Etude de l'extraction de l'élaboration

Université de Pau et des Pays de l’Adour

IUT des Pays de l’Adour, IPREM

Docteur de l’Université de Pau et des Pays de l’Adour

Etude de l’extraction de

l’élaboration

Rapporteur :

M. J. Labidi

M. J. Van Acker

Examinateur :

M. A. Sesbou

M. A. Pizzi

Directeurs de thèses :

Mme F. Charrier – El Bouhtoury

M. B. Charrier

Université de Pau et des Pays de l’Adour

IPREM-EPCP, UMR 5254 CNRS/UPPA

THESE

Pour obtenir le grade de


Avec le label doctorat européen

Discipline : Chimie

Spécialité : Chimie des polymères

Par

Lucie CHUPIN

xtraction de tanins d’écorce de pin maritime pour

l’élaboration de colles tanins-lignosulfonate

Soutenue le 7 novembre 2014

Docteur, Université du Pays Basque, San Sebastian

Professeur, Université de Gent, Gent

Professeur, ENFI, Salé

Professeur, LERMAB-ENTSTIB, Nancy

El Bouhtoury Maître de Conférences HDR, IPREM-EPCP, Mont de Marsan

Professeur, IPREM-EPCP, Mont de Marsan


tanins d’écorce de pin maritime pour

lignosulfonates

Docteur, Université du Pays Basque, San Sebastian

EPCP, Mont de Marsan

EPCP, Mont de Marsan

Remerciements

J’aimerais tout d’abord remercier mes deux directeurs de thèse, Bertrand Charrier et

Fatima Charrier-El Bouhtoury. Tous deux m’ont accompagné tout au long de ce travail de

recherche, ont su me conseiller, m’orienter, m’ont appris à être autonome. Je les remercie

de m’avoir aussi permis de présenter mon travail au cours de ces trois années dans de

nombreux colloques internationaux.

Je remercie le Professeur Sesbou de m’avoir fait l’honneur de présider le jury de ma

thèse, le Docteur Labidi et le Professeur Van Acker d’en avoir été les rapporteurs et le

Professeur Pizzi d’avoir été rapporteur. Je les remercie pour le temps qu’ils ont consacré à la

lecture de ce travail et à leurs remarques constructives.

Je remercie Olivier Donnard, Directeur de l’Institut pluridisciplinaire de recherche sur

l’environnement et les matériaux (IPREM) et Christophe Derail, Directeur de l’Equipe de

physique et chimie des polymères (EPCP), de l’Université de Pau et des Pays de l’Adour, qui

m’ont accueilli au sein de leurs équipes.

Je remercie Pascal Stouff, Directeur de l’Institut Universitaire de Technologie (IUT)

des Pays de l’Adour, au sein de laquelle s’est déroulée la plus grande part de mes travaux et,

à travers lui, toutes les équipes enseignantes et administratives, pour l’accueil et les

conditions de travail privilégiées qui m’ont été offertes.

De manière plus personnalisée, je tiens à remercier Stéphanie Reynaud, pour son

aide pour les extractions au micro-onde ; Jean-Pierre Girard, technicien du département

Science et Génie des Matériaux (SGM) pour avoir découpé les panneaux dont j’avais besoin

pour mes expériences. Je remercie aussi Olivier Gilbert qui, au sein de l’Equipe de physique

et chimie des polymères (EPCP), s’est chargé avec une grande réactivité et beaucoup de

compréhension de tous mes ordres de missions et autres formalités administratives.

Une partie de ma recherche s’est déroulée à l’Université d’Helsinki, en Finlande. Je

tiens à remercier le Professeur Sikka Liisa Maunu, à laquelle j’associe le Professeur Heikki

Tenhu, Directeur du laboratoire de chimie des polymères (Polymeerikemian laboratorio) de

l’Université d’Helsinki et toute l’équipe du laboratoire pour leur accueil et leurs conseils

précieux.

A l’occasion de cette période passée en Finlande, j’ai pu apprécier l’attention que le

Ministère des Affaires Etrangères porte aux chercheurs à l’étranger et je remercie tout

particulièrement Sandrine Testaz, Attachée de coopération scientifique et universitaire à

l’Ambassade de France en Finlande pour son accueil très amical à mon arrivée.

Ce travail n’aurait pu être conduit et mené à bien sans le concours de divers

financeurs qui m’ont permis de vivre et travailler dans de bonnes conditions et de participer

à de nombreuses rencontres professionnelles en France et à l’étranger, notamment l’Ecole

doctorale des Sciences exactes et leurs applications de l’Université de Pau et des Pays de

l’Adour et le Ministère des Affaires étrangères et l’ANR-10-EQPX-16 Xyloforest. Je remercie

tout particulièrement le Conseil général des Landes pour le financement de ma thèse.

Je remercie en propre Monsieur Henri Emmanuelli, Président du Conseil général des

Landes et Madame Geneviève Darrieussecq, Maire de la ville de Mont de Marsan, pour

l’intérêt qu’ils ont bien voulu montrer à mes travaux. A travers ces deux personnalités

politiques, je rends un hommage chaleureux à la population de Mont de Marsan et du

département des Landes, auprès de qui j’ai passé trois années dans un cadre personnel

épanouissant.

Je remercie mes amis et collègues : Coralie, Florian, Aurélie, Annabelle, Juliette,

Mickaël, Mathilde, Houda, Mathie, Divya, Laura, avec qui se sont passées ces années de

travail mais aussi de convivialité et de complicité.

Mes remerciements vont aussi à ma famille, toujours proche, et à mes amis qui sont

restés fidèles et présents malgré l’éloignement géographique, et ont su maintenir ma

motivation tout au long de ma thèse : Jeanne, Manon, Florian, Rathiga, Benjamin, David,

Audrey, Nicolas.

Ce travail m’a enrichi considérablement sur le plan personnel. J’ai appris le métier de

chercheur, fait de patience, d’intuition et de forts moments de mobilisation où tout devient

urgent. Cela m’a conforté dans ma vocation de poursuivre dans cette direction. J’ai aussi

travaillé de mes mains, enseigné, donné des conférences, travaillé en équipe, et j’y ai pris

plaisir. Toute cette expérience m’a muri, apporté beaucoup de confiance et un sens sûr de la

direction que je veux prendre. Que tous ceux qui ont contribué à cela, et qui ne sont pas

cités ci-dessus, en soient remerciés.

1

Sommaire

LISTE DES FIGURES ................................................................................................. 5

LISTE DES TABLEAUX ............................................................................................ 9

LISTE DES ABBREVIATIONS ............................................................................... 11

INTRODUCTION GENERALE ............................................................................... 13

CHAPITRE I Etat de l’art ......................................................................................... 17

I.1. PRESENTATION DES EXPECES UTILISEES DANS LE CADRE DE L'ETUDE .................. 17

I.1.1 Le bois de mimosa (acacia mearnsii) ....................................................... 17

I.1.2 Le pin maritime (pinus pinaster) .............................................................. 18

I.1.3 L’écorce de pin maritime ......................................................................... 19

I.2. LES EXTRACTIBLES .............................................................................................. 20

I.2.1 Les flavonoïdes ......................................................................................... 20

I.2.2 Les tanins .................................................................................................. 21

I.3. LES PROCESSUS D'EXTRACTION DES TANINS ........................................................ 26

I.3.1 Extraction à l’eau chaude ........................................................................ 26

I.3.2 Extraction au Soxhlet ............................................................................... 26

I.3.3 L’extraction assistée par micro-ondes (EAM) ......................................... 27

I.3.4 L’extraction assistée par ultrasons .......................................................... 27

I.3.5 Extraction par fluide supercritique .......................................................... 28

I.3.6 Extraction par liquide pressurisé ............................................................. 28

I.4. LES LIGNINES ...................................................................................................... 29

I.4.1 Les lignines Kraft ..................................................................................... 30

I.4.2 Les lignines organosolves ........................................................................ 30

I.4.3 Les lignosulfonates ................................................................................... 30

I.5. COLLES POUR PANNEAUX DE BOIS ....................................................................... 31

I.5.1 Historique ................................................................................................. 31

I.5.2 Urée-formaldéhyde (UF) .......................................................................... 32

I.5.3 Phénol-formaldéhyde (PF) ....................................................................... 33

I.5.4 Isocyanates ............................................................................................... 33

I.5.5 Colle à base de lignines ........................................................................... 33

I.5.6 Colles à base de tanins ............................................................................. 34

2

I.6. REFERENCES ........................................................................................................ 35

CHAPITRE II Extraction de tanins ......................................................................... 43

II.1. INTRODUCTION AU CHAPITRE II ......................................................................... 43

II.2. CARACTERISATION PAR DES DOSAGES COLORIMETRIQUES, IRTF ET HPLC DES

TANINS D'ECORCE DE PIN MARITIME (PINUS PINASTER) EXTRAITS SOUS DIFFERENTES

CONDITIONS .. ........................................................................................................................ 44

II.2.1 Introduction .............................................................................................. 45

II.2.2 Materials and methods ............................................................................. 46

II.2.3 Results and discussion .............................................................................. 50

II.2.4 Conclusion ................................................................................................ 57

II.2.5 Acknowledgements ................................................................................... 58

II.2.6 References ................................................................................................ 58

II.3. EXTRACTION ASSISTEE PAR MICRO-ONDES D'ECORCE DE PIN MARITIME (PINUS

PINASTER) : IMPACT DE LA TAILLE DES PARTICULES ET CARACTERISATION ........................... 61

II.3.1 Introduction .............................................................................................. 63

II.3.2 Materials and methods ............................................................................. 64

II.3.3 Results ...................................................................................................... 68

II.3.4 Discussion ................................................................................................ 74

II.3.5 Conclusion ................................................................................................ 76

II.3.6 Acknowledgements ................................................................................... 76

II.3.7 References ................................................................................................ 76

II.4. CONCLUSION AU CHAPITRE II ............................................................................. 80

CHAPITRE III Elaboration des colles tanin-lignine .............................................. 81

III.1. INTRODUCTION AU CHAPITRE III ....................................................................... 81

III.2. ETUDE DES PROPRIETES DE DURABILITE THERMIQUE DES COLLES TANIN-

LIGNOSULFONATE .................................................................................................................. 83

III.2.1 Introduction .............................................................................................. 85

III.2.2 Material and methods ............................................................................... 86

III.2.3 Results and discussion .............................................................................. 88

III.2.4 Conclusion .............................................................................................. 100

III.2.5 Acknowledgments ................................................................................... 100

III.2.6 References .............................................................................................. 101

3

III.3. ETUDE DES PROPRIETE DE DURABILITE THERMIQUE DE COLLES POUR PANNEAUX

DE PARTICULES A BASE DE TANINSEXTRAITS D'ECORCE DE PIN MARITIME ET DE

LIGNOSULFONATES .............................................................................................................. 104

III.3.1 Introduction ............................................................................................ 106

III.3.2 Material and methods ............................................................................. 107

III.3.3 Results and discussion ............................................................................ 110

III.3.4 Conclusion .............................................................................................. 116

III.3.5 Acknowledgments ................................................................................... 116

III.3.6 References .............................................................................................. 116

III.4. GLYOXALATION DE LIGNOSULFONATES DE SODIUM ET D'AMMONIUM POUR

L'ELABORATION DE COLLES TANIN-LIGNINE POUR PANNEAUX DE PARTICULES .................... 121

III.4.1 Introduction ............................................................................................ 120

III.4.2 Materials and methods ........................................................................... 122

III.4.3 Results and discussion ............................................................................ 125

III.4.4 Conclusion .............................................................................................. 137

III.4.5 Acknowledgment ..................................................................................... 138

III.4.6 References .............................................................................................. 138

III.5 CONCLUSION AU CHAPITRE III ......................................................................... 144

CONCLUSION GENERALE ET PERSPECTIVES ............................................ 147

PRODUCTIONS SCIENTIFIQUES ...................................................................... 151

ANNEXES ................................................................................................................ 153

5

Liste des figures

Chapitre I Etat de l’art

Figure 1 Répartition géographique du pin maritime en Europe [4,5] P18

Figure 2 Schéma de la structure de l'écorce [14] P20

Figure 3 Structure chimique de base du noyau flavane P21

Figure 4 Exemple d’ellagitanin : castalagine (R1 = H, R2 = OH) ; vescalagine (R1 =

OH, R2 = H) [26]

P22

Figure 5 Exemple de gallotanin : B-1, 2 3 4 6 – pentagalloyl – O D – glucose [26] P22

Figure 6 Unité de base des tanins condensés [28] P23

Figure 7 Unités de base des tanins condensés principaux d'ecorce de pin maritime (a)

catéchine, (b) épicatéchine, (c) épigallocatéchine, (d) épicatéchine gallate

P24

Figure 8 Schéma d'un extracteur Soxhlet [80] P27

Figure 9 Schéma du principe de l'extraction par liquide pressurisé [93] P29

Figure 10 Unités de base de la lignine : (a) alcool p-coumarylique, (b) alcool

conférylique, (c) alcool sinapylique

P29

Figure 11 Unité de base des lignosulfonates de sodium P31

Figure 12 Réaction d'hydroxyméthylolation de l'urée avec le formaldéhyde [102] P32

Chapitre II Extraction de tanins

II-2 Caractérisation par des méthodes spectroscopiques IRTF et HPLC des tanins

d’écorce de pin maritime (Pinus pinaster) extraits sous différentes conditions

Figure 1 FTIR spectra of a: P1T1 (1% NaOH, 80°C); b: P2T1 (5% NaOH, 80°C); c:

P3T1 (1% NaOH, 70°C)

P54

Figure 2 FTIR spectra of tannins extracted with the third method for five different P55

6

trees; a: P3T1; b: P3T2; c: P3T3 ; d: P3T4; e: P3T5

Figure 3 RP-HPLC chromatogram of P3T5 after thiolysis at 280 nm (Cat: catechin,

ECG: epicatechin gallate, GA: gallic acid, EC: epicatechin)

P56

II-3 Extraction assistée par micro-onde d’écorce de pin maritime (Pinus pinaster) : impact de la taille des particules et caractérisation

Figure 1 Chemical structure of condensed tannins (R1: H or R; R2: OH or gallic acid

ester; R3: H or OH)

P63

Figure 2 FTIR spectra of the MAE: MO1 (––); MO2 (––); MO3 (—); MO4 (––);

MO5 (—).

P72

Figure 3 1H NMR spectrum of MO2 P73

Figure 4 1H-13C HSQC NMR spectrum of MO1 P74

Figure 5 Principal component analysis of the microwave-assisted extracts for five

particle size distributions

P75

Chapitre III Elaboration des colles tanin-lignine

III-2 Etude des propriétés de durabilité thermique des colles tanins- lignosulfonates

Figure 1 (a) TG curves and (b) DTG curves of (—) non glyoxalated NaLSP, (- - -)

glyoxalated NaLSP, (— ·) non glyoxalated NH4LSP and (— - -)

glyoxalated NH4LSP recorded at 10°C min-1

P90

Figure 2 DSC curves of NaLSL (—), NH4LSL (- - -), glyoxalated NaLSL (— ·) and

glyoxalated NH4LSL (— - -) recorded at 10°C min-1

P91

Figure 3 FTIR spectra of (—) non glyoxalated NaLSL, (- - -) glyoxalated NaLSL, (— ·) non glyoxalated NH4LSP and (— - -) glyoxalated NH4LSP

P92

7

Figure 4 Module of elasticity of average curves of a pine joint as a function of temperature obtained by TMA testing when bonded with mimosa tannins – glyoxalated NaLSP cured resins: (—) 20 mass % tannins; (- - -) 40 mass % tannins; (— ·) 50 mass % tannins; (— · ·) 60 mass % tannins recorded at 10°C min-1

P93

Figure 5 Module of elasticity of average curves of a pine joint as a function of temperature obtained by TMA testing when bonded with (—) mimosa tannins – glyoxalated NaLSL; (- - -) mimosa tannins – glyoxalated NaLSP; (— ·) mimosa tannins – glyoxalated NH4LSL; (— · ·) mimosa tannins – glyoxalated NH4LSP recorded at 10°C min-1

P95

Figure 6 TG curves of mimosa tannins – glyoxalated NaLSP cured resins: (—) 20 mass % tannins; (- - -) 40 mass % tannins; (— ·) 50 mass % tannins; (— - -) 60 mass % tannins recorded at 10°C min-1

P96

Figure 7 TG curves of mimosa tannins – glyoxalated lignosulfonates with 40 mass % tannins cured resins: (—) mimosa tannins – glyoxalated NaLSL; (- - -) mimosa tannins – glyoxalated NaLSP; (— ·) mimosa tannins – glyoxalated NH4LSL; (— - -) mimosa tannins – glyoxalated NH4LSP recorded at 10°C min-1

P97

Figure 8 DTG curves of (—) mimosa tannins, (— - -) glyoxalated NH4LSP and (- - -) mimosa tannins – glyoxalated NH4LSP recorded at 10°C min-1

P98

Figure 9 DSC curves of mimosa tannins – glyoxalated lignosulfonates with 40 mass % tannins cured resins: (—) mimosa tannins – glyoxalated NaLSL; (- - -) mimosa tannins – glyoxalated NaLSP; (— ·) mimosa tannins – glyoxalated NH4LSL; (— - -) mimosa tannins – glyoxalated NH4LSP recorded at 10°C min-1

P99

Figure 10 FTIR spectra of (—) mimosa tannins, (- - -) glyoxalated NaLSL, (— ·) mimosa tannins – glyoxalated NaLSL and (— - -) hexamine

P100

III-3 Etude des propriétés de durabilité thermique de colles pour panneaux de particules a base de tanins extraits d’écorce de pin maritime et de lignosulfonates

Figure 1 Module of elasticity of average curves of a pine joint as a function of temperature obtained by TMA testing when bonded with (—) P3 tannins – glyoxalated NaLSL; (—) P3 tannins – glyoxalated NaLSP; (—) P3 tannins – glyoxalated NH4LSL; (—) P3 tannins – glyoxalated NH4LSP recorded at 10°C/min

P111

Figure 2 FTIR spectra of (—) P3 tannins, (—) glyoxalated NaLSL, (—) mimosa tannins – glyoxalated NaLSL and (—) hexamine

P112

Figure 3 (a) TG curves and (b) DTG curves of P3 tannins – glyoxalated lignosulfonates with 40 mass % tannins cured resins: (—) P3 tannins – glyoxalated NaLSL; (—) P3 tannins – glyoxalated NaLSP; (—) P3 tannins – glyoxalated NH4LSL; (—) P3 tannins – glyoxalated NH4LSP recorded at 10°C/min

P114

8

Figure 4 DTG curves of (—) P3 tannins, (—) glyoxalated NH4LSP and (—) P3 – glyoxalated NH4LSP recorded at 10°C/min

P115

Figure 5 DSC curves of P3 tannins – glyoxalated lignosulfonates with 40 mass % tannins cured resins: (—) P3 tannins – glyoxalated NaLSL; (—) P3 tannins – glyoxalated NaLSP; (—) P3 tannins – glyoxalated NH4LSL; (—) P3 tannins – glyoxalated NH4LSP recorded at 10°C/min

P116

III-4 Glyoxalation de lignosulfonates de sodium et d’ammonium pour l’élaboration de colles tanin-lignine pour panneaux de particules

Figure 1 CPGM spectra of lignosulfonates after T1: (—) NaLSP, (—) NaLSL, (—) NH4LSL, (—) NH4LSP

P128

Figure 2 CPGM spectra of (a): (—) NaLSL, (—) NaLSL after T1, (—) NaLSL after T2 and of (b): (—) NH4LSL, (—) NH4LSL after T1, (—) NH4LSL after T2

P129

Figure 3 (a) TG curves and (b) DTG curves of (—) NaLSL, (—) NaLSL after T1, (—) NaLSL after T2 recorded at 10°C/min

P131

Figure 4 Effect of the press time on the internal bond of particleboards bonded with mimosa tannin-NaLSP T1 resin (■) and with mimosa tannin-NaLSP T2 resin (■) at a tannin-lignin ratio of 60-40

P136

Figure 5 Effect of the press time on the internal bond of particleboards bonded with mimosa tannin-NaLSP T1 resin (■) and with mimosa tannin-NaLSP T2 resin (■) at a tannin-lignin ratio of 50-50

P137

Figure 6 Effect of the lignosulfonate on the internal bond of particleboards bonded with mimosa tannin-NaLSP T1 resin, with mimosa tannin-NaLSP T2 resin and mimosa tannin-NH4LSL T1 resin at a tannin-lignin ratio of 40-60 and a press time of 7.5 min

P138

Figure 7 Effect of the tannin-lignin ratio on the internal bond of particleboards bonded with mimosa tannin-NaLSP T1 resin (■) and with mimosa tannin-NaLSP T2 resin (■) for a press time of 7.5 min

P139

9

Liste des tableaux

Chapitre II Extraction de tanins

II-2 Caractérisation par des méthodes spectroscopiques FTIR et HPLC des tanins d’écorce de pin maritime (Pinus pinaster) extraits sous différentes conditions

Table 1 Extraction methods P47

Table 2 Total polyphenolic content extracted with three different conditions P50

Table 3 Total polyphenolic content extracted from the bark of five different maritime pine trees

P51

Table 4 Composition of maritime pine bark extracts determined by RP-HPLC after thiolysis

P57

II-3 Extraction assistée par micro-onde d’écorce de pin maritime (Pinus pinaster) : impact de la taille des particules et caractérisation

Table 1 Characteristics of the MAE (average of at least three replicates) P68

Table 2 Characteristics of MO1 and P3 extracts (average of at least three replicates) P69

Chapitre III Elaboration des colles tanin-lignine

III-2 Etude des propriétés de durabilité thermique des colles tanins- lignosulfonates

Table 1 Lignosulfonates characteristics P86

Table 2 Adhesive formulations P87

Table 3 Module of elasticity values for all the adhesive formulations P93

III-3 Etude des propriétés de durabilité thermique de colles pour panneaux de particules a base de tanins extraits d’écorce de pin maritime et de lignosulfonates

Table 1 Characteristics of the lignosulfonates P108


Table 3 Particleboard characteristics P116

III-4 Glyoxalation of sodium and ammonium lignosulfonates to for tannin lignin adhesives for particleboards

Table 1 Characteristics of lignosulfonates P125

Table 2 Proportions of the reactants used to prepare glyoxalated lignins with the P125

10

treatment 1 (T1) and treatment 2 (T2)

Table 3 Adhesive formulations P126

Table 4 Results from thermal analysis (DSC, TGA) of lignosulfonates before treatment and after T1 and after T2

P132


Table 6 Results from thermal analysis (TG) of cured resins produced with lignosulfonates after treatment 1 (T1) and after treatment 2 (T2)

P135

Erreur ! Argument de commutateur inconnu.

List of abbreviations in English:

DMSO: dimethyl sulfoxide

DSC: differential scanning calorimetry

DTG: derivative thermogravimetric analysis

FTIR: Fourier transformed infrared spectroscopy

GA: gallic acid

Hexamine: hexamethylenetetramine

HSQC: heteronuclear single quantum correlation spectroscopy

IB: internal bond

IR: infrared

MAE: microwave assisted extraction

MOE: modulus of elasticity

PCA: principal components analysis

pMDI: polymeric isocyanate

RP-HPLC: reverse phase high pressure liquid chromatography

RP-HPLC-DAD: reverse phase high pressure liquid chromatography diode array detection

TG: thermogravimetric analysis

TGA: thermogravimetric analysis

TMA: thermomechanical analysis

TS: thickness swelling

UF: urea-formaldehyde

Liste des abréviations en français

ATG : Analyse thermogravimétrique

ATM : Analyse thermomécanique

DSC: calorimétrie différentielle à balayage

EAM : extraction assistée par micro-ondes

Hexamine: héxaméthylènetetramine

IRTF : Infrarouge à transformée de Fourier

PF : phenol-formaldéhyde

pMDI: méthyle diphényle diisocyanate

UF : urée formaldéhyde

13

Introduction générale

L’industrie des panneaux de particules utilise, dans sa fabrication, des produits qui

peuvent présenter une certaine toxicité pour la santé des travailleurs exposés et les utilisateurs

finaux. Le secteur de l’ameublement qui utilise les panneaux de particules en grande quantité

est particulièrement concerné par ce problème.

Les colles utilisées aujourd’hui dans la fabrication de panneaux, pour la plupart, des

colles urée-formaldéhyde et phénol-formaldéhyde, produisent des émissions de formaldéhyde

que le Centre International de Recherche sur le Cancer (CIRC) a classé en 2005 en 1A

« cancérogène pour l’homme ». Les producteurs de panneaux de particules sont donc

confrontés à des pressions environnementales et doivent faire face à des obligations légales et

réglementaires pour fabriquer des produits plus respectueux de l’environnement et de la santé.

En effet, les réglementations concernant les émissions de formaldéhyde sont de plus en plus

strictes. L’émission de formaldéhyde pour l’air intérieur en France sera limitée à 30 µg/m3 en

2015 et à 10 µg/m3 en 2023 [1].

Le formaldéhyde est utilisé dans la production et la composition de produits industriels

depuis à peu près 150 ans et c’est une matière première utilisée pour environ 85 industries

comme désinfectant ou résine par exemple. Au niveau mondial, la production annuelle de

formaldéhyde a été de 29 millions de tonnes en 2010, dont la moitié est utilisée pour la

fabrication de résines à base de formaldéhyde [2]. Ces résines sont utilisées comme adhésifs

dans la majorité des panneaux à base de bois. Sur le plan économique, la colle peut

représenter jusqu’à 40 à 60% du coût total d’un produit fini [3]. Le formaldéhyde présente un

avantage considérable par rapport aux autres substituts chimiques. Une étude de 2007 portant

sur les bénéfices sociaux-économiques du formaldéhyde (Formacare) montre que le

consommateur européen aurait eu à dépenser 29,4 milliards d’euros supplémentaires par an si

le formaldéhyde était remplacé par d’autres substituts. Par ailleurs, les produits alternatifs

présentent une performance inférieure à celle du formaldéhyde [4].

Les entreprises de fabrication de panneaux de particules sont donc incitées à réduire la

quantité de formaldéhyde émis ou à utiliser des produits de substitution dans la formulation de

leurs colles. Ainsi, les adhésifs à faible émission de formaldéhyde et les colles bio-sourcées

(ou colles « vertes ») représentent un domaine de recherche grandissant et de nombreux

projets associant industriels et universitaires sont menés sur ce sujet depuis quelques années.

14

Les solutions dégagées doivent apporter des réponses aux contraintes réglementaires,

économique et de qualité de cette industrie.

La région Aquitaine possède la plus grande forêt de pin maritime (Pinus pinaster)

d’Europe. Le pin maritime est majoritairement cultivé pour son bois qui entre dans la

fabrication de palettes, parquets-lambris, bois de charpente, panneaux de bois et pâte. Cette

activité génère près de 20000 emplois en Aquitaine et est à l’aval d’une filière qui emploie

environ 34000 personnes en Aquitaine, dans des industries de sciages, panetières et papetières

par exemple [5]. Son écorce reste peu valorisée, bien qu’elle soit riche en tanins, et qu’il ait

été démontré que ces derniers peuvent servir à l’élaboration de mélanges collants. Une autre

source de tanins qui peut être utilisée dans l’élaboration de colles est l’écorce de bois de

mimosa (Acacia mearnsii). La lignine, notamment papetière, est également une substance

organique polymère qui peut être valorisée dans la formulation des colles bio-sourcées. C’est

dans ce contexte que nous cherchons à substituer le formaldéhyde par des produits naturels

issus des industries du panneau et de la papeterie d’Aquitaine.

Les travaux de thèse présentés dans ce mémoire ont eu pour objectif principal

d’optimiser l’extraction de tanins condensés, dans l’optique de les inclure dans la formulation

de colles à base de tanins et de lignines pour la fabrication de panneaux à base de bois. Des

méthodes d’extractions les moins consommatrices en solvant et en énergie ont été

recherchées. Les tanins ainsi extraits devaient être analysés et servir à la réalisation de colles

les plus vertes possible. Plus précisément, l’objectif a été de réaliser des formulations de

colles naturelles avec différentes proportions de tanins et de lignines, ces colles étant

composées à plus de 99% de composés naturels, le reste étant le glyoxal et de l’hexamine,

utilisés comme durcisseur. Une fois les formulations réalisées, ces colles ont été testées pour

leurs aptitudes pour la fabrication de panneaux de particules.

La présentation des travaux de recherche est organisée en 3 chapitres. Le premier

chapitre est une introduction bibliographique qui décrira les essences de bois utilisés dans

cette étude, présentera les caractéristiques des tanins et des lignines et présentera les

différentes méthodes d’extraction, ainsi que les différentes colles utilisées jusqu’à présent

dans la fabrication des panneaux de particules.

Le « Chapitre 2 » exposera le travail effectué sur l’extraction des tanins. Pour

l’obtention de ces colles, différents protocoles d’extraction de tanins d’écorce de pin maritime

et de mimosa ont été mis en œuvre. Une méthode innovante – l’extraction assistée par micro-

ondes a été mise en œuvre – et la caractérisation des extraits obtenus sera détaillée.

15

Le « Chapitre 3 » présentera le travail d’élaboration de colles tanins-lignines effectué.

Des formulations de colles avec des tanins d’écorce de pin maritime et des lignosulfonates

seront présentées. Des lignosulfonates de sodium ainsi que des lignosulfonates d’ammonium

ayant subi une glyoxalation afin d’accroître leurs réactivités ont été utilisés. Les températures

de réticulation des colles ont été déterminées et les propriétés thermiques des colles réticulées

ont été étudiées. Une étude des propriétés de durabilité thermique des colles tanins-

lignosulfonates sera présentée et les différentes formulations seront comparées. La fabrication

de panneaux de particules avec les colles bio-sourcées a été réalisée. Les conditions de

pressage ont été optimisées pour des colles avec des tanins de mimosa et des lignosulfonates.

Les principaux résultats seront soulignés et mis en perspective dans une « Conclusion

générale ».

Références Introduction Générale

1. Ministère de l’Ecologie du DD des T et du L. Décret no 2011-1727 du 2 décembre 2011 relatif aux valeurs-

guides pour l’air intérieur pour le formaldéhyde et le benzène. J. Off. 2011;0281:4.

2. Zhang J. Synthèse de formaldéhyde par oxydation directe du méthane en microréacteur. Intsitut National

Polytechnique de Lorraine; 2011. p. 308.

3. Navarrete P. Adhésifs naturels à base de tanin, tanin/lignine et lignine/gluten pour la fabrication de panneaux

de bois. Université Henri Poincaré; 2011. p. 315.

4. EPF, CEI-Bois, EFBWW. Réduction de l’exposition au formaldéhyde des travailleurs de l'industrie du bois.

2010 p. 28.

5. CG 40. Les industries du bois en Aquitaine. http://www.landes.org/les-industries-bois-en-aquitaine. Vu le 8

Septembre 2014.

17

CHAPITRE I Etat de l’art

Ce chapitre sera consacré à la présentation des bois utilisés dans cette étude, à la

présentation et à la caractérisation physico-chimique des tanins et des lignines et présentera

les différentes méthodes d’extraction existantes, ainsi que les différentes colles utilisées dans

la fabrication des panneaux de particules.

La formulation de colles bio-sourcées nécessite tout d’abord la connaissance des

produits qui les constituent, des différentes méthodes d’extraction de ces produits et des

différents types de colle. Ainsi ce chapitre sera dédié à la présentation du pin maritime et plus

particulièrement des extractibles de son écorce (les tanins), des lignines, des méthodes

d’extraction et des différentes colles à bois (plus particulièrement les colles pour panneaux de

bois). Une présentation sommaire en entrée de chapitre sera également faite du bois de

mimosa, puisque des comparaisons entre des extraits de mimosa et du pin maritime ont été

effectuées.

I.1 Présentation des espèces utilisées dans le cadre de l’étude

I.1.1 Le bois de mimosa (Acacia mearnsii)

Une comparaison entre les propriétés et les comportements des tanins de pin maritime

et ceux des tanins de mimosa a été effectuée. Ces derniers tanins provenaient d’Acacia

mearnsii (mimosa).

L’importance du bois de mimosa pour les industries faisant appel à des tanins et à des

opérations de tannage est liée au développement mondial, dès la fin du XIXème siècle, de la

consommation de cuirs dont le tannage était jusque là effectué à partir de végétaux indigènes

(écorce de chêne puis de châtaignier en France). La pénurie des produits des matières

tannantes locales, du fait d’une exploitation intensive, a conduit à l’utilisation d’autres

essences, d’origine étrangère, et notamment le mimosa.

Le mimosa (ou acacia noir ou black wattle) est une espèce d’acacia qui fait partie de

l’ordre des fabales et de la famille des fabaceae (on utilise encore le nom de mimosacées).

Présent aujourd’hui dans toutes les régions tropicales et subtropicales, c’est une essence

originaire d’Australie et de Tasmanie. Il est notamment cultivé en Afrique, mais aussi en

Amérique du Sud. L’espèce, découverte lors de l’arrivée des britanniques en Australie, a été

rapidement importée en Europe et, dès le XIXème siècle, on en avait recensé environ 400

espèces. Si son introduction en France est liée à l’ornement, il a été introduit dans d’autres

18

contrées, notamment sur l’île de la Réunion et en Afrique, pour ses tanins. Son écorce

contient en effet de 30 à 40% de tanins, utilisés notamment pour le tannage du cuir. Il s’agit

d’un arbre de croissance rapide, pouvant atteindre jusqu’à 20 mètres. Sa longévité varie de 20

à 40 ans.

Ainsi, Acacia mearnsii est devenu la principale source mondiale d’écorce à tanin.

Mais la volonté des entreprises implantées en Europe, et principalement en France, est

d’utiliser des produits locaux. A un siècle de distance, on assiste à un retournement

économique et culturel qui voit un avantage dans l’exploitation d’une production locale,

génératrice de valeur ajoutée.

I.1.2 Le pin maritime (Pinus pinaster)

Pinus pinaster (pin maritime) qui fait partie de la classe des pinidés (pinidae) et de la

famille des pinacés (pinaceae), pousse dans un certain nombre de pays dans la partie

occidentale du Bassin méditerranéen : des pays européens, tel que la France, le Portugal et

l’Espagne, et certains pays de l’Afrique du Nord-Ouest (Figure 1) [1–3]. C’est l’arbre

caractéristique du massif forestier des Landes où il est présent depuis l’antiquité.

Figure 1: Répartition géographique du pin maritime en Europe [4,5]

Il s’agit d’un arbre à la croissance rapide (6 mètres en 20 ans), qui peut atteindre entre

20 et 30 mètres de haut (jusqu’à 40 mètres) et jusqu’à 5 mètres de circonférence à la base. Le

19

pin maritime atteint sa maturité à 40-50 ans. Sa longévité est assez limitée (autour d’un

siècle).

Lors des reboisements artificiels du XIXe siècle et du début du XXe siècle, le pin

maritime était l’espèce la plus utilisée [6]. Dès le XVIIIe siècle, l’ensemencement de pin

maritime dans le Département des Landes a permis de stabiliser et d’assainir les sols et

d’arrêter la progression des sables. Le développement de la forêt des Landes prend

véritablement son essor sous la période du second empire, Napoléon III imposant, dès 1857,

l’ensemencement du pin maritime.

Aujourd’hui, le massif forestier des Landes de Gascogne (couvrant les départements

aquitains de la Gironde, des Landes et du Lot et Garonne) fournit un exemple réussi d’un

reboisement artificiel. Couvrant près d’un million d’hectares et représentant 8% de la surface

forestière du pays, le massif est la plus grande forêt artificielle d’Europe [6]. La production en

bois y est importante. Ainsi, la forêt des Landes de Gascogne fournissait 16% du volume

produit dans les années 1960 en France en bois d’œuvre ou d’industrie.

Récemment, en 1999 (tempête Martin du 27 décembre) et 2009 (tempête Klaus du 27

janvier) des tempêtes ont causé d’énormes dégâts à ce massif forestier, la tempête Klaus de

2009 ayant dévasté près d’un cinquième de la forêt. Le travail de reboisement considérable

engagé avec la mobilisation de l’Etat et de la filière montre l’enjeu de cette production pour la

Région Aquitaine et pour la valorisation de l’espace forestier français.

I.1.3 L’écorce de pin maritime

Le pin maritime de la forêt des Landes est exploité sous différentes formes. Le plus

souvent, lors de l’exploitation forestière, le bois abattu est écorcé. De ce conditionnement du

bois résultent des produits dérivés dont l’écorce. L’écorce est épaisse dans cette espèce. Elle

se fissure au fur et à mesure de la croissance de l’arbre et prend une couleur rouge-noir chez

un sujet adulte. L’écorce constitue, sur un peuplement de 26 ans, de l’ordre de 12% de la

biomasse aérienne [7]. La forêt des Landes représente donc une réserve d’écorce très

importante. L’écorce est actuellement surtout vendue en horticulture, mais de nombreuses

autres possibilités d’emploi de l’écorce de pin sont citées dans la littérature scientifique. Son

enfouissement dans le sol de la forêt après l’abattage des arbres permettrait de restituer une

importante quantité de matière organique et d’augmenter la fertilité du sol [8]. Le fait que des

propriétés anti-radical libre, anti-oxydant, et anti-inflammatoire sont attribuées à ses

extractibles [1,9–11] a fait que certains d’entre eux sont utilisés dans la fabrication de

suppléments alimentaires et d’un médicament en phytothérapie, commercialisé sous le nom

20

de Pycnogenol® (tanins d’écorce de pin maritime des Landes). L’écorce de pin maritime peut

également être utilisée dans la production de colles.

Il y a plusieurs types d’extractibles dans l’écorce de pin maritime qui est riche en

composés phénoliques [12] et plus particulièrement riche en tanins condensés [2], la quantité

la plus significative de ces tanins se trouve dans la partie interne de l’écorce (phloème)

(Figure 2) [13]. Nous nous intersserons en particulier aux flavanoïdes et aux tanins.

Figure 2: Schéma de la structure de l'écorce [14]

I.2 Les extractibles

Les extractibles correspondent à une gamme de composés présents dans la structure

poreuse de toutes les plantes [15]. Ce sont des molécules que l’on peut facilement extraire

avec un solvant organique ou de l’eau. Les extractibles du bois se trouvent en majorité dans

l’écorce. Parmi les extractibles présents dans l’écorce de bois, on peut citer les polyphénols

dont les tanins, les flavonoïdes, des acides gras, des terpènes, des matières grasses, des

huiles… Dans le pin maritime et d’autres espèces de pin, on trouve également des acides

phénoliques comme l’acide cafféique, l’acide ferulique et l’acide p-hydroxybenzoïque, des

dérivés de glucopyranosyl, des sucres et des huiles [10,11,15,16].

I.2.1 Les flavonoïdes

Les flavonoïdes sont des composés polyphénoliques avec une structure flavane (Figure

3). Ils sont présents dans beaucoup de plantes. Selon Routray et Orsat [17], ce sont des « anti-

oxydants qui agissent comme récepteurs de radicaux libres, comme de potentiels agents

réducteurs, et qui protègent des réactions d’oxydation qui ont lieu dans le corps humain ». En

fonction du régime alimentaire, on consomme entre 25 mg et 1g de flavonoïdes par jour [18].

21

Figure 3: Structure chimique de base du noyau flavane

I.2.2 Les tanins

Stevanovic et Perrin [15] définissent les tanins comme « des composés phénoliques

solubles dans l’eau, dont la masse molaire se situe entre 500 et 3000 g/mol, et sont de plus

capables de précipiter les alcaloïdes, la gélatine et les autres protéines ». Les tanins sont des

composés polyphénoliques présents dans les plantes. L’écorce des arbres en contient une

quantité importante. Les tanins protègent la plante contre des champignons et d’autres

parasites.

Les tanins sont divisés en trois catégories: les tanins hydrolysables, les tanins

condensés (aussi appelé proanthocyanidines) et les phlorotanins que l’on trouve dans les

algues brunes [19]. Ces derniers ne se trouvant pas dans les écorces d’arbre, ils ne seront pas

présentés.

I.2.2.1 Tanins hydrolysables

Les tanins hydrolysables (ellagitanins et gallotanins) ont déjà été utilisés dans la

formulation de colles à bois [20–22] mais leur faible réactivité et disponibilité font qu’ils sont

peu employés dans l’industrie du bois [23]. Ce sont des esters de l’acide gallique qui peuvent

être divisés en deux catégories : les ellagitanins et les gallotanins.

Les ellagitanins se décomposent en acide ellagique et différents dérivés d’acide

hexahydroxydiphénique et d’acides phénoliques (Figure 4). On trouve des ellagitanins dans

des espèces d’arbres comme le châtaignier ou le bois de chêne ou bien de fruits comme les

fraises [24–26]

22

Figure 4: Exemple d’ellagitanin : castalagine (R1 = H, R2 = OH) ; vescalagine (R1 = OH, R2 =

H) [27]

Les gallotanins sont des esters d’acide gallique avec D-glucose (Figure 5).

Théoriquement, des formulations de colles gallotanin-formaldéhyde peuvent être de bonne

qualité. En effet, le formaldéhyde en réagissant avec des cycles galloylés en position ortho des

gallotanins permettrait la formation de structures tridimensionnelles. Toutefois, l’hydrolyse

des gallotanins pendant la réticulations réduit leurs réactivités [28]. On trouve des gallotanins

dans de nombreuses plantes telles que dans le tara, dans le sumac, dans Castanopsis cuspidata

et dans l’érable à sucre [15,25,28].

Figure 5: Exemple de gallotanin : B-1, 2 3 4 6 – pentagalloyl – O D – glucose [27]

I.2.2.2 Tanins condensés

Les tanins condensés (ou proanthocyanidines) sont des polyphénols présents dans

toutes les plantes. Leur unité structurale fondamentale est le noyau flavane.

23

Ce sont des oligomères flavan-3-ol qui possèdent une liaison flavanoïde C4-C6 ou C4-

C8. Ils peuvent contenir des acides galliques esters. L’anneau aromatique A est composé de

resorcinol ou de phloroglucinol et l’anneau aromatique B consiste de catéchol ou de

pyragallol (Figure 6).

Figure 6: Unité de base des tanins condensés [29]

La liaison interflavanoïde est spécifique à chaque espèce. Le quebracho présente une

proportion élevée de liaisons interflavanoïdes C4-C8 tandis que le mimosa présente une

prédominance de liaisons interflavanoïdes C4-C8 [30]. Les principales structures des tanins

extraits d’écorce de pin maritime sont les catéchine/épicatéchine, épigallocatéchine et

épicatéchine gallate (Figure 7). On trouve des liaisons flavonoïdes C4-C6 et C4-C8 dans les

tanins d’écorce de pin maritime [2].

24

Figure 7: Unités de base des tanins condensés principaux d'ecorce de pin maritime (a) catéchine,

(b) épicatéchine, (c) épigallocatéchine, (d) épicatéchine gallate

D’après Stevanovic et Perrin [15], on trouve des tanins condensés « dans les tissus

épidermiques des plantes, dans les feuilles, l’écorce et le phloème de l’écorce et sont en règle

générale absents du bois d’aubier et de cœur ».

I.2.2.3 Valorisation des tanins

Traditionnellement, les tanins s’emploient dans le processus de tannage du cuir. La

capacité des tanins à précipiter des protéines telles que le collagène permet de générer ainsi un

matériau non-putrescible sous des conditions d’humidité et de chaleur [31].

Aujourd’hui, en plus des secteurs traditionnels d’utilisations tels que celui du tannage

et également celui de l’œnologie, les tanins sont utilisés dans de nombreuses applications :

œnologie, pharmacologie, purification de l’eau, cosmétique…

(d)

(c)

(b)

25

En œnologie, quantifier et connaitre les tanins présents dans le vin, notamment le vin

rouge, est très important. En effet, les tanins hydrolysables, extraits des tonneaux ou bien

ajoutés pendant la vinification, permettent d’éliminer des protéines indésirables [32]. Les

tanins condensés ont un effet sur l’astringence, la sensation en bouche et en partie sur la

stabilisation de la couleur du vin [32,33]. L’astringence est la sensation de sécheresse et de

rugosité du palais. Les tanins sont à l’origine de la réticulation de chaînes polypeptides de

protéines riches en proline, ce qui précipite ces dernières et donne la sensation d’astringence

[34].

Les tannins présentent des propriétés anti-oxydantes, anti-radicaux libres et anti-

inflammatoires et constituent de bons suppléments alimentaires pour l’homme et les animaux

[10]. Les tanins condensés ont un effet direct sur l’abondance de nématodes gastro-intestinaux

et indirects sur l’amélioration de l’apport en protéine des animaux, ce qui améliore la

digestion des protéines et les performances des animaux [35].

Ces dernières années, des mousses rigides à base de tanins condensés ont été réalisées

[36–38]. Tondi et al. [39] cherchent à utiliser ce type de mousses pour la dépollution en

métaux d’eaux usées industrielles. En effet, les tanins ont la capacité de piéger les ions

métalliques. Peng et Zhong [40] ont fabriqué un hydrogel à base d’acides tanniques ayant la

capacité d’adsorber des ions Cu (II).

Des tanins et des acides tanniques ont également été utilisés afin de réaliser des

hydrogels capables d’inhiber la croissance de bactéries et de champignons [41].

Les tanins peuvent également être utilisés dans la fabrication de colles [30]. Des tanins

extraits d’un grand nombre de plantes différentes ont été utilisés dans la production de colles à

bois : le marc de raisin [42], la châtaigne [43], la noix de pécan [44], le mimosa [45,46] et le

pin maritime [47]. Jusqu’au milieu des années 2000 des tanins ont été utilisés pour remplacer

le phénol dans des résines formaldéhyde – phénol [48–51]. Pichelin et al. [52] ont démontré

que l’héxaméthlènetetramine (hexamine) est un durcisseur qui possède des propriétés

équivalentes au formaldéhyde ou au paraformaldéhyde et des adhésifs à base de tanins et de

l’hexamine avec de bonnes propriétés mécaniques ont été produites [53,54]. Des adhésifs à

base de tanins et de lignines ont également été développés. Dans ce cas, les tanins utilisés

étaient extraits du bois de mimosa [46,55,56].

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I.3 Les processus d’extraction des tanins

De nombreuses méthodes d’extraction sont employées pour obtenir des tanins

condensés, telles que l’extraction Soxhlet, l’extraction assistée par micro-ondes, l’extraction

assistée par ultrasons, par fluide supercritique, et par liquide pressurisé et l’extraction à base

d’eau chaude [57–61].

I.3.1 Extraction à l’eau chaude

Les extractions à l’eau chaude sont depuis longtemps utilisées pour extraire des tanins

condensés dans le but de faire des adhésifs ou pour leurs propriétés anti-oxydantes [16,47,62–

66]. Des sels, tels que le sulfite de sodium, le bisulfite de sodium, le carbonate de sodium,

sont ajoutés afin de limiter des réactions d’auto-condensations des tanins et de ce fait réduire

la viscosité et d’empêcher la formation de phlobaphènes [64,67]. C’est une méthode simple,

peu coûteuse et qui n’utilise pas de solvants.

I.3.2 Extraction au Soxhlet

L’extraction au Soxhlet est considérée comme une extraction conventionnelle et est

souvent cité comme extraction témoin dans la littérature [3,57,61,68,69]. Cette méthode est

utilisée pour extraire des végétaux, entre autres, des extractibles [70,71], des lignines [72], des

lipides [73] et autres corps gras [74]. La matière végétale est placée dans une cartouche de

cellulose et le solvant dans le ballon (Figure 8). Le solvant est porté à ébullition, s’évapore,

condense sur les parois du réfrigérant et retombe dans la cartouche solubilisant les composés à

extraire. Lorsque la cartouche est pleine, le solvant avec les composés extraits tombe dans le

ballon. L’extraction au Soxhlet est très efficace, simple et peu coûteuse. Elle demande

toutefois une grande quantité de solvant, prend beaucoup de temps, et peut dégrader la

matière du fait de la température élevée du solvant.

27

Figure 8: Schéma d'un extracteur Soxhlet [75]

I.3.3 L’extraction assistée par micro-ondes (EAM)

L’EAM consiste en des radiations électromagnétiques de fréquences allant de 0,3 à

300 GHz. Les micro-ondes se propagent sous forme de vagues qui réagissent avec les

molécules polaires dans le matériau. Ces molécules polaires comme l’eau génèrent alors de la

chaleur qui chauffe le matériau à cœur [75]. L’EAM est une méthode d’extraction rapide qui

génère une pression élevée dans le biomatériel qui provoque la rupture des cellules. La

pénétration du solvant d’extraction est ainsi facilitée [76]. Un des emplois principaux de

l’extraction assistée par micro-ondes est la dépollution des sols. Elle permet d’extraire des

pesticides des sols [77,78]. L’EAM des matériaux organiques s’emploie essentiellement dans

le but d’extraire des flavonoïdes simples ou des polyphénols simples [17,79,80]. Cette

méthode a rarement été utilisée pour extraire des tanins condensés [57,81].

I.3.4 L’extraction assistée par ultrasons

L’extraction assistée par ultrasons est une technique simple et peu couteuse. L’effet

mécanique des ultrasons, avec une fréquence supérieure à 20 kHz, abîme les parois cellulaires

de l’échantillon, permettant ainsi une meilleure pénétration du solvant et donc un meilleur

rendement d’extraction. Les ultrasons peuvent parfois détruire les parois cellulaires [75]. De

plus, les ultrasons provoquent des cycles d’expansion et de compression des cellules formant

ainsi des bulles. Les bulles vont grossir jusqu’à exploser et déchirer les parois cellulaires [75].

28

La température d’extraction étant faible, généralement 20-25°C, l’extraction de composés

thermolabiles est possible sans répercussions sur la qualité des extraits [17]. L’extraction

assistée par ultrasons est très utilisée pour l’extraction de flavonoïdes, de polyphénols et de

composés phytosanitaires [57,65,80,82–84].

I.3.5 Extraction par fluide supercritique

L’extraction par fluide supercritique est une méthode qui permet d’extraire de

nombreux composés bioactifs. Le point critique est défini à la température et pression

maximales auxquelles le composé peut exister à l’équilibre solide-liquide. A des températures

et pressions supérieures, un fluide homogène est formé et est appelé fluide supercritique. Ce

fluide est aussi lourd qu’un liquide et a une force de pénétration d’un gaz [85]. Cette méthode

est également appelé extraction par CO2 supercritique car le CO2 est le solvant le plus

fréquemment utilisé du fait de sa facilité d’obtention. En effet, le CO2 a une température

critique faible, 304,1 K, est une pression critique modérée, 7,28 MPa. Le dioxyde de carbone

a également l’avantage de créer un environnement sans oxygène et donc d’éviter d’avoir des

réactions d’oxydation. Toutefois, le CO2 est un fluide non-polaire ce qui limite la solubilité

des composés polaires. L’ajout d’un co-solvant polaire en faible quantité permet d’augmenter

significativement la solubilité des composés polaires [60]. Le méthanol et l’éthanol sont de

bons co-solvants pour l’extraction de composés phénoliques. Ainsi, des composés

phénoliques dont des tanins condensés et des composés lipophiliques ont été extraits d’Acacia

mearnsii, de grignons de raisins et de bois de pin maritime entre autres dans du CO2-Ethanol

(90-10) à des températures modérées, 50°C à 20-25 MPa [60,61,86].

I.3.6 Extraction par liquide pressurisé

L’extraction par liquide pressurisé est utilisée pour extraire des flavonoïdes, des

polyphénols, des polluants de sols, de végétaux et d’aliments [58,87–90]. Il s’agit d’une

méthode rapide (5-15 min) qui utilise des solvants à haute température (40-200°C) et à haute

pression (3,3-20,3 MPa). Une pression élevée est utilisée afin de maintenir les solvants à l’état

liquide. En augmentant la température, la viscosité du solvant diminue, augmentant la

diffusivité du solvant [3]. De plus, les fortes pressions appliquées permettent au solvant de

pénétrer plus facilement dans l’échantillon [85]. Ces deux caractéristiques permettent de

réduire drastiquement le temps d’extraction par rapport à une extraction conventionnelle : 5

min au lieu de 1h pour l’extraction de tanins condensés d’écorces de grenade par exemple

[58]. L’extraction par liquide pressurisé présente également l’avantage de se dérouler à l’abri

de la lumière et sous azote évitant toute réaction d’oxydation

par liquide pressurisé est présenté en

Figure 9: Schéma du principe de l'extraction par liquide pressurisé

I.4 Les lignines

La lignine est un polymère phénolique qui rend les parois cellulaires des plantes

rigides. C’est le polymère naturel le plus abondant après la cellulose et il représente une part

importante de la biomasse terrestre. La structure moléculaire est hétérogène et complexe,

basée sur trois monomères : l’alcool p

sinapylique (Figure 10). Les proportions des trois monomères variant non seulement selon les

plantes mais aussi d’une partie d’une p

Figure 10: Unités de base de la lignine : (a) alcool p

alcool sinapylique

(a)

29

de la lumière et sous azote évitant toute réaction d’oxydation [17]. Le principe de l’extraction

par liquide pressurisé est présenté en Figure 9.

: Schéma du principe de l'extraction par liquide pressurisé [88]




: l’alcool p-coumarylique, l’alcool conifér

). Les proportions des trois monomères variant non seulement selon les

plantes mais aussi d’une partie d’une plante à une autre [15].

base de la lignine : (a) alcool p-coumarylique, (b) alcool conférylique, (c)

(c)

(

(b)

. Le principe de l’extraction




coumarylique, l’alcool coniférylique et l’alcool

). Les proportions des trois monomères variant non seulement selon les

coumarylique, (b) alcool conférylique, (c)

30

En 2004, seuls 2% de la production mondiale de lignines étaient valorisés [91].

I.4.1 Les lignines Kraft

Afin d’obtenir des lignines kraft de la biomasse, une réaction de délignification doit

avoir lieu. La délignification s’effectue par un traitement de la matière végétale dans de

l’hydroxyde de sodium et de sulfure de sodium à forte température, 180°C, forte pression,

0,7-1 MPa, pendant 0,5-2 h [92,93]. On obtient ce que l’on appelle de la liqueur noire.

Les lignines krafts étant insolubles en milieu acide, elles sont précipitées par une

réaction d’acidification au dioxyde de carbone ou à l’acide sulfurique [92]. A haute

température la réaction d’acidification est plus efficace [15]. En 2004, approximativement

100 000 t/an de lignines kraft étaient produites. La majorité de ces lignines sont brûlées afin

de produire de l’énergie, et une infime partie est valorisée après modifications chimiques [91].

I.4.2 Les lignines organosolves

Les lignines organosolves sont obtenues par un traitement de la biomasse par des

solutions aqueuses de solvants organiques. Elles ont habituellement un faible poids

moléculaire [92]. Les solvants le plus souvent utilisés sont l’éthanol, l’acide acétique, l’acide

formique et l’acétate de sodium, le méthanol et le peroxyde d’oxygène ; l’éthyle acétate, le

butanol et le phénol sont également utilisés [42,71,92–95]

I.4.3 Les lignosulfonates

Les lignosulfonates sont obtenus à partir d’un traitement du bois ou de la matière

végétale avec différents sulfites. La biomasse est traitée à pH acide, 1-2, à forte température et

pression dans des solutions de sulfites ou bisulfites de sodium, d’ammonium, de calcium ou

de magnésium (Figure 11). Dans ces conditions, les lignines, les extractibles et des

hémicelluloses sont extraits. Les hémicelluloses de ces « liqueurs sulfitées » se décomposent

en monosaccharides solubles dans l’eau par hydrolyse [92]. En 2004, près de 1 000 000 t/an

de lignosulfonates étaient produites dans le monde. Les lignosulfonates sont généralement

utilisés comme combustible pour produire de l’énergie mais ils sont aussi utilisés comme

agents liants pour l’alimentation végétale et les routes non-goudronnées, et comme des

dispersants pour ciments, bétons et pour le forage de puits de pétrole [91,92].

31

Figure 11:Unité de base des lignosulfonates de sodium

I.5 Colles pour panneaux de bois

La plupart des colles à bois utilisées aujourd’hui sont des amino-résines (urée-

formaldéhyde, mélamine-formaldéhyde, etc.) ou des résines phénol-formaldéhyde ou

resorcinol-formaldéhyde [96]. Le prix élevé des produits issus du pétrole et la volonté de

réduire les émissions de formaldéhyde poussent l’industrie du panneau de particules à

chercher des matériaux naturels qui puissent se substituer, partiellement ou entièrement, aux

composants de ces résines, et plus particulièrement au formaldéhyde.

I.5.1 Historique

L'homme a très tôt utilisé des substances naturelles pour fabriquer des adhésifs. Il est

attesté que les néandertaliens utilisaient aussi des colles à base de brai de bouleau (bétuline)

[97]. Résines de conifères et de pistachier, cire d'abeilles, bitume, sont attestés dès le

néolithique moyen (plus de 35000 ans). Au moyen âge et à la Renaissance, blanc et jaune

d'œuf, miel, servent de liant pour les pigments des peintures et des manuscrits [98,99]. Ainsi,

de tous temps et sous toutes les latitudes, le collage a été un moyen privilégié d'assemblage de

matériaux. On estime qu'il existe aujourd'hui une dizaine de milliers de colles ou d'adhésifs.

A partir du XIXème siècle mais surtout du XXème siècle, des adhésifs synthétiques

sont venus se combiner ou remplacer les colles naturelles. Aujourd'hui, les exigences sur

l'environnement, la santé, conduisent à ouvrir de nombreuses voies de recherche autour de

l'utilisation des biopolymères [100]. Des colles thermodurcissables sont utilisées dans

l’industrie des panneaux de bois. Les résines thermodurcissables sont des polymères à l’état

de liquide visqueux dont la polymérisation se fait sous l’effet de la chaleur, et en présence ou

non d’un durcisseur chimique, d’un catalyseur et d’un accélérateur éventuel, pour former un

O

OH

H (or lignin)

H3C

S

lignin

OH

ONa

O

O

32

réseau tridimensionnel qui durcit de façon irréversible [27]. Les tanins, notamment les tanins

du bois, sont considérés comme un constituant principal pour de nouvelles générations de

colles susceptibles d’être employées dans l’industrie des panneaux de particules pour

remplacer ou réduire l'utilisation des résines dites urée-formaldéhyde (UF), phénol-

formaldéhyde (PF), mélamine-urée-formaldéhyde (MUF) et isocyanates.

I.5.2 Urée-formaldéhyde (UF)

Les résines UF ont été inventées en 1920 mais ce n’est pas avant la fin des années 30

qu’elles furent commercialisées [101]. Elles sont majoritairement utilisées pour l’encollage de

panneaux de particules. La réaction entre l’urée et le formaldéhyde se déroule en deux étapes.

Dans un premier temps, il y a une réaction d’hydroxyméthylolation de l’urée (Figure 12).

Cette réaction forme des urées mono-, di-, et trihydroxyméthyle et a lieu en milieu alcalin, pH

≈ 8-9, afin d’éviter des réactions de condensation. La deuxième étape consiste en la

condensation des urées hydroxyméthylées en polymères de fort poids moléculaire. Cette

réaction a lieu en milieu acide, pH ≈ 5 [102,103].

Figure 12: Réaction d'hydroxyméthylolation de l'urée avec le formaldéhyde [102]

L’inconvénient majeur des résines UF est l’émission de formaldéhyde qui a lieu

pendant le processus de fabrication des résines et par les panneaux collés avec ces résines. Un

autre inconvénient des résines UF est leur faible résistance à l’eau et à l’humidité, ce

problème est souvent résolu par l’ajout de substances comme la mélamine et parfois le phénol

[103]. Plus récemment de l’albumine et de l’huile de tournesol ont été ajoutés à de la résine

UF pour la rendre plus résistante à l’eau [104]. De la farine ou de l’amidon peuvent être

33

incluses dans la résine UF afin de réduire les proportions de formaldéhyde [105]. Des ajouts

de tanins afin de diminuer les quantités de formaldéhyde ont également été réalisés [106] ainsi

qu’en tant que substitut à l’urée [28,107].

I.5.3 Phénol-formaldéhyde (PF)

C’est au début des années 30 que les résines phénoliques pour contreplaqué se sont

développées. Les résines PF sont essentiellement utilisées pour l’encollage de panneaux

contreplaqués et de panneaux de particules. D’après Pizzi [108], les phénols réagissent avec le

formaldéhyde en position ortho et para pour former des phénols hydroxyméthylés ou des

alcools phénoliques. Dans un second temps, les groupes hydroxyméthyles réagissent avec des

phénols ou bien avec des phénols hydroxyméthylés. On obtient alors des polymères linéaires.

Des tanins et des lignines peuvent être ajoutés aux résines PF afin de diminuer la

consommation de PF. Des résines tanins-PF, et lignines-PF pour panneaux de contreplaqué

ont été élaborées et ont montré des propriétés mécaniques performantes [51,109,110]. Des

résines lignines-PF ont également été produites pour l’encollage de panneaux de particules et

les lignines ont pu substituer jusqu’à 30% de PF [111].

I.5.4 Isocyanates

Les résines isocyanates ont été développées pendant la seconde guerre mondiale en

Allemagne. A l’origine, elles ont été conçues afin de coller le caoutchouc au métal [101].

Aujourd’hui, elles sont utilisées principalement sous la forme de 4,4’-méthyle diphényle

diisocyanate (pMDI). Elles sont utilisées pour l’isolation et principalement pour la

formulation d’adhésifs [93]. Le pMDI est généralement ajouté à des formulations de résines

lignines-PF ou bien tanins-formaldéhyde. Selon Ping et al. [112], les tanins condensés et

notamment la catéchine réagissent de deux manières avec le pMDI. Le pMDI réagit avec les

groupes hydroxyméthyles et il y a formation de ponts méthylènes entre les tanins et le pMDI.

En présence de lignines et de PF, le pMDI accélère la réticulation et l’améliore [113]. Il y a

formation de ponts méthylènes entre lignine-lignine, lignine-phénol et phénol-phénol et de

ponts uréthanes entre les groupes hydroxyméthyles des lignines et de la résine PF avec le

pMDI [114].

I.5.5 Colle à base de lignines

Plusieurs méthodes de prétraitement des lignines ont été mises au point afin de palier à

la faible réactivité des lignines : la sulfonation, la methylolation, le greffage, la phénolation, la

carboxymethylation, la copolymérisation avec l’anhydride maléique [92,115–118].

Récemment une nouvelle méthode, la glyoxalation, a été mise au point [114].

34

La structure de la lignine, grâce à la présence de groupes aliphatique hydroxyle et

phénolique, en fait un bon substitut partiel pour le phénol dans des colles à base de phénol et

de formaldéhyde [94,109,119–123]. Récemment des résines lignine –phénolique – pMDI ont

été produites avec des lignines glyoxalées [114,124]. La glyoxalation des lignines s’est avérée

un alternatif à la préméthylolation qui augmente la réactivité des lignines [114]. Le glyoxal est

un aldéhyde non-volatile, non-toxique [125] qui a été utilisé en tant que durcisseur pour des

colles à base de tanin dans la fabrication de panneaux de particules, leur apportant de bonnes

propriétés mécanique d’adhésion [126]. Des colles à base de tanins et de lignine ont

également été développées. Les tanins employés étaient des tanins de mimosa avec soit des

lignines organosolv soit des lignines kraft purifiées ou non [46,55,56]. Plusieurs études ont

montré que des formulations de colles tanins-lignines en présence d’hexamine donnent de

bons panneaux de particules avec au moins 40% de tanins [42,46,127].

I.5.6 Colles à base de tanins

A l’origine, les tanins avaient pour but de se substituer au phénol dans les résines PF.

Des ponts méthylènes sont formés entre l’anneau A des tanins condensés et le formaldéhyde

[107].Dès le début des années 70, des adhésifs tanin-formaldéhyde ont été produits de

manière industrielle et commercialisés [30]. Les tanins ont tout d’abord été ajoutés à des

résines UF et PF pour remplacer en partie le phénol, en utilisant du paraformaldéhyde comme

durcisseur [51,128,129]. A la fin des années 90 et pendant les années 2000, des investigations

ont été menées afin de trouver un durcisseur autre que le paraformaldéhyde. Des études sur

l’utilisation comme durcisseur du tris(hydroxyl)nitrométhane, de l’hexamine, du glyoxal, du

sulfate d’ammonium, de l’épichlorhydrine, etc ont été menées [23,52,126,130,131].

Aujourd’hui, le durcisseur le plus communément utilisé est l’hexamine [42,67,93,127,132–

134]. Pour l’encollage de panneaux de contreplaqué des charges comme de la farine peuvent

être ajoutées [106]. Pour l’encollage des panneaux de particules, un des inconvénients des

adhésifs à base de tanins peut être leur viscosité importante. La température et le pH au

moment du mélangeage des formulations de colle notamment influent sur la viscosité des

colles [23].

35

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43

CHAPITRE II Extraction de tanins

II.1 Introduction au chapitre II

Les tanins sont obtenus de la matière végétale par extraction. En fonction de la

méthode d’extraction choisie, la composition des extraits varie ainsi que les quantités

extraites. Le temps d’extraction et les solvants utilisés varient selon les différentes extractions.

Dans l’optique de réduire la consommation de solvants, de temps, d’énergie consommée et les

coûts d’extraction, nous avons décidé de comparer expérimentalement deux techniques

d’extraction de tanins d’écorce de pin maritime.

L’extraction à l’eau chaude a l’avantage de ne pas consommer de solvants et d’être

facilement mise en œuvre. Nous avons cherché à optimiser l’extraction de tanins d’écorce de

pin maritime par cette méthode. Toutefois, on n’extrait pas uniquement des tanins condensés

par cette technique. De façon à quantifier et à identifier les tanins condensés extraits, des

dosages colorimétriques, de la chromatographie liquide à haute pression et de l’infrarouge à

transformée de Fourier (IRTF) ont été réalisés.

Une technique d’extraction innovante, l’extraction assistée par micro-ondes, a

également été réalisée. C’est une technique qui permet d’effectuer une extraction rapidement

avec peu de solvants. Pour notre étude, un solvant peu toxique, l’éthanol, a été utilisé. Cette

technique n’ayant, à notre connaissance, jamais été utilisée pour l’extraction de tanins

condensés une étude de l’impact de la granulométrie sur les rendements d’extractions et sur la

nature des extraits a été réalisée. Comme pour l’extraction à l’eau chaude, les extraits ont été

caractérisés par des dosages colorimétriques, IRTF et également par 1H RMN et 2D HSQC

RMN.

Les deux techniques d’extraction ont été comparées afin de mieux comprendre les

avantages et inconvénients de chaque méthode.

44

II.2 Caractérisation par des dosages colorimétriques, IRTF et HPLC des

tanins d’écorce de pin maritime (Pinus pinaster) extraits sous

différentes conditions

Publié dans : Industrial Crops and Products, 49, p897-903, 2013.

Résumé

Pour la première fois une caractérisation extensive des tanins condensés d’écorce de

pin maritime (Pinus pinaster) a été conduite. Des extractibles ont été obtenus à partir d’écorce

de pin maritime provenant du sud-ouest de la France en utilisant 1% NaOH et 5% NaOH à

70°C et 80°C. Pour chaque extraction, le rendement d’extraction, le contenu total en

polyphénols (Folin-Ciocalteu), en tanins condensés et la réactivité au formaldéhyde (nombre

de Stiasny) ont été quantifiés. Les extraits ont été caractérisés par spectroscopie infra-rouge à

transformée de Fourier (IRTF) et par chromatographie liquide à haute pression en phase

inverse (RP-HPLC). Les taux les plus élevés de rendement d’extraction provenaient d’une

extraction utilisant 5% NaOH à 80°C. Les extraits obtenus en utilisant 1% NaOH à 70°C

contenaient les quantités les plus élevées de polyphénols, de tanins condensés et ont donné la

plus grande réactivité au formaldéhyde. Des pics d’absorbance à 1260 cm-1, à 1440 cm-1 et à

1495 cm-1 n’ont été détectés que pour des extraits obtenus en utilisant 1% NaOH. RP-HPLC a

révélé que la catéchine est le principal tanin condensé contenu dans les extraits ; des quantités

moins élevées d’épicatéchine et d’épicatéchine gallate ont également ont été mesurées.

Mots-clés : IRTF, polyphénols, tanins condensés, Pinus pinaster, RP-HPLC

45

Characterisation of maritime pine (Pinus pinaster) bark tannins extracted

under different conditions by spectroscopic methods, FTIR and HPLC

L. Chupina*, C. Motillona, F. Charrier - El Bouhtourya, A. Pizzib, B. Charriera

a EPCP-IPREM, IUT des Pays de l’Adour, 371 Rue du Ruisseau, BP 201, 40004 Mont de

Marsan, France

b ENSTIB-LERMAB, University of Lorraine, 27 Rue Philippe Seguin, BP 1041, 88051

Epinal, France

*Corresponding Author: EPCP-IPREM, IUT des Pays de l’Adour, 371 Rue du Ruisseau, BP

201, 40004 Mont de Marsan, France; tel: +33 558513722; fax: +33 558513737; email:

[email protected]

Abstract

For the first time an extensive characterisation of maritime pine (Pinus pinaster) bark

condensed tannins was conducted. Extractives were obtained from the bark of maritime pine

from the south west of France using 1% NaOH and 5% NaOH at 70°C and 80°C. For each

extraction, extraction yield, total polyphenols (Folin-Ciocalteu), condensed tannins and

reactivity to formaldehyde (Stiasny number) were quantified. The extracts were characterised

by Fourier transformed infrared spectroscopy (FTIR) and reverse phase high pressure liquid

chromatography (RP-HPLC). The higher extraction yields originated from an extraction with

5% NaOH at 80°C. The extracts obtained with 1% NaOH at 70°C had the highest amounts of

total polyphenols, condensed tannins and reactivity to formaldehyde. Absorbance at 1260 cm-

1, 1440 cm-1 and 1495 cm-1 were only detected for extracts obtained with 1% NaOH. RP-

HPLC revealed that catechin was the main condensed tannin in the extracts, lower quantities

of epicatechin and epicatechin gallate were also measured.

Keywords: FTIR, Polyphenols, condensed tannins, Pinus pinaster bark, RP-HPLC

II.2.1 Introduction

Pinus pinaster (maritime pine) can be found in some Mediterranean countries, such as

France, Portugal and Spain, and in some north western African countries [1–3]. They are

46

mostly cultivated for their timber. The pine bark is a lumber industry sub-product that is

produced when wood is transformed.

Pine bark is rich in phenolic compounds [4]. The main tannin structures found in

maritime pine bark are catechin/epicatechin, epigallocatechin and epicatechin gallate [2].

Many possible applications for pine bark are cited in the scientific literature. Its extracts have

been reported to have antiradical, antioxidant and anti-inflammatory properties [1,5–7] and

are marketed as a food supplement and a herbal-based medication, Pycnogenol®. Tannins

from many different plants have been used to make wood adhesives, such as grape pomace

[8], chestnut [9], pecan nut [10], mimosa [11,12] and maritime pine [13]. Tannins from pine

bark have also been used to make foams [14,15].

Traditionally, tannins are used in the tanning process of leather. The capacity of

tannins to precipitates proteins such as collagen, thus generating a non-putrescible material

under moist and warm conditions. The best procedure to obtain good quality tannins for

leather is with an acetone-water-bisulfite procedure [16,17].

In order to increase the extraction of procyanidins without the use of too many organic

solvents, research has been done to find new environmentally-friendly extraction conditions

such as microwave assisted extraction, ultrasound assisted extraction or maceration [18].

This study aims to optimise tannin extraction form maritime pine bark and characterise

these tannins in order to make wood adhesives and foams. Three extraction conditions were

compared. For the first time an extensive characterisation of maritime pine bark condensed

tannins extracted was conducted. The total polyphenolics and condensed tannins of the

extracts are evaluated. The extracts are characterised by several colorimetric analyses, by their

reactivity to formaldehyde, by Fourier transformed infrared spectroscopy (FTIR) and by

reverse-phase high pressure liquid chromatography (RP-HPLC).

II.2.2 Materials and methods

II.2.2.1 Reagents

(+) Catechin hydrate, (-) epicatechin gallate and cysteamine hydrochloride were

purchased from Sigma-Aldrich (St Louis, USA). Gallic acid, trifluoroacetic acid and HPLC

grade acetonitrile were obtained from Fisher Scientific (Waltham, USA). (-) Epicatechin was

obtained from LGC Standard (Teddington, UK) and vanillin was purchased from VWR

(Radnor, USA).

47

II.2.2.2 Samples

Bark from five different trees of maritime pine was collected in the Landes forest

(Uchacq, 40). The trees’ diameters ranged from 31 to 35.5 cm and were 27 to 32 years old.

The barks were air-dried and ground in a mill (Retsch) and sieved to select particles smaller

than 1 mm. The ground pine bark was kept in sealed bags.

II.2.2.3 Extraction

Tannins were extracted in different conditions described in Table 1. Bark, water,

NaOH, Na2SO3 and NaHSO3 were stirred to the desired temperature. NaOH was used in the

extraction in order to be in high alkalinity conditions and increase the extraction yield.

Na2SO3 and NaHSO3 were added to lessen the viscosity of the extracts and to stabilize them.

This was kept stirring and heating for 120 min. The supernatant was filtered through

Whatman paper no. 1 and the residue was rinsed with water. The filtrates were dried in an

oven at 40°C. The extraction yield was calculated as the percentage of the amount of extract

recovered in mass compared to the initial mass of dry bark.

Table 1: Extraction methods

Extraction

method Solvent

Temperature

(°C)

Ratio

(s/l)

1 Water + 1% NaOH + 0.25% Na2SO3 + 0.25% NaHSO3 80 1/9



II.2.2.4 Determination of total polyphenolic content

Total polyphenolic content was determined with the Folin-Ciocalteu method [19]. 2.5

mL of Folin reagent (diluted 10 times) was added to 0.5 mL of aqueous extract. 2 mL of

sodium carbonate (75 g.L-1) was then added. The mixture was then put in a water bath at 50°C

for 5 min before the absorbance was read at 760 nm. A calibration curve was done with a

solution of gallic acid (80 ppm, Jenway 6300 Spectrophotometer). The results were obtained

as mg of gallic acid equivalent (GAE) per g of dry bark (mg GAE / g bark).

II.2.2.5 Determination of condensed tannins

Condensed tannin content was determined with the vanillin method as described by

Broadhurst and Jones [20]. 3 mL of vanillin (4% in methanol) was added to 0.5 mL of the

48

aqueous extract. 1.5 mL of highly concentrated HCl was then added. The mixture was then

kept in the dark for 15 min at 20°C. The absorbance was read at 500 nm. A calibration curve

was prepared with a solution of catechin (30 ppm, Jenway 6300 Spectrophotometer). The

results were obtained in mg of catechin equivalent per g of dry bark (mg CE / g bark).

II.2.2.6 Proanthocyanidin content

Proanthocyanidin content was obtained with a BuOH/HCl test as described by

Scalbert et al. [21]. 5 mL of an acidic ferrous solution (77 mg FeSO4.7H2O in 500 ml

HCl/BuOH (2/3)) was added to 0.5 mL of the aqueous extract. The tubes were covered and

put in a water bath at 95°C for 15 min. The absorbance was read at 530 nm (Jenway 6300

Spectrophotometer). Results were expressed as mg of cyanidin per g of dry bark (mg Cya / g

bark) (εmol=34700 L.mol-1.cm-1).

mg CyaE / g bark = (A x V x D x M x 1000) / (ε x l x m) (1)

A: absorbance at 530 nm

V: volume of the reaction

D: dilution factor

M: cyanidin molar mass

ε: molar extinction coefficient

l: the path length

m: mass of dry bark

II.2.2.7 Stiasny number determination

The reactivity of the extracts to formaldehyde was determined by measuring the

Stiasny number as described by Voulgaridis et al. [22]. A solution of extract at a

concentration of 4 g.L-1 was prepared. 25 mL of this solution was put in a round bottom flask

and 5 mL of formaldehyde 37% and 2.5 mL of HCl 10M were added. The mixture was heated

under reflux for 30 min. The residue was filtered through a sintered glass n°2 or 4. The

precipitate was washed with water and dried at 105°C until constant weight. The reactivity

was calculated with the formula:

SI = A / B x 100 (2)

SI: Stiasny number

A: dry weight of the precipitate

49

B: dry weight of extract

II.2.2.8 FTIR Spectroscopy

FTIR spectra were recorded on a Perkin Elmer Spectrum One equipped with an ATR-

FTIR unit. A few milligrams of ground extract sample were placed on a crystal (diamond /

ZnSe). The spectra were obtained with a resolution of 4 cm-1 and 10 co-addition scans in a

wavelength range of 650-4000 cm-1. The spectra were collected and analysed using Spectrum

software (Perkin Elmer).

II.2.2.9 Thiolysis and RP-HPLC

The composition of the proanthocynidins in the extracts was determined by RP-HPLC.

The extracts were dissolved in methanol (10 g.L-1). An aliquot (400 µL) was added to 400 µL

of acidified methanol (3.3% HCl) and 800 µL of cysteamine hydrochloride (50 g.L-1 in

methanol). The mixture was put in a waterbath at 40°C for 30 min. The proanthocyanidins

were depolymerised by thiolysis with cysteamine as described by Torres and Selga (2003).

The samples were analysed by RP-HPLC-DAD (Ultimate 3000, Thermo Scientific), equipped

with a acclaim 120 C18, 250 x 4.6 mm, 5µm column. Elution: [A] 0.1% (v/v) aqueous TFA,

[B] acetonitrile. The solvent gradient was as follow: 0—12.6 min, 97.7A/2.3B (v/v); 12.6—

15.6 min, 97A/3B; 15.6—18 min, 92A/8B; 18—28 min, 84A/16B; 28—58 min, 100B; 58—

83 min, 100B. Detection was done at 220, 254, 272 and 280 nm. The chromatograms were

analysed with Chromeleon software.

II.2.2.10 Statistical analysis

The data are presented as mean ± SD values. The extraction yield, the total

polyphenolic content, the condensed tannins content, the cyanidin equivalent content and the

Stiasny number for the different trees and different extraction methods were compared using

Fisher Snedcor test, Aspin Welch test, Mann-Whitney-Wilcoxon test, student test and the

difference of mean test used when required. All the statistical analyses were carried out at

P<0.05 significance level.

50

II.2.3 Results and discussion

II.2.3.1 Extraction

Table 2: Total polyphenolic content extracted with three different conditions

Extraction

method

Extraction

yield (%)1,3

Total

polyphenolics

(mg GAE / g

bark)2,3

Vanillin test

(mg CE / g

bark) 2,3

BuOH-HCl

(mg CyaE /

g bark) 2,3

Stiasny

Number (%)2,3 pH1,3

1 22.06 ± 7.79a 62.21 ± 22.54a 4.73 ± 1.67a 5.65 ± 3.15a 48.97 ± 9.56a 9.52 ± 0.33 a

2 31.30 ± 10.42b 22.01 ± 4.91b 2.18 ± 0.57b 1.20 ± 0.44b 17.92 ± 9.49b 10.87 ± 0.13 b

3 22.07 ± 4.46a 54.23 ± 19.36a 5.15 ± 2.97a 5.43 ± 3.34a 53.98 ± 12.48a 9.89 ± 0.31 a 1 Standard deviations are of at least ten replicates 2 Standard deviations are of at least eighteen replicates 3 The means were statistically analysed with the Student test or the Welch test when required at P<0.05 significance level a, b Group of values with significant differences between each group for each parameters

Three extraction methods are performed on maritime pine bark. For each method the

extraction yield is presented in Table 2. The most important yield is obtained with the highest

concentration of NaOH and the highest temperature (extraction method 2, Table 1). We obtain

the highest extraction yield, 31.30%, with 5% NaOH at 80°C. It is significantly higher than

for the other protocols (Welch and Student tests). With 1% NaOH we obtain 22.06% and

22.07% at 80°C and 70°C (extraction method 1 and 3, Table 1). There is no significant

difference between the two (Welch test). The more alkaline the solution is the more extracts

are recovered. Similar extraction yields are obtained by Yazaki and Collins [23], whilst

extracting maritime pine bark tannins with a succession of water extraction and water at pH

8.3 at 100°C for 15 min. Vazquez et al. [24,25] also find similar results when extracting

maritime pine bark tannins in water with 2% and 5% NaOH at 100°C and 90°C respectively

for 30 min. Smaller amounts of extractives are recovered when the solvent used are

dichloromethane, ethanol and urea [3,26,27].

II.2.3.2 Total polyphenolic content

The total polyphenolic content for the different extraction methods and for different

maritime pine trees are presented in Table 2. The total phenol content does not significantly

change between maceration at 70°C and 80°C for the same amount of NaOH (54.23 and

62.21 mg GAE / g bark respectively) (extraction method 1 and 2, Table 1), (Welch and

Student tests). However a lower amount of polyphenols are extracted with 5% NaOH (22.01

51

mg GAE / g bark) than with 1% NaOH. A significantly lower amount of total polyphenols is

extracted with the second extraction method than for the first and third extraction method. For

the second extraction method, the total polyphenolic content is very low compared to the

extraction yield, which means with a higher amount of NaOH an important quantity of non-

tannin material is recovered.

Table 3: Total polyphenolic content extracted from the bark of five different maritime

pine trees

Extraction

yield (%)1,3

Total

polyphenolics

(mg GAE / g

bark) 2,3

Vanillin test

(mg CE / g

bark) 2,3

BuOH-HCl

(mg Cya / g

bark) 2,3

Stiasny Number

(%)2,3 pH1,3

P1T1 26.09 ± 4.02a 61.81 ± 11.62a 5.38 ± 0.84a 5.67 ± 1.62a 48.54 ± 11.71a 9.81 ± 0.33

P1T5 22.84±12.31a 64.09 ± 31.60a 4.08 ± 2.08a 5.63 ± 4.30a 49.40 ± 7.53a 9.23 ± 0.08

P2T1 33.73 ± 3.4d 24.68 ± 3.34d 2.58 ± 0.30 d 1.39 ± 0.32a 17.09 ± 11.42a 10.90± 0.03

P2T5 20.48 ± 5.44e 19.34 ± 4.90e 1.77 ± 0.48 1.01 ± 0.49a 18.75 ± 7.71a 10.75± 0.10

P3T1 18.39 ± 3.29d 48.50 ± 10.43a 3.42 ± 0.64a 3.86 ± 0.96a 48.82 ± 16.15a 10.36 ± 0.04

P3T2 26.94 ± 3.04a 96.81 ± 1.32 d 10.62 ± 1.66 d 11.73 ± 1.32 d 47.51 ± 3.49a 9.65 ± 0.08

P3T3 19.86 ± 2.46b 37.21 ± 4.08 e 2.81 ± 0.48a 3.00 ± 0.68 e 58.13 ± 15.44abc 9.78 ± 0.08

P3T4 24.70 ± 2.31a 56.25 ± 3.35ab 4.78 ± 0.39 e 4.74 ± 0.24a 52.74 ± 10.05ab 9.56 ± 0.09

P3T5 20.95 ± 4.11b 60.33 ± 8.54b 4.10 ± 0.67a 3.81 ± 0.66a 64.24 ± 5.42c 10.06 ± 0.08 1 Standard deviations are of five replicates 2 Standard deviations are of nine replicates 3 The means were statistically analysed with the Student test, the Mann-Whitney-Wilcoxon test and the Welch test when required at P<0.05 significance level a, b, c, d, e Group of values with significant differences between each group for each parameters

ab No significant differences between a and b group

abc No significant differences between a, b and c group

Five different bark trees are submitted to an extraction at 70°C, giving a range in

polyphenolic content from 37.21 to 96.81 mg GAE / g bark (Table 3). This can be due to the

difference in age of the trees. Alonso-Amelot et al. [28] show the trees at the same altitude

don’t have the same amount of phenols in their fronds. The trees with greater exposure to the

sun have higher concentrations of phenols than the shaded trees.

II.2.3.3 Condensed tannins content

Results of the vanillin assay for the different extraction methods are presented in Table

2 and Table 3. The highest extractions of condensed tannins are obtained with 1% NaOH at

70°C and at 80°C (5.15 mg CE / g bark and 4.73 mg CE / g bark respectively), (Welch and

52

Mann-Whitney-Wilcoxon tests). We extract the least amount of condensed tannins at 80°C

with 5% NaOH (2.18 mg CE / g bark). Ku et al. [29] find similar amounts of condensed

tannins for other pine species such as P. densiflora (19.6 mg CE / g bark), P. rigida (70.4 mg

CE / g bark) and P.radiata (93.7 mg CE / g bark).

The tree two has 10.62 mg CE / g bark. It is significantly higher than the other trees

when the extraction is carried out in the same conditions (Mann-Whitney-Wilcoxon test). The

condensed tannins contents are statistically the same for the trees one, three and five (Mann-

Whitney-Wilcoxon and Student tests). There are more condensed tannins in the fourth tree

than for the first, third and fifth trees (Student and Mann-Whitney-Wilcoxon tests). The bark

was obtained in winter; the tree two might have been less lixiviated which explains the higher

amount of condensed tannins compared to the other trees. Saad et al. [30] notice this

lixiviation of condensed tannins soluble in water for pomegranate peels. The condensed

tannins soluble in water from the trees one, three, four and five have been partly lixiviated by

the rain.

II.2.3.4 Cyanidin equivalent content

The BuOH-HCl assay gives similar results as the vanillin assay (Table 2 and Table 3).

We have the same trend. The highest amounts of cyanidin are obtained with the first and third

method of extraction (5.65 mg CyaE / g bark and 5.43 mg CyaE / g bark, respectively). The

second extraction method presents less cyaniding than the other two (1.20 mg CyaE / g bark)

(Mann-Whitney-Wilcoxon test). The cyanidin contents are not statistically different for the

extraction protocols one and three (Welch test). The cyanidin content for the extraction with

5% NaOH can be due to a mechanism of autocondensation of tannins. Merlin and Pizzi [31]

have noticed that the procyanidins from P. radiata autocondensate in presence of silica at

high alkalinity. In this study silica is not added to the extraction solvent however the presence

of silica as sand attached to the bark is possible.

There is significantly more cyanidin in the tree two than in the others (Mann-Whitney-

Wilcoxon, Student and Welch tests). The trees one, four and five have significantly the same

amount of cyanidin (Mann-Whitney-Wilcoxon and Welch tests). The third tree has the lowest

amount of cyanidin for all the trees (Mann-Whitney-Wilcoxon, Student and Welch tests).

Catechin and cyanidin levels are not significantly different, when looking at the results

obtained with the vanillin assay and the BuOH-HCl assay (difference of mean test). The

vanillin assay measures condensed tannins and simple flavonoids and not only condensed

53

tannins like the BuOH-HCl assay [32]. This can mean that we have extracted mostly

condensed tannins and very few simple flavonoids.

II.2.3.5 Stiasny number

The results are presented in Table 2 and Table 3. The Stiasny number gives us the

reactivity of our extracts to formaldehyde, this information can help us determine if the

extracts can be used as adhesives [9,33,34]. Yazaki and Collins [23] assessed that the

minimum Stiasny value to produce high quality adhesives is 65%. However, Ping et al. [35]

have produced good quality adhesives while obtaining a Stiasny number of 46%. In this

study, we obtain a similar Stiasny value in the case of the first extraction method and the third

extraction method (respectively 48.97% and 53.98%). The minimum value is obtained for the

second extraction method (17.92%), which is significantly lower than the Stiasny number for

the other two protocols (Student test). The Stiasny number decreases for high amounts of

NaOH, similar results were obtained by Vazquez et al. [24] and Voulgaridis et al. [22] for

other pine species. Vazquez et al. [24] obtained more important Stiasny values for maritime

pine bark from Spain with similar extraction conditions; this could be due to the region the

trees come from. In this study, the trees come from the Landes forest in France. Guilley et al.

[36] have noticed that oak trees had different properties when grown in different regions of

France. The Stiasny number is significantly the same for the extraction at 70°C and at 80°C

(Student test). The Stiasny number seems independent for this range of temperature. This

supposition is confirmed with the findings of Vazquez et al. [25].


The IR spectra of the tannins extracted with different conditions are recorded in the

650-4000 cm-1 region and are presented in Fig. 1. These spectra show that there are clear

differences between the extraction method with 5% NaOH (P2T1) and the ones with 1%

NaOH. The spectrum for the extraction with 1% NaOH at 80°C (P1T1) is similar to the

spectrum of the extracts obtained with 1% NaOH at 70°C (P3T1). The analyses of the spectra

are based on the assignments given by Boeiru et al. [37] and Ping et al. [8].

54

Fig. 1: FTIR spectra of a: P1T1 (1% NaOH, 80°C); b: P2T1 (5% NaOH, 80°C); c: P3T1 (1%

NaOH, 70°C)

The band at 3300 cm-1 is assigned as –OH stretch vibration in phenolic and aliphatic

structures. Small peaks at 2930 and 2850 cm-1 originate from –CH stretch vibration in

aromatic methoxyl groups and in methyl and methylene groups of side chains. The band at

1575 cm-1 can not be identified precisely; Soto et al. [38] suggest the peaks between 1400 and

2000 cm-1 show the aromatic nature of the structure. The band at 1370-1380 cm-1 is attributed

to phenolic stretch vibration of –OH and aliphatic –CH deformation in methyl groups. This

band is common to lignins and is more intense for P2T1. It can mean that more lignins are

extracted at high alkalinity. Aromatic –CH bending in plane bending vibration is detected at

1115 cm-1 and a –CO stretch vibration is produced at 1040 cm-1.The lower intensity of the

peaks at 1040 cm-1 and 1115 cm-1 at pH > 10 can be due to an opening of the cyclic ether

structure of polyflavonoids [38]. For peaks at wavelengths smaller than 900 cm-1, aromatic –

CH stretch vibration is detected. Three main bands are detected only for P1T1 and P3T1 at

1495 cm-1 assigned to aromatic squeal vibration, 1440 cm-1 corresponding to –CH

deformation and aromatic ring vibration, and at 1260 cm-1. This last peak is harder to identify,

Vazquez et al. [39] attribute it as C O stretch vibration, whereas Ping et al. [8] assign it to

saturated C—C stretch vibration attributed to CR2—CHR—CR(SO32-). This structure can be

due to the opening of the pyran ring during sulphitation of flavanoid tannins.

Fig. 2: FTIR spectra of tannins extracted with the third method for five different trees; a: P3T1;

b: P3T2; c: P3T3 ; d: P3T4; e: P3T5

The IR spectra of the tannins extracted with the third method for five different trees

are recorded in the 650-4000 cm

the same bands. There is a band at 3300 cm

phenolic and aliphatic structures, two small peaks at 2950 and 2850 cm

stretch vibration in aromatic methoxyl groups and in methyl and methylene groups, a band at

1370-1380 cm-1 due to phenolic stretch vibration of

methyl groups, a peak at 1115 cm

vibration and a band at 1040 cm

can be noted for P3T4 (Fig. 2.d) and P3T5 (Fig. 2.e). P3T4 has a very intense peak at 1115

cm-1, which corresponds to aromatic

other extracts of the other trees. P3T5 is the only tree to present a peak at 1515 cm

originating from aromatic skeletal vibration.

55


b: P3T2; c: P3T3 ; d: P3T4; e: P3T5


4000 cm-1 region and are presented in Fig. 2. All the spectra present

the same bands. There is a band at 3300 cm-1 corresponding to an –OH stretch vibration in

phenolic and aliphatic structures, two small peaks at 2950 and 2850 cm

in aromatic methoxyl groups and in methyl and methylene groups, a band at

due to phenolic stretch vibration of –OH and aliphatic –

methyl groups, a peak at 1115 cm-1 attributed to aromatic –CH bending in plane bending

tion and a band at 1040 cm-1 corresponding to –CO stretch vibration. Small differences


, which corresponds to aromatic –CH bending vibration, compared to the spe

other extracts of the other trees. P3T5 is the only tree to present a peak at 1515 cm

originating from aromatic skeletal vibration.



region and are presented in Fig. 2. All the spectra present

OH stretch vibration in

phenolic and aliphatic structures, two small peaks at 2950 and 2850 cm-1 assigned to –CH

in aromatic methoxyl groups and in methyl and methylene groups, a band at

–CH deformation in

CH bending in plane bending

CO stretch vibration. Small differences


CH bending vibration, compared to the spectra for the

other extracts of the other trees. P3T5 is the only tree to present a peak at 1515 cm-1

56

II.2.3.7 RP-HPLC

Fig. 3: RP-HPLC chromatogram of P3T5 after thiolysis at 280 nm (Cat: catechin, ECG:

epicatechin gallate, GA: gallic acid, EC: epicatechin)

The nature of the polyphenols extracted can be determined by thiolysis followed by

RP-HPLC [40]. Catechin, epicatechin, epicatechin gallate and gallic acid are detected (Fig. 3,

Table 4). These components are also found by Jerez et al. [1,4,5] and Navarrete et al. [2].

Catechin is the main condensed tannin present in the extracts followed by epicatechin and

then epicatechin gallate. When comparing the amounts measured by HPLC and with the

vanillin assay for catechin, we find that there are no significant differences between the two

(difference of mean test). The same amounts of catechin, epicatechin, epicatechin gallate are

obtained for the three extraction methods (Student, Mann-Whitney-Wilcoxon and Welch

tests). Extracts obtained with the second method present more gallic acid than the other

extracts (Student and Welch tests).

57

Table 4: Composition of maritime pine bark extracts determined by RP-HPLC after thiolysis

Catechin

(mg / g bark)3

Epicatechin

(mg / g bark) 3

Epicatechin gallate

(mg / g bark)3

Gallic acid

(mg / g bark)3

P12 3.73 ± 1.46a 1.51 ± 0.78 a 0.68 ± 0.91 a 2.74 ± 1.37 a

P22 4.78 ± 1.45 a 1.75 ± 0.68 a 0.78 ± 1.12 a 5.20 ± 2.01 a

P32 3.82 ± 0.81 a 1.43 ± 0.68 a 0.49 ± 0.13 a 1.43 ± 0.42 a

P1T11 3.86 ± 0.73 a 1.87 ± 0.93 a 0.99 ± 1.05 a 2.77 ± 0.73 a

P1T51 3.59 ± 1.80 a 1.23 ± 0.30 a 0.31 ± 0.10 a 2.72 ± 1.69 a

P2T11 5.05 ± 0.70 a 1.92 ± 0.41 a 0.67 ± 0.54 a 5.50 ± 0.73 a

P2T51 4.56 ± 1.70 a 1.61 ± 0.75 a 0.87 ± 1.33 a 4.97 ± 2.43 a

P3T11 3.02 ± 0.36 a 0.84 ± 0.40 ab 0.43 ± 0.14 ab 2.16 ± 0.46 a

P3T21 4.83 ± 0.42 c 1.79 ± 0.41 c 0.48 ± 0.04 ab 3.31 ± 0.36 b

P3T31 3.23 ± 0.42 a 0.93 ± 0.18 a 0.60 ± 0.13 b 2.62 ± 0.38 a

P3T41 4.05 ± 0.30 b 1.56 ± 0.69b 0.51 ± 0.15 ab 3.36 ± 0.27 b

P3T51 3.67 ± 0.71 ab 1.80 ± 0.71 ab 0.40 ± 0.07 ab 3.08 ± 0.66 ab 1 Standard deviations are of at least three replicates 2 Standard deviations are of at least nine replicates 3 The means were statistically analysed with the Student test, the Mann-Whitney-Wilcoxon test and the Welch test when required at P<0.05 significance level a, b, c Group of values with significant differences between each group for each parameters

ab No significant differences between a and b group

II.2.4 Conclusion

In this study, the third extraction method, with 1% NaOH at 70°C, presented the

highest amounts of polyphenolics, of condensed tannins and the highest reactivity to

formaldehyde. Smaller amounts of polyphenolics and condensed tannins were obtained when

we increased the extraction temperature or the concentration of NaOH. High concentration of

NaOH gave better extraction yields but less tannin is extracted. The main condensed tannins

units present in the extracts were catechin units. Epicatechin, epicatechin gallate and gallic

acid were also detected but in lower quantities. Further studies need to be conducted since all

the components present were not identified.

The FTIR spectra highlighted the presence of other polyphenolic compounds in the

extracts such as lignins. The second method of extraction, with 5% NaOH at 80°C, had more

lignins than the other extraction methods. Therefore the higher the alkalinity of the extraction

solvent the more lignins were extracted.

Complementary studies will be needed in order to evaluate the possibility of using the

tannins extracted in this study to prepare a wood adhesive.

58

II.2.5 Acknowledgements

We gratefully acknowledge the financial support of the “Conseil Général des Landes”

and this work was funded by ANR-10-EQPX-16 Xyloforest.

II.2.6 References

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61

II.3 Extraction assistée par micro-ondes d’écorce de pin maritime (Pinus

pinaster) : impact de la taille des particules et caractérisation

Soumis dans « Industrial Crops and Products », le 15 Septembre 2014

Résumé

L’effet de la taille des particules sur l’extraction de tanins par extraction assistée par

micro-onde (EAM) a été étudié pour la première fois. Des écorces de pin maritime (Pinus

pinaster) en provenance du Sud-Ouest de la France ont été tamisées et séparés en cinq

gammes de granulométrie. Une extraction a été réalisée dans une solution éthanol-eau. Pour

chaque extraction, le rendement d’extraction, le contenu total en polyphénols, le contenu en

tanins condensés et le contenu en sucres ont été quantifiés. Les extraits ont été caractérisés par

leur réactivité au formaldéhyde, par infrarouge à transformée de Fourier (IRTF), par 1H RMN

(résonance magnétique nucléaire) et par 2D HSQC RMN (2 dimensions heteronuclear single

quantum correlation RMN). L’EAM a également été comparée à une extraction utilisant de

l’eau chaude. La granulométrie n’a pas affecté le type de tanins extraits. 2D HSQC RMN a

révélé la présence de catéchine/ épicatéchine, épicatéchine gallate, epigallocatéchine, et

epigalocatéchine gallate. Des quantités plus élevées de tanins condensés, de sucres et de

flavonoïdes simples ont été obtenues par EAM que par une extraction par eau chaude.

Mots-clés : Extraction assistée par micro-ondes, écorce de pin maritime, tanin, granulométrie

62

Microwave assisted extraction of maritime pine (Pinus pinaster) bark:

impact of particle size and characterisation

L. Chupina*, S. L. Maunub, S. Reynaudc, A. Pizzid, B. Charriera, F. Charrier - El Bouhtourya

a IUT des Pays de l’Adour, IPREM, UMR 5254 CNRS/UPPA, 371 Rue du Ruisseau, BP 201,

40004 Mont de Marsan, France

b Laboratory of Polymer Chemistry, P.O. Box 55, FIN-00014 University of Helsinki,

Helsinki, Finland

c Université de Pau et des Pays de l’Adour (UPPA), IPREM, UMR 5254 CNRS/UPPA,

Hélioparc Pau Pyrénées, 2 Avenue P. Angot, 64053 PAU cedex 09, France

d ENSTIB-LERMAB, University of Lorraine, 27 Rue Philippe Seguin, BP 1041, 88051

Epinal, France

* IUT des Pays de l’Adour, IPREM, UMR 5254 CNRS/UPPA, 371 Rue du Ruisseau, BP 201,

40004 Mont de Marsan, France; tel: +33 558513722; fax: +33 558513737; email:

[email protected]

Abstract

The effect of particle size on the extraction of tannins by microwave assisted

extraction (MAE) was studied for the first time to our knowledge. Maritime pine (Pinus

pinaster) bark from the South West of France was sieved into five ranges of particle size

before extraction was carried out in an ethanol-water solution. For each extraction, the

extraction yield, the total polyphenolic content, the condensed tannins content and the sugar

content were quantified. The extracts were characterised by their reactivity to formaldehyde,

Fourier transformed infrared spectroscopy (FTIR), 1H NMR and heteronuclear single

correlation spectroscopy 2D NMR (HSQC 2D NMR). The MAE was also compared to a hot

water based extraction. The effect of the particle size on the extraction efficiency was studied.

The HSQC 2D NMR revealed that (epi)catechin, epicatechin gallate, epigallocatechine,

epigallocatechine gallate were obtained. Higher amounts of condensed tannins, sugars and

simple flavonoids were extracted by MAE extraction.

Keywords: Microwave assisted extraction, maritime pine bark, tannin, particle size

II.3.1 Introduction

Pinus pinaster (maritime pine) can be found in some Mediterranean countries, such as

France, Portugal, Spain, and in some North African countries

cultivated for their timber. The pine bark is a lumber sub

is transformed. It is rich in phenolic compounds

tannins [2].

Condensed tannins are

ol oligomers that have C4-C6

esters (Fig. 1). The aromatic A ring consists of resorcinol or phloroglucinol and the aromatic

B ring of catechol or pyrogallol. The interflavonoid linkage is specific for each species.

Quebracho presents a high proportion of C

presents a predominance of C4

the tanning process of leather. Tannins have the capacity to precipitate proteins such as

collagen, thus generating a non

Condensed tannins have proven to have antimycotic, antiradical, antioxidant and anti

inflammatory properties [6–9]

maritime pine tannin extracts as a food supplement and herbal

Pycnogenol®. Tannin extracts from many plants have also been used to make wood adhesives,

such as grape pomace, chestnut, mimosa and maritime pine

have also been used to make foams

Fig. 1: Chemical structure of condensed tannins (R

or OH)

Many extraction methods have been used to obtain condensed tannins su

extraction, ultrasound-assisted extraction, microwave

extraction, supercritical fluid extraction or hot water based extraction

63

(maritime pine) can be found in some Mediterranean countries, such as

France, Portugal, Spain, and in some North African countries [1–3]. They are mostly

cultivated for their timber. The pine bark is a lumber sub-product that is produced when wood

is transformed. It is rich in phenolic compounds [1] and more particularly in condensed

Condensed tannins are polyphenols present in a majority of plants. They are flavan

6 or C4-C8 interflavonoid linkage and may contain gallic acid


f catechol or pyrogallol. The interflavonoid linkage is specific for each species.

Quebracho presents a high proportion of C4-C8 interflavonoid linkage, whereas mimosa

4-C6 interflavonoid linkage [4]. Traditionally, tannins are used in


collagen, thus generating a non-putrescible material under moisture and warm conditions

Condensed tannins have proven to have antimycotic, antiradical, antioxidant and anti

9]. These properties have led to the commercialisation of purified

maritime pine tannin extracts as a food supplement and herbal-

. Tannin extracts from many plants have also been used to make wood adhesives,

such as grape pomace, chestnut, mimosa and maritime pine [10–14]. Tannins from pine bark

have also been used to make foams [15,16].

Fig. 1: Chemical structure of condensed tannins (R1: H or R; R 2: OH or gallic acid ester; R

Many extraction methods have been used to obtain condensed tannins su

assisted extraction, microwave-assisted extraction, pressurised liquid

extraction, supercritical fluid extraction or hot water based extraction [17

(maritime pine) can be found in some Mediterranean countries, such as

. They are mostly

product that is produced when wood

and more particularly in condensed

polyphenols present in a majority of plants. They are flavan-3-

interflavonoid linkage and may contain gallic acid


f catechol or pyrogallol. The interflavonoid linkage is specific for each species.

interflavonoid linkage, whereas mimosa

raditionally, tannins are used in


putrescible material under moisture and warm conditions [5].

Condensed tannins have proven to have antimycotic, antiradical, antioxidant and anti-

. These properties have led to the commercialisation of purified

-based medication,

. Tannin extracts from many plants have also been used to make wood adhesives,

. Tannins from pine bark

: OH or gallic acid ester; R3: H

Many extraction methods have been used to obtain condensed tannins such as soxhlet

assisted extraction, pressurised liquid

[17–22]. Microwave-

64

assisted extraction (MAE) is a fast extraction method that generates a high pressure in the

biomaterial that leads to a rupture of the cells thus improving the penetration of the extracting

solvent [23]. One of the main uses of MAE is for soil depollution as it extracts pesticide from

soils [24,25]. MAE of plant material has mostly been used to extract simple flavonoids or

polyphenols [26-28]. MAE is a promising extraction method for it is fast, obtains high

extraction rates, uses low amounts of solvent and can extract both polar and non-polar

compounds. It has rarely been used to extract condensed tannins [22,29].

This study aims to assess the effect of particle size on a microwave assisted extraction

of maritime pine bark and to characterise the extracts. Since microwave assisted extraction

has not already been used on maritime pine species, the results obtained were compared to a

method of extraction in hot water, in order to determine the advantages or drawbacks of this

process to a conventional extraction.

II.3.2 Materials and methods

II.3.2.1 Reagents

(+) Catechin hydrate (98%) was purchased from Sigma-Aldrich (St Louis, USA).

Gallic acid (98%), Folin reagent, sodium carbonate (99%), methanol (99.8%), ethanol (95%),

butanol (99.4%), hydrochloric acid (37%), sulphuric acid (95%) and sodium hydroxide (97%)

were obtained from Fisher Scientific (Waltham, USA). Vanillin (99%), iron sulphate (97%),

phenol and glucose were purchased from VWR (Radnor, USA) and the formaldehyde (37%)

from Acros Organics (Geel, Belgium).

II.3.2.2 Samples

Bark from maritime pine was collected in the Landes forest (Uchacq, 40). The tree

was 32 years old and had a diameter of 33 cm. The bark was air-dried at 20°C until the mass

was constant and ground in a mill (Retsch) and sieved to select particles smaller than 1 mm.

The ground pine bark was kept in sealed bags.

Tannins were also extracted under different conditions from maritime pine bark

particles with a particle size smaller than 1 mm. The extraction method is described in Chupin

et al. (2013). The extracts are referred to as P3.

II.3.2.3 Microwave-assisted extraction

Tannins were extracted from maritime pine bark. The bark was grounded and sieved to

select particles between 1 mm and 0 mm (MO1), between 1 and 0.8 mm (MO2), between 0.8

and 0.4 mm (MO3), between 0.4 and 0.1 mm (MO4) and between 0.1 and 0.05 mm (MO5).

65

The tannins were extracted in ethanol-water (80-20, v-v) at a solid-liquid ratio of 1-10 (w-v)

in a Prolabo Microdigest 3.6 apparatus equipped with an open reflux column. The irradiation

occurred at a microwave power of 100 W for 3 min. The microwave assisted extraction

(MAE) was performed three times for each particle size distribution.

II.3.2.4 Determination of total polyphenolic content

Total polyphenolic content was determined with the Folin-Ciocalteu method [30]. 2.5

mL of Folin reagent (diluted 10 times) was added to 0.5 mL of aqueous extract. 2 mL of

sodium carbonate (75 g.L-1) was then added. The mixture was then heated up to 50°C for 5

min before the absorbance was read at 760 nm. A calibration curve was done with a solution

of gallic acid (80 mg.L-1, Jenway 6300 Spectrophotometer). The results were obtained as mg

of gallic acid equivalent (GAE) per g of dry bark (mg GAE / g bark) and as mg of GAE per g

of dry extract (mg GAE / g ext).

II.3.2.5 Determination of condensed tannins

Condensed tannin content was determined with the vanillin method as described by

Broadhurst and Jones [31]. 3 mL of vanillin (4% in methanol) was added to 0.5 mL of the

aqueous extract of the sample. 1.5 mL of highly concentrated HCl (37%) was then added. The

mixture was kept in the dark for 15 min at 20°C. The absorbance was read at 500 nm. A

calibration curve was prepared with a solution of catechin (30 mg.L-1, Jenway 6300

Spectrophotometer). The results were obtained in mg of catechin equivalent per g of dry bark

(mg CE / g bark) and as mg of catechin equivalent per g of dry extract (mg CE / g ext).

II.3.2.6 Proanthocyanidin content

Proanthocyanidin content was obtained with a BuOH/HCl test as described by

Scalbert et al. [32]. 5 mL of an acidic ferrous solution (77 mg FeSO4.7H2O in 500 ml

HCl/BuOH (2/3, v/v)) was added to 0.5 mL of the aqueous extract of the sample. The tubes

were covered and put in a water bath at 95°C for 15 min. The absorbance was read at 530 nm

(Jenway 6300 Spectrophotometer). Results were expressed as mg of cyanidin per g of dry

bark (mg Cya / g bark) (εmol=34700 L.mol-1.cm-1) and as mg of cyanidin per g of dry extract

(mg Cya / g ext).

mg CyaE / g bark = (A x V x D x M x 1000) / (ε x l x m) (1)

mg CyaE / g ext = (A x V x D x M x 1000) / (ε x l x me) (2)

A: absorbance at 530 nm

V: volume of the reaction

66

D: dilution factor

M: cyanidin molar mass

ε: molar extinction coefficient

l: the path length

m: mass of dry bark

me: mass of dry extract

II.3.2.7 Total sugars quantification

The total sugars quantification was determined by the phenol/sulphuric acid method

after hydrolysis of the polysaccharides [33]. 10 mL of HCl 0.2 M was added to 0.02 g of

tannin extracts and was put in a water bath at 85°C for 2h. The solution was cooled down and

neutralised with NaOH 33%. The solution was then filtered and adjusted to 10 mL with

dionised water. To 0.5 mL of the filtrate, 0.5 mL of phenol 5% was added. The solution was

mixed and 2.5 mL of concentrated H2SO4 was added. The solution was put in a water bath at

100°C for 15 min and then kept in the dark for 15 min. The absorbance was read at 490 nm. A

calibration curve was prepared with a glucose solution (1 g.L-1, Jenway 6300

Spectrophotometer). The results are obtained in mg of glucose equivalent per g of dry bark

(mg GE / g bark) and as mg of glucose equivalent per g of dry extract (mg GE / g ext).

II.3.2.8 Stiasny number determination

The reactivity of the extracts to formaldehyde was determined by measuring the

Stiasny number as described by Voulgaridis et al. [34]. A solution of extract at a

concentration of 4 g.L-1 was prepared. 25 mL of this solution was put in a round bottom flask

and 5 mL of formaldehyde 37% and 2.5 mL of HCl 10M were added. The mixture was heated

under reflux for 30 min. The residue was filtered through a sintered glass n°2 or 4. The

precipitate was washed with water and dried at 105°C until constant weight. The reactivity

was calculated with the formula:

SI = A / B x 100 (3)

SI: Stiasny number

A: dry weight of the precipitate

B: dry weight of extract

67







II.3.2.10 NMR analysis

NMR experiments were performed on a Bruker Avance III 500 spectrometer. Samples

were dissolved in either DMSO-d6 or D2O (for the MAE 10 mg of material were dissolved in

1 mL of DMSO d6; for P3 10 mg of material were dissolved in 1 mL of D2O). For the 1H-

NMR spectra, a relaxation delay of 1.5 s and an acquisition time of 2 s were used to collect 86

scans. The 2D heteronuclear single quantum correlation spectroscopy (HSQC) spectra were

acquired employing a pulse sequence with a 0.073 s acquisition time, 2.5 s pulse delay were

used to collect 192 scans.

II.3.2.11 Data analysis

The data are presented as mean ± SD values. The extraction yield, the total

polyphenolic content, the condensed tannins content, the cyanidin equivalent content, the total

sugar content and the Stiasny number for the different trees and different extraction methods

were compared using Fisher Snedcor test, Aspin Welch test, Mann-Whitney-Wilcoxon test,

student test and the difference of mean test used when required. All the statistical analyses

were carried out at P<0.05 significance level. Principal components analysis (PCA) was

performed using R software.

68

II.3.3 Results

Table 1: Characteristics of the MAE (average of at least three replicates)1

Yield (%) Total polyphenolics

(mg GAE/g bark)

Vanillin test (mg

CE/g bark)

BuOH-HCl (mg

CyaE/g bark)

Total sugars (mg

GE/g bark)

MO1 9.24 ± 0.117b 28.30 ± 2.937b 37.14 ± 3.447b 7.66 ± 1.335c 3.51 ± 1.117a

MO2 3.78 ± 0.572a 7.52 ±1.120a 10.05 ± 3.285a 1.63 ± 0.450a 1.09 ± 0.139b

MO3 3.51 ± 0.150a 8.46 ± 1.604a 10.53 ± 1.181a 2.12 ± 0.416b 0.93 ± 0.080c

MO4 6.49 ± 1.574c 18.07 ± 3.821c 38.50 ± 0.636b 3.83 ± 1.677ab 1.85 ± 0.464d

MO5 13.16 ±0.0002d 39.52 ± 0.495d 48.98 ± 0.567c 11.99 ± 0.386d 4.53 ± 0.066e

1 The means were statistically analysed with the Student test, the Mann-Whitney-Wilcoxon and the Welch test when required at P < 0.05 significance level. 2 Extraction performed only once. ab No significant differences between a and b group. a, b, c, d, e Group of values with significant differences between each group for each parameter.

II.3.3.1 Extraction

Four different particle size of maritime pine bark and a mixture of all particle size

were submitted to MAE. For each extraction, the extraction yield is measured as the

percentage of the amount of extract recovered in mass compared to the initial mass of dry

bark and is presented in Table 1. The most important yield is obtained with MO5, the smallest

particle size. MO1’s yield is significantly lower than MO5s, 9.24% and 13.16% respectively.

When the particle size increases to 400-100 µm (MO4), the extraction yield is lower than for

MO5 and MO1, 6.49. MO2 and MO3 give smaller extraction yield, 3.78% and 3.51%, than

the extraction with smaller particle sizes. There are no significant difference between the

yields for MO2 and MO3.

MO1 is also compared to maritime pine bark extracts, obtained by maceration [17].

The extraction yields are presented in Table 2. The more traditional extraction, by maceration,

has a higher extraction yield than MO1.

69

Table 2: Characteristics of MO1 and P3 extracts (average of at least three replicates)1

Yield Total

polyphenolics

Vanillin test BuOH-HCl Total sugars Stiasny

number pH

(%)

(mg

GAE/g

ext)

(mg

GAE/g

bark)

(mg

CE/g

ext)

(mg

CE/g

bark)

(mg

CyaE/g

ext)

(mg

CyaE/g

bark)

(mg

GE/g

ext)

(mg

GE/g

bark) (%)

MO1

9.24

±

0.101a

306.48

±

33.147a

28.30

±

2.937a

402.52

±

41.723a

37.14

±

3.447a

82.96 ±

14.187a

7.66 ±

1.335a

378.65 ±

117.670a

3.51

±

1.117a

48.99 ±

9.115a

4.73 ±

0.11a

P3

22.07

±

4.46b

236.11

±

51.185b

54.23

±

19.36b

22.81

±

9.444b

5.15

±

2.97b

24.05 ±

11.169b

5.43 ±

3.34a

216.23 ±

18.161b

45.95

±

8.98b

53.98 ±

12.48a

9.89 ±

0.31b 1 The means were statistically analysed with the Student test and the Welch test when required at P < 0.05 significance level. a, b Group of values with significant differences between each group for each parameter.

II.3.3.2 Total polyphenolic content

The total polyphenolic content for the MAE are presented in Table 1. The highest

amounts of total polyphenols are extracted with the smallest particle size of bark, MO5. MO1

gives statistically lower amounts of total polyphenols than MO5. The total polyphenolic

content of MO4 is statistically lower than for MO5 and MO1, 18.07, 29.52 and 28.30 mg

GAE/g bark respectively. When the particle size is higher than 400 µm, the total polyphenolic

content extracted is lower than with a particle size smaller than 400 µm. MO2 and MO3 have

statistically lower amounts of total polyphenols than MO1, MO4 and MO5. There are no

significant differences between the total polyphenolic content of MO2 and MO3, 7.52 and

8.46 mg GAE/g bark respectively.

Two different types of extraction are performed on a mixture of all particle sizes.

More polyphenols are extracted by P3 than by MO1 for the same amount of dry bark, 54.23

and 28.30 mg GAE/g bark respectively (Table 2). MO1 extracts have statistically more total

polyphenols than P3 extracts, 306.48 and 236.11 mg GAE/g ext respectively.

II.3.3.3 Condensed tannins content

Results of the vanillin assay for the different MAE are presented in Table 1. The

highest extraction of condensed tannins is obtained with MO5. There are no significant

differences between MO1 and MO4. There are less condensed tannins extracted with MO1

and MO4 than with MO5, 37.14, 38.50 and 48.98 mg CE/g bark respectively. We extract the

70

least amount of condensed tannins with MO2 and MO3, 10.05 and 10.53 mg CE/g bark.

There are no significant differences between the two.

More condensed tannins are extracted by MO1 than P3 for the same amount of dry

bark, 37.14 and 5.15 mg CE/g bark respectively (Table 2). Almost 20 times more condensed

tannins are present in MO1 extracts than in P3 extracts, 402.52 and 22.81 mg CE/g ext.

II.3.3.4 Cyanidin equivalent content

The BuOH-HCl assay has the same trend of result as the vanillin assay (Table1). The

highest amount of cyanidin are obtained with the smallest particle size, MO5. MO1 gives

statistically lower amounts of cyanidin, 7.66 compared to 11.99 mg CyaE/g bark for MO5.

The cyanidin content of MO4 is lower than for MO5 and MO1. MO3 has a lower cyanidin

content than MO5, MO1 and MO4 but it is higher than for MO2. The smaller the initial

particle size of bark is, the more cyanidin are extracted.

More cyanidin are extracted with a MAE than with P3, 7.66 and 5.43 mg CyaE/g bark

respectively (Table 2). For the same amount of extract, MO1 has approximately 3.5 times

more cyanidin than P3, 82.96 and 24.05 mg CyaE/g ext.

II.3.3.5 Total sugar content

The total sugars content of the MAE are presented in Table 1. The most important

quantity of total sugars is extracted by MO5, 4.53 mg GluE/g bark. A significantly lower

amount of total sugars is extracted by MO1, 3.51 mg GluE/g bark. MO4 has lower amounts of

sugars than MO5 and MO1 but it extracts more sugars than MO3 and MO2. The lowest

amounts of sugars are extracted by MO3, 0.93 mg GluE/g bark.

More sugars are extracted by P3 than MO1 for the same amount of dry bark, 45.95 and

3.51 mg GluE/g bark respectively (Table 2). MO1 extracts have statistically more sugars than

P3 for the same weight of dry extract, 378.65 and 216.23 mg GluE/g ext.

II.3.3.6 Stiasny number

The Stiasny test was performed for MO1 and P3; the results are presented in Table 2.

Similar Stiasny values are obtained in the case of a MAE and an extraction by maceration

with 1% NaOH (respectively 48.99% and 53.98%).

II.3.3.7 FTIR spectroscopy

The spectra of the tannins extracted were recorded in the 650-4000 cm-1 region and are

presented in Fig. 2. The analyses of the spectra are based on the assignments given by Boeriu

et al. [35], Ping et al. [36] and Soto et al. [37].

71

The band at 3300 cm-1 is assigned to a – OH stretch vibration in phenolic and aliphatic

structures. The peaks at 2925 cm-1 and 2850 cm-1 originate from a – CH stretch vibration in

aromatic methoxy groups and in methyl and methylene groups of side chains. The band at

1695 cm-1 corresponds to the conjugated carbonyl-carbonyl stretching. The bands at 1605 cm-

1, 1515 cm-1 and at 1440 cm-1 correspond to aromatic skeleton vibrations and to a –CH

deformation at 1440 cm-1. The band at 1370–1380 cm−1 is attributed to phenolic stretch

vibration of OH and aliphatic CH deformation in methyl groups. This band is common to

lignins. The band at 1260 cm-1 is identified by Vázquez et al. [38] as a C–O stretch vibration,

whereas Ping et al. [36] assign it to saturated C–C stretch vibration attributed to CR2 CHR

CR(SO32−). The bands at 1275 cm-1 and at 1245 cm-1 are assigned to a C–O–C asymmetric

stretch vibration [38]. A C–O stretching vibration is detected at 1200 cm-1 [5]. The bands at

1155 cm-1 and 1105 cm-1 are attributed to an aromatic CH in-plane bending vibration. A C–O

stretching vibration is detected at 1050 cm-1. The band at 1035 cm-1 can be assigned to

aliphatic C–O stretching [37]. Boeriu et al. [35] identified it as an aromatic C–H deformation

associated with the C–O, C–C stretching and C–OH bending in polysaccharides. The band at

970 cm-1 is assigned as an aliphatic C–OH stretching vibration. Aromatic –CH stretch

vibrations are detected for peaks at wavelengths smaller than 900 cm-1.

All the spectra for the MAE are similar. MO1, MO2 and MO3 present a band at 2925

cm-1 and 2850 cm-1 which are not present for the other extracts and MO2 and MO3 present

bands at 1385 cm-1 and at 1365 cm-1 which are not present for the other extracts. The non

presence of these bands for the other MAE can be due to the low intensity of the signals.

There are no other differences between the MAE. The particle size of the bark powder has no

consequences on the types of tannins extracted.

72

Fig. 2: FTIR spectra of the MAE: MO1 (––); MO2 (—); MO3 (—); MO4 (—); MO5 (—).

The FTIR spectra of MO1 and P3 [17] have also been compared. All the peaks

detected for P3 are also present for MO1. MO1 presents peaks at 1695, 1445, 1155, 970, 865

and 770 cm-1 that are not detected for P3.

II.3.3.8 NMR 1H NMR spectrum of the MO2 extracts is presented in Fig. 3. All the MAE showed

identical 1H NMR spectra. The assignments were given based on the work of Ramires and

Frollini [39]. The analysis of the 1H NMR spectra confirms the presence of condensed

tannins. A very intense peak at 2.5 ppm is observed, corresponding to the solvent. Another is

detected at 3.3 ppm, which is characteristic of a chemical shift of water impurities in the

sample [40]. The peaks around 1.2 ppm are related to protons of aliphatic carbons. The peaks

at 2.6 and 2.8 ppm are related to the protons linked to carbon 4, with R1 = H. All the small

peaks in the 3.0-5.5 ppm range confirm the presence of sugars in the sample [41]. In that

region, the peaks around 4.7 ppm might correspond to protons linked to carbon 5. The peaks

at 4.8 ppm might be related to protons linked to the aliphatic carbons 2 and 3, with R2 = OH,

as well as related to sugars. The peaks around 5.8-5.5 ppm indicate the presence of

epicatechin gallate and epigallocatechin gallate, for they correspond to protons linked to the

aliphatic carbons 2 and 3. The chemical shifts in the 6.2-7.3 ppm region are related to

aromatic protons. The peaks from the 7.9-7.1 ppm region could correspond to protons from

0,84

0,86

0,88

0,9

0,92

0,94

0,96

0,98

1

5001000150020002500300035004000

% T

rans

mitt

ance

Wavenumbers [1/cm]

73

hydroxyl groups linked to the carbons 3’, 4’, 5’ of epigallocatechin and epicatechin gallate.

The broad peak at 9.0-8.5 ppm could correspond to protons from hydroxyl groups linked to

aromatic rings.

Fig. 3: 1H NMR spectrum of MO2.

1H NMR was performed on all the MAE and on maritime pine bark extracts obtained

by maceration (figure not shown for P3). When comparing all the 1H NMR spectra, it is

possible to make the following observations: P3 1H NMR spectra present sharper peaks than

MO2 at 1.8 ppm, 3.0 ppm, 8.3 ppm, which are related to the protons linked to carbon 4, with

R1 = H, sugars and to phenol type OH respectively.

The HSQC 2D NMR spectrum of MO1 extract is presented in Fig. 4. Since several

types of condensed tannins are extracted, the chemical shifts were broad and separated in five

groups of signal. The signal around H: 0.5-1.5 / C: 10-40 ppm reveals the presence of

aliphatic carbons. The signal around H: 3-4 / C: 55-80 ppm is attributed to the aliphatic

carbons from sugars. The presence of epicatechin gallate and epigallocatechin gallate is

confirmed by the signals around H: 6 / C: 95 ppm that correspond to the aromatic carbons of

the gallate ring, the broad signal is coming from polymeric structures and the sharp one from

gallic acid. The signals around H: 6.5-7 / C: 110-125 ppm are related to the aromatic carbons,

2’, 3’ and 4’, and other aromatics. The signal around 6.6 ppm might be due to C4-C6

interflavonoid linkage, a better resolution spectrum is needed to confirm it [42]. Similar

results were found for P3 (figure not shown).

74

Fig. 4: 1H-13C HSQC NMR spectrum of MO1.

II.3.4 Discussion

The particle size has a direct effect on the quantity of polyphenols, condensed tannins

and sugars extracted. PCA was applied to improve and highlight the main information

contained in the data (Fig 5). Four clusters are obtained corresponding to MO2 and MO3, to

MO4, to MO1 and to MO5. The extraction yield and the spectrocolorimetric tests show the

same trend, the smaller the particle size of ground bark, the more is extracted up to 400 µm

[43–45]. Approximately the same amounts of extracts and polyphenols are extracted with

particles between 1000 and 400 µm; MO2 and MO3 are thus both in the same cluster. The

specific surface area is more important with small particles, which will allow more contact

between the solvent and the particles and a better penetration of the microwaves.

75

Fig. 5: Principal component analysis of the microwave-assisted extracts for five particle size

distributions.

More extracts are obtained with an extraction by maceration at high pH than with

MAE. However more condensed tannins are extracted with MAE. The vanillin and BuOH-

HCl tests are both usually used to quantify the amount of condensed tannins extracted [29,46–

49]. The vanillin assay measures condensed tannins and simple flavonoids and not only

condensed tannins like the BuOH-HCl assay [50]. For the P3 extraction, the results obtained

are similar for both tests which means only condensed tannins are extracted. With the MAE,

the results obtained with the vanillin test are much higher than what is measured with the

BuOH-HCl assay. The MAE extracts are rich in simple flavonoids, condensed tannins and

sugars. The long process time of the P3 extraction can lead to a thermal degradation of the

simple flavonoid.

The 1H-NMR spectra show the presence of condensed tannins. Several peaks are

specific to catechin/epicatechin, epigallocatechin, epicatechin gallate and epigallocatechin

gallate. The presence of sugars has also been confirmed.

The HSQC 2D NMR spectra might show the presence of C4-C6 interflavonoid linkage

of maritime pine bark condensed tannins for the different extraction methods. It is in

agreement with previous observations done by Navarrete et al. [2], who showed by MALDI-

TOF analysis that industrial maritime pine bark tannins also have an interflavonoid linkage at

C4-C6 and also at C4-C8. The extraction method has no effect on the location of the

interflavonoid linkage. The interflavonoid linkage in C4-C6 of maritime pine bark tannins can

explain the relatively low reactivity to formaldehyde due to steric hindrances.

76

II.3.5 Conclusion

The particle size of the bark has a direct effect on quantities of extracts obtained. The

smaller the particle size is, the more extracts are extracted, under 400 µm we have observed

an improvement of the extraction. There is no impact on the nature of the extract and on the

types of condensed tannins extracted.

The MAE has many advantages compared to a hot water based extraction. The process

is less time consuming (3 min instead of 2 h) and higher amounts of condensed tannins,

flavonoids and sugars are obtained (3.5, 9.6 and 1.8 times more than for a hot water based

extraction respectively). The structure of the condensed tannins extracted remains the same

hence the reactivity of the extracts is not modified. The C4-C6 interflavonoid linkage of the

condensed tannins extracted could explain the low reactivity of the maritime pine condensed

tannins observed by the Stiasny test.

II.3.6 Acknowledgements


and the ANR-10-EQPX-16 Xyloforest.

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80

II.4 Conclusion au chapitre II

Dans ce chapitre, l’extraction de tanins de pin maritime a été réalisée et les extraits

caractérisés. Le meilleur rendement d’extraction en extractibles pour l’extraction à l’eau

chaude est obtenu pour un pH proche de 11. Toutefois, plus de tanins condensés sont extraits

à un pH légèrement inférieur à 10. Si on observe la nature des extraits, on constate que 20%

des extraits sont composés de polyphénols. Des sucres et très peu de flavonoïdes simples sont

extraits ; on obtient donc surtout des tanins condensés. De la catéchine, de l’épicatéchine, de

l’épicatéchine gallate et de l’épigallocatéchine sont extraits. Les meilleurs conditions

d’extraction à l’eau chaude sont obtenues à 70°C avec 1% NaOH, 0,25% Na2SO3 et 0,25%

NaHSO3 avec un ratio solide-liquide de 1-9 pendant 2 h.

L’extraction assistée par micro-ondes a un rendement plus faible que l’extraction à

l’eau chaude mais elle est 40 fois plus rapide. Comme pour l’extraction à l’eau chaude, des

sucres, des flavonoïdes simples et des tanins condensés sont extraits. Toutefois des quantités

supérieures de tous ces composés sont extraites ; près de 30% des extraits sont des composés

polyphénoliques. En outre, plus la granulométrie des poudres d’écorce est fine, plus d’extraits

sont obtenus. De 400 µm à 1 mm, la granulométrie ne joue pas sur les rendements

d’extraction. Quelle que soit la granulométrie, la nature des extraits n’est pas modifiée. Les

tanins condensés extraits sont de la catéchine, l’épicatéchine, l’épicatéchine gallate et

l’épigallocatéchine. La 2D HSQC RMN montre qu’il y a probablement des liaisons

interflavonoides en C4-C6, ce qui pourrait expliquer la faible réactivité des extraits au

formaldéhyde due à des gênes stériques.

81

CHAPITRE III Elaboration des colles tanin-lignine

III.1 Introduction au chapitre III

Depuis plus d’une cinquantaine d’années, des colles à base de tanins et des colles à

base de lignines sont étudiées. Les tanins et les lignines sont souvent ajoutés à des résines UF

et PF afin d’augmenter les proportions de matière renouvelable. Depuis quelques années, des

colles à base de tanins et de lignines combinés sont réalisées avec des tanins de différentes

sources et des lignines krafts ou des lignines organosolves ayant subies un traitement au

glyoxal.

Le traitement au glyoxal des lignosulfonates de sodium et d’ammonium qui n’a, à

notre connaissance, jamais été étudié auparavant a été réalisé. Un premier traitement au

glyoxal a été mis en œuvre et les propriétés thermiques des lignosulfonates ont été étudiées

avant et après traitement. Afin de comprendre les réactions chimiques ayant lieu durant le

traitement au glyoxal, une étude en IRTF a été menée. Ces lignosulfonates glyoxalés sont

rentrés dans la formulation de colles tanin-lignine. Ces colles ont été analysées par IRTF afin

d’avoir des pistes concernant les réactions chimiques se déroulant pendant la réticulation. Des

tanins de mimosa ont été utilisés afin de déterminer le meilleur ratio tanin-lignine avec un

tanin déjà commercialisé comme composant de colles à base de tanins. L’optimisation du

ratio tanin-lignine a été obtenue en fonction des propriétés thermiques des colles, pendant et

après réticulation.

Ce ratio a servi à la formulation de colles avec des tanins d’écorce de pin maritime

extrait précédemment et de lignosulfonates de sodium et d’ammonium glyoxalés. De

nouveau, la réticulation des colles a été étudiée par IRTF et les températures de réticulations

ont été mesurées par analyse thermomécanique. Les propriétés thermiques des colles

réticulées ont été analysées afin de savoir si la température de pressage de panneaux de

particules peut dégrader nos colles. Des panneaux de particules avec une colle à base de

tanins d’écorce de pin maritime et de lignosulfonates de sodium glyoxalés, avec une colle à

base de tanins de mimosa et de lignosulfonates de sodium glyoxalés et avec une résine UF ont

été produits. Les émissions de formaldéhyde et la cohésion interne des panneaux ont été

mesurées.

Un deuxième traitement au glyoxal a été réalisé en modifiant notamment les

proportions d’eau utilisées pendant le traitement. Ce traitement a été comparé au premier

82

traitement. Les lignosulfonates de sodium et d’ammonium ont subi les deux traitements et ont

été analysés par RMN bas champs, analyse thermogravimétrique et calorimétrie différentielle

à balayage. Tous les lignosulfonates glyoxalés sont rentrés dans la formulation de colles

tanins de mimosa-lignine. Les propriétés thermiques de ces colles ont été analysées pendant et

après réticulation. Des panneaux de particules ont été encollés avec des colles avec des

lignosulfonates ayant subi les deux traitements, avec des lignosulfonates de sodium et

d’ammonium et avec des colles ayant des ratios tanin-lignine différents.

83

III.2 Etude des propriétés de durabilité thermique des colles tanins-

lignosulfonates

Soumis dans « Journal of Thermal Analysis and Calorimetry », le 25 Mars 2014

Résumé

L’article vise à étudier certains aspects de la préparation de colles tanins-

lignosulfonates. Des tanins de mimosa et des lignosulfonates ont été utilisés dans la

préparation de colles à bois à la place de résines à base de formaldéhyde. Deux

lignosulfonates d’ammonium et deux lignosulfonates de sodium ont été glyoxalés pour les

rendre plus réactifs. La stabilité thermique des lignosulfonates avant et après glyoxalation a

été analysée par analyse thermogravimétrique (TGA) et par calorimétrie différentielle à

balayage (DSC). La proportion des tanins de mimosa et des lignosulfonates varie de 20% de

tanins à 60% de tanins. 40% des tanins de mimosa ont été ajoutés soit à des lignosulfonates de

sodium glyoxalés soit à des lignosulfonates d’ammonium. Les propriétés thermiques des

résines ont été étudiées par analyse thermogravimétrique (ATG), par calorimétrie

différentielle à balayage (DSC) et par analyse thermomécanique (ATM). Les résultats ont

démontré que la résine contenant 40%m de tanins était la plus efficace. Les résultats de

l’ATM ont démontré que la réticulation des résines a commencé entre 100° et 110°C. Les

résultats ATG et DSC des résines réticulées ont démontré une stabilité thermique des colles

jusqu’à approximativement 200°C.

Mots clés: tanin de mimosa; lignosulfonate; colle tanin-lignine glyoxalée; glyoxalation.

84

Study of thermal durability properties of tannin lignosulfonate adhesives

Lucie Chupina*, Bertrand Charriera, Antonio Pizzib, Arturo Perdomoc, Fatima Charrier – El

Bouhtourya



b ENSTIB-LERMAB, University of Lorraine, 27 rue Philippe Seguin, BP1041, 88051 Epinal,

France

c Tembec, 1154 avenue du Général Leclerc, 40400 Tartas, France

*Corresponding Author: IUT des Pays de l’Adour, IPREM, UMR 5254 CNRS/UPPA, 371

Rue du Ruisseau, BP 201, 40004 Mont de Marsan, France; tel: +33 558513722; fax: +33

558513737; email: [email protected]

Abstract

This paper aims to study certain aspects of the preparation of tannin – lignin

adhesives. Mimosa tannins and lignosulfonates were used in wood adhesives formulation to

substitute resins based on formaldehyde. Two ammonium lignosulfonates and two sodium

lignosulfonates were glyoxalated to be more reactive. The thermal stability of the

lignosulfonates before and after glyoxalation was analysed by thermogravimetric analysis

(TG) and differential scanning calorimetry (DSC). The proportion of mimosa tannins and

sodium lignosulfonates varied from 20% tannins to 60% tannins. 40% mimosa tannins were

mixed with either glyoxalated sodium lignosulfonates or glyoxalated ammonium

lignosulfonates. The thermal properties of the resins were studied by TG, DSC and

thermomechanical analysis (TMA). The results showed that after glyoxalation, the

degradation of lignosulfonates started at 125°C instead of 171°C for the non glyoxalated

lignosulfonates. The results obtained showed that the 40 mass % tannins resin was the most

efficient. The TMA results showed that the curing of the resins started at 100-110°C. The TG

and DSC results of the cured resins showed a thermal stability of the adhesives up to

approximately 200°C.

Keywords: mimosa tannin; lignosulfonate; tannin-glyoxalated lignin adhesive; glyoxalation.

85

III.2.1 Introduction

Most wood adhesives used today are amino resins (urea-formaldehyde, melamine-

formaldehyde, etc), phenol-formaldehyde and resorcinol formaldehyde resins [1,2]. With

increasing oil prices and the desire to reduce formaldehyde emissions, natural materials have

been used to partially or totally substitute formaldehyde.

Lignin is the second most abundant natural polymer after cellulose. The structure of

lignin, with the presence of aliphatic hydroxyl groups and phenolic groups, has made it a good

partial replacement of phenol for phenol-formaldehyde adhesives [3–9]. In recent years,

lignin-phenolic resins – pMDI (polymeric isocyanate) were produced with glyoxalated lignins

[10,11]. The glyoxalation of the lignins was found to be an alternative to premethylolation of

lignins, which increases the reactivity of lignins [11]. Glyoxal is a non volatile, non-toxic

aldehyde [12]. It has been used to make lignin-based adhesives [11] and tannin-lignin

adhesives [13]. Glyoxal has also been used as a hardener for tannin based adhesives for

particle board bonding with good mechanical properties [14]. Fourier transformed infrared

spectroscopy (FTIR) has been used in order to understand the reaction of glyoxalation for

organosolv and kraft lignins [15,16]

Tannins from many different plants have been used to make wood adhesives, such as

grape pomace [17], chestnut [18], pecan nut [19], mimosa [20,21], and maritime pine [22].

Until the mid 2000s tannins were used to substitute phenol in phenol formaldehyde resins

[23–26]. Pichelin et al. [27] showed that hexamine is a hardener with equivalent properties to

formaldehyde or paraformaldehyde. Tannin – hexamine based adhesives have been

formulated and presented good mechanical properties that were evaluated with

thermomechanical analysis [28,29].

In recent years, tannin – lignin based adhesives have been developed. The tannins used

were mimosa tannins with either organosolv lignins or kraft lignins [13,21,30]. FTIR has been

used to characterise resins since it shows the appearance or disappearance of chemical groups

after curing of the resins [24]. Thermal analysis of resins with differential scanning

calorimetry and thermogravimetric analysis monitors the thermal stability of the resins and

the pure tannins and lignins that gives the maximum temperature at which they can been used

and an insight on the major chemical structures of the compounds [7,24,31].

This study aims to prepare tannin – lignin adhesives from mimosa tannins and

glyoxalated ammonium lignosulfonates and sodium lignosulfonates. For the first time, two

ammonium lignosulfonates and two sodium lignosulfonates were glyoxalated and their

86

thermal stability were studied before and after treatment by thermogravimetric analysis and

differential scanning calorimetry. These lignosulfonates were used to make tannin – lignin

adhesives with different proportions of tannins and lignosulfonates and with the different

lignosulfonates. The thermal properties of these adhesives were evaluated by

thermogravimetric analysis, differential scanning calorimetry and thermomechanical analysis.

III.2.2 Material and methods

III.2.2.1 Material

Lignins used in this work were two different sodium lignosulfonates (NaLSP and

NaLSL) types and two different ammonium lignosulfonates (NH4LSL and NH4LSP) types

delivered by Tembec (Tartas, France). The mimosa tannin extracts were provided by

Silvateam (Italy). The sodium hydroxide, hexamethylene tetramine (hexamine) and the

glyoxal (40%) were purchased from Fisher Scientific (Waltham, USA).

III.2.2.2 Glyoxalation of Lignosulfonates

The glyoxalation of the four lignosulfonates was carried out as described by El

Mansouri et al. [10]. 29.5 parts by mass of dry lignin were slowly added to 38.4 parts of

water. Sodium hydroxide solution (33%) was added in order to keep the pH of the solution

between 12 and 12.5 for better dissolution of the lignin. A 800 mL flat bottom flask equipped

with a condenser and a magnetic stirrer bar was charged with the above solution and heated to

58°C. 17.5 parts by mass glyoxal (40% in water) were added and the lignin solution was then

continuously stirred with a magnetic stirrer/hot plate for 8 hours. The solid contents for all

glyoxalated lignin were around 43%. The lignosulfonates characteristics are presented in

Table 1.

Table 1 Lignosulfonates characteristics

Identification Type Aspect pH

NaLSP Sodium lignosulfonate Brown powder 8.5

NaLSL Sodium lignosulfonate Dark liquid 8.3

NH4LSL Ammonium lignosulfonate Dark liquid 3.6

NH4LSP Ammonium lignosulfonate Brown powder 5.6

87

III.2.2.3 Adhesive Formulation

A 45% mimosa tannin solution in water was prepared. A sodium hydroxide solution

(33%) was added in order to keep the pH of the solution at 10.4. The pH was chosen for the

hardener performs at its best at that pH and tannins dissolve at high pH. 5% of a

hexamethylenetetramine (hexamine) (solid mass of hexamine on solid mass of tannins) was

added as hardener. The hexamine was added as a solution at a concentration of 30% in water.

In this paper we studied four different ratios of tannin – glyoxalated lignin adhesive

formulations for the sodium lignosulfonate NaLSP. Mimosa tannins – glyoxalated lignins

adhesive formulations were also produced with the other lignosulfonates for one ratio. The

different formulations are described in Table 2.

Table 2 Adhesive formulations

Formulations Tannins/mass % Glyoxalated lignins/mass % pH

Mimosa-NaLSP 20 80 9.08

40 60 10.10

50 50 9.84

60 40 10.33

NaLSL 40 60 10.10

NH4LSL 40 60 9.97

NH4LSP 40 60 10.05

III.2.2.4 Thermomechanical Analysis (TMA)

The curing kinetics and mechanical properties of the adhesives were measured by

TMA by monitoring the rigidity of a bonded wood joint as a function of temperature. The

analyses were performed on a Mettler Toledo TMA SDTA 840. 30 mg of resin was placed

between two plys of maritime pine wood to form a joint of 17 x 5 x 1.2 mm. The bonded

wood joint were submitted to three points bending on a span of 14 mm and subjected to an

alternating force of 0.1 / 0.5 N with a 6 s / 6 s cycle. The heating rate was of 10°C min-1 from

25°C to 250°C. For each formulation, five replicates were done. The results were analysed

using STARe software.

88

III.2.2.5 Thermogravimetric Analysis (TG)

The thermal stability of the cured resins and of the raw materials was determined by

thermogravimetric analysis, TA Instrument TGA Q500. The samples were made of cured

resin and analysed from 30°C to 600°C at a heating rate of 10°C min-1 under 40-60 mL min-1

N2. The results were analysed with Universal Analysis software.

III.2.2.6 Differential Scanning Calorimetry (DSC)

DSC data were obtained with a TA Instrument DSC Q20. The samples were made of 5

to 8 mg of cured resin and of glyoxalated and non – glyoxalated lignosulfonates and were put

in an aluminium crucible. The temperature scanned from 30°C to 250°C at heating rate of

10°C min-1 under 50 mL min-1 N2. The results were analysed with Universal Analysis

software.

III.2.2.7 Fourier Transformed Infrared (FTIR)






III.2.2.8 Statistical Analysis

The data are presented as mean ± SD values. The TMA values were analysed with the

Bartlett test, the student test and the ANOVA test. All the statistical analyses were carried out

at P < 0.05 significance level.

III.2.3 Results and discussion

III.2.3.1 Analysis of lignosulfonates before and after glyoxalation

The thermal decomposition of the lignosulfonates before and after glyoxalation was

determined by TG. The TG curve presents the mass loss of the lignosulfonates in relation to

the temperature and the first derivative of that curve (DTG) shows the corresponding rate of

mass loss. The TG curves and DTG curves for the lignosulfonates NaLSP and NH4LSP are

presented in Fig. 1. NaLSP and NaLSL present similar curves and NH4LSL and NH4LSP

also present similar curves.

In a first step, which occurs up to approximately 110°C, the mass loss is due to the

elimination of any residual water. In a second step, the non glyoxalated ammonium

lignosulfonates start to decompose at 171 – 176°C. The non glyoxalated sodium

89

lignosulfonates start to decompose at 186 – 202°C. The decomposition occurs up to 375 –

400°C with a mass loss of 30 – 40%, except for NaLSL which is submitted to the degradation

up to 345°C and 23% mass loss. The glyoxalated lignosulfonates are less stable to

temperature, the second step started at 125 – 135°C and terminated at 330 – 345°C. This early

mass loss is caused by the link formed by the glyoxal that is less stable. This might be due to

the low reactivity of glyoxal, which introduces much earlier sites of tridimensional cross-

linking in the network. The network is weaker and degradation starts at lower temperatures.

Another hypothesis is that there is formation of glyoxal – O – glyoxal unstable bridges that on

heating should rearrange to a simple glyoxal bridge, such as formaldehyde does. However due

to the low reactivity of glyoxal it is unable to rearrange to a single bridge causing instability.

The mass loss of glyoxalated lignosulfonates during this second step is of 30 – 33%. After

heating to 600°C, there are still up to 45 – 60% of the samples mass that has not been

volatilised. According to Tejado et al. [32], the mass loss occurring after 500°C is due to the

decomposition of aromatic rings.

The DTG max appears at approximately 240 – 245°C for all the lignosulfonates and

glyoxalated lignosulfonates (Fig. 1b). Khan et al. [6] attributed this mass loss to a breakdown

of side chains present in lignin. Non glyoxalated sodium lignosulfonates present a DTG peak

at 300°C and the non glyoxalated ammonium lignosulfonates present a DTG peak at 360°C.

After glyoxalation the ammonium lignosulfonates show a DTG peak at 300°C whereas the

sodium lignosulfonates at 310°C. Between 300 and 450°C, a pyrolytic degradation occurs

which leads to the fragmentation of the intermolecular bonding and the release of monomeric

phenols [4,32]. After glyoxalation, a mass loss appears between 130 – 170°C with a peak at

145°C. This mass loss does not appear with alkaline rice straw lignin [31].

90

(a)

(b)

Fig. 1 (a) TG curves and (b) DTG curves of (—) non glyoxalated NaLSP, (- - -) glyoxalated

NaLSP, (— ·) non glyoxalated NH4LSP and (— - -) glyoxalated NH4LSP recorded at 10°C min-1

DSC analyses were also performed on all lignosulfonates (Fig. 2). Prior to the

analysis, the lignosulfonates were dried at 60°C until the mass had stabilised. The non

glyoxalated lignosulfonates are stable to temperature. An endothermic transition occurs at

136°C for glyoxalated sodium lignosulfonates. The glyoxalated ammonium lignosulfonates

show an endotherm at 145 – 149°C. These transitions correspond to the glass transition of the

lignosulfonates and are found in the same range of temperature as kraft lignins or organosolv

lignins [4,32]. The endotherms that take place at 190-200°C and at 210-235°C are present for

all the glyoxalated lignosulfonates and are more important for glyoxalated NaLSL. They

correspond to the degradation of the degradation of the lignosulfonates.

20

40

60

80

100

0 100 200 300 400 500 600

Mas

s/%

Temperature/°C

20 220 420

DT

G/%

°C-

1

Temperature/°C

91

Fig. 2 DSC curves of NaLSL (—), NH4LSL (- - -), glyoxalated NaLSL (— ·) and glyoxalated

NH4LSL (— - -) recorded at 10°C min-1

All the lignosulfonates, before and after glyoxalation were studied by FTIR in the 650-

2000 cm-1 region (Fig. 3). The analyses of the spectra are based on the assignments given by

Boeriu and Bravo [33]. The two ammonium lignosulfonates were identical so as the two

sodium lignosulfonates. When comparing two ammonium lignosulfonates and two sodium

lignosulfonates before treatment, we can see small differences between the two types of

lignosulfonates. The ammonium lignosulfonates show bands at 1075 cm-1, 860 cm-1 and 808

cm-1 that are not present in sodium lignosulfonates. These bands correspond to carbohydrate

vibration, C—H out of plane vibrations from guaiacyl units and other aromatic C—H stretch

vibrations respectively. After glyoxalation, when comparing ammonium lignosulfonates to

sodium lignosulfonates, the FTIR spectra are similar. The only differences can be found in the

1680-1715 cm-1 region. Glyoxalated ammonium lignosulfonates have a band at 1680 cm-1

and glyoxalated sodium lignosulfonates present a band at 1715 cm-1 which are assigned to

conjugated carbonyl/carboxyl stretching. After glyoxalation, ammonium lignosulfonates show

a shift of the band at 1155 cm-1 to 1140cm-1 which is assigned to C—O stretch vibration.

Peaks at 1715-1680 cm-1, 1350 cm-1 and 1140 cm-1 are present only after glyoxalation and

are attributed to carbonyl/carboxyl stretching, phenolic hydroxyl groups and C—O stretch

vibration respectively.

25 75 125 175 225

←E

ndo

Hea

t Flo

w/W

g-1

Temperature/°C

92

Fig. 3 FTIR spectra of (—) non glyoxalated NaLSL, (- - -) glyoxalated NaLSL, (— ·) non

glyoxalated NH4LSP and (— - -) glyoxalated NH4LSP

III.2.3.2 Analysis of the resin’s curing

The three bending point mode enabled us to get the deflection curves from which the

modulus of elasticity (MOE) of the resins is determined. The MOE is a good indicator of the

adhesive behaviour during curing and its wood-joint strength.

The influence of four proportions of tannins – glyoxalated lignosulfonates on the

adhesive properties is studied. The curves of MOE as a function of temperature are given in

Fig. 4. The tannin proportions varied from 20 mass %, 40 mass %, 50 mass % and 60 mass %.

All the adhesives show an increase of the MOE starting 100 – 110°C. According to Ping et al.

[34], this corresponds to a first cross-linking reaction through formation of a non stable cross-

linker. The highest MOE values are obtained at 160 – 170°C. There is a decrease in the MOE

at 170 – 190°C that corresponds to the degradation of wood components and of the adhesive.

A comparison of the TMA profiles shows that the less tannins is added the lower the MOE is.

The maximum values of MOE are presented in Table 3. There are statistically no significant

differences between the MOE of the adhesive formulations containing 40 mass %, 50 mass %

and 60 mass % of mimosa tannins. However, the MOE of the adhesive formulation containing

20 mass % of mimosa tannins is statistically lower than the others.

600100014001800

Tra

nsm

ittan

ce/A

.U.

Wavenumber/cm-1

93

Table 3 Module of elasticity values for all the adhesive formulations

Formulation Maximum MOE/MPa* Temperature/°C

Mimosa-NaLSP (20-80) 1905 ± 99ac 159

Mimosa-NaLSP (40-60) 2264 ± 110bcd 168

Mimosa-NaLSP (50-50) 2294 ± 308bc 157

Mimosa-NaLSP (60-40) 2497 ± 285bc 165

Mimosa-NaLSL (40-60) 2483 ± 394bd 165

Mimosa-NH4LSL (40-60) 2325 ± 260bd 165

Mimosa-NH4LSP (40-60) 2185 ± 65bd 176

* Standard deviation of five replicates a,b Group of values with significant differences between each group c The means were analysed with the Student test at P < 0.05 d The means were analysed with the Anova test at P < 0.05

Fig. 4 Module of elasticity of average curves of a pine joint as a function of temperature obtained

by TMA testing when bonded with mimosa tannins – glyoxalated NaLSP cured resins: (—) 20

mass % tannins; (- - -) 40 mass % tannins; (— ·) 50 mass % tannins; (— · ·) 60 mass % tannins

recorded at 10°C min-1

0

500

1000

1500

2000

2500

20 60 100 140 180 220

MO

E/M

Pa

Temperature/°C

94

As lignosulfonates are co-products in pulp mills and there are no significant

differences between adhesive formulations with 40 mass % tannins and 60 mass % tannins, it

was decided that adhesives with NaLSL, NH4LSL and NH4LSP lignosulfonates would have

40 mass % mimosa tannins and 60 mass % of glyoxalated lignosulfonates. The curves of

MOE as a function of temperature, obtained by TMA for resins with the different

lignosulfonates, are given in Fig. 5. All curves increase at 110°C, which corresponds to a first

cross-linking reaction through formation of a non stable cross-linker. The maximum MOE

values are obtained between 160°C and 180°C. The maximum values of MOE are presented

in Table 3. There are statistically no significant differences between the MOE of the adhesive

formulations with 40 mass % mimosa tannins and 60 mass % NaLSP, 60 mass % NaLSL, 60

mass % NH4LSL, 60 mass % NH4LSP. The wood joint bonded with the adhesive

formulation with NaLSL has a slightly higher MOE between 137°C and 208°C compared to

the adhesive formulations with NaLSP, NH4LSL and NH4LSP. The adhesive formulations

with NaLSP and NH4LSL have a higher MOE between 135°C and 250°C compared to the

adhesive with NH4LSP. The adhesive formulation with NaLSP shows a slower cross linking

reaction than the adhesives with NaLSL, NH4LSL and NH4LSP. Similar results have been

found for mimosa tannins – glyoxalated organosolv lignin – pMDI (polymeric 4,4’-diphenyl

methane diisocyante) adhesives [16]. Navarrete et al. [21] found similar results for mimosa

tannins – glyoxalated organosolv lignins using hexamine as a hardener for a tannin – lignin

ratio of 50 – 50, however resins with a 60 – 40 ratio presented higher MOE.

95

Fig. 5 Module of elasticity of average curves of a pine joint as a function of temperature obtained

by TMA testing when bonded with (—) mimosa tannins – glyoxalated NaLSL; (- - -) mimosa

tannins – glyoxalated NaLSP; (— ·) mimosa tannins – glyoxalated NH4LSL; (— · ·) mimosa

tannins – glyoxalated NH4LSP recorded at 10°C min-1

III.2.3.3 Thermal analysis of cured resins

The TG curves of the cured resins with glyoxalated NaLSP in different proportions are

presented in Fig. 6. The initial decomposition temperature and the final decomposition

temperature of the resins were observed. In a first stage, the resins are submitted to the

elimination of water, in which 8 to 15% mass loss occurs depending on the initial water

content of the samples. In a second stage, the resin with 20 mass % mimosa tannins starts to

decompose at 238°C, the one with 40 mass % starts its degradation at 242°C, the cured resin

with 50 mass % tannins at 258°C and the adhesive with 60 mass % tannins decomposes at

222°C. When the proportion of tannins increase the initial decomposition starts at higher

temperatures up to 50 mass % of tannins after which the decomposition starts at lower

temperatures. This decomposition could be the result of a partial breakdown of the

intermolecular bonding [24] and occurs up to approximately 377 – 392°C. The mass loss

progressively increases with the temperature up to 600°C where the mass loss starts to

stabilise. The mass loss ranges from 30% to 36%. Similar mass losses were found for mimosa

tannin resins [24].

0

500

1000

1500

2000

2500

20 60 100 140 180 220

MO

E/M

Pa

Temperature/°C

96

Fig. 6 TG curves of mimosa tannins – glyoxalated NaLSP cured resins: (—) 20 mass % tannins;

(- - -) 40 mass % tannins; (— ·) 50 mass % tannins; (— - -) 60 mass % tannins recorded at 10°C

min-1

The TG curves of the cured resins with 40 mass % mimosa tannins with four

glyoxalated lignosulfonates are presented in Fig. 7. The initial decomposition temperature and

the final decomposition temperature of the resins were observed. In a first stage, the resins are

submitted to the elimination of water, in which 3 to 12% mass loss occurs depending on the

initial water content of the samples. In a second stage, the resin with NaLSL starts to

decompose at 191°C, the one with NaLSP starts its degradation at 242°C, the cured resin with

NH4LSL at 197°C and the adhesive with NH4LSP decomposes at 201°C.The resin with

NaLSP is the most stable to temperature. This decomposition could be the result of a partial

breakdown of the intermolecular bonding and occurs up to approximately 369 – 379°C. The

mass loss progressively increases with the temperature up to 600°C where the mass loss starts

to stabilise. The mass loss ranges from 31% to 41%. Similar mass losses were found for

mimosa tannin resins, valonia tannin resins [24].

50

60

70

80

90

100

0 100 200 300 400 500 600

Mas

s/%

Temperature/°C

97

Fig. 7 TG curves of mimosa tannins – glyoxalated lignosulfonates with 40 mass % tannins cured

resins: (—) mimosa tannins – glyoxalated NaLSL; (- - -) mimosa tannins – glyoxalated NaLSP;

(— ·) mimosa tannins – glyoxalated NH4LSL; (— - -) mimosa tannins – glyoxalated NH4LSP

recorded at 10°C min-1

The DTG curves of mimosa tannins, glyoxalated NH4LSP and of the adhesive

formulation with NH4LSP are presented in Fig. 8. Four major mass losses are present for

mimosa tannins. The first one, at 56°C corresponds to the elimination of water. At 200°C, the

mass loss corresponds to the degradation of hydroxyl, ether and ester groups [35]. The mass

loss at 253°C is due to decarboxylation of the mimosa tannins. At 355°C, the mass loss

corresponds to further degradation of hydroxyl groups [36]. The mimosa tannins and the

cured resins have no common peaks, the mimosa has completely reacted during the curing

process. The DTG curve of the cured resin has several peaks which correspond to the

degradation of the lignosulfonates that did not react during the curing process, at 243°C, and

from approximately 335°C to 600°C. The cured resin shows a DTG peak at 243°C, which

corresponds to a breakdown of side chains present in the remaining lignosulfonates. The

disappearance of the degradation peaks in the cured resin at 147°C and at 300°C which

correspond to the glass transition NH4LSP and to inter-unit degradation of the glyoxalated

lignosulfonate NH4LSP respectively confirm that most of the glyoxalated NH4LSP has

reacted.

50

60

70

80

90

100

0 100 200 300 400 500 600

Mas

s/%

Temperature/°C

98

Fig. 8 DTG curves of (—) mimosa tannins, (— - -) glyoxalated NH4LSP and (- - -) mimosa

tannins – glyoxalated NH4LSP recorded at 10°C min-1

The DSC results for cured resins with 40 mass % mimosa tannins are presented in Fig.

9. The adhesive formulation with NaLSP 40 mass % mimosa tannins is stable up to 233°C,

the resin with NaLSL is stable until 198°C, the resin with NH4LSL is stable until 216°C and

the resin with NH4LSP is stable until 210°C, where an endothermic reaction takes place. This

reaction is a consequence of the degradation of the resins. The DSC and TG results both show

that all the resins are stable to temperature up to at least 190°C. The resins with the sodium

lignosulfonate NaLSP are more stable and are not submitted to a thermal degradation until

222°C.

20 120 220 320 420 520

DT

G/%

°C-

1

Temperature/°C

99

Fig. 9 DSC curves of mimosa tannins – glyoxalated lignosulfonates with 40 mass % tannins

cured resins: (—) mimosa tannins – glyoxalated NaLSL; (- - -) mimosa tannins – glyoxalated

NaLSP; (— ·) mimosa tannins – glyoxalated NH4LSL; (— - -) mimosa tannins – glyoxalated

NH4LSP recorded at 10°C min-1

In order to understand the chemical reactions that take place during the curing of the

resins, FTIR analyses were carried out, the spectra of the mimosa tannins – glyoxalated

NaLSL, mimosa tannins, glyoxalated NaLSL and hexamine are presented in Fig. 10. When

comparing the cured adhesive to the raw materials used, we can notice that the hardener

(hexamine) has totally reacted. The cured resin spectrum is very similar to the glyoxalated

NaLSL spectrum. The only bands not shared are at 1740 cm-1, 1715 cm-1, which are assigned

to carbonyl/carboxyl groups and at 840-835 cm-1 and at 765 cm-1, which correspond to

aromatic C—H “oop” stretch respectively. The carbonyl/carboxyl groups from the

glyoxalated lignosulfonates react during curing and disappear in the resin. The cured resin and

the mimosa tannins spectra only have a few bands in common that are not present in the

glyoxalated NaLSL at 840-835 cm-1 and at 765 cm-1.

Autocondensation of mimosa tannins occurs at high alkalinity, pH 10 or higher [35].

At lower pH, mimosa tannins crosslink with hexamine. As our adhesive solutions are at

approximately pH 10, mimosa tannins are subjected to both autocondensation reactions and

crosslinking reactions with hexamine and the glyoxalated lignosulfonates.

25 75 125 175 225

←E

ndo

Hea

t Flo

w/W

g-1

Temperature/°C

100

Fig. 10 FTIR spectra of (—) mimosa tannins, (- - -) glyoxalated NaLSL, (— ·) mimosa tannins –

glyoxalated NaLSL and (— - -) hexamine

III.2.4 Conclusion

In this study, we monitored the thermal stability of sodium and ammonium

lignosulfonates before and after glyoxalation. After glyoxalation, the lignosulfonates have a

glass transition at 136°C for sodium lignosulfonates and at 145-149°C for ammonium

lignosulfonates. The glyoxalation reaction lessens the thermal durability of all

lignosulfonates.

We assessed the optimum proportion of mimosa tannins – glyoxalated NaLSP. The

resins with 40 mass %, 50 mass % and 60 mass % mimosa tannins are statistically the same

and give more efficient adhesives than the adhesive formulation with 20 mass % mimosa

tannins. The adhesive formulations with sodium lignosulfonates or ammonium

lignosulfonates gave the same maximum of modulus of elasticity.

The cured resins with glyoxalated NaLSL, NH4LSL and NH4LSP are stable up to

190-200°C. The adhesive formulations with NaLSP are more thermally stable than the ones

with the other lignosulfonates. They are stable up to 222-258°C. Further analysis by TG-MS

or TG-FTIR would give more information on the chemical structures of the cured resins.

III.2.5 Acknowledgments


and of ANR-10-EQPX-16 Xyloforest. We also thank Tembec company (Tartas 40 - France)

for providing lignosulfonate samples.

600100014001800

Tra

nsm

ittan

ce/A

. U

.

Wavenumber/cm-1

101

III.2.6 References


2. Gomez-Bueso J, Haupt R. Chapter 8: Wood composite adhesives. In: Pilato L, editor. Phenolic Resins A

Century Prog. Berlin, Heidelberg: Springer Berlin Heidelberg; 2010. p. 155–87.




2013;44:315–22.







23.

7. Khan MA, Ashraf SM. Studies on thermal characterization of lignin: Substituted phenol formaldehyde resin as

wood adhesives. J. Therm. Anal. Calorim. 2007;89:993–1000.

8. Matuana LM, Riedl B, Barry AO. Caractérisation cinétique par analyse enthalpique différentielle des résines

phenol-formaldehyde à base de lignosulfonates. Eur. Polym. J. 1993;29:483–90.



2013;45:296–302.


Roh- und Werkst. 2007;65:65–70.


Polym. Sci. 2007;103:1690–9.





2010;24:1597–610.





16. Lei H, Pizzi A, Du G. Environmentally friendly mixed tannin / lignin wood resins. J. Appl. Polym. Sci.

2008;107:203–9.



102



2009;29:364–70.










und Werkst. 2002;60:303–10.



24. Özacar M, Soykan C, Şengil İA. Studies on synthesis, characterization, and metal adsorption of mimosa and






2006;84:651–4.


wood adhesives. Holz als Roh- und Werkst. 1999;57:305–17.



2013;47:675–83.





2011;25:1549–60.

31. El Mansouri NE, Yuan Q, Huang F. Study of chemical modification of alkaline lignin by the glyoxalation

reaction. Bioresources. 2011;6:4523–36.

32. Tejado A, Peña C, Labidi J, Echeverria JM, Mondragon I. Physico-chemical characterization of lignins from

different sources for use in phenol-formaldehyde resin synthesis. Bioresour. Technol. 2007;98:1655–63.





103




104

III.3 Etude des propriétés de durabilite thermique de colles pour panneaux

de particules à base de tanins extraits d’ecorce de pin maritime et de

lignosulfonates

Soumis dans « European Journal of Wood and Wood Products », le 2 Septembre 2014

Résumé

Des colles à base de lignines et de tanins ont été étudiées. Elles contenaient des tanins

extraits d’écorce de pin maritime ainsi que deux lignosulfonates d’ammonium glyoxalés

différents et deux lignosulfonates de sodium. Pour mieux comprendre la réaction chimique

qui a lieu pendant la réticulation des colles tanins-lignosulfonates, la réaction de réticulation a

été étudiée par spectroscopie infra-rouge à transformée de Fourier. Les propriétés mécaniques

des colles ont été déterminées par analyse thermomécanique. Les propriétés thermiques des

colles ont été étudiées par analyse thermogravimétrique et par calorimétrie différentielle à

balayage. La réticulation des résines commence à 85-95°C et la température de réticulation

optimum est à 150-170°C. Le module d’élasticité maximum de la résine contenant le

lignosulfonate de sodium liquide glyoxalé est considérablement plus élevé que pour les

résines contenant les autres lignosulfates. Toutes les résines réticulées sont stables jusqu’à

200°C. Des panneaux de particules ont été réalisés à partir de résines de tanins-

lignosulfonates glyoxalés (tanins extraits d’écorce de pin maritime et tanins extraits de

mimosa). Ils ont été comparés à des panneaux de particules utilisant une résine urée-

formaldéhyde (cohésion interne et émission de formaldéhyde). Les colles tanins-

lignosulfonates glyoxalés n’ont pas rempli les conditions requises par la Norme Européenne

EN-312. Les panneaux de particules fabriqués à partir de ces colles bio-sourcées émettent très

peu de formaldéhyde.

105

Study of thermal durability properties of maritime pine bark tannin-

lignosulfonate adhesives for particleboards

Lucie Chupina*, Fatima Charrier – El Bouhtourya, Antonio Pizzib, Arturo Perdomoc, Bertrand

Charriera



b ENSTIB-LERMAB, University of Lorraine, 27 rue Philippe Seguin, BP1041, 88051 Epinal,

France

c Tembec, 1154 avenue du Général Leclerc, 40400 Tartas, France




Abstract

Tannin – lignin adhesives for particleboards were studied. They contained maritime

pine bark tannins, two different glyoxalated ammonium lignosulfonates and two different

sodium lignosulfonates. In order to understand the chemical reaction that takes place during

the tannin – lignosulfonate adhesive curing, the curing reaction was investigated by Fourier

transformed infrared spectroscopy. The mechanical properties of the adhesives were

monitored by thermomechanical analysis. The thermal properties of the adhesive were studied

by thermogravimetrical analysis and differential scanning calorimetry. The curing of the

adhesives starts at 85-95°C and the optimum curing temperature is 150-170°C. The maximum

modulus of elasticity of an adhesive with glyoxalated liquid sodium lignosulfonate was

significantly higher than that of the adhesives with the other glyoxalated lignosulfonates. All

the cured adhesives were stable up to 200°C. Particleboards with tannin – glyoxalated

lignosulfonate were produced with maritime pine bark tannins and mimosa tannins. They

were compared to particleboards bonded with a urea – formaldehyde resin (internal bond and

formaldehyde emission). The tannin – glyoxalated lignosulfonate adhesives did not fulfil the

requirements for internal bond requested by the European Norm EN-312. The particleboards

with the bio – based adhesives had a very low level of formaldehyde emission.

106














particle board bonding with good mechanical properties [14]. Fourier transformed infrared

spectroscopy (FTIR) has been used in order to understand the reaction of glyoxalation for

organosolv and kraft lignins [15,16]













the pure tannins and lignins and gives the maximum temperature at which they can been used

and an insight into the major chemical structures of the compounds [7,24,31].

This study aims to prepare tannin-lignin adhesives from maritime pine bark tannins

and glyoxalated ammonium lignosulfonates and sodium lignosulfonates for particleboards.

For the first time to our knowledge, maritime pine bark tannins, sodium and ammonium

107

lignosulfonates were used to make particleboard adhesives. The thermal properties of these

adhesives were evaluated by therrmogravimetric analysis, differential scanning calorimetry

and thermomechanical analysis. The curing reaction was studied by Fourier transformed

infrared spectroscopy. Particleboards with tannin – glyoxalated lignosulfonates were prepared

and their performances were compared to an industrial urea – formaldehyde adhesive.

III.3.2 Material and methods

III.3.2.1 Material


NaLSL) and two different ammonium lignosulfonates (NH4LSL and NH4LSP) delivered by

Tembec (Tartas, France). The mimosa extracts were provided by Silvateam (Italy). The wood

particles and urea – formaldehyde were provided by Egger (Rion des Landes, France). The

sodium hydroxide, hexamethylene tetramine (hexamine), the glyoxal (40%), the glacial acetic

acid, the sulphuric acid (95%) and the formaldehyde (37%) were purchased from Fisher

Scientific (Waltham, USA). The acetylacetone was purchased from Sigma-Aldrich (St Louis,

USA). The ammonium acetate (98%) and the sodium thiosulfate were purchased from

AppliChem GmbH (Darmstadt, Germany).

III.3.2.2 Samples

Bark from maritime pine was collected in the Landes forest (Uchacq, 40). The tree

was 32 years old and had a diameter of 33 cm. The bark was air-dried at 20°C until the mass

was constant, ground in a mill (Retsch) and sieved to select particles smaller than 1 mm. The

ground pine bark was kept in sealed bags.

Tannins were extracted from maritime pine bark particles with a particle size smaller

than 1 mm. The extraction method is described in Chupin et al. [32]. The extracts are referred

to as P3.



Mansouri et al. [10]. 29.5 parts by mass of dry lignin were slowly added to 38.4 parts of

water. Sodium hydroxide solution (33%) was added in order to keep the pH of the solution

between 12 and 12.5 for better dissolution of the lignin. A 800 mL flat bottom flask equipped

with a condenser and a magnetic stirrer bar was charged with the above solution and heated to

58°C. 17.5 parts by mass glyoxal (40% in water) were added and the lignin solution was then


108

glyoxalated lignin were around 43%. The characteristics of the lignosulfonates are presented

in Table 1.

Table 1: Characteristics of the lignosulfonates

Identification Type Aspect pH

NaLSP Sodium lignosulfonate Brown powder 8.5

NaLSL Sodium lignosulfonate Dark liquid 8.3

NH4LSL Ammonium lignosulfonate Dark liquid 3.6

NH4LSP Ammonium lignosulfonate Brown powder 5.6

III.3.2.4 Adhesive Formulation

A 45% P3 tannin solution in water was prepared. A sodium hydroxide solution (33%)

was added in order to keep the pH of the solution at 10.4. This pH was chosen for the

hardener performs at its best at this pH and tannins dissolve at high pH. 5% of a



In this paper, P3 tannin – glyoxalated lignin adhesive formulations were produced

with 40 wt% tannins and 60 wt% glyoxalated lignosulfonates. The different formulations all

had a pH of approximately 10.





between two plies of maritime pine wood to form a joint of 17 x 5 x 1.2 mm. The bonded

wood joint were submitted to three points bending on a span of 14 mm and subjected to an

alternating force of 0.1 / 0.5 N with a 6 s / 6 s cycle. The heating rate was 10°C/min from

25°C to 250°C. For each formulation, three replicates were done. The results were analysed





109

resin and analysed from 30°C to 600°C at a heating rate of 10°C/min under 40-60 mL/min N2.

The results were analysed with Universal Analysis software.




in an aluminium crucible. The DSC scans were recorded from 30°C to 250°C at a heating rate

of 10°C/min under 50 mL/min N2. The results were analysed with Universal Analysis

software.

III.3.2.8 Fourier Transformed Infrared (FTIR)






III.3.2.9 Particleboard Preparation

Three-layer particleboards of 25 x 25 x 2 cm were prepared using a mixture of wood

particles. The resin ratio was 10-12-10. The mat was pressed for 7.5 min at 180°C, the

pressures applied were 35 kg/cm2, 25 kg/cm2, 15 kg/cm2 and 5 kg/cm2. The particleboards

were conditioned in a climate chamber at a relative humidity of 65% at 20°C.

III.3.2.10 Particleboard Characterisation

Nine particleboard samples were tested for their internal bond (IB) strength according

to the European Norm EN 319 [33].

The formaldehyde emission of the particleboards was determined according to an

adaptation of the desiccator method, ISO/DC 12460-4 [34].



Fisher Snedecor test, the student test, the Aspin-Welch test and the Mann-Whitney-Wilcoxon

test. All the statistical analyses were carried out at P < 0.05 significance level.

110


III.3.3.1 Analysis of the curing of the resins



adhesive behaviour during curing and its wood-joint strength.

Table 2: Module of elasticity values for all the adhesive formulations

Formulation Maximum MOE (MPa)*c Temperature (°C)

P3-NaLSP (40-60) 1695 ± 546.7 168

P3-NaLSL (40-60) 2310 ± 321.9 175

P3-NH4LSL (40-60) 1422 ± 488.8 152

P3-NH4LSP (40-60) 2118 ± 88.8 157

* Standard deviation of three replicates c The means were analysed with the Student test at P < 0.05

The curves of MOE as a function of temperature, obtained by TMA for resins with the

different lignosulfonates, are given in Fig. 1. All curves start to increase at 85-95°C, which

corresponds to the evaporation of water and a first cross-linking reaction through formation of

a non stable cross-linker. The maximum MOE values are obtained between 150°C and 170°C.

The maximum values of MOE are presented in Table 2. There are statistically no significant

differences between the MOE of the adhesive formulations prepared with NaLSP, NaLSL,

NH4LSP and NH4LSL. The wood joint bonded with the adhesive formulation with NaLSL

and NH4LSP tend to have a higher MOE than the adhesive formulations with NaLSP and

NH4LSL. The adhesive formulation with NH4LSP has a higher MOE from 125°C to at least

250°C compared to the adhesive with NaLSP and NH4LSL. The adhesive with NaLSP

presents a higher MOE than the adhesive with NH4LSL from 110°C. The adhesive

formulation with NaLSL shows a faster cross linking reaction than the adhesives with NaLSP,

NH4LSL and NH4LSP. The adhesive with NaLSL gave results similar to what is found for

mimosa tannin – glyoxalated organosolv lignin – pMDI (polymeric 4,4’-diphenyl methane

diisocyante) adhesives and for mimosa tannin – glyoxalated sodium and ammonium

lignosulfonates [16]. Navarrete et al. [21] found similar results for mimosa tannins –

glyoxalated organosolv lignins using hexamine as a hardener for a tannin – lignin ratio of 50 –

50, however resins with a 60 – 40 ratio presented higher MOE.

111

Fig. 1: Module of elasticity of average curves of a pine joint as a function of temperature

obtained by TMA testing when bonded with (—) P3 tannins – glyoxalated NaLSL; (—) P3

tannins – glyoxalated NaLSP; (—) P3 tannins – glyoxalated NH4LSL; (—) P3 tannins –

glyoxalated NH4LSP recorded at 10°C/min

In order to understand the chemical reactions that take place during the curing of the

resins, FTIR analyses were carried out. The spectra of the P3 tannins – glyoxalated NaLSL,

P3 tannins, glyoxalated NaLSL and hexamine are studied in the 650-2000 cm-1 region and

presented in Fig. 2. The analyses of the spectra are based on the assignments given by Boeriu

et al. [35]. When comparing the cured adhesive to the raw materials used, we can notice that

the hardener (hexamine), the tannins and the lignosulfonates have totally reacted. The resin

has a few bands in common with the tannins and glyoxalated lignosulfonates at 1580-1590

cm-1 and 1030-1040 cm-1, which are attributed to aromatic skeleton vibration and to –CO

stretching vibration respectively. They also share a band at 1262 cm-1 that according to

Özacar et al. [24] corresponds to –CO stretching of the benzene ring and the dimethylene

ether bridges formed during curing. Ping et al. [17] assign it to saturated C–C stretch

vibration. The cured resin and the glyoxalated lignosulfonates also have a band at 1505-1511

cm-1 in common, which corresponds to aromatic skeleton vibration. The resin presents peaks

at 1450-1460 cm-1, 1135 cm-1 and 1080 cm-1, which correspond to aromatic ring vibration

with C–H deformation, C–H in plane deformation and C–O deformation respectively. The

glyoxalated NaLSL presents a band at 1715 cm-1, which is assigned to carbonyl/carboxyl

groups. The tannins and the glyoxalated lignosulfonates share a band at 1440-1445 cm-1 that

is attributed to –CH deformation and aromatic ring vibration and at 1115 cm-1 that

corresponds to aromatic –CH in plane bending vibration. The tannins present bands at 1495

0

500

1000

1500

2000

2500

25 75 125 175 225

MO

E (

MP

a)

Temperature (°C)

112

cm-1 that is assigned to aromatic skeleton vibration and at 1380 cm-1, which is attributed to

phenolic –OH stretch vibration and aliphatic –CH deformation in methyl groups. The bands at

875 cm-1, 865 cm-1, 816 cm-1 and 768 cm-1 correspond to aromatic C–H “oop” stretch

vibration. The carbonyl/carboxyl groups from the glyoxalated lignosulfonates react during

curing and disappear in the resin.

Autocondensation of tannins occurs at high alkalinity, pH 10 or higher [36]. At lower

pH, tannins crosslink with hexamine. As our adhesive solutions are at approximately pH 10,

P3 tannins are subjected to both autocondensation reactions and crosslinking reactions with

hexamine and the glyoxalated lignosulfonates.

Fig. 2: FTIR spectra of (—) P3 tannins, (—) glyoxalated NaLSL, (—) mimosa tannins –

glyoxalated NaLSL and (—) hexamine

III.3.3.2 Thermal analysis of cured resins

The TG and DTG curves of the cured resins with 40 mass % mimosa tannins with four

glyoxalated lignosulfonates are presented in Fig. 3. The initial decomposition temperature and

the final decomposition temperature of the resins were observed. In a first stage, the resins are

submitted to the elimination of water, in which 3 to 12% mass loss occurs depending on the

initial water content of the samples. In a second stage, the resin with NaLSL starts to

decompose at 202°C, the one with NaLSP starts its degradation at 208°C, the cured resin with

NH4LSL at 206°C and the adhesive with NH4LSP decomposes at 199°C. This decomposition

is the result of a partial breakdown of the intermolecular bonding and occurs up to

approximately 370 – 380°C and takes place in two stages. The major mass losses occur at

240-260°C and at 305-315°C. In a third stage, from approximately 370-380°C to 510-520°C,

60011001600

Tra

nsm

ittan

ce (

A.

U.)

Wavenumber (cm-1)

113

the maximum resin degradation takes place at 440°C and is caused by the decomposition of

aromatic rings [37]. For resins with sodium lignosulfonates, there is also a degradation step

between 465-475°C, which corresponds to the degradation of a high quantity of aromatic

rings. The mass loss progressively increases with the temperature up to 600°C where the mass

loss starts to stabilise. The mass loss ranges from 34% to 47%. Similar mass losses were

found for mimosa tannin resins, valonia tannin resins [24].

(a)

(b)

Fig. 3: (a) TG curves and (b) DTG curves of P3 tannins – glyoxalated lignosulfonates with 40

mass % tannins cured resins: (—) P3 tannins – glyoxalated NaLSL; (—) P3 tannins –

glyoxalated NaLSP; (—) P3 tannins – glyoxalated NH4LSL; (—) P3 tannins – glyoxalated

NH4LSP recorded at 10°C/min

The DTG curves of P3 tannins, glyoxalated NH4LSL and of the adhesive formulation

with NH4LSL are presented in Fig. 4. Three major mass losses are present for P3 tannins. At

55

60

65

70

75

80

85

90

95

100

20 220 420

Mas

s (%

)

Temperature (°C)

20 220 420

DT

G (

%/°

C)

Temperature (°C)

114

237°C, the mass loss corresponds to the decarboxylation of the tannins. At 299°C, the mass

loss corresponds to further degradation of hydroxyl groups that occurs up to 355°C [38]. At

449°C, the oxidation of carbon residue and aromatic rings decomposition occurs. The P3

tannins, the glyoxalated NH4LSL and the cured resins have two common peaks, at 237-

241°C, which corresponds to decarboxylation and at 299-304°C, which corresponds to the

degradation of hydroxyl groups and to inter-unit degradation. The DTG curve of the

glyoxalated NH4LSL presents peaks at 143°C, 169°C and 207°C, which correspond to the

glass transition of NH4LSL, and to the degradation of the lignocellulosic materials. The

disappearance of the degradation peaks up to 220°C of the glyoxalated NH4LSL in the cured

resin shows that the glyoxalated NH4LSL has reacted with the hexamine and tannins.

Fig. 4: DTG curves of (—) P3 tannins, (—) glyoxalated NH4LSP and (—) P3 – glyoxalated

NH4LSP recorded at 10°C/min

The DSC results for cured resins with 40 mass % P3 tannins are presented in Fig. 5.

The adhesive formulation with NaLSP is stable up to 227°C; the resin with NaLSL is stable

up to 215°C; the resin with NH4LSL is stable up to 192°C and the resin with NH4LSP is

stable up to 185°C, where an endothermic reaction takes place. This reaction is a consequence

of the degradation of the resins. The DSC and TG results both show that all the resins are

stable at temperatures up to at least 185°C. The resins with sodium lignosulfonates are more

stable and are not submitted to a thermal degradation until the temperature reaches 215°C.

20 220 420

DT

G (

%/°

C)

Temperature (°C)

115

Fig. 5: DSC curves of P3 tannins – glyoxalated lignosulfonates with 40 mass % tannins cured

resins: (—) P3 tannins – glyoxalated NaLSL; (—) P3 tannins – glyoxalated NaLSP; (—) P3

tannins – glyoxalated NH4LSL; (—) P3 tannins – glyoxalated NH4LSP recorded at 10°C/min

III.3.3.3 Particleboard characterisation

Table 3 Particleboard characteristics

Internal bond (N/mm2) Formaldehyde emission

(mg/L) Density (kg/m3)

P3 – glyoxalated NaLSL 0.02 ± 0.006a 0.003 ± 0.0003d 570 ± 34

Mimosa – glyoxalated

NaLSL 0.15 ± 0.028b 0.004 ± 0.0008d 599 ± 19

UF 0.50 ± 0.153c 0.007 ± 0.0007e 595 ± 34

a, b, c, d, e Group of values with significant differences between each group for each parameter c The means were analysed with the Aspin-Welch test and the Mann-Whitney-Wilcoxon test at P < 0.05

As the highest MOE is obtained with NaLSL, particleboards bonded with tannin –

lignin adhesive were produced using the glyoxalated lignosulfonate NaLSL and P3 extracts.

Particleboards with mimosa tannins and urea-formaldehyde adhesives were also made in

order to compare the type of tannins used to an industrial adhesive. The IB and the

formaldehyde emission of the particleboards were measured and are presented in Table 3 for

the three adhesives. The IB strength of the particleboard samples bonded with UF resin is

above 0.3 N/mm2, which is the requirement for the P2 type of particleboard [39], which

corresponds to particleboards for interior fitments for use in dry conditions. The mimosa

25 75 125 175 225

←E

ndo

Hea

t Flo

w (

W/g

)

Temperature (°C)

116

tannin – glyoxalated NaLSL particleboards have a significantly lower IB strength than the UF

particleboards. The P3 tannin – glyoxalated NaLSL particleboard’s IB strength is significantly

lower than those of the mimosa tannins – glyoxalated NaLSL particleboards and of the UF

particleboards. Neither of the tannin – glyoxalated lignosulfonate particleboards comply with

the EN 312 requirements. By increasing the density of the particleboards, mimosa tannin –

organosolv lignin adhesive particleboards comply with the EN 312 requirements [39].

All particleboards have formaldehyde emissions lower than 0.3 mg/L, which is the

Japanese standard limit F**** required. The emissions of formaldehyde of the UF

particleboards are significantly higher than for the tannin – glyoxalated NaLSL, and emit

approximately twice as much.

III.3.4 Conclusion

In this study, maritime pine bark extracts were used in the formulation of tannin –

glyoxalated lignosulfonate adhesives. Four different lignosulfonates were used. The curing of

the resins starts at 85-95°C with the evaporation of water. The resins are fully cured at 150-

170°C. The adhesive with glyoxalated NaLSL is the most efficient. During curing, P3 tannins,

glyoxalated lignosulfonates and hexamine react totally. The tannins crosslink with hexamine

and the glyoxalated lignosulfonates and autocondensation reactions occur. There is a

disappearance of carbonyl/carboxyl groups during curing. The resins with sodium

lignosulfonates are stable up to 215-225°C, when they start to degrade and the resins with

ammonium lignosulfoantes up to 185-195°C. Tannin – glyoxalated lignosulfonate adhesives

did not produce particleboards that can be used for interior fitments for a density of 570-600

kg/m3. The particle boards have a very low level of formaldehyde emission.

III.3.5 Acknowledgments




III.3.6 References







2013;44:315–22.

117







23.






cane bagasse: Production of lignin–phenol-formaldehyde wood adhesive. Ind. Crops Prod.;45:296–302.


Roh- und Werkst. 2007;65:65–70.


Polym. Sci. 2007;103:1690–9.





2010;24:1597–610.





16. Lei H, Pizzi A, Du G. Environmentally friendly mixed tannin / lignin wood resins. J. Appl. Polym. Sci.

2008;107:203–9.





2009;29:364–70.








118



und Werkst. 2002;60:303–10.









2006;84:651–4.





2013;47:675–83.





2011;25:1549–60.



32. Chupin L, Motillon C, Charrier-El Bouhtoury F, Pizzi A, Charrier B. Characterisation of maritime pine

(Pinus pinaster) bark tannins extracted under different conditions by spectroscopic methods, FTIR and

HPLC. Ind. Crops Prod. 2013;49:897–903.

33. EN 319. (1993): Particleboards and fibreboards – Determination of tensile strength perpendicular to the plane

of the board. European standard, June 1993.

34. ISO/CD 12460-4. (2006). Wood-based panels – Determination of formaldehyde release – Part 4 – Desiccator

method. International Organisation of Standardisation, August 2006.





37. Ramires EC, Frollini E. Tannin–phenolic resins: Synthesis, characterization, and application as matrix in

biobased composites reinforced with sisal fibers. Compos. Part B 2012;43:2851–60.


39. EN 312. (2010): Particleboards - Specifications. European Standard, November 2010.

119

III.4 Glyoxalation de lignosulfonates de sodium et d’ammonium

pour l’élaboration de colles tanin-lignine pour panneaux de particules

Soumis dans « European Journal of Wood and Wood Products », le 2 Septembre 2014

Résumé

Deux types de traitements au glyoxal ont été effectués sur deux lignosulfonates de

sodium et deux lignosulfonates d’ammonium pour les rendre plus réactifs. La distribution des

protons des lignosulfonates a été analysée par 1H RMN à bas champ. Leurs propriétés

thermiques ont été établies par analyse thermogravimétrique (ATG) et par calorimétrie

différentielle à balayage. Les lignosulfonates traités ont été utilisés pour réaliser des colles à

base de lignines et de tannins de mimosa. Les effets du traitement au glyoxal, du ratio tanin-

lignine des colles et du type de lignosulfonate utilisé ont été analysés par analyse

thermoméchanique et par ATG. Des panneaux de particules ont été préparés à partir de

lignosulfonates de sodium et d’ammonium ayant subi les deux traitements au glyoxal. Le ratio

tanin-lignine et le temps de pression des panneaux de particules ont été optimisés. Une bonne

cohésion interne a été obtenue pour les panneaux avec 60%m et ayant été pressés pendant 7,5

minutes.

120

Glyoxalation of sodium and ammonium lignosulfonates for tannin lignin

adhesives for particleboards

Lucie Chupin*a, Bertrand Charriera, Arturo Perdomob, Antonio Pizzic, Fatima Charrier – El

Bouhtourya



b Tembec, 1154 avenue du Général Leclerc, 40400 Tartas, France

c ENSTIB-LERMAB, University of Lorraine, 27 rue Philippe Seguin, BP1041, 88051 Epinal,

France




Abstract

Two types of glyoxal treatments were performed on two sodium lignosulfonates and

two ammonium lignosulfonates to make them more reactive. The lignosulfonates proton

distribution was analysed by low resolution 1H NMR. Their thermal properties were assessed

by thermogravimetric analysis (TG) and by differential scanning calorimetry. The treated

lignosulfonates were used to make mimosa tannin-lignin adhesives. The effect of the glyoxal

treatment, of the tannin-lignin ratio and of the type of lignosulfonate was analysed by

thermomechanical analysis and by TG analysis. Particleboards were prepared with sodium

and ammonium lignosulfonates that underwent both glyoxal treatments. The tannin-lignin

ratio and the press time of the particleboards were optimised. Good IB strength was obtained

for particleboards with 60 wt% tannins and pressed for 7.5 min.






121









particle board bonding with good mechanical properties [14].













the pure tannins and lignins and gives the maximum temperature at which they can been used

and an insight on the major chemical structures of the compounds [7,22,29].

Low resolution 1H NMR relaxation is a fast, non-destructive and non-invasive

technique that has been used to characterise pore size distribution of porous media, to

determine oil and water content and phase changes, for example from liquid to solid during

the curing of resins [30–33]. Medical applications have been found by profiling human skin

[34]. It has also been extensively used to characterise food quality by analysing the water

distribution of the food products, such as ramen soup powders, potatoes, pears, blueberries

and cakes [35–40]. As proton transverse relaxation time of oil molecules is shorter than that

of water molecules, low resolution NMR is a good method of detection of oils, for example in

oil contaminated soils [41].

122

This study aims to compare two glyoxal treatments of two different sodium

lignosulfonates and of two different ammonium lignosulfonates and to optimise the

preparation of particleboards prepared with mimosa tannin-lignin adhesives. The

lignosulfonates before and after treatment were analysed by low resolution 1H NMR. The

thermal properties of the lignosulfonates before and after treatment were evaluated by

therrmogravimetric analysis and differential scanning calorimetry. The adhesives were

analysed by thermomechanical analysis and therrmogravimetric analysis. The tannin-lignin

ratio of the adhesives and the press time were optimised for the production of particleboards.

III.4.2 Materials and methods

III.4.2.1 Material


NaLSL) and two different ammonium lignosulfonates (NH4LSL and NH4LSP) delivered by

Tembec (Tartas, France). The mimosa extracts were provided by Silvateam (Italy). The wood

particles and urea – formaldehyde were provided by Egger (Rion des Landes, France). The

sodium hydroxide, hexamethylene tetramine (hexamine), the glyoxal (40%), the glacial acetic

acid, the sulphuric acid (95%) and the formaldehyde (37%) were purchased from Fisher

Scientific (Waltham, USA). The acetylacetone was purchased from Sigma-Aldrich (St Louis,

USA). The ammonium acetate (98%) and the sodium thiosulfate were purchased from

AppliChem GmbH (Darmstadt, Germany).



Mansouri [10]. 29.5 parts by mass of dry lignin were slowly added to 38.4 parts of water.

Sodium hydroxide solution (33%) was added in order to keep the pH of the solution between

12 and 12.5 for better dissolution of the lignin. An 800 mL flat bottom flask equipped with a

condenser and a magnetic stirrer bar was charged with the above solution and heated to 58°C.

17.5 parts by mass of glyoxal (40% in water) were added and the lignin solution was then


glyoxalated lignin were around 43%. The lignosulfonates characteristics are presented in

Table 1. Another glyoxal treatment of lignosulfonates was prepared with different water

proportions. The quantities used in this formulation are indicated below in Table 2.

123

Table 1: Characteristics of lignosulfonates

Identification Type Aspect pH Solid content

(%)

NaLSP Sodium lignosulfonate Brown powder 8.5 95

NaLSL Sodium lignosulfonate Dark liquid 8.3 55

NH4LSL Ammonium

lignosulfonate Dark liquid 3.6 60

NH4LSP Ammonium

lignosulfonate Brown powder 5.6 95

Table 2: Proportions of the reactants used to prepare glyoxalated lignins with the

treatment 1 (T1) and treatment 2 (T2)

T1 T2

Lignin 29.5 29.5

Glyoxal 40% in water 17.5 17.5

Water 38.4 26.2

III.4.2.3 Low resolution 1H NMR

Proton relaxation measurements were performed on Artec system DIASPEC42003

low resolution spectrometer (Artec system, Annonay, France) at a resonance frequency of

7.982 MHz. The probe head temperature was 28°C. Transverse relaxation curves were

measured with a Carr-Purcel-Meiboom-Gill (CPMG) pulse sequence (for a spin-spin

relaxation time, T2, between 0.1 and 10000 ms). The pulse widths of the 90° pulse and the

180° pulse were 8.4 µs and 16.7 µs respectively. The 90-180° pulse spacing (τ) was 300 µs.

50 scans were made with a repeat delay of 1 s. For untreated NaLSP and NH4LSP, 1000

scans were made with a repeat delay of 3 s and a 90-180° τ of 150 µs.

I II.4.2.4 Adhesive Formulation

A 45% mimosa tannin solution in water was prepared. A sodium hydroxide solution

(33%) was added in order to keep the pH of the solution at 10.4. This pH was chosen for the

hardener performs at its best at this pH and tannins dissolve at high pH. 5% of a



124

In this paper we study four different ratios of tannin – glyoxalated lignin adhesive

formulations for the sodium lignosulfonate NaLSP. Mimosa tannins – glyoxalated lignins

adhesive formulations were also produced with the other lignosulfonates for one ratio. The

different formulations are described in Table 3.

Table 3: Adhesive formulations

Formulations Tannins/mass

%

Glyoxalated lignins/mass

%

pH Solid content (%)

T1 T2 T1 T2

Mimosa-

NaLSP 20 80

9.08 8.67 48.12 55.05

40 60 10.10 9.77 46.99 49.59

50 50 9.84 10.05 47.73 48.72

60 40 10.33 10.08 39.45 45.49

NaLSL 60 40 10.4 10.45 43.98 43.69

NH4LSL 60 40 10.2 10.55 41.77 41.47

NH4LSP 60 40 10.2 10.61 42.95 40.44





between two plies of maritime pine wood to form a joint of 17 x 5 x 1.2 mm. The bonded

wood joint was submitted to three points bending on a span of 14 mm and subjected to an

alternating force of 0.1 / 0.5 N with a 6 s / 6 s cycle. The heating rate was 10°C min-1 from

25°C to 250°C. For each formulation, five replicates were done. The results were analysed





resin and analysed from 30°C to 600°C at a heating rate of 10°C min-1 under 40-60 mL min-1

N2. The results were analysed with Universal Analysis software.

125




in an aluminium crucible. The DSC scans were recorded from 30°C to 250°C at a heating rate

of 10°C/min under 50 mL/min N2. The results were analysed with Universal Analysis

software.

III.4.2.8 Particleboard Preparation

Three-layer particleboards of 25 x 25 x 2 cm were prepared using a mixture of wood

particles. The resin ratio was 10-12-10. The mat was pressed for 5.5, 7.5 and 9.5 min at

180°C, the pressures applied were 35 kg/cm2, 25 kg/cm2, 15 kg/cm2 and 5 kg/cm2. The

particleboards were conditioned in a climate chamber at a relative humidity of 65% at 20°C.

III.4.2.9 Particleboard Characterisation

Nine particleboard samples were tested for their internal bond (IB) strength according

to the European Norm EN 319 [42].

The formaldehyde emission of the particleboards was determined according to an

adaptation of the desiccator method, ISO/DC 12460-4 [43].



Fisher-Snedecor test, the student test, the Aspin-Welch test and the Mann-Whitney-Wilcoxon

test. The IB results were analysed with the Fisher-Snedecor test, the Wilcoxon test, the

student test, the Aspin-Welch test and the Mann-Whitney-Wilcoxon test. All the statistical

analyses were carried out at P < 0.05 significance level.


III.4.3.1 Study of two glyoxal treatments of lignosulfonates

Figure 1 shows the distribution of transverse relaxation times obtained for sodium and

ammonium lignosulfonates after T1. The water distribution of sodium and ammonium

lignosulfonates after T1 is distinct. NH4LSL show a bimodal distribution with peaks centred

at about 3.5 and 70 ms. The first component is assigned to non exchanging protons of

ammonium lignosulfonates. The longest component is assigned to extra-granular mobile

protons. The other T1 lignosulfonates present one peak at 40-50 ms for sodium

lignosulfonates and at 70-90 ms for ammonium lignosulfonates. The extra-granular protons of

T1 ammonium lignosulfonates have a higher mobility than that of T1 sodium lignosulfonates.

126

Fig 1 CPGM spectra of lignosulfonates after T1: (—) NaLSP, (—) NaLSL, (—) NH4LSL, (—)

NH4LSP

Figure 2 shows the distribution of transverse relaxation times obtained for sodium

lignosulfonates (Fig 2 (a)) and ammonium lignosulfonates (Fig 2 (b)) before treatment, after

T1 and after T2. After T1 and T2, both types of lignosulfonates show a shift of the mobile

protons; to 45-50 ms instead of 25 ms for NaLSL and to 55-70 ms instead of 15 ms for

NH4LSL. The shift is due to an increase in the solvent volume which induces a higher

mobility for the proton populations [40].

0,1 1 10 100 1000 10000

Sig

nal a

mp

litud

e (a

. u.)

T2 (ms)

NaLSP

NaLSL

NH4LSL

NH4LSP

127

(a)

(b)

Fig 2 CPGM spectra of (a): (—) NaLSL, (—) NaLSL after T1, (—) NaLSL after T2 and of (b):

(—) NH4LSL, (—) NH4LSL after T1, (—) NH4LSL after T2

The thermal properties of the lignosulfonates before and after T1 and T2 were

analysed by TG and DSC (Table 4). The TG and the first derivative of the TG (DTG) curves

of NaLSL before and after T1 and T2 are presented in Figure 3. In a first step, which occurs

up to approximately 110°C, the mass loss is due to the elimination of any residual water. In a

second step, the non treated NaLSL starts to decompose at 200°C up to 345°C with a mass

loss of 23%. NaLSL decomposes in three peaks at 213°C, at 253°C and the maximum loss

rate occurs at 300°C. The mass loss corresponding to the peaks at 213°C and 253°C are

attributed to a breakdown of side chains present in lignins. Between 300 and 450°C, a

pyrolytic degradation occurs, which leads to the fragmentation of the intermolecular bonding

and the release of monomeric phenols. NaLSL after T1 and T2 have similar decomposition

0,1 1 10 100 1000 10000

Sig

nal a

mp

litud

e (a

. u.)

T2(ms)

NaLSL

NaLSL T1

NaLSL T2

0,1 1 10 100 1000 10000

Sig

nal a

mp

litud

e (a

. u.)

T2 (ms)

NH4LSL

NH4LSL T1

NH4LSL T2

128

temperature and mass loss. NaLSL T1 and NaLSL T2 first eliminate residual water present in

the samples. In a second step, from 124°C and 129°C respectively, four peaks of

decomposition are present at 137-140°C, 158-169°C, 238-239°C and 311-314°C. Up to

200°C, the mass loss is caused by the link formed by the glyoxal that is less stable. This might

be due to the low reactivity of glyoxal, which introduces much earlier sites of tridimensional

cross-linking in the network. The network is weaker and degradation starts at lower

temperatures. Another hypothesis is that there is formation of glyoxal – O – glyoxal unstable

bridges that on heating should rearrange to a simple glyoxal bridge, as formaldehyde does.

However due to the low reactivity of glyoxal, it is unable to rearrange to a single bridge

causing instability.

All lignosulfonates have degradation trends similar to NaLSL (figures not shown). The

non treated sodium lignosulfonates start to decompose at 186-202°C and the ammonium

lignosulfonates at 171-176°C. The major mass losses occur up to 375-400°C with a mass loss

of 30-40% for NaLSP, NH4LSL and NH4LSP.

At 600°C, non treated sodium lignosulfonates have a residue corresponding to

approximately 60 wt% of the initial dry matter of the samples and non treated ammonium

lignosulfonates of approximately 45%. After T1 and T2, sodium and ammonium

lignosulfonates have up to 55 wt% of the samples that have not been volatilised. Before

treatment, sodium lignosulfonates are more stable to temperature than ammonium

lignosulfonates; they start to decompose higher temperatures and there are more residues at

600°C. After T1 and T2, all the lignosulfonates have the same thermal properties.

129

(a)

(b)

Fig 3 (a) TG curves and (b) DTG curves of (—) NaLSL, (—) NaLSL after T1, (—) NaLSL after

T2 recorded at 10°C/min

40

50

60

70

80

90

100

30 230 430

Wei

ght (

%)

Temperature (°C)

NaLSL

NaLSL T1

NaLSL T2

30 230 430

DT

G (

%/°

C)

Temperature (°C)

NaLSL

NaLSL T1

NaLSL T2

130

Table 4: Results from thermal analysis (DSC, TGA) of lignosulfonates before treatment and

after T1 and after T2

Samples Tg (°C) DTGmax (°C) Residue at 600°C

(%)

NaLSP 300 59.1

NaLSP T1 141 311 55.8

NaLSP T2 140 313 54.5

NaLSL 300 60.0

NaLSL T1 140 311 55.5

NaLSL T2 144 313 56.5

NH4LSP 240 43.7

NH4LSP T1 145 245 57.3

NH4LSP T2 130 248 52.2

NH4LSL 244 45.9

NH4LSL

T1 149 242 55.6

NH4LSL

T2 161 248 55.3

Tg: glass transition temperature, DTGmax: maximum of thermal decomposition temperature,

Residue: unvolatilised weight fraction at 600°C

The glass transition temperatures (Tg) of the lignosulfonates were determined by DSC

(Table 4). For untreated lignosulfonates, no Tg are found. The Tg obtained for sodium

lignosulfonates after T1 and T2 are of 140-144°C. For ammonium lignosulfonates the Tg

range from 130°C to 160°C. These Tg are in the same range of temperatures as glyoxalated

kraft lignins or glyoxalated organosolv lignins [4,44].

III.4.3.2 Analysis of the adhesives



adhesive behaviour during curing and its wood-joint strength. The maximum MOE of mimosa

tannin – lignosulfonates after T1 and T2 are presented in Table 5.

All the adhesives show an increase of the MOE at 100-110°C. This corresponds to a

first cross-linking reaction through formation of a non stable cross linker and the elimination

of water [45]. The highest MOE values are obtained at 160-180°C. There is a decrease in the

131

MOE at 170-190°C corresponding to the decomposition of wood components and of the

adhesive. There are no significant differences between the adhesive with lignosulfonates after

T1 and T2 (student test at P < 0.05; Mann-Whitney-Wilcoxon test when comparing adhesive

with NaLSL after T1 and T2). Only the formulation with NaLSP with 20 wt% tannins has a

lower MOE after T2 than after T1 (student test at P < 0.05). A comparison of adhesive made

with mimosa tannins and NaLSP after T1 with tannin-lignin ratios of 20 wt% tannins, 40 wt%

tannins, 50 wt% tannins and 60 wt% tannins shows that the less tannin is added, the lower the

MOE. There are statistically no differences between the maximum MOE of the adhesive

formulations containing 40 wt%, 50 wt% and 60 wt% tannins (student test, P < 0.05). The

MOE of the adhesive formulation containing 20 wt% tannins is statistically lower than the

others (student test, P < 0.05). TMA results obtained for adhesives with NaLSP T2 with

different tannin-lignin ratios confirm that the less tannin is used, the lower the maximum

MOE of the adhesive. The formulation with 20 wt% tannins gives lower MOE values than the

formulations with more tannins (student test, P < 0.05). The formulation with 50 wt% tannins

is not statistically different from the formulations with 40 wt% and 60 wt% tannins (student

test, P < 0.05). The formulation with 40 wt% tannin is less efficient than the formulation with

60 wt% tannins (student test, P < 0.05).

Since the higher the tannin content in the adhesive formulation, the higher the MOE

values obtained, it was decided that adhesives with the other lignosulfonates would be made

with a tannin-lignin ratio of 60-40. There are no statistical differences between the maximum

MOE values of adhesives with NaLSP T1, NaLSL T1, NH4LSP T1 and NH4LSL T1 and a

tannin-lignin ratio of 60-40 (student test, P < 0.05; Mann-Whitney-Wilcoxon test at P < 0.05

when comparing with NaLSL). There are no statistical differences between the maximum

MOE values of adhesives with NaLSP T2, NaLSL T2, NH4LSP T2 and NH4LSL T2 and a

tannin-lignin ratio of 60-40 except for NaLSP and NH4LSP, which are statistically different

(Aspin-Welch test, P < 0.05; student test at P < 0.05 when comparing NaLSL with NH4LSL

and NaLSP with NH4LSP). The cross linking reaction starts at a higher temperature for the

adhesive formulations with NaLSP T2 and NH4LSL T2, 120°C and 130°C respectively.

132

Table 5: Module of elasticity values for all the adhesive formulations

Formulation Maximum MOE (MPa)*e Temperature (°C)

T1 T2 T1 T2

Mimosa-NaLSP (20-80) 1905 ± 98.7a 1706 ± 123.5c 161 164

Mimosa-NaLSP (40-60) 2264 ± 110.5b 2264 ± 154.2d 168 169

Mimosa-NaLSP (50-50) 2294 ± 308.3b 2484 ± 197.3bd 160 177

Mimosa-NaLSP (60-40) 2497 ± 284.9b 2499 ± 92.6b 165 164

Mimosa-NaLSL (60-40) 2374 ± 339.2b 2086 ± 449.0cd 163 162

Mimosa-NH4LSL (60-40) 2122 ± 364.6ab 2382 ± 350.1bd 171 180

Mimosa-NH4LSP (60-40) 2335 ± 147.4b 2329 ± 103.1d 169 161

* Standard deviation of five replicates a, b, c, d Group of values with significant differences between each group ab, bd, cd Group of values significantly different one from the other at P < 0.05 e The means were analysed with the student test, the Aspin Welch and the Mann-Whitney-Wilcoxon at P < 0.05 when required

The thermal properties of the cured tannin-lignin adhesives were analysed by TG, the

results are presented in Table 6. All the formulations show three decomposition steps. The

first step occurs up to 110-120°C and corresponds to the evaporation of residual water present

in the sample. In a second step, from 210-265°C to 377-400°C, the maximum DTG occurs.

Adhesives with lignosulfonates after T1 and T2 have similar thermal properties, their DTGmax

is found at approximately the same temperature. Formulations with NaLSP after T1 and T2

have a DTG max at a higher temperature compared to formulations with other

lignosulfonates, 280-288°C instead of approximately 270°C. This decomposition can be the

result of a partial breakdown of the intermolecular bonding [22]. The third step occurs up to

600°C and corresponds to further degradation of the intermolecular bonding. At 600°C the

mass of the sample starts to stabilise and the mass loss ranges from 33% to 40%. For cured

adhesives with NaLSP after T1 and T2 with several tannin-lignin ratios, we notice that when

the proportion of tannins increases up to 50 wt% tannins, the initial decomposition

temperature increases.

133

Table 6: Results from thermal analysis (TG) of cured resins produced with

lignosulfonates after treatment 1 (T1) and after treatment 2 (T2)

Samples Ti DTGmax

(°C) Residue at 600°C (%)

T1 T2 T1 T2 T1 T2

Mimosa-NaLSP 20-80 238 236 284 281 65.9 65.7

Mimosa-NaLSP 40-60 242 243 280 280 66.2 65.7

Mimosa-NaLSP 50-50 258 266 288 282 64.3 64.2

Mimosa-NaLSP 60-40 222 209 280 282 62.2 60.5

Mimosa-NaLSL 60-40 227 220 273 271 62.9 60.3

Mimosa-NH4LSP 60-40 225 243 266 267 63.0 62.7

Mimosa-NH4LSL 60-40 227 225 273 269 62.3 62.0

Ti: initial decomposition temperature

III.4.3.3 Particleboard characterisation

Three layer particleboards were prepared with tannin-lignin adhesives at 180°C and

they were characterised by their IB. The effect of the press time was evaluated for adhesive

formulations with NaLSP after T1 and T2 and a tannin-lignin ratio of 60-40 (Fig 4).

Particleboards with NaLSP T1 were produced for a press time of 7.5 min and 9.5 min. There

are no significant differences between the IB for the two press times (Aspin-Welch test, P <

0.05). Particleboards with NaLSP T2 were prepared with a press time of 5.5 min, 7.5 min and

9.5 min. For a press time of 5.5 min, the IB is significantly lower than that of the

particleboards pressed for 7.5 and 9.5 min (student test and Aspin-Welch test, P < 0.05). For

higher press time, no differences are measured (student test, P < 0.05). For mimosa tannins-

glyoxalated lignosulfonates, the optimum press time is of 7.5 min. Particleboards prepared

with a press time of 7.5 min and 9.5 min with NaLSP T1 and T2 display significant

differences between the two treatments (student test, P < 0.05). Particleboards prepared with

adhesives with NaLSP T2 have lower IB than the ones prepared with NaLSP T1. Even though

adhesives with T1 and T2 lignosulfonates had similar thermal properties, the particleboards

produced with T1 lignosulfonates have better bonding properties.

134

Fig 4: Effect of the press time on the internal bond of particleboards bonded with mimosa

tannin-NaLSP T1 resin (■) and with mimosa tannin-NaLSP T2 resin (■) at a tannin-lignin ratio

of 60-40

The effect of the press time was evaluated for adhesive formulations with NaLSP after

T1 and T2 and a tannin-lignin ratio of 50-50 (Fig 5). Particleboards were produced for a press

time of 5.5 min and 7.5 min. For particleboards with NaLSP T1, there are no significant

differences between the IB for the two press times (Mann-Whitney-Wilcoxon test, P < 0.05).

Particleboards with NaLSP T2 obtain a higher IB for a press time of 7.5 min (Aspin-Welch

test, P < 0.05). When comparing particleboards pressed during the same amount of time with

NaLSP T1 and T2, we see no differences between the two when pressed for 7.5 min (student

test, P < 0.05). Particleboards with NaLSP T2 have lower IB than the ones with NaLSP T1

(Mann-Whitney-Wilcoxon test, P < 0.05). The results obtained for 50-50 tannin-lignin ratio

are similar to the ones obtained for a 60-40 tannin-lignin ratio.

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

5.5 7.5 9.5

Inte

rnal

bo

nd (

N/m

m²)

Press time (min)

135

Fig 5: Effect of the press time on the internal bond of particleboards bonded with mimosa

tannin-NaLSP T1 resin (■) and with mimosa tannin-NaLSP T2 resin (■) at a tannin-lignin ratio

of 50-50

The effect of the lignosulfonates was evaluated further by comparing the IB of

particleboards made with adhesive formulations with NaLSP T1, NaLSP T2 and NH4LSL T1

at a tannin-lignin ratio of 40-60 and pressed for 7.5 min (Fig 6). Particleboards with NaLSP

T2 gave the lowest IB (Aspin-Welch and student test, P < 0.05). Ammonium lignosulfonates

after T1 obtained significantly higher IB than sodium lignosulfonates after T1 (student test, P

< 0.05).

0,00

0,05

0,10

0,15

0,20

0,25

0,30

0,35

0,40

0,45

0,50

5.5 7.5

Inte

rnal

bo

nd (

N/m

m²)

Press time (min)

136

Fig 6: Effect of the lignosulfonate on the internal bond of particleboards bonded with mimosa

tannin-NaLSP T1 resin, with mimosa tannin-NaLSP T2 resin and mimosa tannin-NH4LSL T1

resin at a tannin-lignin ratio of 40-60 and a press time of 7.5 min

The effect of the tannin-lignin ratio was analysed by comparing the IB of

particleboards made with adhesive formulations with NaLSP T1 and NaLSP T2 at a press

time of 7.5 min (Fig 7). The adhesives used had a tannin-lignin ratio of 40-60, 50-50 and 60-

40. For particleboards with NaLSP T1, the lowest IB is obtained with a 40-60 tannin-lignin

ratio (Aspin-Welch test, P < 0.05). Particleboards with a 50-50 ratio adhesive have an IB

higher than for 40-60 but lower than for a 60-40 tannin-lignin ratio (Aspin-Welch test and

student test, P < 0.05). For particleboards with NaLSP T2, the lowest IB is also obtained with

a 40-60 tannin-lignin ratio (student test, P < 0.05). There are no significant differences

between the IB of particleboards with a 50-50 and a 60-40 tannin-lignin ratio (student test, P

< 0.05). As previously, particleboards with NaLSP T2 obtain similar or lower IB than those

with NaLSP T1 (Aspin-Welch test and student test, P < 0.05).

0,00

0,05

0,10

0,15

0,20

0,25

0,30

0,35

0,40

NaLSP T1 NaLSP T2 NH4LSL T1

Inte

rnal

bo

nd (

N/m

m²)

137

Fig 7: Effect of the tannin-lignin ratio on the internal bond of particleboards bonded with

mimosa tannin-NaLSP T1 resin (■) and with mimosa tannin-NaLSP T2 resin (■) for a press

time of 7.5 min

The IB of the particleboards prepared ranged from 0.06 to 0.55 N/mm². The European

standard EN 312 [46] requires that particleboards for interior fitments for use in dry

conditions have an IB above 0.3 N/mm². The particleboards with a mimosa tannin-NaLSP T1

ratio of 60-40 and pressed for 7.5 min and 9.5 min have an IB that is strictly higher than 0.3

N/mm², 0.48 and 0.55 N/mm² respectively (student test, P < 0.05). All the other particleboards

have lower or equal to 0.3 N/mm² IB (student test, P < 0.05; except for the particleboard with

a 50-50 ratio, pressed for 5.5 min with NaLSP T1 where it was the Wilcoxon test, P < 0.05).

III.4.4 Conclusion

In this study, sodium and ammonium lignosulfonates were submitted to two glyoxal

treatments. The results obtained by low resolution NMR showed that the proton distribution is

different for the two types of lignosulfonates and that after treatment there is a shift in the

distribution of protons that are more mobile. The two treatments gave similar results for low

resolution NMR, TG and DSC. After treatment, the lignosulfonates present a Tg between

140-145°C for sodium lignosulfonates and between 130-160°C for ammonium

lignosulfonates. The lignosulfonates are less stable to temperature after treatment.

The optimum curing temperature of tannin-lignin adhesives made with the treated

lignosulfonates was at 160-170°C. The cured adhesives were stable up to 210-265°C. The

more tannins used in the adhesive, the better the MOE obtained is.

0,00

0,10

0,20

0,30

0,40

0,50

0,60

0,70

40-60 50-50 60-40

Inte

rnal

bo

nd (

N/m

m²)

Tannin-lignin ratio

138

Particleboards with lignosulfonates after T2 were less efficient than the ones with

lignosulfonates after T1. Adhesives with ammonium lignosulfonates after T1 produce more

resistant particleboards than with treated sodium lignosulfonates. The most efficient

particleboards are produced with an adhesive with 60-40 tannin-lignin ratio and for a press

time of 7.5 min or more.

III.4.5 Acknowledgment




III.4.6 References







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142

III.5 Conclusion au chapitre III

Des lignosulfonates de sodium et des lignosulfonates d’ammonium ont subi deux

traitements au glyoxal. Le traitement avec la plus grande proportion d’eau s’est avéré

légèrement plus efficace. Après glyoxalation, des groupes carbonyle/carboxyle, des

hydroxyles phénoliques et des groupes C—O apparaissent. Les lignosulfonates sont moins

stables thermiquement après glyoxalation, ils se dégradent à partir de 125-135°C au lieu de

186-202°C pour les lignosulfonates de sodium et de 171-176°C pour les lignosulfonates

d’ammonium. Les lignosulfonates d’ammonium sont moins stables que les lignosulfonates de

sodium à la température. Les protons des lignosulfonates de sodium sont moins mobiles que

ceux des lignosulfonates d’ammonium.

Des colles à base de tanins de mimosa et de lignosulfonates de sodium et d’ammonium

ont été produites. Tout d’abord, l’optimisation du ratio tanin-lignine a été déterminée pour des

ratios de tanin-lignine de 20-80, 40-60, 50-50 et 60-40 en masse. Les analyses en ATM ont

montré que plus il y a de tanins dans les formulations de colles, plus les colles sont résistantes

et que les températures optimales de réticulations sont de 157-168°C. Les colles réticulées

sont stables jusqu’à 220°C.

Lorsque l’on compare les différents lignosulfonates pour des colles avec des tanins de

mimosa, on ne mesure pas de différence entre les maximums de module d’élasticité. Après

réticulation, les colles sont stables jusqu’à approximativement 190°C sauf pour un des

lignosulfonates de sodium.

Les formulations de colles avec des tanins d’écorce de pin maritime extrait à l’eau

chaude et des lignosulfonates glyoxalés sont généralement moins efficaces que les

formulations avec les tanins de mimosa, qui sont plus réactifs. Les formulations de colles avec

un des lignosulfonates de sodium et un des lignosulfonates d’ammonium ont des propriétés

mécaniques similaires qu’avec les tanins de mimosa. La réaction de réticulation démarre plus

tôt que les colles avec des tanins de mimosa, 85-95°C contre 110°C. Les températures

optimales de réticulation sont comprises entre 150 et 175°C en fonction des lignosulfonates.

Les colles avec les tanins d’écorce de pin maritime sont stables jusqu’à approximativement

200°C quelles que soient les lignines.

Durant la réticulation de colles tanin-lignine, les tanins subissent des réactions

d’autocondensation et réagissent avec l’hexamine. Les lignosulfonates réagissent en partie

mais étant en excès, les réactions chimiques des lignosulfonates ne sont pas identifiables.

143

L’optimisation des conditions de pressage de panneaux de particules à 180°C avec des

colles à base de tanins de mimosa et de lignosulfonates d’ammonium et de sodium a révélé

que les meilleures conditions sont :

- une colle avec 60% en masse de tanins et 40% en masse de lignosulfonates de

sodium ayant subi le traitement au glyoxal 1 (avec la plus grande proportion d’eau),

- un temps de pressage de 7,5 min, une densité de 750 kg/m3.

En respectant ces conditions de pressage, on obtient un panneau de particules

respectant la norme européenne pour les panneaux pour agencements intérieurs en milieux

secs. Les panneaux de particules encollés avec une colle tanin-lignine n’émet presque pas de

formaldéhyde contrairement à un panneau avec une résine industrielle UF. Pour conclure,

nous avons réussi à faire une colle tanin-lignosulfonate qui présente des propriétés

mécaniques similaire à des colles avec des lignines organosolves en utilisant davantage de

lignosulfonates permettant ainsi de baisser les coups de production au niveau industriel.

145

Conclusion générale et perspectives

Ce travail s’inscrit dans la volonté de proposer aux industriels de l’industrie de

panneaux de particules des Landes une alternative aux résines UF. Nous avons cherché à

utiliser des produits locaux, dans notre cas le pin maritime comme source de composés pour

nos colles. Les tanins condensés sont des extractibles présents dans l’écorce de pin maritime

et sont connus pour rentrer dans la formulation de colles bio-sourcées pour panneaux de bois.

Malgré cela, les écorces sont majoritairement considérées simplement comme un coproduit de

l’industrie du panneau et papetière. C’est dans cette optique que nous avons cherché à mettre

en œuvre des méthodes d’extraction simples, écologiques et peu coûteuses qui seraient

facilement transférables aux industriels.

Les lignosulfonates sont également un coproduit de l’industrie papetière et sont

produits en grande quantité. Des lignosulfonates d’ammonium et des lignosulfonates de

sodium de pin maritime ont été récupérés d’une bio-raffinerie landaise. Nous avons donc

cherché à valoriser ces deux coproduits en les combinant dans la formulation de colles pour

panneaux de bois, plus particulièrement pour panneaux de particules.

Pour ce faire, nous avons cherché à extraire des extractibles et notamment des tanins

condensés d’écorce de pin maritime. Tout d’abord, les extractibles ont été obtenus par une

extraction à base d’eau chaude. La méthode d’extraction a été optimisée en faisant varier la

température et les quantités de sels. Les meilleurs conditions d’extraction sont avec 70°C avec

1% NaOH, 0,25% Na2SO3 et 0,25% NaHSO3, un ratio solide-liquide de 1-9 et une durée

d’extraction de 2 h. Par la suite, des EAM ont été réalisées avec un ratio solide liquide de 1-

10, pendant 3 min dans de l’éthanol eau (80-20, l-l). Pour la première fois, cette technique

d’extraction a été utilisée sur du pin maritime. L’impact de la granulométrie sur l’extraction

d’extractibles a été mesuré par des dosages colorimétriques. La caractérisation des extraits est

une étape déterminante pour pouvoir par la suite mieux comprendre les possibles applications

de nos extraits. Il était important de savoir s’il y avait des tanins condensés dans nos extraits

ainsi que leur quantité et leur nature. Ces informations sont essentielles pour pouvoir réaliser

des colles de bonne qualité et dégager des pistes quant aux réactions chimiques ayant lieu

pendant la réticulation. Nous avons mesuré davantage de tanins condensés, de flavonoïdes

simples et de sucres dans les extraits obtenus par EAM que par extraction à base d’eau

chaude. En outre, plus la granulométrie des poudres d’écorce est fine, plus d’extractibles sont

obtenus. Les tanins condensés des extraits obtenus par les deux types d’extraction ont été

146

identifiés comme étant de la catéchine, de l’épicatéchine, de l’épicatéchine gallate et de

l’épigallocatéchine ; ce qui explique que les deux types d’extrait avait la même réactivité au

formaldéhyde, 50%.

Comme pour les tanins, il est important de connaître les quatre différents

lignosulfonates que nous utilisons. Les lignosulfonates ne sont pas naturellement très réactifs.

Afin de les rendre plus réactifs, nous leurs avons fait subir une glyoxalation. Deux traitements

au glyoxal ont été administrés aux lignosulfonates ; un des traitements s’est révélé plus

efficace. La glyoxalation a apporté des groupes carbonyle/carboxyle, hydroxyles phénoliques

et C—O. Toutefois après traitement, la stabilité thermique des lignosulfonates diminue.

Nous avons ensuite cherché à nous consacrer à la formulation de colles bio-sourcées

tanin-lignine. Dans un premier temps, n’ayant pas une quantité suffisante de tanins d’écorce

de pin maritime, nous avons fait le choix d’utiliser des tanins industriels de mimosa déjà

utilisés dans la formulation de colles pour panneaux à base de tanins. Le lignosulfonate ayant

la plus grande stabilité thermique, le lignosulfonate de sodium en poudre a été utilisé afin

d’optimiser le ratio tanin-lignine des colles. Plus il y a de lignines dans la colle moins la colle

est efficace, mais il n’y a pas de différence de propriétés mécaniques entre des colles ayant de

40% à 60% en masse de lignosulfonates. Les tests mécaniques nous informent sur la

résistance en flexion trois points de nos adhésifs utilisés pour coller des plaquettes de bois.

Les colles réticulent le plus rapidement à 157-168°C. Les colles ne se dégradent qu’à partir de

220°C. Afin de déterminer l’effet de la nature des lignosulfonates sur les adhésifs, des colles à

40% et 60% en masse de lignosulfonates ont été réalisées. Cette étude a montré que les

propriétés mécaniques des lignosulfonates sont similaires et confirme qu’il n’y a pas de

différence entre les adhésifs avec 40% et 60% en masse de lignosulfonates. La nature des

lignosulfonates n’affecte pas les propriétés mécaniques des colles. Dans un second temps,

grâce à ces résultats, des tanins d’écorce de pin maritime et des lignosulfonates sont rentrés

dans la formulation de colles bio-sourcées. Deux formulations ont des propriétés mécaniques

et thermiques similaires aux formulations avec des tanins de mimosa.

Finalement, puisque nous cherchions à substituer nos colles aux résines UF utilisés

pour le collage de panneaux de particules, nous avons fabriqué des panneaux de particules

avec nos colles bio-sourcées. L’optimisation des conditions de pressage de panneaux de

particules à 180°C avec des colles à base de tanins de mimosa et de lignosulfonates

d’ammonium et de sodium a révélé que les meilleures conditions sont :

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- une colle avec 60% en masse de tanins et 40% en masse de lignosulfonates de

sodium ayant subi le traitement au glyoxal 1 (avec la plus grande proportion d’eau),

- un temps de pressage de 7,5 min, une densité de 750 kg/m3.

Nous avons obtenu des panneaux de particules respectant la norme européenne pour

les panneaux pour agencements intérieurs en milieux secs. Les panneaux de particules

encollés avec une colle tanin-lignine n’émettent presque pas de formaldéhyde, contrairement

à un panneau avec une résine industrielle UF.

Tous ces travaux, ont permit de montrer qu’il est possible, à l’échelle du laboratoire,

d’extraire facilement et de manière écologique des tanins d’écorce de pin maritime. De

bonnes propriétés mécaniques et thermiques ont été mesurées sur des formulations de colles

avec ces tanins. Des panneaux de particules encollés avec ces colles ont émis très peu de

formaldéhyde et étaient bien en-dessous de la norme japonaise, une des plus strictes au

monde, sur les émissions de formaldéhyde. Des panneaux de particules encollés avec des

colles bio-sourcées à base de tanins de mimosa et de lignosulfonates de sodium respectent les

normes européennes sur la résistance en traction des panneaux et sur l’émission de

formaldéhyde en milieu intérieur sec.

Au regard des résultats obtenus, d’autres études pourraient être menées sur l’extraction

de tanins condensés. La caractérisation des extraits pourrait être approfondie en déterminant le

degré de polymérisation des tanins condensés par MALDI-TOF et en identifiant les sucres

présents par chromatographie liquide à haute pression. Cette étude a été réalisée sur de petits

volumes d’extractions en laboratoire. Il serait intéressant de regarder l’effet de l’augmentation

des volumes d’extraction sur les rendements et sur la nature des extraits, si l’on veut pouvoir

transférer les méthodes d’extractions à l’industrie. Les flavonoïdes et les tanins condensés

sont connus pour avoir des propriétés anti-oxydantes. Les propriétés anti-oxydantes de nos

extraits pourraient être vérifiées. Ainsi, d’autres applications de nos extraits pourraient être

envisagées.

Il serait également intéressant de purifier et de séparer les tanins condensés de nos

extraits et de purifier les lignosulfonates. En faisant des colles avec ces composés purifiés, on

pourrait essayer de décrire dans le détail les mécanismes réactionnels qui ont lieu pendant la

réticulation. De même, des analyses par analyse thermogravimétrique couplée à un

spectroscope de masse ou couplée à une IRTF des colles permettraient d’avoir plus

d’informations sur les structures chimiques des résines réticulées. Dans un but similaire,

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l’étude des gaz émis pendant la réticulation et pendant leur dégradation nous éclairerait sur les

réactions chimiques se déroulant durant ces deux étapes.

La réticulation des colles dans les panneaux de particules pourrait être modélisée en

ATM en faisant des cycles de pression, de température et de temps similaire à ce qui est

réalisé pendant le pressage industriel. Les panneaux de particules encollés avec une colle à

base de tanins d’écorce de pin maritime n’ont pas respecté les normes européennes de

résistance en traction. Une optimisation des conditions de pressage pour des panneaux avec

des colles à base de tanins de pin maritime est à envisager. De plus, pour cette étude, le taux

d’encollage des panneaux n’a pas été modifié, une optimisation de ce taux d’encollage dans

les couches externes et dans la couche interne pourrait augmenter les propriétés mécaniques

des panneaux. Il serait judicieux d’aproffondir les études sur l’amélioration de la résistance à

l’humidité de nos panneaux, par l’ajout d’un plastifiant, du glycérol ou de cire par exemple.

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Productions scientifiques

Articles parus dans un journal avec comité de lecture

Chupin, L, Motillon, C, Charrier – El Bouhtoury, F, Pizzi, A, Charrier, B (2013).

Characterisation of maritime pine (Pinus pinaster) bark tannins extracted under different

conditions by spectroscopic methods, FTIR and HPLC. Industrial Crops and Products, 49, p.

897-903.

Chupin, L, Charrier, B, Pizzi, A, Charrier – El Bouhtoury, F. Study of thermal durability

properties of tannin lignosulfonate adhesives. Journal of Thermal Analysis and Calorimetry

(article soumis).

Communications avec actes dans des conférences internationales

Chupin, L, Charrier – El Bouhtoury, F, Pizzi, A, Charrier, B. Extraction of maritime pine bark

tannins for tannin – lignosulfonate adhesives, International Conference on Bioinspired and

Biobased Chemistry & Materials, Nice, France, October 2014

Chupin, L, Charrier, B, Pizzi, A, Charrier – El Bouhtoury, F. Particle board panel adhesive

without formaldehyde emission, PFT PTBI, Kuchl, Austria, September 2014

Chupin, L, Charrier – El Bouhtoury, F, Charrier, B. Particleboard adhesives from maritime

pine bark tannins and lignosulfonate, Thèses des Bois, Bordeaux, France, July 2014 (Prize

from the foundation of the University of Bordeaux)

Chupin, L, Charrier, B, Pizzi, A, Charrier – El Bouhtoury, F. Thermal durability and

biodegradation of tannin lignin adhesives, IRG/WP45, St Georges, Utah, USA, Mai 2014

(Ron Cockcroft Award).

Présentations par affiches (posters)

Chupin, L, Charrier – El Bouhtoury, F, Charrier, B. Maritime pine bark extraction for tannin-

lignin adhesives, IUFRO, Salt Lake City, Utah, USA, October 2014

Chupin, L, Reynaud, S, Charrier, B, Charrier – El Bouhtoury, F. Microwave assisted

extraction of condensed tannins from maritime pine (Pinus pinaster) bark, IUFRO, Salt Lake

City, Utah, USA, October 2014

Chupin, L, Charrier – El Bouhtoury, F, Charrier, B. Extraction of maritime pine bark tannins

for the formulation of green-adhesives, GDR Bois, Marne la Vallée, France, November 2013

150

Chupin, L, Motillon, C, Charrier-El Bouhtoury, F,Pizzi, A, Charrier B. Study of maritime

pine (Pinus pinaster) bark tannins extracted in alkaline conditions by spectroscopic methods

and FTIR, Woodchem, Nancy, France, September 2013

Chupin, L, Charrier – El Bouhtoury, F, Charrier, B. Extraction and characterisation of

maritime pine (Pinus pinaster) bark tannins in order to make novel tannin-lignin adhesives,

Doctoral Students’ Day, Pau, France, July 2013

Chupin, L, Charrier-El Bouhtoury, F, Allal, A, Charrier, B. Study of the performances of

green adhesives, made with tannins and lignins, GDR Bois, Montpellier, France, November

2012

Chupin, L, Allal, A, Charrier-El Bouhtoury, F, Charrier, B. Study of green-adhesives

performances, made with tannins and lignins, in order to make particle boards, Transborder

Doctorials, Anglet, France, October 2012

Chupin, L, Allal, A, Charrier-El Bouhtoury, F, Charrier, B. Study of green-adhesives

performances, made with tannins and lignins, in order to make particle boards, Graines

d’Adhésion, Toulon, France, July 2012. (Best Poster Award).

Annexes

Annexe 1 : Thermal durability and biodegradation of tannin lignin adhesives

Annexe 2 : Particleboard panel adhesive without formaldehyde emission

Etude de l’extraction de tanins d’écorce de pin maritime pour l’élaboration de colles

tanin-lignosulfonate

Cette étude a deux objectifs principaux : l’extraction de tanins condensés d’écorces de pin maritime et la formulation de colles tanin-lignosulfonate. Deux méthodes d’extraction ont été étudiées. La première est une extraction à l’eau chaude ; c’est une technique simple, peu coûteuse, sans solvant. La deuxième est une extraction assistée par micro-ondes ; c’est une technique innovante, rapide et peu consommatrice en solvant. L’optimisation des conditions d’extraction à l’eau chaude a été réalisée. Les extraits ont été caractérisés par des dosages colorimétriques, leur réactivité au formaldéhyde, par infrarouge à transformée de Fourier (IRTF), par chromatographie en phase liquide à haute pression, par 1H RMN et 2D HSQC RMN. L’impact de la granulométrie sur l’extraction de polyphénols et particulièrement de tanins condensés par extraction assistée par micro-ondes a été étudié pour la première fois. Les deux types d’extraction ont été comparés. L’extraction assistée par micro-ondes a un rendement en extractibles inférieur à l’extraction à l’eau chaude. Mais elle extrait plus de tanins condensés, de flavonoïdes simples et plus de sucres. Quelle que soit la méthode d’extraction, les tanins condensés majoriatires extraits de l’écorce de pin maritime sont de la catéchine, de l’épicatéchine, de l’épicatéchine gallate et de l’épigallocatéchine.

Des colles tanin-lignosulfonate ont été produites en utilisant l’héxaméthylènetetramine comme durcisseur. Dans un premier temps, des tanins de mimosa ont été utilisés avec des lignosulfonates de sodium et des lignosulfonates d’ammonium. Les lignosulfonates ont subi deux traitements au glyoxal qui ont été comparés par analyse thermogravimétrique (ATG), par calorimétrie différentielle à balayage (DSC), par les propriétés thermiques et mécaniques de colles et de panneaux de particules avec des lignosulfonates ayant subi les deux traitements ont également été étudiées. L’optimisation du ratio tanin de mimosa-lignosulfonate glyoxalé a été menée et les propriétés thermiques des colles mesurées. L’optimisation des conditions de pressage de panneaux de particules a été réalisée. Des panneaux de particules avec de bonnes performances mécaniques ont été fabriqués.

Des colles à base de tanins d’écorce de pin maritime et de lignosulfonates ont été réalisées avec 40% de tanins et 60% de lignosulfonates. Ces colles ont été caractérisées par IRTF, analyse thermomécanique, ATG et DSC. Ces colles sont rentrées dans la fabrication de panneaux de particules. Les émissions de formaldéhyde et la cohésion interne des panneaux ont été mesurées et comparées à des panneaux encollés avec une colle tanin de mimosa-lignosulfonate et une résine urée-formaldéhyde. Grâce à ces résultats, il a été possible de montrer que les panneaux de particules fabriqués à partir de colles bio-sourcées n’émettaient pas de formaldéhyde.

Mots-clés : tanins condensés ; extraction ; extraction assistée par micro-ondes ; écorce de pin maritime ; lignosulfonates ; colle tanin-lignine ; glyoxalation ; panneaux de particules

Study of maritime pine bark extraction for the preparation of tannin-lignosulfonate

adhesives

This study has two main objectives: the extraction of condensed tannins from maritime

pine bark and the preparation of tannin-lignosulfonate adhesives. Two extraction methods

were studied. The first is hot water extraction which is a simple, cheap method without the

use of an organic solvent. The second is microwave-assisted extraction which is a fast,

innovative method using only a small amount of solvent. Optimum extraction conditions were

determined for hot water extraction. The extracts were characterised by their reaction to

formaldehyde and by using colorimetric tests, Fourier Transformed Infrared spectroscopy

(FTIR), high pressure liquid chromatography, 1H NMR and heteronuclear single quantum

correlation 2D NMR.. The two types of extraction were compared. It was found that

microwave-assisted extraction produced a lower yield of extractibles than the hot water

method but that it produced more condensed tannins, simple flavonoids and sugars. The

condensed tannins extracted from maritime pine bark are catechin, epicatechin, epicatechin

gallate and epigallocatechin, whatever the extraction method used.

Tannin-lignosulfonate adhesives were produced using hexamethylenetetramine as a

hardener. First, mimosa tannins were used with sodium lignosulfonates and ammonium

lignosulfonates. The lignosulfonates underwent two glyoxal treatments which were compared

using thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), and by

determining the thermal and mechanical properties of the adhesives and of the particle boards

made using the lignosulfonates resulting from the two treatments. The optimum mimosa

tannin-glyoxalated lignosulfonate ratio was determined and the thermal properties of the

adhesives were measured. The optimum conditions of pressing the particle boards were

determined. Particle boards which recorded a good mechanical performance were produced.

Adhesives using maritime pine bark tannins and lignosulfonates were prepared with

40% tannins and 60% lignosulfonates. These adhesives were characterised using FTIR,

thermomechanical analysis, TGA and DSC. These adhesives were used to produce particle

boards. The emission of formaldehyde and the internal bond of the boards were measured and

compared to those of boards made with a mimosa tannin-lignosulfonate adhesive and to those

of boards made with a urea-formaldehyde resin. Thanks to these results, we were able to

produce particleboards with bio-based adhesives that didn’t emit formaldehyde.

Keywords: condensed tannins ; extraction ; microwave assisted extraction ; maritime pine bark ; lignosulfonates ; tannin-lignin adhesive ; glyoxalation ; particleboard

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