Surface Properties of Suberin

The surface energy of suberin was determined by four different methods, namely, (i) contact angle measurements, (ii) Wilhelmy plate measurements, (iii) maximum bubble pressure, and (iv) inverse gas chromatography (IGC). The first three methods gave a gammasub value in the range 40-50 mN m-1 at room temperature. The major component of this value reflects the dispersive contribution. The IGC measurements showed a higher dispersive term, which is common with this method of characterization. The surface acid (A)/base (B) properties were also evaluated, and the results indicated that suberin has an acidic character.


INTRODUCTION
applications, particularly in the field of novel polymeric materials. The precise knowledge of the surface properties of The cork from Quercus suber L. is used in many industrial suberin is particularly relevant to its possible use as an addiapplications such as cork stoppers and insulating boards, tive in coating formulations like printing inks and varnishes, among others. During cork transformation, large amounts of a research topic presently in progress in our laboratory which cork powder are produced. This cork fraction has no indusdictated the systematic approach described here. trial interest due to the small size of its particles and is frequently burned for energy production. However, cork powder can be an important source of chemicals, viz. mainly

MATERIALS AND METHODS
suberin (1) which is its most important constituent amounting to 30 to 50% by weight. In this context, the Isolation of Suberin development of suitable applications for suberin could constitute an interesting way to valorize large amounts of a As mentioned above, the suberin used in this work was byproduct of the cork industry.
obtained by the alkaline alcoholysis of a sample of high-The structure of suberin in cork is not yet fully understood. quality cork, kindly supplied by the Champcork Company. Kolattukudy has proposed that suberin, as isolated from vari-This procedure consisted on successive soxhlet extractions ous vegetal products like potatoes, is a network containing of the cork powder with methylene chloride, ethanol, and both phenolic and long aliphatic moieties which are linked water. The extractive-free cork was dried and submitted to through ester groups, as shown in Scheme 1 (2): The phenoalkaline methanolysis with a solution of 0.1 M KOH in lic part displays features similar to those of lignins and the MeOH, for 5 h with a solvent/cork ratio of 10/1 v/w. The aliphatic one is composed of fatty acid polyesters bearing reaction medium was neutralized, extracted with chloroform free OH groups. In situ suberin is insoluble in all solvents and dried. After evaporation of chloroform, the residual orbut can be decrosslinked by alkaline hydrolysis or methaganic products were considered as suberin which was a waxy nolysis. The main products resulting from this operation paste-like material containing both a liquid and a microcrys-SCHEME 1 characterization of this product will be described in detail liquids, namely their surface tension g L and the correspondelsewhere (5) as well as those of cork lignin (6).
ing dispersive contribution g D L .

Contact Angle Measurements Wilhelmy Plate Measurements
The contact angle measurements were carried out with a A Dognon Abribat Wilhelmy plate tensiometer ({0.5 mN goniometer constructed in our laboratory equipped with a m 01 ) was used in order to measure the surface tension of CCD camera working at up to 200 images per second as liquid suberin. The experiments were carried out between described in a previous publication (7). The results were 50 and 120ЊC, i.e., above the melting temperature of suberin, collected with a video-card and treated by image analysis after 2 h of thermal stabilization. In that temperature domain software. The reproducibility was about {1Њ. Two reference the viscosity was sufficiently low (h Å 0.47 Pa s at 50ЊC) surfaces, namely, glass and polyethylene, both thoroughly to ensure reliable results. cleaned and submitted to a 6 h Soxhlet extraction with methanol, were used as alternative supports for suberin. Whereas Maximum Bubble Pressure the glass surface was coated with a 40 mm layer of suberin using a spreading rod, the thickness of the layer on polyethyl-A Sensadyne 6000 maximum bubble pressure tensiometer ({0.1 mN/m) was used to measure the surface tension of ene (PE) was much higher, viz. ca. 500 mm, because of its bad wettability with suberin. After that, the surfaces were suberin. The bubble frequency was about 1 Hz, which corresponds to ''semi-dynamic'' conditions. The experiments placed in a thermostated oven at 60ЊC for 30 min in order to induce a smooth and flat surface of substrate. Drops of were carried out at temperatures ranging from 57 to 86ЊC with inverted capillary probes in order to minimize possible various test liquids were deposited on these conditioned suberin surfaces in order to acquire values of contact angle artefacts arising from the relatively high viscosity of the medium. At each temperature, the tensiometer was recali-relative to polar and non-polar as well as acidic or basic substances. Table 1 gives the specific properties of these brated with water and methanol.

IGC Measurements Principle of IGC Calculations
The dispersive component of the surface energy of a solid IGC experiments were carried out using a DELSI 121 surface, g D S , and its acidic, K A , and basic, K B , numbers were DFL chromatograph equipped with a flame ionization detecobtained following the classical procedure described in detail tor and a 28 cm 1 4 mm pyrex column. About 1 g of suberin elsewhere (9). Only the general approach will be presented was adsorbed on chromosorb W sililated powder (60/80 here. mesh) and the column filled and conditioned overnight at The dispersive component of the surface energy, g D S , is 100ЊC in a stream of dry nitrogen. The dispersive component given by Eq. [1]: of the surface energy of suberin was obtained from results related to the injection of a series of n-alkane probes. The acid/base properties were estimated using tetrahydrofuran RT ln Vn Å 2N(g D S ) 1/2 a(g D L ) 1/2 , [1] (basic), chloroform (acidic) and ethyl acetate (amphoteric) as probes. The relevant characteristics of these probes, i.e., where R is the gas constant, T the working temperature, Vn the dispersive component of their surface tension in the liqthe retention volume, N Avogadro's number, a the probe's uid state g D L , their molecular surface (a) and their Gutmann's molecular surface, and g D L the dispersive component of surdonor (DN) and acceptor (AN) numbers (8), are given in face tension of the probe in the liquid state. g D S values are Table 1. Propane was used as a marker, and the carrier gas calculated accordingly. was pure nitrogen.
K A and K B were obtained by carrying out experiment at Zero coverage conditions were reached by injecting 5 ml least at three different temperatures. First, the specific free of vapors of the different probes. In these conditions the energy DG sp for the interaction between the polar probe and interactions between adsorbate molecules themselves are the suberin surface was determined using Eq. [2]: negligible and the thermodynamic parameters, calculated using the retention time of each probe, depend only on the DG sp Å RT ln Vn 0 RT ln Vn ref , [2] adsorbate/adsorbent interactions. Experiments were carried out repeatedly (and reproducibly) at five different temperawhere Vn is the retention volume of the polar probe and tures, namely 50, 60, 70, 80, and 90 { 0.5ЊC.
Vn ref the retention volume related to the n-alkanes. Then, the specific enthalpy DH sp was obtained as the slope Probes and Solvents of the plot DG sp /T vs 1/T (Eq. [3]).
All probes and solvents used for the different surface characterizations described above were commercial products of [3] very high purity. The glass surface was a microscope slide and the polyethylene was a special sample without any additive kindly prepared for us by BP Chemicals, Lavera, France.
Finally, K A and K B were calculated from the slope and  (7). Figure 2 shows the typical evolution within these short spans of the contact angle of four different liquids on a suberin layer coated on DH sp AN [4] glass and PE.
The first observation related to these fast data-acquisition mode, is that the decrease in the u values thus recorded Here DN and AN are, respectively, Gutmann's (8) donor always began after 30 to 50 ms, depending on the liquid. and acceptor numbers corresponding to the polar probes.
Because the suberin layer was flat and non-porous, the evolution of the contact angle as a function of time could be

RESULTS AND DISCUSSION
attributed to the following phenomena: (i) deformation of Contact Angle Measurements the contact line because of the low Young modulus of the material, (ii) diffusion of the probe into the suberin layer. The apparent contact angles of water droplets were re- The possible evaporation of the liquids is not taken into corded over a period of time of 100 s, with a frequency of account here because of their low volatility and the short 1 image/s. Figure 1 shows a typical time evolution and time scale of the experiments. Furthermore, considering the the standard deviation observed over three wetting kinetics relatively high surface tension of the probes used in this experiments. A major common feature is that this evolution study (water, 72.8 mN m 01 ; formamide, 58 mN m 01 ; areflected a decrease of about 25Њ within 100 s. When such bromonaphthalene, 44.6 mN m 01 ; diiodomethane, 50.8 mN a phenomenon is observed, it is common to extrapolate the m 01 ), spontaneous spreading on suberin, which is likely to data at t Å 0 and then apply the Young equation. But with be a low surface-energy material, is not expected to occur such a steep decrease of u with time ( Fig. 1), it was difficult on thermodynamic grounds. to extrapolate our data reliably to t Å 0. This is why it was This assumption was indeed verified because suberin was decided to carry out some experiments at very short times after the drop deposition, namely, every 5 ms after contact found to be insoluble in water and negligibly soluble in formamide and diiodomethane within the times required for position of the support cannot be envisaged in the present context because of the large thickness of the suberin layer the measurements. Thus, the decrease in contact angle observed for these liquids (Fig. 2) was attributed to the defor-(40 mm on glass and 500 mm on PE) which was more than sufficient to screen the interactions between the liquid drops mation of the contact line (10) and was confirmed by a close observation by optical microscopy of the interface. and the support material. Furthermore, if these interactions had been relevant, the shift observed for water would have The modulus of suberin was measured by a dynamic mechanical test on a Metravib apparatus working at room tem-been inverted because the glass surface is much more hydrophilic than the PE surface and, moreover, here the thickness perature between 5 and 100 Hz, and was found to be in the range of 10 5 N m 02 . This value is 4 orders of magnitude of the suberin layer was lower on glass. The fact that u remained higher with a glass support rules out the possibility lower than that associated with the onset of a rigidity sufficient to avoid deformation of the contact line (10). The of long-range interactions between water (and therefore also other liquids) and the base materials through the suberin possible role of the difference in density between the various liquids deposited and suberin was not taken into account, layer.
The systematic downshifts of about 2Њ for the thicker su-but we feel that it should not be too important.
Suberin was found to be soluble in a-bromonaphthalene. berin layer is most likely attributed to a better ability of a thicker layer of that soft material to be deformed by an The decrease in the contact angle observed with this solvent was thus attributed both to the deformation of the suberin external stress.
Before proceeding to calculate surface energies by different layer near the contact line and to the diffusion of the solvent in the substrate. This interpretation is reinforced by the fact classical methods, one final question has to be answered: Which contact angle should be taken as the ''real'' Young that the slope of the u vs time plot obtained with a-bromonaphthalene was much higher than those registered with the contact angle? An enticing idea is to take the value of the wavy plateau observed during the first 30-50 ms, but this other liquids (Fig. 2).
The second observation made in the short-time experi-requires a preliminary check that the droplet has had sufficient time to reorganize itself in terms of thermodynamic criteria. ments was that there is a systematic shift of about 2Њ between the contact angles measured on the suberin layer coated on To verify this point we proceeded as follows: (i) the approximative self-diffusion coefficients of the different solvents glass and those recorded from coatings done on PE. The explanation based on a possible effect of the chemical com-were calculated according to the Einstein-Stokes equation as (in cm 2 s 01 ) 2 1 10 05 for water, 4 1 10 06 for a-bromonaph-Fowkes' Approach thalene, 3 1 10 06 for formamide, and 2 1 10 05 for diiodo-Although a-bromonaphthalene would have been the most methane; (ii) the oscillations of bouncing drops of the four adequate probe in this context for a purely dispersive liquid, liquids on a high-modulus material, viz. anodized aluminium the fact that it showed good solvent properties for our suboxide contaminated by atmospheric impurities, were recorded. strate made the corresponding values of contact angle suspi-Each drop was slowly generated at the tip of a microsyringe cious. We therefore preferred to use the data obtained with needle and maintained at a distance of about 1 mm from that diiodomethane, assuming in the first approximation that this substrate during volume increase, until it detached itself under liquid was essentially dispersive in character (see Table 1). its own weight. The volume of the detached drops was in the Thus, the adhesion energy W a between these materials could range of 5 to 10 ml. The oscillations of the newly deposited be expressed as the geometric mean involving the dispersive drop are produced by the kinetic energy acquired during its contribution to the surface energy of suberin and the actual fall. The characteristic oscillation periods recorded with our surface tension of diiodomethane, viz.: liquids were from 10 to 15 ms (Fig. 3). By comparison with the molecular movements associated with the data in given W a Å g L (1 / cos u) Å 2( g D sub g L ).
[5] above, it was therefore concluded that the drops have enough time to reach thermodynamic equilibrium with their environment. This is also true of course of drops falling on suberin, Table 2 gives the values of g D sub obtained by this procedure which produce much more attenuated oscillations as shown applied to the two different supports. in Fig. 2 because their kinetic energy is more readily shared The value of 80Њ obtained for water suggests some hydrowith that soft material. philic interactions at the interface. Fowkes' non-dispersive interaction parameter, I sl , reflecting the non-dispersive con-These verifications justify the decision of taking as the Young tribution to the energy of adhesion, can be calculated as contact angle the mean value observed for each liquid on the follows: oscillating plateau during the first 30-50 ms. Moreover, since the thick layer of suberin was certainly more subject to deformation near the contact line, the values obtained on the thin layer deposited on the glass support were considered as more reliable Å g H 2 O (1 / cos u) 0 2( g D sub g D H 2 O ). [6] in the following calculations and discussions. diiodomethane according to Fowkes' equation, the polarity The values obtained for this parameter are reported in of the suberin surface was found again to be relatively high, Table 2. First of all, it became obvious that the differences confirming the non-negligible Fowkes' I sl value calculated observed between experiments performed on the thin layer with water ( Table 3). The total surface energy of suberin (coated on glass) and the thick one (coated on PE) are reached a value of 41.5 mN m 01 , which seems reasonable negligible, namely less than 4%.
considering the structure of the suberin macromolecules (see The I sl value obtained with water represents more than Chart 1) with its occasional polar groups. one third of the total energy of adhesion. This important contribution is clearly related to the presence of some OH Van Oss Approach and COOH functions in the structure of suberin, which can The latter two approaches did not discriminate between establish occasional hydrogen bonds with water. polar and acid/base interactions. It seemed interesting to verify whether suberin, which contains some carboxylic Owens-Wendt's Approach groups, displayed a certain surface acidity. In that context, Owens and Wendt extended Fowkes' approach to the nonthe experimental values of the contact angles, obtained with dispersive (polar) part of the energy of adhesion. They astwo polar probes (water and formamide) and a non-polar sumed that the geometric mean approach is also applicable liquid (diiodomethane), were processed according to Van to that specific contribution, i.e.: Oss' approach, namely: This point is questionable because the geometric mean where g LW sub is the Lifshitz-Van der Waals contribution and seems to be irrelevant for short-distance interactions like g AB sub the acid-base contribution to the surface energy (14). hydrogen bonding. Some examples of aberrations observed with this approach were given by Fowkes (11). Despite   TABLE 3 their controversial character (11)(12)(13), it seemed interesting to the four probes. value obtained for g D sub is lower than that calculated with Thus, the surface under investigation is characterized by value after 200 s, whereas those of the neutral and the acidic solutions decreased within that time to a constant value of three parameters g LW sub , g 0 sub , and g / sub . g LW sub takes into account 50Њ. This result is direct proof of the predominance of not only the London dispersive interactions, but also both Brønsted acidic sites on the suberin surface, which is hardly dipole/dipole and dipole/induced-dipole interactions. The surprising given the presence of COOH functions in the energy of adhesion is therefore expressed here as structure of this material. Moreover, the OH groups, also present in its macromolecules, contribute, albeit to a minor W a Å g L (1 / cos u) Å W LW a / W AB a extent, to this acidic character, as with cellulose (15). Å 2 g LW sub g LW L / 2 g 0 sub g / L / 2 g / sub g 0 L . [9] Wilhelmy Plate Measurements The use of three liquids gave the solution for the three The surface tension of melted suberin showed a linear unknowns through the corresponding three equations. The decrease with temperature as shown in Fig. 6, with a slope results of this procedure are summarized in Table 2. g AB sub of 00.14 mN m 01 ЊC 01 , which is a classical value for organic reached a significant value of 3.6 mN m 01 , arising mostly liquids (16). The extrapolation of these data to 25ЊC gives from a basic character. This result seems highly questionable a value of 37 mN m 01 for the surface tension of suberin, both with respect to the chemical properties of suberin and which is slightly lower than that obtained by the contact in view of the results obtained with water at different pHs angle method. (see below). Furthermore, a total surface energy of 51.2 mN m 01 seems excessive for such an essentially non-polar Maximum Bubble Pressure material.
The surface tension of liquid suberin as a function of The Role of pH temperature was also measured by the maximum bubble pressure technique. Figure 7 shows again a linear decrease Three aqueous solutions of pH 3, 7, and 12, respectively, were prepared. Their surface tension were found to be inde-with a slope of 00.13 mN m 01 ЊC 01 , which confirmed the results obtained with the Wilhelmy plate. However, the value pendent of pH. The evolution of the contact angle of these solutions was recorded using both the short-and long-time of the surface tension extrapolated to 25ЊC was now 45 mN m 01 . The difference of 8 mN m 01 between the two methods modes as shown in Fig. 5. Within the short-time range, the plateau values were similar, with a slight decrease for the could probably arises from one of the following factors (or from both): (i) the Wilhelmy plate technique is highly sensi-basic solution, whereas within the longer time range, the decrease in contact angle with time, observed for all pHs, tive to surface contaminations of the liquid, whereas the maximum bubble pressure is not; (ii) the first method in-was drastically enhanced in the case of the basic solution. In fact, in that instance the contact angle reached a zero volves static measurements, that is to say all specific orienta- tions or migrations of the more surface-active parts of the namic equilibrium involving the predominant presence of non-polar moieties. We are inclined to privilege the latter suberin macromolecules have been completed, whereas the second involves dynamic measurements during which the explanation. air/suberin interface is created on a time scale of about one second and this might not be sufficient for molecular IGC Measurements reorientation. Thus, such fresh suberin surfaces would contain more polar groups (and therefore possess a higher en-Before recording the results of IGC measurements applied to the surface characterization of suberin, the validity of ergy) than those which have had the time to reach thermody-  to the absence of problems related to the actual morphol-DG sp , DH sp , and DS sp for the Suberin Surface, ogy of the liquid / solid interface.

as Determined by IGC
The acid -base properties of the suberin surface were evaluated from its interaction with polar probes at five cork ( 38 mN m 01 ) ( 4 ) indicates that the portions of the macromolecules laying on its surface tend to be the long non-polar chains of suberin, a fact that is responsible for the proverbial water repellency of stopcorks. the application of this method had to be confirmed. Two phenomenological contributions had to be proved negligible CONCLUSION with the experimental conditions chosen, namely (i) bulk sorption and (ii) diffusion of probe compounds into the The results gathered in this multiple approach show a material. This is verified if: satisfactory coherence which enabled us to draw the conclu-• The chromatographic peaks for polar as well as nonsion that the suberin we isolated from cork as a paste-like polar probes are sharp and symmetrical.
polymer is a substance possessing a rather high surface en-• The retention times of the polar and non-polar probes ergy attributed to the various polar functions identified in chosen, as well as of propane (the marker), are reproducible. its structure. Work is in progress to apply the consequences • The retention times of the probes repeated at the end of of these results to the correct formulation of novel types of an experiment, i.e., after the use of different probes, remain coating materials in which the presence of extracted suberins unchanged, thus excluding permanent surface contaminacould play an important surface role. tion.