Formation and Characterization of Spread Lignin Layers at the Air

UMR de Chimie Biologique, INRA/INA PG, 78850 Thiverval Grignon, France. Received December 7 ... they are not chemically modified at the air/water inte...
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Langmuir 2002, 18, 5190-5196

Formation and Characterization of Spread Lignin Layers at the Air/Water Interface Ve´ronique Aguie´-Be´ghin,*,† Ste´phanie Baumberger,‡ Bernard Monties,† and Roger Douillard† Equipe Parois Ve´ ge´ tales et Mate´ riaux Fibreux, UMR-FARE INRA/URCA, Centre de Recherche Agronomique, 2 Esplanade Roland Garros, BP 224, 51686 Reims Cedex 2, France, and UMR de Chimie Biologique, INRA/INA PG, 78850 Thiverval Grignon, France Received December 7, 2001. In Final Form: March 25, 2002 The structure and surface properties of spread layers of lignins at the air/water interface have been studied by neutron reflectivity, spectroscopic ellipsometry, and static and dynamic tensiometry. The layers hold 70% water, and the surface concentration of lignin measured by neutron reflectivity is less than the amount deposited. When the spread amount increases from 1 to 16 mg m-2, 65-12.5% of the deposited lignin is recovered at the interface. The absolute value of the Brewster ellipticity decreases slowly after spreading and tends toward quasi-equilibrium after 30 h, a fact which could be explained by a slow desorption from the interfacial layer to the bulk. After a compression of the interfacial layers, the surface pressure and the Brewster ellipticity exhibit a strong relaxation due, at least in part, to a desorption. However, the diffusion of lignin molecules from the layer to the substrate is limited because at equilibrium the neutron reflectivity and ellipsometry data show that a significant layer covers the interface. Thus, the desorption is limited as shown also by the large values of the dilational modulus at quasi-equilibrium, suggesting that large interactions occur between adsorbed molecules at the interface. The refractive index and extinction coefficient spectra of lignin layers were calculated by a point by point numerical resolution of the ellipsometric data assuming isotropic and homogeneous layers and using the thickness obtained by neutron reflectivity. The absorption coefficient spectrum calculated from the extinction coefficient of the interfacial layer compares well with the bulk absorption spectrum of lignin solutions in a dioxane/water mixture. Thus, spectroscopic ellipsometry measurements are convenient for the characterization of the adsorbed lignins and show that they are not chemically modified at the air/water interface.

Introduction Lignin is the second most abundant biopolymer in plant cell walls, after cellulose.1 It is a very complex natural aromatic polymer, based on phenylpropane monomers, which are phenylalanine derivatives.2 Phenylpropane monomers are cross-linked through many bond types such as C-C and C-O-C and form a large variety of threedimensional structures.2-5 These molecules exhibit fewer hydroxyl groups than cellulose and hemicellulose and are considered to be “hydrophobic”. They interact with polysaccharides to form complexes in the cell walls with improved mechanical properties and resistance to microbiological or chemical treatments.2 The knowledge of physical properties and solid-state organization of extracted and model lignins was improved by physical techniques in solution and at the air/water interface.6-12 Light scattering * Corresponding author. Tel: (33) 3 26 77 35 95. Fax: (33) 03 26 77 35 99. E-mail: [email protected]. † Equipe Parois Ve ´ ge´tales et Mate´riaux Fibreux, UMR-FARE INRA/URCA, Centre de Recherche Agronomique. ‡ UMR de Chimie Biologique, INRA/INA PG. (1) Monties, B.; Fukushima, K. In Biopolymers; Steinbu¨chel, A., Ed.; Wiley: New York, 2001; Vol. 1, pp 1-64. (2) Whetten, R.; Sederoff, R. Plant Cell 1995, 7, 1001-1013. (3) Monties, B. Ann. Sci. For. 1989, 46, 848-855. (4) Shigematsu, M.; Morita, M.; Sakata, I. Makromol. Chem. 1992, 193, 133-142. (5) Campbell, M. M.; Sederoff, R. R. Plant Physiol. 1996, 110, 3-13. (6) Luner, P.; Kempf, U. Tappi 1970, 53, 2069-2076. (7) Luner, P.; Roseman, G. Holzforschung 1986, 40, 61-66. (8) Gilardi, G.; Cass, A. E. Langmuir 1993, 9, 1721-1726. (9) Constantino, C. J. L.; Juliani, L. P.; Botaro, V. R.; Balogh, D. T.; Pereira, M. R.; Ticianelli, E. A.; Curvelo, A. A. S.; Oliviera, J. Thin Solid Films 1996, 191, 284-285. (10) Oliviera, O. N. J.; Constantino, C. J. L.; Balogh, D. T.; Curvelo, A. A. S. Cellul. Chem. Technol. 1994, 28, 541-549.

measurements have shown that organosolve spruce lignin in dioxane-water (5/1, v/v) can form aggregates with a large size polydispersity at concentrations above 0.5 g L-1, which would correspond to the critical micellar concentration in aqueous solutions.8 Luner and his collaborators6,7 showed that lignins can form interfacial layers at the air/aqueous solution interface. Significant hysteresis is observed when the monolayer is compressed beyond a certain pressure, and the hysteresis energy varies with the nature of the lignin sample according to its affinity for water. At zero surface pressure, the area occupied by a monomer lies between 12 and 17 Å and the thickness of the film is in the range between 12 and 18 Å in Langmuir-Blodgett monolayers, a value which corresponds approximatively to the height of a phenyl propane unit (≈11 Å).6 In Langmuir-Blodgett films, it was shown by measurement of the refractive index by ellipsometry that the films with a thickness of 60 Å possess a large free volume, a fact which suggests that lignin molecules assume a three-dimensional network incorporating a large proportion of solvent.9 Recent results with models of lignins (DHPs, dehydrogenation polymers) spread at the air/water interface show that the layer has a nonhomogeneous DHP distribution going from a dense structure at the air side to a dilute one at the water side and that this distribution is a function of the molecular structure of the DHPs.12 Thus, it was suggested that in the plant cell wall, the chemical composition of lignins has an important role in the control of the water content. However, the spreading (11) Cathala, B.; Aguie´-Be´ghin, V.; Douillard, R.; Monties, B. Polym. Degrad. Stab. 1997, 59, 77-80. (12) Cathala, B.; Lee, L. T.; Aguie´-Be´ghin, V.; Douillard, R.; Monties, B. Langmuir 2000, 16, 10444-10448.

10.1021/la011766v CCC: $22.00 © 2002 American Chemical Society Published on Web 05/21/2002

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procedure of DHPs is not quantitative and the conclusions cannot be completely unequivocal. First, the aim of this study is to explain the supramolecular organization of lignin in an anisotropic environment like the air/water interface which may be taken as a model of contact areas between macromolecular layers in the plant cell wall. Second, the spreading of cell wall extracted lignin at the air/water interface is tentatively quantitated. The characterization of lignin layers after spreading is assessed by measurement of the concentration profile and of the optical and the rheological properties. Material and Methods Lignin Preparation and Characterization. Lignin fractions were extracted under reflux by hydrochloric dioxane from wheat straw13 (Triticum aestivum) (LD) and milled wild cherry wood after ultragrinding and extraction in dioxane-water14 (Prunus avium L.) (LM). The extracted lignins were freeze-dried and dissolved in a deuterium oxide/dioxane mixture (1/9, v/v) for the reflectivity measurements and in a water/dioxane mixture (1/9, v/v) for the ellipsometric and Langmuir trough experiments. Deuterium oxide (99.9% pure) was obtained from Eurisotop (CEA, Saclay, France), hydrogenated dioxane (99.5% pure) was obtained from SDS (France), and ultrapure water with a resistivity of 0.055 Ω-1 cm-1 was prepared using a Maxima-HPLC (USFELGA, France). It was verified that deuterium oxide, dioxane, and ultrapure water do not contain surface active impurities. The main chemical properties of the fractions were determined previously and are summarized in Table 1. Neutron Reflectivity. Neutron reflectivity, R, at the air/ liquid interface was determined as a function of the wave vector, q ) (4π sin θ/λ). The range of q was determined by the range of wavelength, λ (polychromatic source). R is the ratio of the intensity of the specularly reflected beam to the intensity of the incident beam. The reflectivity is a function of the refractive index profile of the interfacial region.15 The interpretation of the experimental data is achieved by comparing them with the calculated reflectivity of a model where the refractive index profile, n(z), in the direction z perpendicular to the interface plane is defined by a number of uniform layers. The Fresnel reflection and transmission coefficients are calculated for each interface and combined to give the total reflectivity of the model adsorption layer.16 The refractive index, n(z), is related to the scattering length density, Nb(z), by15

n(z) ) 1 - (λ2 Nb(z))/2π

Table 1. Chemical and Structural Characteristics of Lignin Samples (References 13 and 14)a lignin fraction LD elementary compositionb Mnc Mwc empiric formulad densitye (g cm-3) Nbf (10-6 Å-2) (one-proton exchange) Nbf (10-6 Å-2) (two-proton exchange) sugar contentg total esterified phenolic acidsh absorption coefficienti (L g-1 cm-1) thioacidolysis yieldj (µmol g-1 lignin) relative molar ratio of S/Gk

C, 59.2%; H, 6.1%; O, 31.7% 3600 21000 {C9H9O2(OCH3)1-2}n 1.419 2.597

LM

1.41

3.046 3 wt % 3.5 wt %

n.d.

20.0

11.0

726 1.13

1.78

a

LD, lignin fraction extracted from wheat straw; LM, lignin fraction extracted from milled wild cherry wood. b Determined by the “Service Central d’Analyse” of the Centre National de la Recherche Scientifique (Grenoble, France). c Mn, number-average molecular weight, is determined by high-performance size exclusion chromatography. Mw, weight-average molecular weight, is determined by an ultracentrifuge method. d The 1-2 index specifies the number of methoxylated groups on the monomeric units of lignins. One methoxylated group at position 3 of the aromatic cycle corresponds to the guaiacyl monomer (G), and two methoxylated groups at positions 3 and 5 of the aromatic cycle correspond to the syringyl monomer (S). In natural lignin, there is a mixture of G and S monomers. e Average of the density of lignins extracted in dioxane (ref 6). f Nb is the coherent scattering length density of lignin taking into account the isotopic exchange of one and two mobile protons. g Total oligo- and polysaccharides linked to lignin, determined by the colorimetric phenol sulfuric method. h Ferulic and p-coumaric acids released during alkaline hydrolysis. n.d., not detected. i The absorption coefficient is determined in the dioxane/water mixture (9/1, v/v) at 280 nm. j C6C3 monomers (µmoles) released by cleavage of C-O-C ether links per gram of lignin. This relatively high value reflects the low degradation level of lignin during the chemical extraction process. k The relative molar ratio shows the relative amounts of syringyl and guaiacyl monomeric units of extracted lignins.

(1)

when Φ(z) is the volume fraction of the adsorbed molecule.

Experiments have been performed at the “time of flight” reflectometer DESIR in the Orphe´e reactor (Le´on Brillouin Laboratory, Saclay) at a grazing incidence angle of 1.317°. The useful neutron wavelength ranged from 3 to 18 Å. The reflectivity measurements were performed using a Teflon trough, 6.5 × 13.5 × 0.3 cm, in a cell thermostated at 20 °C enclosed in a second cell which helps to keep constant the temperature of the air surrounding the first cell. The trough was filled up with 8 mL of substrate which is pure deuterium oxide (D2O) with a scattering length density of 6.37 × 10-6 Å-2. According to its chemical composition (Table 1), the lignin preparation is mainly composed by guaiacyl (G) and syringyl (S) units of C9H9O2(OCH3) and C9H9O2(OCH3)2 with one or two exchangeable protons. Thus, taking into account isotopic exchange of one or two mobile protons, the scattering length density of lignin was in the range of 2.6 × 10-6 to 3.05 × 10-6 Å-2. Surface layers were formed by droplet deposition (1 µL) of solubilized lignin on the surface of pure deuterium oxide, so that the deposited surface concentration ranges from 1 to 16 mg m-2. Neutron reflectivity spectra were recorded as 2 h runs. The first run is slightly different from the others. A good statistics was obtained by combining three successive runs (a 6 h measurement). Background noise was determined from each spectrum between 2 and 3 Å and was of

(13) Baumberger, S.; Aguie´-Be´ghin, V.; Douillard, R.; Lapierre, C.; Monties, B. Ind. Crops Prod. 1997, 6, 259-263. (14) Tollier, M. T.; Monties, B.; Lapierre, C.; du Penhoat, H.; Rolando, C. Holzforschung 1986, 40, 75-79.

(15) Penfold, J.; Thomas, R. K. J. Phys.: Condens. Matter 1990, 2, 1369-1412. (16) Lee, L. T.; Mann, E. K.; Langevin, D.; Farnoux, B. Langmuir 1991, 7, 3076-3080.

where N is the atomic number density and b is the average coherent scattering length. The product Nb is the scattering length density of the medium. Since the scattering lengths of hydrogen and deuterium have opposite signs, it is possible to obtain large differences of refractive index between the deuterated solvent and adsorbed hydrogenated molecules in the interface layer. According to Snell’s law and eq 1, θc defines the critical wavelength, λc, beyond which total reflection occurs,

λc ) θc(π/Nb)1/2

(2)

and consequently the critical transfer wave vector, qc (qc ) 4(πNb)1/2). Thus, the reflectivity, R, is analyzed below λc. With the values of the scattering length densities of the solvent, Nbs, and of the adsorbed macromolecule, Nbm, the composition of each layer can be deduced from

Nb(z) ) Φ(z) (Nbm) + [1 - Φ(z)](Nbs)

(3)

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Aguie´ -Be´ ghin et al.

the order of 1 count s-1 . It was assumed constant over the whole spectrum and subtracted before reflectivity calculation. Ellipsometry. Ellipsometry is one of the most sensitive techniques to measure the optical properties of interfacial thin layers.17,18 It measures the change of polarization between the incident and the reflected beams, leading to the determination of the complex ellipticity, F, ratio of the Fresnel reflection coefficients, r// and r⊥. r// is the ratio of the electric field amplitudes parallel to the incidence plane after and before reflection of light. r⊥ is the same ratio for light with the electric field perpendicular to the plane of incidence:

F ) r///r⊥ ) tan Ψ ei∆ ) F˜ + iFj

(4)

The ellipsometric angles Ψ limited to the range [0, π/2] and ∆ in the range [0, 2π] determine the changes in amplitude and phase of the electric field. F˜ and Fj are the real and imaginary parts of the complex ellipticity F. From Fresnel coefficients and Snell’s law and when reflection occurs at an ideal interface between two media 0 and 1 with refractive indexes n0 and n1, one gets

n1 ) n0 sin θ0 [1 + ((1 - F)/(1 + F))2 tan2 θ0]1/2

(5)

where θ0 is the angle of incidence. At a particular angle θB (Brewster angle), the reflection of the wave with the electric field parallel to the incidence plane is null, F ) 0. From eq 5, θB is defined as

tan θB ) n1/n0

(6)

The complex ellipticity measured at this angle is very sensitive to any structure present at the interface. Experimentally, the coefficient r// is not zero and the ellipticity passes through a positive minimum value which is due to the roughness of the interface (capillary waves on a liquid surface for instance). In the case where an adsorption layer occurs with a small thickness, d, such as d , λ, at the Brewster angle, the coefficient of ellipticity, FjB, is equal to

FjB ≈ Fjd ≈ tan Ψ sin ∆

(7)

where Fjd is the negative contribution of the adsorption layer. The sign ≈ means that the roughness and the anisotropy are negligible compared to the contribution of the adsorption layer.19-21 The propagation of the wave in an isotropic absorbing medium is described by the complex index of refraction, N ) n - ik, where n is the refractive index and k is the extinction coefficient of the medium. The wave amplitude (A) of the elliptic electric field decays exponentially along the direction of propagation:18

A(d) ) A(0) e-2πkd/λ

(8)

where A(0) is the wave amplitude at z ) 0 and A(d) is that at z ) d. Since the intensity of the wave, I, is equal to A2, one gets

I(d) ) A2(0) e-Rd

(9)

R ) 4πk/λ

(10)

where

is called the absorption coefficient (cm-1).22 In a three-phase (air-layer-substrate) system, the measured Ψ and ∆ quantities (or equivalently, F) are determined by the parameters of the system: (17) Drude, P. Ann. Phys. Chem. (Leipzig) 1891, 43, 126-157. (18) Azzam, R. M. A.; Bashara, N. M. Ellipsometry and Polarized Light; North-Holland: Amsterdam, 1987. (19) Meunier, J. J. Phys. 1987, 48, 1819-1831. (20) Manning-Benson, S.; Bain, C. D.; Darton, R. C. J. Colloid Interface Sci. 1997, 189, 109-116. (21) Mann, E. K.; Lee, L. T.; He´non, S.; Langevin, D.; Meunier, J. Macromolecules 1993, 26, 7037-7045. (22) Muller, R. H. Surf. Sci. 1969, 16, 14-33.

tan Ψ ei∆ ) F (N0, N1, N2, d1, θ0, λ)

(11)

where N0, N1, and N2 are the complex indexes of refraction of air, of the interfacial layer, and of the substrate, d1 is the thickness of the adsorption layer, and θ0 is the angle of incidence in air. Air and the substrate are transparent, and N0 and N2 are real. A solution of eq 11 may be obtained for n1 and k by a numerical resolution at each λ using a value of d1 determined otherwise. All of the measurements have been performed using a spectroscopic phase modulated ellipsometer (Uvisel, Jobin Yvon, Arpajon, France). It is equipped with a xenon arc lamp, a polarizer and an analyzer which are set to the 45° configuration angle. A photoelastic modulator, activated at a resonance frequency of 50 kHz, is set to the 0° orientation configuration. This configuration allows the measurements of sin 2Ψ and ∆ simultaneously at each wavelength. The spectroscopic measurements were monitored between 240 and 820 nm, and the angle of incidence was 53°4. The fixed wavelength chosen for the kinetics measurements in the Brewster conditions of the substrate was 413 nm. All measurements were done at the air/water interface using a glass trough in a closed cell and in an air-conditioned room at 20 °C. The trough was filled up with 20 mL of substrate, and the surface layer was formed by droplet deposition as for neutron reflectivity measurements. The spectra were measured in quasi-equilibrium conditions after spreading. The absorption coefficent, R, was calculated (eq 10). The surface concentration (Γ) was calculated according to de Feijter23 (Γ ) (nlayer - nsubstrate)d/(dn/dc)) where d is the thickness of the interfacial layer and a specific refractive index increment for lignin, dn/dc, of 0.173 was determined in a concentration range between 0.4 and 1.6 g L-1 in dioxane/water (9/1) using a Dawn DSP differential refractometer (Wyatt Technology, USA). Langmuir Trough Experiments. Surface layers were formed by the method used for neutron reflectometry or for ellipsometry measurements. The trough was 6.5 × 48 × 0.5 cm. Surface pressure was measured at 20 ( 1 °C with a Wilhelmy plate. The compression/relaxation isotherms were obtained by moving a barrier with a rate proportional to the surface in such a way that τ (τ ) -∆t/∆Ln(A), where A is the surface area) was kept to 2600 s during the compression where the surface area was reduced by 50%.13 Relaxation of the surface pressure was monitored during 20 h after the end of the compression. The dilational modulus, , was measured after 3 h of relaxation by oscillating the barrier with an amplitude of 4 mm at a frequency of 17 mHz and calculated according to  ) -dπ/d ln(A).24 The simultaneous measurements of the surface tension and of the coefficient of ellipticity were performed using a Teflon trough, 7.5 × 36.3 × 0.6 cm (KSV, Finland), with two moving barriers. The maximum experimental surface area was 272 cm2.

Results and Discussion 1. Kinetics of Surface Properties after Spreading. The properties of the spread layers were analyzed after their formation by neutron reflectivity, by ellipsometry, and by surface tension measurements. All three methods clearly evidence a time course of surface properties evolution. The absolute value of the Brewster ellipticity decreases rapidly in the first hour and then progressively stabilizes although a true equilibrium situation is probably not reached before 60-70 h (Figure 1a). These signal variations may be explained by (i) a release of lignin from the layer (desorption), (ii) a change of the refractive index of the molecules in the layer (due to oxidation for instance), or (iii) a change of the spread layer structure (modification of the refractive index profile). The surface pressure measured in the same conditions also shows a fast evolution during the first half hour and a slow decrease thereafter which does not level off before (23) De Feijter, J. A.; Benjamins, J.; Veer, F. A. Biopolymers 1978, 17, 1759-1772. (24) Lucassen, J.; van den Tempel, M. Chem. Eng. Sci. 1971, 27, 1283-1291.

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Figure 2. Reflectivity spectra of a spread lignin layer at the air/water interface. The amount of deposited lignin was 8 mg m-2. Reflectivity spectrum of the substrate (without lignin) (- - -). Reflectivity spectrum with a lignin layer (O) measured between 2 and 8 h after spreading. Adjusted reflectivity spectrum (s) using the one-layer model.

Figure 1. Ellipticity (a) and surface pressure (b) kinetics of a lignin layer at the air/water interface after spreading. The amount of spread lignin was 4 mg m-2; ellipsometry measurements were performed in Brewster conditions. The inset of (a) shows the total kinetics.

10 h (Figure 1b). These data may also be due to (i) a desorption of molecules from the interface or to (ii) a kind of aggregation process which may reduce the number of independent objects in the interfacial layer. However, the neutron reflectivity data show that during the first 2 h, the calculated surface concentration is not very different from that obtained in the next 2 h measurements. This may be due to the weak resolution of the neutron reflectivity method which does not allow observation of weak and fast modifications of the structure of the layer. Thus, the ellipsometric and surface pressure results may indicate (i) most likely a larger surface concentration during the first run and consequently a desorption occurring mostly during that period, (ii) a change of the concentration profile, or both. After 2 h, desorption does not seem to be a major phenomenon. Putting together the results of these two surface technique measurements, it seems likely that a significant desorption of lignin occurs in the first hour and that phenomena such as (i) concentration profile change, (ii) chemical modification of the molecules (oxidation), or (iii) aggregation happen then during a time course of several hours or tens of hours. A characterization of the quasiequilibrium properties of the interfacial layers may afford arguments in favor (or not) of these hypotheses. 2. Equilibrium Properties of Lignin Layers. Structure and Surface Concentration of the Interfacial Layer. Neutron reflectivity spectra were performed for deposits ranging from 1 to 16 mg m-2 and were obtained in the q range from 0.016 to 0.1 Å-1. A typical reflectivity spectrum shows a large deviation from the Fresnel reflectivity curve and can be fitted by a one-layer model (Figure 2 and Table 2). A model with two layers does not improve significantly the χ2 of the adjustment, except for the 16 mg m-2 spread amount (Table 3). The volume fraction of lignin in the layers is around 0.3-0.4 for all concentrations (Tables 2 and 3). In the case of the 16 mg m-2 lignin deposit, the water side layer has a very low volume fraction of 0.015 with the two-layer model (Table 3). This low value is indicative of a large dilution of lignin by water. Even though lignins are usually considered to be hydrophobic,

Table 2. Neutron Reflectivity Parameters of the Lignin Interfacial Layers at the Air/Water Interface Using a One-Layer Modela D d (Å) (mg m-2) ((0.1) 1 2 4 8 16

15.5 20.5 24.0 30.5 36.5

one- proton exchange

two-proton exchange

Φ ((0.01)

Γ (mg m-2)

Φ ((0.01)

Γ (mg m-2)

0.3 0.3 0.3 0.3 0.4

0.6 ( 0.02 0.9 ( 0.03 1.0 ( 0.04 1.2 ( 0.04 2.0 ( 0.06

0.3 0.4 0.3 0.3 0.4

0.7 ( 0.03 1.0 ( 0.03 1.1 ( 0.04 1.4 ( 0.05 2.2 ( 0.06

a D is the amount of spread lignin at the air/water interface, d is the thickness, Φ is the volume fraction, and Γ is the calculated surface concentration of the interfacial layer.

Table 3. Neutron Reflectivity Parameters of the Interfacial Layer Formed from a Spread Lignin Amount of 16 mg m-2 at the Air/Water Interface Using One-, Two-, and Three-Layer Modelsa layer (no.)

Φ

d (Å)

Γ (mg m-2)

Γtotal (mg m-2)

χ2

2

0.38

36.5

One Layer 2.0 ( 0.06

2.0 ( 0.06

0.0181

2 3

0.40 0.015

Two Layers in the Substrate 34.6 2.0 ( 0.05 2.2 ( 0.2 111.8 0.2 ( 0.16

1 2

0.51 0.36

Two Layers with One in Air 8.4 0.6 ( 0.02 2.3 ( 0.07 33.9 1.7 ( 0.05

1 2 3

0.55 0.36 0.013

Three Layers with One in Air 9.1 0.7 ( 0.02 2.6 ( 0.07 32.9 1.7 ( 0.05 109.2 0.2 ( 0.02

0.0163

0.0178

0.0163

a d is the thickness, Φ is the volume fraction, and Γ is the surface concentration of the interfacial layer. The indexes 1, 2, 3, and t refer to the layer 1 (in the air), the layers 2 and 3 (between layer 1 and the bulk), and the total adsorption layer (Γt ) Γ1 + Γ2 + Γ3), respectively. The values are calculated with a scattering length density of lignin of 2.597 × 10-6 Å-2 (one-proton exchange).

this seems to confirm that these molecules may form strong associations with water molecules and can “trap” them in aggregates as previously suspected in the case of lignins8 and of their chemical models, DHPs.12 The thickness of the one-layer model increases with the spread amount from 15 to 37 Å (Table 2). This thickness in the case of the smallest deposit of lignin is not far from 11 Å, the height of a phenyl propane unit.6 At the spread concentration of 16 mg m-2, the two-layer model gives an air side layer with a thickness of 35 Å and a water side layer with a

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Aguie´ -Be´ ghin et al. Table 4. Optical Parameters of Lignin Interfacial Layers at the Air/Water Interface Using One Isotropic and Homogeneous Layer Modela spread lignin (mg m-2) d (Å) 1 2 4 8 16

Figure 3. Absolute value of the ellipticity of deposited lignin amounts ranging from 1 to 16 mg m-2 at the air/water interface. The ellipticity was measured in Brewster conditions of the substrate with an angle of incidence of 53°4.

thickness of 112 Å (Table 3). Models of lignins (DHPs) studied by neutron reflectivity exhibit the same general behavior with a volume fraction of lignin of roughly 0.10.5 in the interfacial layer in the case of spread amounts in the range between 3 and 48 mg m-2 of DHPs.12,25 Moreover, it was observed that at a spread concentration between 12 and 18 mg m-2 a two-layer model is necessary to fit the reflectivity data for DHPs GS and G. For DHP GS, a water side layer with a large thickness (116 Å) and a low volume fraction (0.02) gives a good fit. This is not true for DHP G where the water side layer has a smaller thickness (24 Å) and a larger volume fraction (0.17).12 Thus, the interfacial behavior of lignin is closer to that of DHP GS than to that of DHP G. This is consistent with the chemical composition of the LD lignin where the ratio of the S to the G monomers is 1.13 (Table 1). However, the interpretation of these data is restricted by the physical significance of the so-called “two- and three-layer models” (Table 3). In fact, in the case of a polymer adsorption layer the concentration profile is continuous,26 but the precision of the experimental data is too low to calculate accurately this profile which is approximated by a multilayer structure. The absolute value of Brewster ellipticity FjB measured close to the equilibrium state, 12 h after lignin deposition, also increases when the lignin deposit increases (Figure 3). This is indicative of an increasing amount of adsorbed molecules.20 However, the surface concentration obtained by ellipsometry is somewhat larger than that obtained by neutron reflectivity (Tables 2 and 4). This probably reflects the various approximations made in both cases. Nevertheless, neither technique affords a surface concentration corresponding to the spread amount and the discrepancy increases with the amount. To check if this problem may arise from a “wrong model adjustment” as it was noticed with polymers27-29 or β-casein30,31 forming a layer which is partly in air, a model with a layer in air was tested for (25) Cathala, B.; Puff, N.; Aguie´-Be´ghin, V.; Douillard, R.; Monties, B. In Lignin: Historical, Biological, and Materials Perspectives; Glasser, W. G., Northey, R. A., Schultz, T. P., Eds.; American Chemical Society: Washington, 1999; pp 278-290. (26) Guiselin, O.; Lee, L. T.; Farnoux, B.; Lapp, A. J. Chem. Phys. 1991, 95, 4632-4640. (27) An, S. W.; Thomas, R. K.; Baines, F. L.; Billingham, N. C.; Armes, S. P.; Penfold, J. J. Phys. Chem. B 1998, 102, 5120-5126. (28) An, S. W.; Thomas, R. K.; Baines, F. L.; Billingham, N. C.; Armes, S. P.; Penfold, J. Macromolecules 1998, 31, 7877-7885. (29) An, S. W.; Su, T. J.; Thomas, R. K.; Baines, F. L.; Billingham, N. C.; Armes, S. P.; Penfold, J. J. Phys. Chem. B 1998, 102, 387-393. (30) Aschi, A.; Gharbi, A.; Bitri, L.; Calmettes, P.; Daoud, M.; Aguie´Be´ghin, V.; Douillard, R. Langmuir 2001, 17, 1890-1904. (31) Harzallah, B.; Aguie´-Be´ghin, V.; Douillard, R.; Bosio, L. Int. J. Biol. Macromol. 1998, 23, 73-84.

15.5 20.5 24.0 30.5 36.5

nlayer

Φ

Γ (mg m-2)

k

 (L g-1 cm-1)

1.4304 1.4577 1.4993 1.5369 1.5340

0.38 0.48 0.65 0.79 0.78

0.8 1.4 2.2 3.4 4.1

0.071 0.071 0.085 0.104 0.099

26.0 20.3 18.1 18.0 17.4

a The thickness, d, is extracted from neutron reflectivity measurements. The layer (nl) and substrate refractive indices are measured at 589 nm. For the substrate, the refractive index is 1.3375. Γ is calculated according to ref 23 with a specific refractive index increment, dn/dc ) 0.173 g-1 cm3. Φ is the volume fraction. k is the extinction coefficient, and  (L g-1 cm-1) is the absorption coefficient calculated by eq 10 at 280 nm.

Figure 4. Relation between the spread surface concentration (D) and the measured surface concentration (Γ) determined by neutron reflectivity.

the neutron data (Table 3). As expected, this model increases significantly the calculated surface concentration but the values remain far from the spread ones. Finally, from these surface concentration data, it can be concluded that the amount of lignin found at quasiequilibrium is significantly smaller than that spread. Thus, the kinetics data (Figure 1) probably reflect the end of a desorption process which begins at the moment of spreading. However, the measured surface concentration always increases with the spread one (Figure 3 and Table 4) showing that the quasi-equilibrium is the result of two processes: one of desorption and one of reorganization leading to an interfacial layer. The hypothesis that the lignin sample is heterogeneous in molecular mass and size is hardly to be expected because the ratio of the spread to the measured concentrations tends to 1 when the spread amount tends to zero (Figure 4). Moreover, the determination of the surface concentration of spread DHPs by neutron reflectivity has led to the same conclusion that roughly 10% of the DHPs remain at the interface.12,25 Thus, the spreading procedure leads not to the interfacial deposition of the whole amount of lignin but to the reproducible formation of a layer with a smaller amount of molecules. Moreover, when the spread amount increases largely, a saturation of the interfacial layer seems to occur (Figure 3). Optical Coefficients of Lignin Layers. Optical properties of lignin layers were calculated from ellipsometric spectra assuming the thickness determined by neutron reflectivity. A point per point numerical resolution was applied to obtain the refractive index (n) and the extinction coefficient (k) of the interfacial layer as a function of the wavelength (Figure 5a,b). The refractive index spectra increase between 1.430 and 1.630 when the spread amount of lignin increases (Figure 5a). The extinction coefficient

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Langmuir, Vol. 18, No. 13, 2002 5195

Figure 5. Refractive index n (a) and extinction coefficient k (b) of spread lignin layers at the air/water interface calculated by the point per point resolution. The spread lignin amount ranges from 1 to 16 mg m-2 : 1 mg m-2 (O), 4 mg m-2 (]), and 16 mg m-2 (4).

Figure 6. Comparison of the bulk absorption coefficient  (L g-1cm-1) and the absorption coefficient of lignins determined in the air/water spread layer by spectroscopic ellipsometry. The spread lignin amount was 16 mg m-2 for wheat straw lignin, LD (O), and milled wild cherry, LM (4).

spectra of the interfacial layers of lignin (Figure 5b) show two maxima at 280 and 320 nm. They have the same pattern as that of the absorption spectrum measured in a dioxane/water 90/10 (v/v) solution (Figure 6). The first maximum comes from nonconjugated phenolic groups in lignin, and the second one from conjugated phenolic groups in p-coumaric and ferulic acid.32 The absorption coefficient  (L g-1 cm-1) is calculated from R (eq 10) according to eq 12:

 (L g-1 cm-1) ) R/2.3C

(12)

where R is expressed in cm-1 and C is the volume concentration in g L-1 in the interfacial layer. At 280 nm, the absorption coefficient calculated from ellipsometric data is 21.5 ( 4.5 L g-1 cm-1 for all the deposits (Table 4). It compares well with the bulk absorption coefficient of lignin in the dioxane/water mixture determined by spectrophotometry using the Beer-Lambert absorption law which is equal to 19.5 ( 0.5 L g-1 cm-1 (Figure 6). The same comparison between the absorption properties of spread lignin at an interface and solubilized lignin in bulk was performed with lignin isolated from wild cherry. The spectra are different from those obtained with wheat straw lignin (Figure 6). The spectrum of spread lignin (32) Scalbert, A.; Monties, B. Holzforschung 1986, 40, 119-127.

Figure 7. Variations of the surface pressure π (O) and of the ellipticity |FjΒ| (4) during a compression and the subsequent relaxation. The spread lignin amount was 3 mg m-2. The compression was performed 1 h after spreading, and the relaxation was measured during 16 h after the end of the compression. The ellipticity was measured in the Brewster conditions of the substrate with an angle of incidence of 53°4. The inset shows the surface pressure against the Brewster ellipticity of spread lignin layers during the compression (O) and the relaxation (4). The two spread lignin amounts were 3 and 8 mg m-2.

shows one maximum at 280 nm coming from nonconjugated phenolic groups, and the absorption coefficient is equal to 11 L g-1 cm-1. It also compares well with the bulk absorption coefficient (Figure 6). Moreover, the same pattern of bulk and interfacial absorption spectra of the lignins and the lack of a maximum of absorption around 400 nm33 indicates that the lignin molecules are not chemically modified by oxidation neither in the bulk nor at the interface. These good agreements between the bulk and interfacial absorption coefficients must be taken with caution since they are obtained in two distinct sets of conditions. The surface concentration determined by ellipsometry is calculated by extrapolating bulk data of dn/dc measured in the range of 0.4-1.6 g L-1, while within the adsorbed layer, the mass concentration is in the range of 50-1120 g L-1 (Table 4). By comparison, in the bulk, the concentration is between 0 and 0.25 g L-1. Perturbation of the Quasi-Equilibrium. Compression/ Relaxation Isotherms. A quasi-equilibrium layer was formed with a spread amount of 3 mg/m2 after a time course of 1 h. After that period, the surface pressure was 0.2 mN/m. The surface area was reduced to half of its initial value by a constant rate compression. During that compression, π and |FjB| have the same relative evolution, showing that the surface pressure and the surface concentration are proportional one to the other (Figure 7). At the end of the compression, the values of π and |FjB| are 9 mN/m and 0.014, respectively. Then, a relaxation is observed during about 20 h. During that relaxation, the same time course of the surface pressure and of |FjB| is also observed, indicating a proportionality of the two quantities and probably a limited desorption. However, at the end of the relaxation, the values of π () 5.5 mN/m) and |FjB| () 0.011) are lower than at the initial quasi-equilibrium state, showing that a new quasi-equilibrium state has been reached. The relations between π and |FjB| are linear until about 10 mN/m (Figure 7 inset). When the surface pressure reached at the end of the compression is higher, because of a larger spread amount (8 mg/m2), the relaxation process is different for π and for |FjB|: in a first (33) Billa, E. Thesis, Institut National Agronomique Paris-Grignon, Paris-Grignon, France, 1994, 197 pp.

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Table 5. Surface Pressure and Dilational Modulus of the Lignin Interfacial Layers at the Air/Water Interface πmax πrel π0 D0 (mN m-1) (mN m-1) (mN m-1) rel Dmax -2 -2 ((0.1) ((0.1) ((0.1) (mg m ) (mg m ) (mN m-1) 3.1 4.2 5.2 6.3

6.3 8.4 10.6 12.6

1.0 3.2 3.9 6.6

4.9 14.9 23.0 33.4

0.3 4.5 5.7 10.9

17.0 26.2 38.2 168.6

a D is the amount of deposited lignin at the air/water interface, π is the surface pressure, and  is the dilational modulus. The indexes 0, max, and rel refer to an early stage of spreading, to the end of compression (area divided by 2), and to a time after 3 h of the subsequent relaxation, respectively.

step, the relaxation concerns mostly π, from 27 to 15 mN/ m, while the |FjB| parameter exhibits only a very limited time change (close to 0.022); in the second step, π is nearly constant between 12 and 15 mN/m and |FjB| decreases significantly from 0.022 to 0.018. These two steps could correspond first to a reorganization of the layer and then to a desorption while π is determined by the uppermost layer which does not change much in the second step. It can be concluded that when the perturbation is large enough, reorganization and desorption are not synchronous while for small perturbations the two phenomena happen simultaneously. In all of the cases, the final state is different from the initial one and an increase in π and |FjB| is noticed. Dilational Modulus. The dilational modulus was measured after 3 h of relaxation. It ranges from 17 to 169 mN m-1 when the spread lignin concentration ranges from 6.3 to 12.6 mg m-2 at the end of the compression (Table 5). These values are slightly smaller than those observed previously where the dilational modulus ranges from 13.5 to 71.6 mN/m when the spread lignin concentration ranges from 2.2 to 7.3 mg/m2.13 This difference can be explained by the fact that the previous values were obtained after 1 h of relaxation instead of 3 h in the present study. Moreover, the dilational modulus values are higher than those usually observed for small surfactants34 and proteins35 which are around 15 mN/m. The long relaxation time and the large dilational modulus are in favor of strong interactions between the lignin molecules in an aqueous and anisotropic environment. Conclusion Several conclusions can be drawn from the neutron reflectivity, ellipsometry, and Langmuir trough data. The first one is that a significant loss of lignin molecules occurs in the bulk during the first hour after spreading. This loss decreases largely after that and is proportionally more (34) Lucassen, J. In Anionic Surfactants: Physical Chemistry of Surfactant Action; Schick, M. J. a. F., Ed.; Surfactant Science Series, Vol. 11; Marcel Dekker: New York, 1981; pp 217-265. (35) Benjamins, J.; Cagna, A.; Lucassen-Reynders, E. H. Colloids Surf. 1996, 114, 245-254.

important when the spread amount increases. Nevertheless, an increase of the spread surface concentration leads to an increase of the thickness and of the surface concentration in quasi-equilibrium conditions. As a consequence, the spreading procedure used in this study does not lead to the deposition of the whole amount of lignin at the air/water interface. The second is that after spreading or compression, the kinetics of ellipticity and surface pressure imply a reorganization of the layers. This phenomenon is more pronounced when the surface concentration is large. Moreover, it is associated with a moderate desorption which implies that in addition to the surface layer, lignin exists in the bulk in a colloidal or aggregation state. The third is that spectroscopic ellipsometry allows the determination of the absorption spectrum of lignin in the layer. This spectrum is very similar to that in the bulk, favoring the idea that the covalent structure of the adsorbed molecules is not different from that of molecules in solution. Thus, the equilibrium state from neutron reflectivity data (maximal limit value of Γ ≈ 2 mg/m2) or from ellipsometry data (maximal limit value of |FjB| ≈ 0.025) and the high value of the dilational modulus prove that the lignin molecules reach a quasi-equilibrium state in the interfacial layer. One can finally emphasize that the possibilities of organization of lignin layers, after spreading at the air/ water interface reported here, could be of some practical importance for example in relation to pulp and air-drying of related cellulosic fibers. The occurrence of considerably higher lignin concentration (aromatic cycles and double bounds) on the surface of pulp fibers than in the bulk material has been recently confirmed in the case of both standard kraft pulp36 and isothermal cooked pulps.37,38 Changes in both fibers’ surface morphology and surface chemical composition were emphasized by the last authors who compared freeze-dried and air-dried fiber bundles. Air-dried fibers showed a significantly higher lignin surface content than freeze-dried fibers. Thus, the surface properties of lignin observed at the air/water interface may be significant in more complex systems including liquid, solid, and gas phases. Acknowledgment. Thanks are due to Dr. B. Chabbert for wild cherry lignin samples, to L. T. Lee (LLB, CEA, Saclay, France) for help in neutron reflectivity, to Dr. B. Cathala for discussions concerning lignins and their models, and to M. Stchakovsky (Jobin Yvon, Arpajon, France) and G. Zalczer (SPEC, CEA Saclay, France) for the ellipsometry experiments and interpretations. LA011766V (36) Westermark, U. Proceedings of the 10th International Symposium on Wood and Pulping Chemistry; Yokohama: London, 1999; Vol. 1. (37) Duchesne, I. Ph.D. Thesis, Swedish University of Agricultural Science, Uppsala, Sweden, 2001, 47 pp. (38) Duchesne, I.; Daniel, G.; van Leerdam, G. C.; Basta J. Pulp Pap. Sci., submitted.