry, Center for Advanced Microscopy and Center for Supramolecular Sciences, University of Miami, Coral Gables, FL 33124-0431, USA b Department of Biomedical Engineering, School of Engineering, University of Miami, Coral Gables, FL 33146, USA c Department of Microbiology and Immunology, University of Miami, Coral Gables, FL 33146, USA d Faculty of Physical Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, FR, Yugoslavia e Center for Multidisciplinary Studies, University of Belgrade, Slobodana Penezica 53, 11000 Belgrade, FR, Yugoslavia f Department of Physiology and Biophysics, School of Medicine, University of Miami, Miami, FL 33136, USA Received 15 June 2000; in ®nal form 14 June 2001
Abstract By combining atomic force microscopy (AFM) force and environmental scanning electron microscopies (ESEMs), herein we present an evidence for the existence of strong intermolecular forces, which are responsible for holding lignin globules together in higher ordered structures. Based on this observation, we provide a support for the hypothesis that lignin globules consist of at least two individual spherical layers, with space in between ®lled with solvent or gas. Ó 2001 Published by Elsevier Science B.V.
1. Introduction Lignin, the main structural polymer of the higher plant cell wall, is the second most abundand organic matter in the world. In living plant cells, lignin is highly cross-linked with cellulose and other polysaccharides. As during the process of lignin extraction or craft pulping, chemical bonds between lignin and cellulose are broken and lignin and cellulose gets separated; nobody has ever seen unaltered 3D structure of pure lignin. Knowledge of lignin spatial structure
*
Corresponding authors. Fax: +1-305-284-1880. E-mail addresses: [email protected] (M. [email protected] (R.M. Leblanc).
Micic),
is important not only to understand the biological role of lignin in a plant cell, but also to envision its possible future applications such as polymer additives, cosmetics and arti®cial fuels. Very important physical properties of macromolecules, which are closely connected with their shape, are rheological and mechanical properties. More importantly, knowledge of the rheological properties of lignin can be applied toward optimization of the kraft pulping and papermaking process. Based on the results obtained by studying the extracted lignin model compound using the high resolution transmission electron microscopy (TEM) [1], scanning electron microscopy (SEM) [2], environmental scanning electron microscopy (ESEM) [3] and atomic force microscopy (AFM) [4], it is to be believed that lignin probably
0009-2614/01/$ - see front matter Ó 2001 Published by Elsevier Science B.V. PII: S 0 0 0 9 - 2 6 1 4 ( 0 1 ) 0 1 0 2 2 - 3
42
M. Micic et al. / Chemical Physics Letters 347 (2001) 41±45
consists of globular shaped macromolecular assemblies, which then assemble larger semi-ordered superstructures. Based on the gel permeation chromatography [5] and electron spin resonance (ESR) studies of lignin [6,7], it has been indicated that individual lignin globule consists of regions with dierent mobilities, which could be explained in terms of the fragmented structure of an individual globule. Armative results for the above-mentioned hypothesis were recently con®rmed by the scanning tunneling microscopy (STM) [8], and with the computational study of lignin formation [9]. In this Letter, we are examining the lignin model compound, created by the enzymatic polymerization of coniferyl alcohol (DHP), using the contact mode AFM in the force spectroscopic mode. The goal of this research was to obtain mechanical and rheological properties of individual globules of the lignin model compound, and to explore forces of interaction between lignin globules. This objective will answer the question regarding the assembly of semi-ordered structures, as observed by various imaging methods, to be purely a result of random collision of the globules, or if they were orchestrated by the intermolecular forces that hold the lignin superstructure together. Applied methodology has already been successfully demonstrated in the investigation of protein± protein intermolecular forces [10] and in protein unfolding and secondary and quaternary structure studies.
2.2. Substrate preparation A drop of suspension has been placed on the bottom of a Petri dish and let it dry at room temperature. After the lignin ®lm was dried, Petri dish was ®lled out with neutral phosphate buer solution, pH 7.0. 2.3. AFM cantilever functionalization with lignin Unsharpened, Si3 N cantilever tips (Thermomicroscopes, MLCT-AUHW, Sunnywale, CA) were incubated with a lignin suspension during 12 h at 37 °C. Afterwards, the cantilevers were rinsed and dried at 37 °C. No additional treatment has been performed, as a strong adhesion between the tip surface and lignin occurs. The scanning electron micrography in Fig. 1 showed the lignin assemblies bound to the tip surface. 2.4. AFM All force measurements were done using the custom-made atomic force microscope [12], running in the force scan mode. Cantilevers were calibrated by thermal ¯uctuation analysis and had a spring constant of 0:010 N m 1 . Measurements have been done in the neutral phosphate buer solution, pH 7.0, in order to reduce the charging eect artifacts.
2. Materials and methods 2.1. Enzymatically polymerized lignin synthesis A solution of 5 10 3 M coniferyl alcohol, 5 10 3 M H2 O2 and 2:5 10 8 M horseradish peroxidase in 5 10 2 M phosphate buer pH 7.6 (all reagents, Fluka Chem. Corp., NY) has been prepared. Lignin was polymerized in solution and at room temperature, 25 °C. The reaction took 48 h to complete. Water suspension of lignin has been prepared using ultrasound bath (Astrason, Hert Systems, NY) for 60 s. A detailed synthetic procedure can be found in [11].
Fig. 1. Lignin adsorbed onto AFM cantilever tip as observed by SEM. (High vacuum mode, Pd-coated, magni®cation 21 961, accelerating voltage 10.0 kV).
M. Micic et al. / Chemical Physics Letters 347 (2001) 41±45
43
2.5. SEM Functionalized AFM cantilever tips were examined using the FeiCo Phillips-Electroscan FEG XL-30 ®eld emission gun ESEM microscope, in order to con®rm lignin adhesion to the tip surface. Due to high charging eect of the cantilever surface, which induces its bending and destruction, as previously reported in our protein studies [13], we were not capable of obtaining images in the ESEM, wet mode. We were forced to coat the cantilever with the palladium coating in the sputter coater (Anatech, VA) and to run imaging in the high-vacuum, SEM mode. 2.6. ESEM imaging of lignin on mica substrate A drop of lignin suspension has been deposited on the mica substrate. After drying at room temperature, substrates were examined using the ESEM microscope, running in the environmental, wet mode. We used 1 Torr of water pressure as imaging gas. No additional sample treatment has been performed, thus avoiding introduction of possible specimen coating artifacts. 3. Results and discussion Within this study, we have been running two dierent sets of experiments to: (1) examine the mechanical and rheological properties of the individual lignin model compound globular structure; and (2) to explore the existence and nature of interactions between two macromolecules. For the purpose of determination of mechanical and rheological properties of the globule, a force scan with clean cantilever tip has been done. Typical result of such a force scan is presented in Fig. 2. Very small hysteresis indicates strong elastic behavior of the material, which at the higher force is turning visco-elastic. Such behavior is in compliance with the macroscopic observation, and with a molecular mechanics simulation of elastic molecular movements in lignin oligomers under the applied force [14]. With this experiment, we have not observed events which could be described as structure alteration such as unwinding of lignin globules or
3 nN 200 nm
Fig. 2. Typical atomic force scan with non-functionalized, clean AFM tip.
®lament pulling. From the shape of the approach curve, we can only observe a slight existence of the repulsive double-layer electrostatic force combined with the repulsive hydration force. These two forces are responsible for rounding up the shape of curve near the point of contact between the clean tip and lignin surface. Measured adhesion force was in the range 5±7 nN. From the shape of interaction in retraction, we can say that we have adhesion due to van der Waals forces and indentation of sample by tip. Much more interesting results are obtained with the use of a lignin functionalized tip. Some of the characteristic force scans are presented in Fig. 3. During the cantilever approaches, which correspond to compression, we can observe complex interactions in the form of a non-linear shape of the approaching curve. These interactions are attractive in form, and they are responsible for holding the lignin superstructure together. By this shape we can speak about hydrophobic interaction, as we have a gradual pull-o contact. Most probably lignin monomer p±p interactions, and other non-hydrogen bonding are shown in this approach stage. Also, there are nevertheless secondary structure unfolding and reorganization which also contribute to the shape of this part of the approach curve. Non-linear event observed during the approach and retraction of the functionalized cantilever, in the proximity to the substrate, can be described as a fusion of two globules or globule secondary structure alteration by a large deformation followed by fusion. On all force scans we can clearly observe the existence of three cohesion peaks during the cantilever retraction, within the distance which corresponds to the di-
44
M. Micic et al. / Chemical Physics Letters 347 (2001) 41±45
linked secondary structure could unwind under the in¯uence of external stimuli, either as another macromolecule or by mechanical force. The shape of retraction peaks, which indicates jump-o contact, expresses a strong hydrophobic van der Waals and hydrogen bonding kind of interaction. The AFM force scan results, combined with our previous observation by ESEM, of an open-shell lignin globule (Fig. 4), lead us to conclude that the lignin globule consists of at least two layered spherical, `onion-like' structures, which are composed of individual macromolecular subunits. Interior between these shells may be ®lled with water or combined with water and air in order to accommodate hydrophobic and hydrophilic regions of lignin macromolecules. Such an organization of lignin globules may have physiological importance, as it reduces the mass and amount of materials necessary to create the lignin globule, as well as making such a globule act as a dumper in response to outside mechanical stress to the plant cell. Such a structure also supports lignin mediated mechanism of water transport through plants proposed by Laschimke [16] and is in excellent agreement with electron spin resonance observations of the existence of area of dierent mobilities in lignin. Moreover, non-linear shape of peaks indicates complex interactions between lignin macromolecules in the globular assemblies, and the existence of a plethora of small peaks that look Fig. 3. Atomic force scans with the lignin functionalized tip.
ameter of individual lignin globules, in the range 400±800 nm. These peaks with the force intensity range 1±8 nN are repetitive but their exact position and intensity vary. However, their appearance is always the same. Most signi®ciant variation is in the intensity and position of the central peak, which in all scans are smaller than the peripherals. As lignin is the most enthropic macromolecule in terms of randomness of its structure found in nature, we believe that discrepancy in the position and intensity of peaks could be associated by different ways that two lignin macromolecules or macromolecular assemblies can interact between themselves or by many ways the random cross-
Fig. 4. ESEM micrography of the open-shell lignin globular structures. (Wet mode, 0.9 Torr, magni®cation 20 480, accelerating voltage 10.0 kV.)
M. Micic et al. / Chemical Physics Letters 347 (2001) 41±45
45
scale range expresses elastic±viscoelastic properties. Using the lignin-coated AFM tip, we were able to con®rm the existence of strong attractive intermolecular forces between two individual lignin macromolecular globules. However, there is a large non-linearity involved in the process of globule attractions. The existence of three dierent distinctive cohesion peaks within the range of globule diameters indicates that the lignin globule possesses an onion-like, layered structure. Much of the space within the lignin globules is ®lled either with surrounding solvent or gas. Fig. 5. Example of the lignin gigantic supramolecular structures. (Wet mode, 0.9 Torr, magni®cation 25 295, accelerating voltage 10.0 kV.)
like noise indicates ®lamentous pulling±unwinding of the individual fragments from the globule. The existence of intermolecular attractive forces between two lignin globules gives a clue that semiordered higher structures, such as channel and pore-like hexagonal structures (Fig. 5), are not generated by the random collision of individual globules, as earlier suggested ([15] and references cited therein) but are orchestrated by the actions of both hydrophilic and hydrophobic intermolecular attractive forces such as p±p interactions, hydrogen bonding and van der Waals forces. Observed elastic property of lignin and its ability to reshape the supramolecular structure under mutual interaction of globules partially explains lignin's adaptability to grow directionally within geometrically constrained plant cell wall matrix. 4. Conclusions The lignin model compound expresses dierent levels of structural organization. Based on the combined electron microscopy and AFM studies, herein we explore the mechanical properties, intermolecular assemblies' interactions and structure of a lignin model compound. Lignin at the nano-
Acknowledgements We would like to acknowledge Dr. Matthew Lynn, Center for Advanced Microscopy, University of Miami, for his technical assistance. References [1] V.D. Fengel, G. Wegener, J. Feckl, Holzforschung 35 (1981) 111. [2] B. Kosikova, L. Zakutna, D. Joniak, Holzforschung 32 (1978) 15. [3] M. Micic, M. Jeremic, K. Radotic, M. Mavers, R.M. Leblanc, Scanning 22 (2000) 288. [4] S.M. Shevchenko, G.W. Bailey, Y. Shane Yu, L.G. Akim, Tappi 79 (1996) 227. [5] M. Wayman, T.I. Obiaga, Can. J. Chem. 52 (1974) 2102. [6] J.J. Lindberg, I. Bulla, P. Tormala, J. Polym. Sci., Polym. Symp. Edit. 53 (1975) 167. [7] P. Tormala, J.J. Lindberg, S. Lehtinen, Pap. Puu 57 (1975) 601. [8] K. Radotic, J. Simic-Krstic, M. Jeremic, M. Trifunovic, Biophys. J. 66 (1994) 1763. [9] L. Jurasek, J. Pulp Pap. Sci. 21 (1995) J274. [10] E.L. Florin, V.T. Moy, H.E. Gaub, Science 264 (1994) 415. [11] N.G. Lewis, J. Newman, G. Just, J. Ripmeister, Macromolecules 20 (1988) 1752. [12] V.T. Moy, E.L. Florin, H.E. Gaub, Science 265 (1994) 257. [13] M. Micic, A. Chen, R.M. Leblanc, V.T. Moy, Scanning 21 (1999) 394. [14] J.P. Simon, K.E.L. Erikson, Tappi 83 (2000) 87. [15] S.I. Falkehag, Appl. Polym. Symp. 28 (1975) 247. [16] R. Laschimke, Thermochim. Acta 151 (1989) 35.
Chemical Physics Letters 347 (2001) 41±45
www.elsevier.com/locate/cplett
Probing the lignin nanomechanical properties and lignin±lignin interactions using the atomic force microscopy Miodrag Micic a,*, Ivan Benitez b,f, Melanie Ruano b,f, Melissa Mavers c,f, Milorad Jeremic d, Ksenija Radotic e, Vincent Moy f, Roger M. Leblanc a,* a
Deptartment of Chemistry, Center for Advanced Microscopy and Center for Supramolecular Sciences, University of Miami, Coral Gables, FL 33124-0431, USA b Department of Biomedical Engineering, School of Engineering, University of Miami, Coral Gables, FL 33146, USA c Department of Microbiology and Immunology, University of Miami, Coral Gables, FL 33146, USA d Faculty of Physical Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, FR, Yugoslavia e Center for Multidisciplinary Studies, University of Belgrade, Slobodana Penezica 53, 11000 Belgrade, FR, Yugoslavia f Department of Physiology and Biophysics, School of Medicine, University of Miami, Miami, FL 33136, USA Received 15 June 2000; in ®nal form 14 June 2001
Abstract By combining atomic force microscopy (AFM) force and environmental scanning electron microscopies (ESEMs), herein we present an evidence for the existence of strong intermolecular forces, which are responsible for holding lignin globules together in higher ordered structures. Based on this observation, we provide a support for the hypothesis that lignin globules consist of at least two individual spherical layers, with space in between ®lled with solvent or gas. Ó 2001 Published by Elsevier Science B.V.
1. Introduction Lignin, the main structural polymer of the higher plant cell wall, is the second most abundand organic matter in the world. In living plant cells, lignin is highly cross-linked with cellulose and other polysaccharides. As during the process of lignin extraction or craft pulping, chemical bonds between lignin and cellulose are broken and lignin and cellulose gets separated; nobody has ever seen unaltered 3D structure of pure lignin. Knowledge of lignin spatial structure
*
Corresponding authors. Fax: +1-305-284-1880. E-mail addresses: [email protected] (M. [email protected] (R.M. Leblanc).
Micic),
is important not only to understand the biological role of lignin in a plant cell, but also to envision its possible future applications such as polymer additives, cosmetics and arti®cial fuels. Very important physical properties of macromolecules, which are closely connected with their shape, are rheological and mechanical properties. More importantly, knowledge of the rheological properties of lignin can be applied toward optimization of the kraft pulping and papermaking process. Based on the results obtained by studying the extracted lignin model compound using the high resolution transmission electron microscopy (TEM) [1], scanning electron microscopy (SEM) [2], environmental scanning electron microscopy (ESEM) [3] and atomic force microscopy (AFM) [4], it is to be believed that lignin probably
0009-2614/01/$ - see front matter Ó 2001 Published by Elsevier Science B.V. PII: S 0 0 0 9 - 2 6 1 4 ( 0 1 ) 0 1 0 2 2 - 3
42
M. Micic et al. / Chemical Physics Letters 347 (2001) 41±45
consists of globular shaped macromolecular assemblies, which then assemble larger semi-ordered superstructures. Based on the gel permeation chromatography [5] and electron spin resonance (ESR) studies of lignin [6,7], it has been indicated that individual lignin globule consists of regions with dierent mobilities, which could be explained in terms of the fragmented structure of an individual globule. Armative results for the above-mentioned hypothesis were recently con®rmed by the scanning tunneling microscopy (STM) [8], and with the computational study of lignin formation [9]. In this Letter, we are examining the lignin model compound, created by the enzymatic polymerization of coniferyl alcohol (DHP), using the contact mode AFM in the force spectroscopic mode. The goal of this research was to obtain mechanical and rheological properties of individual globules of the lignin model compound, and to explore forces of interaction between lignin globules. This objective will answer the question regarding the assembly of semi-ordered structures, as observed by various imaging methods, to be purely a result of random collision of the globules, or if they were orchestrated by the intermolecular forces that hold the lignin superstructure together. Applied methodology has already been successfully demonstrated in the investigation of protein± protein intermolecular forces [10] and in protein unfolding and secondary and quaternary structure studies.
2.2. Substrate preparation A drop of suspension has been placed on the bottom of a Petri dish and let it dry at room temperature. After the lignin ®lm was dried, Petri dish was ®lled out with neutral phosphate buer solution, pH 7.0. 2.3. AFM cantilever functionalization with lignin Unsharpened, Si3 N cantilever tips (Thermomicroscopes, MLCT-AUHW, Sunnywale, CA) were incubated with a lignin suspension during 12 h at 37 °C. Afterwards, the cantilevers were rinsed and dried at 37 °C. No additional treatment has been performed, as a strong adhesion between the tip surface and lignin occurs. The scanning electron micrography in Fig. 1 showed the lignin assemblies bound to the tip surface. 2.4. AFM All force measurements were done using the custom-made atomic force microscope [12], running in the force scan mode. Cantilevers were calibrated by thermal ¯uctuation analysis and had a spring constant of 0:010 N m 1 . Measurements have been done in the neutral phosphate buer solution, pH 7.0, in order to reduce the charging eect artifacts.
2. Materials and methods 2.1. Enzymatically polymerized lignin synthesis A solution of 5 10 3 M coniferyl alcohol, 5 10 3 M H2 O2 and 2:5 10 8 M horseradish peroxidase in 5 10 2 M phosphate buer pH 7.6 (all reagents, Fluka Chem. Corp., NY) has been prepared. Lignin was polymerized in solution and at room temperature, 25 °C. The reaction took 48 h to complete. Water suspension of lignin has been prepared using ultrasound bath (Astrason, Hert Systems, NY) for 60 s. A detailed synthetic procedure can be found in [11].
Fig. 1. Lignin adsorbed onto AFM cantilever tip as observed by SEM. (High vacuum mode, Pd-coated, magni®cation 21 961, accelerating voltage 10.0 kV).
M. Micic et al. / Chemical Physics Letters 347 (2001) 41±45
43
2.5. SEM Functionalized AFM cantilever tips were examined using the FeiCo Phillips-Electroscan FEG XL-30 ®eld emission gun ESEM microscope, in order to con®rm lignin adhesion to the tip surface. Due to high charging eect of the cantilever surface, which induces its bending and destruction, as previously reported in our protein studies [13], we were not capable of obtaining images in the ESEM, wet mode. We were forced to coat the cantilever with the palladium coating in the sputter coater (Anatech, VA) and to run imaging in the high-vacuum, SEM mode. 2.6. ESEM imaging of lignin on mica substrate A drop of lignin suspension has been deposited on the mica substrate. After drying at room temperature, substrates were examined using the ESEM microscope, running in the environmental, wet mode. We used 1 Torr of water pressure as imaging gas. No additional sample treatment has been performed, thus avoiding introduction of possible specimen coating artifacts. 3. Results and discussion Within this study, we have been running two dierent sets of experiments to: (1) examine the mechanical and rheological properties of the individual lignin model compound globular structure; and (2) to explore the existence and nature of interactions between two macromolecules. For the purpose of determination of mechanical and rheological properties of the globule, a force scan with clean cantilever tip has been done. Typical result of such a force scan is presented in Fig. 2. Very small hysteresis indicates strong elastic behavior of the material, which at the higher force is turning visco-elastic. Such behavior is in compliance with the macroscopic observation, and with a molecular mechanics simulation of elastic molecular movements in lignin oligomers under the applied force [14]. With this experiment, we have not observed events which could be described as structure alteration such as unwinding of lignin globules or
3 nN 200 nm
Fig. 2. Typical atomic force scan with non-functionalized, clean AFM tip.
®lament pulling. From the shape of the approach curve, we can only observe a slight existence of the repulsive double-layer electrostatic force combined with the repulsive hydration force. These two forces are responsible for rounding up the shape of curve near the point of contact between the clean tip and lignin surface. Measured adhesion force was in the range 5±7 nN. From the shape of interaction in retraction, we can say that we have adhesion due to van der Waals forces and indentation of sample by tip. Much more interesting results are obtained with the use of a lignin functionalized tip. Some of the characteristic force scans are presented in Fig. 3. During the cantilever approaches, which correspond to compression, we can observe complex interactions in the form of a non-linear shape of the approaching curve. These interactions are attractive in form, and they are responsible for holding the lignin superstructure together. By this shape we can speak about hydrophobic interaction, as we have a gradual pull-o contact. Most probably lignin monomer p±p interactions, and other non-hydrogen bonding are shown in this approach stage. Also, there are nevertheless secondary structure unfolding and reorganization which also contribute to the shape of this part of the approach curve. Non-linear event observed during the approach and retraction of the functionalized cantilever, in the proximity to the substrate, can be described as a fusion of two globules or globule secondary structure alteration by a large deformation followed by fusion. On all force scans we can clearly observe the existence of three cohesion peaks during the cantilever retraction, within the distance which corresponds to the di-
44
M. Micic et al. / Chemical Physics Letters 347 (2001) 41±45
linked secondary structure could unwind under the in¯uence of external stimuli, either as another macromolecule or by mechanical force. The shape of retraction peaks, which indicates jump-o contact, expresses a strong hydrophobic van der Waals and hydrogen bonding kind of interaction. The AFM force scan results, combined with our previous observation by ESEM, of an open-shell lignin globule (Fig. 4), lead us to conclude that the lignin globule consists of at least two layered spherical, `onion-like' structures, which are composed of individual macromolecular subunits. Interior between these shells may be ®lled with water or combined with water and air in order to accommodate hydrophobic and hydrophilic regions of lignin macromolecules. Such an organization of lignin globules may have physiological importance, as it reduces the mass and amount of materials necessary to create the lignin globule, as well as making such a globule act as a dumper in response to outside mechanical stress to the plant cell. Such a structure also supports lignin mediated mechanism of water transport through plants proposed by Laschimke [16] and is in excellent agreement with electron spin resonance observations of the existence of area of dierent mobilities in lignin. Moreover, non-linear shape of peaks indicates complex interactions between lignin macromolecules in the globular assemblies, and the existence of a plethora of small peaks that look Fig. 3. Atomic force scans with the lignin functionalized tip.
ameter of individual lignin globules, in the range 400±800 nm. These peaks with the force intensity range 1±8 nN are repetitive but their exact position and intensity vary. However, their appearance is always the same. Most signi®ciant variation is in the intensity and position of the central peak, which in all scans are smaller than the peripherals. As lignin is the most enthropic macromolecule in terms of randomness of its structure found in nature, we believe that discrepancy in the position and intensity of peaks could be associated by different ways that two lignin macromolecules or macromolecular assemblies can interact between themselves or by many ways the random cross-
Fig. 4. ESEM micrography of the open-shell lignin globular structures. (Wet mode, 0.9 Torr, magni®cation 20 480, accelerating voltage 10.0 kV.)
M. Micic et al. / Chemical Physics Letters 347 (2001) 41±45
45
scale range expresses elastic±viscoelastic properties. Using the lignin-coated AFM tip, we were able to con®rm the existence of strong attractive intermolecular forces between two individual lignin macromolecular globules. However, there is a large non-linearity involved in the process of globule attractions. The existence of three dierent distinctive cohesion peaks within the range of globule diameters indicates that the lignin globule possesses an onion-like, layered structure. Much of the space within the lignin globules is ®lled either with surrounding solvent or gas. Fig. 5. Example of the lignin gigantic supramolecular structures. (Wet mode, 0.9 Torr, magni®cation 25 295, accelerating voltage 10.0 kV.)
like noise indicates ®lamentous pulling±unwinding of the individual fragments from the globule. The existence of intermolecular attractive forces between two lignin globules gives a clue that semiordered higher structures, such as channel and pore-like hexagonal structures (Fig. 5), are not generated by the random collision of individual globules, as earlier suggested ([15] and references cited therein) but are orchestrated by the actions of both hydrophilic and hydrophobic intermolecular attractive forces such as p±p interactions, hydrogen bonding and van der Waals forces. Observed elastic property of lignin and its ability to reshape the supramolecular structure under mutual interaction of globules partially explains lignin's adaptability to grow directionally within geometrically constrained plant cell wall matrix. 4. Conclusions The lignin model compound expresses dierent levels of structural organization. Based on the combined electron microscopy and AFM studies, herein we explore the mechanical properties, intermolecular assemblies' interactions and structure of a lignin model compound. Lignin at the nano-
Acknowledgements We would like to acknowledge Dr. Matthew Lynn, Center for Advanced Microscopy, University of Miami, for his technical assistance. References [1] V.D. Fengel, G. Wegener, J. Feckl, Holzforschung 35 (1981) 111. [2] B. Kosikova, L. Zakutna, D. Joniak, Holzforschung 32 (1978) 15. [3] M. Micic, M. Jeremic, K. Radotic, M. Mavers, R.M. Leblanc, Scanning 22 (2000) 288. [4] S.M. Shevchenko, G.W. Bailey, Y. Shane Yu, L.G. Akim, Tappi 79 (1996) 227. [5] M. Wayman, T.I. Obiaga, Can. J. Chem. 52 (1974) 2102. [6] J.J. Lindberg, I. Bulla, P. Tormala, J. Polym. Sci., Polym. Symp. Edit. 53 (1975) 167. [7] P. Tormala, J.J. Lindberg, S. Lehtinen, Pap. Puu 57 (1975) 601. [8] K. Radotic, J. Simic-Krstic, M. Jeremic, M. Trifunovic, Biophys. J. 66 (1994) 1763. [9] L. Jurasek, J. Pulp Pap. Sci. 21 (1995) J274. [10] E.L. Florin, V.T. Moy, H.E. Gaub, Science 264 (1994) 415. [11] N.G. Lewis, J. Newman, G. Just, J. Ripmeister, Macromolecules 20 (1988) 1752. [12] V.T. Moy, E.L. Florin, H.E. Gaub, Science 265 (1994) 257. [13] M. Micic, A. Chen, R.M. Leblanc, V.T. Moy, Scanning 21 (1999) 394. [14] J.P. Simon, K.E.L. Erikson, Tappi 83 (2000) 87. [15] S.I. Falkehag, Appl. Polym. Symp. 28 (1975) 247. [16] R. Laschimke, Thermochim. Acta 151 (1989) 35.