Furfural production from d-xylose and xylan by using stable Nafion NR50 and NaCl in a microwave-assisted biphasic reaction

molecules A icle Fu u al P oduc ion om D-Xylose and Xylan by Using S able Na ion NR50 and NaCl in a Mic owa e-Assis ed Biphasic Reac ion Sa ah Le Guenic 1, Da id Ge gela 2, Clai e Ceballos 3, F ede ic Delbecq 3and Ch is ophe Len 1,* 1Uni e si é de Technologie de Compiègne (UTC), CS 60319, 60203 Compiègne Cedex, F ance; [email p o ec ed] 2Depa men o Chemis y, Facul y o Technology, 760 01 Zlin, Czech Republic; Da id.Ge g[email p o ec ed] 3Ecole Supé ieu e de Chimie O ganique e Miné ale (ESCOM), 1 ue du Réseau Jean-Ma ie Buckmas e , 60200 Compiègne, F ance; [email p o ec ed] (C.C.); [email p o ec ed] (F.D.) *Co espondence: [email p o ec ed]; Tel.: +33-344-238-828 Academic Edi o : De ek J. McPhee Recei ed: 9 July 2016; Accep ed: 10 Augus 2016; Published: 22 Augus 2016 Abs ac : Pen ose dehyd a ion and di ec ans o ma ion o xylan in o u u al we e pe o med in a wa e -cyclopen yl me hyl e he (CPME) biphasic sys em unde mic owa e i adia ion. Hea ed up be ween 170 and 190 ◦ C in he p esence o Na ion NR50 and NaCl, D-xylose, L-a abinose and xylan ga e u u al wi h maximum yields o 80%, 42% and 55%, espec i ely. The in luence o empe a u e and eac ion ime on he eac ion kine ics was discussed. This s udy was also comple ed by he su ey o di e en eac an a ios, such as o ganic laye -wa e o ca alys -ino ganic sal a ios. The exchange be ween p o on and ca ion induced by an excess o NaCl was moni o ed, and a syne ge ic e ec be ween he emaining p o ons and he eleased HCl was also disco e ed. Keywo ds: u u al; D-xylose; xylan; Na ion NR50; biphasic sys em; mic owa e-assis ed dehyd a ion 1. In oduc ion Because o he deple ion o uel-based esou ces, he e is an u gen need o ind al e na i e eeds ocks o he chemical indus y. Lignocellulosic biomass, plan was es o example, appea s o be he bes al e na i e o ge access o a ious bio-based chemicals, such as u an de i a i es. One o hese op- alue ma e ials is u u al, la gely p oduced om he dehyd a ion o D-xylose, he main monome o hemicellulose- ich biomass. Fu u al is a key compound o he p epa a ion o many in e media es [ 1 ]. Fo example, u u yl alcohol is a p ecu so o comme cially a ailable esins (namely poly u u yl alcohol o PFA) and can be o med om u u al by he educ ion o i s aldehyde unc ion. Fu u al is also use ul o he p epa a ion o o ganic sol en s, such as e ahyd o u an (THF), me hyl e ahyd o u an (MeTHF) o he syn hesis o bioac i e compounds. The indus ial p oduc ion o u u al is ealized by ba ch and con inuous acid hyd olysis o lignocellulosic biomass [ 2 ] in o D-xylose ollowed by i s dehyd a ion leading o u u al. Bo h acid hyd olysis and subsequen dehyd a ion o lignocellulosic biomass a e usually ca ied ou in he same eac o , and ha dehyd a ion is ecognized as he a e-limi ing s ep. The eac ion is usually ca ied ou in a ange o empe a u es ound be ween 170 and 185 ◦ C unde a con en ional hea ing. Pe o med in wa e , hese p ocesses equi e s ong mine al acids, such as HCl [ 3 , 4 ], H 2 SO 4 [ 5 ] o H 3 PO 4 [ 6 ], causing co osion, sa e y and handling p oblems. Reco e y o u u al equi es dis illa ion o he sol en and o he ea men s, such as neu aliza ion, which lead o la ge was e s eams and high-ene gy consump ion. Besides, in a monophasic sys em, u u al yields and selec i i y a e gene ally limi ed by he o ma ion o by-p oduc s, humin, a black insoluble Molecules 2016,21, 1102; doi:10.3390/molecules21081102 www.mdpi.com/jou nal/molecules Molecules 2016,21, 1102 2 o 11 ca bonaceous solid, de i ed om he c oss-polyme iza ion be ween u u al and pen ose in e media es, o om he condensa ion o u u al on i sel . Today, he use o easy- o-handle solid sul ona ed polyme s ecognized o hei s ong B øns ed acidi y, such as p o ona ed Ambe lys [ 7 – 11 ] o Na ion [ 12 ], shows p omising esul s in he ield o g een chemis y. Na ion NR50 is a pe luo oalkane sul onic esin ha could be an al e na i e o o he ypes o he e ogeneous ca alys s, such as zeoli es [ 13 ] o he e opolyacids [ 14 ]. Acco ding o he p oduce da a, Na ion NR50 has a maximum ope a ing empe a u e o 220 ◦ C. Recen ly, Lam e al. demons a ed he po en ial o he solid Na ion 117 o he C5 suga dehyd a ion [ 15 ]. They epo ed he o ma ion o u u al wi h a maximum yield o 60% a 150 ◦ C in dime hylsul oxide (DMSO). Howe e , du ing he p ocess, Na ion was also subjec ed o he deposi ion o a humin laye . This laye almos co e ed he en i e ac i e su ace o he solid ca alys and limi ed he numbe o ca aly ic uns. Na ion NR50 esin was also epo ed as an e icien supe acid ca alys in he p esence o ionic liquids o he hyd olysis o cellobiose in o D-glucose monome s [ 16 ]. To inhibi he o ma ion o side p oduc s like humins, one o he mos p omising app oaches is o add an o ganic co-sol en , such as cyclopen yl me hyl e he (CPME), a low wa e -miscible eco- iendly sol en [ 17 ]. The use o an o ganic co-sol en allows he ex ac ion o u u al om he aqueous phase in o he o ganic phase and hus imp o es he u u al yield by inhibi ing he gene a ion o undesi ed p oduc s. Wi h ega ds o he scale-up p ocess, in o de o sa e ene gy and a oid undesi able deg ada ions, i is be e o use ex ac i e sol en wi h lowe boiling poin s han ha o solubilized u u al o easily eco e he a ge compound. Acco ding o Weinga en e al. [ 18 ], a biphasic sys em does no al e he undamen al kine ics o i s en i e eac ion compa ed o he classic monophasic sys em. I s only ole is o be he s o age o he hyd ophobic species. Se e al s udies ha e epo ed he use o MIBK, oluene, ni o oluene o dichlo ome hane as e icien co-sol en s [ 19 – 22 ] o he dehyd a ion o D-xylose. In acco dance wi h a ecen wo k o ou g oup on he dehyd a ion o D-xylose wi h homogeneous ca alysis [ 23 ], he p esen wo k ocuses on he use o he Na ion NR50 as a he e ogeneous ca alys in he p esence o NaCl in a biphasic sys em unde mic owa e i adia ion in a single mode ca i y. 2. Resul s and Discussion 2.1. In luence o he Tempe a u e on Fu u al Yield Using he i s esul s ob ained by he g oup, he biphasic expe imen s we e pe o med by employing wo immiscible phases, wa e -CPME wi h a a io 1:3 ( / ) [ 23 ]. D-xylose is no soluble in he o ganic phase, and CPME is qui e s able a high empe a u e. Due o i s low densi y, his ex ac ion sol en was he uppe phase. The s udies o he dehyd a ion o D-xylose we e conduc ed o 1 h be ween 130 and 180 ◦ C in he p esence o NaCl (95 mg) and wo Na ion NR50 pelle s in a biphasic mix u e (wa e -CPME, 1:3, / ) unde mic owa e i adia ion (Figu e 1). A maximum u u al yield o 80% was eached a 170 ◦ C a e one hou o eac ion wi h a quan i a i e con e sion o D-xylose, which is a compe i i e esul by compa ison wi h one o ou mos ecen esul s o a 74% u u al yield ob ained wi h FeCl 3 as he ca alys in a biphasic sys em [ 23 ]. Be ween 130 and 160 ◦ C, emaining D-xylose loading dec eased slowly. I was no ewo hy ha Na ion NR50 pelle s a e subjec ed o a na u al swelling in wa e a oom empe a u e [ 24 ]. In a p elimina y s udy, he same eac ion was pe o med wi hou he use o NaCl. Howe e , Na ion NR50 pelle s u ned o black and exploded, and u u al yields we e no ep oducible. As a consequence, NaCl was necessa y o keep he ac i i y and he in eg i y o Na ion NR50 a 170 ◦ C. The mechanism in ol ed du ing he addi ion o NaCl will be desc ibed la e in he publica ion. Besides, o compa ison, he same eac ion was ca ied ou in a sealed ube illed wi h 4.0 mL o wa e -CPME (1:3, / ) keeping unchanged he cu en subs a e o ca alys a io in he p esence o 95 mg o NaCl. The dehyd a ion was ealized by means o a p ehea ed oil ba h wi h con en ional hea ing and led o a u u al yield o only 55% a 150 ◦ C o 2 h. The eby, hese esul s unde line ha he ca aly ic sys em Na ion NR50-NaCl could be an al e na i e o he common hyd ochlo ic acid aqueous solu ion unde mic owa e ac i a ion a 170 ◦C. Molecules 2016,21, 1102 3 o 11 Molecules 2016, 21, 1102 3 o 11 Figu e 1. E ec o eac ion empe a u e on u u al yield. Reac ion condi ions: Na ion NR50 ( wo pelle s), NaCl (95 mg), D-xylose (1.0 mmol), wa e (1 mL), CPME (3 mL) and MW, 1 h. When he same p ocedu e was applied o L-a abinose, a lowe yield (42%) was ob ained o 1 h a 170 °C, as has been obse ed in he li e a u e [25]. I was epo ed ha L-a abinose was dehyd a ing less apidly han xylose a 170 °C [25,26]. This di e ence is due o he ac ha D-xylose exp essed lowe ac i a ion ene gy o i s dehyd a ion compa ed o L-a abinose, gene ally supe io o 112 kJ·mol−1. The s uc u e o L-a abinose was mo e s able. The con e sion o L-a abinose in o u u al was also epo ed o be mo e empe a u e dependen and o equi e high empe a u es (abo e 220 °C) o accele a e he p ocess [27,28]. 2.2. E ec o D-xylose Loading Va ia ion The in luence o he ini ial D-xylose concen a ion was also s udied in a mix u e o wa e -CPME, 1:3, / , a 170 °C o 1 h unde mic owa e i adia ion (Figu e S1, ESI). Fo a D-xylose concen a ion o 150 g·L−1 in he aqueous phase, he yield eached a alue o 80%. Then, by keeping he op imized eac ion condi ions, he pen ose loading was g adually inc eased un il a singula dec ease o he u u al yield was eco ded. Fo a D-xylose concen a ion o 500 g·L−1, he u u al yield s ill eached a good alue o 72%. Howe e , i he concen a ion we e inc eased o 600 g·L−1, he u u al yield d opped o he lowe alue o 62%. Fi s , he high pen ose concen a ion a o ed he con ac be ween molecules and hus p omo ed condensa ion be ween pen ose in e media es and u u al: la ge amoun s o a da k b own sy up insoluble in bo h laye s we e p oduced. Then, he la ge amoun s o u u al p oduced om he high xylose concen a ion may also sa u a e he o ganic phase and make he u u al ex ac ion no longe e icien . Finally, he iscosi y o he eac ion medium also inc eased wi h inc easing subs a e concen a ion and may ha e led o a non-uni o m hea ing in he mic owa e eac o . 2.3. In luence o he Residence Time on he Fu u al P oduc ion A e de e mining he bes empe a u e condi ion o he dehyd a ion o D-xylose using CPME as he ex ac ion sol en , he e ec o he eac ion ime was s udied a 160 °C, 170 °C and 180 °C (Figu e 2). Consequen ly, di e en ime pe iods ( om 5 o 60 min) we e es ed in o de o de e mine he op imal condi ions. The o he pa ame e s we e unchanged using D-xylose (1.0 mmol), Na ion NR50 loading ( wo pelle s), NaCl (95 mg) and a biphasic a io wa e -CPME, 1:3, / . The end o he D-xylose con e sion cu e did no change compa ed o hose ob ained om a ypical monophasic ba ch ope a ed in he same ange o empe a u es epo ed in Figu e 2. A e 25 min o as consump ion, only 5% o he D-xylose emained in he mix u e. A maximum u u al yield o 80% was eached a e 40 min o eac ion. A e his pe iod, a sligh dec ease o he yield was obse ed. In addi ion, a 170 °C, he selec i i y o he p oduced u u al ne e exceeded 80%, and 0 10 20 30 40 50 60 70 80 90 120 140 160 180 Fu u al yield and xylose con en s (%) Tempe a u e (°C) Fu u al om xylose Remaining xylose Figu e 1. E ec o eac ion empe a u e on u u al yield. Reac ion condi ions: Na ion NR50 ( wo pelle s), NaCl (95 mg), D-xylose (1.0 mmol), wa e (1 mL), CPME (3 mL) and MW, 1 h. When he same p ocedu e was applied o L-a abinose, a lowe yield (42%) was ob ained o 1 h a 170 ◦ C, as has been obse ed in he li e a u e [ 25 ]. I was epo ed ha L-a abinose was dehyd a ing less apidly han xylose a 170 ◦ C [ 25 , 26 ]. This di e ence is due o he ac ha D-xylose exp essed lowe ac i a ion ene gy o i s dehyd a ion compa ed o L-a abinose, gene ally supe io o 112 kJ · mol −1 . The s uc u e o L-a abinose was mo e s able. The con e sion o L-a abinose in o u u al was also epo ed o be mo e empe a u e dependen and o equi e high empe a u es (abo e 220 ◦ C) o accele a e he p ocess [27,28]. 2.2. E ec o D-xylose Loading Va ia ion The in luence o he ini ial D-xylose concen a ion was also s udied in a mix u e o wa e -CPME, 1:3, / , a 170 ◦ C o 1 h unde mic owa e i adia ion (Figu e S1, ESI). Fo a D-xylose concen a ion o 150 g · L −1 in he aqueous phase, he yield eached a alue o 80%. Then, by keeping he op imized eac ion condi ions, he pen ose loading was g adually inc eased un il a singula dec ease o he u u al yield was eco ded. Fo a D-xylose concen a ion o 500 g · L −1 , he u u al yield s ill eached a good alue o 72%. Howe e , i he concen a ion we e inc eased o 600 g · L −1 , he u u al yield d opped o he lowe alue o 62%. Fi s , he high pen ose concen a ion a o ed he con ac be ween molecules and hus p omo ed condensa ion be ween pen ose in e media es and u u al: la ge amoun s o a da k b own sy up insoluble in bo h laye s we e p oduced. Then, he la ge amoun s o u u al p oduced om he high xylose concen a ion may also sa u a e he o ganic phase and make he u u al ex ac ion no longe e icien . Finally, he iscosi y o he eac ion medium also inc eased wi h inc easing subs a e concen a ion and may ha e led o a non-uni o m hea ing in he mic owa e eac o . 2.3. In luence o he Residence Time on he Fu u al P oduc ion A e de e mining he bes empe a u e condi ion o he dehyd a ion o D-xylose using CPME as he ex ac ion sol en , he e ec o he eac ion ime was s udied a 160 ◦ C, 170 ◦ C and 180 ◦ C (Figu e 2). Consequen ly, di e en ime pe iods ( om 5 o 60 min) we e es ed in o de o de e mine he op imal condi ions. The o he pa ame e s we e unchanged using D-xylose (1.0 mmol), Na ion NR50 loading ( wo pelle s), NaCl (95 mg) and a biphasic a io wa e -CPME, 1:3, / . The end o he D-xylose con e sion cu e did no change compa ed o hose ob ained om a ypical monophasic ba ch ope a ed in he same ange o empe a u es epo ed in Figu e 2. A e 25 min o as consump ion, only 5% o he D-xylose emained in he mix u e. A maximum u u al yield o 80% was eached a e 40 min o eac ion. A e his pe iod, a sligh dec ease o he yield was obse ed. In addi ion, a 170 ◦ C, Molecules 2016,21, 1102 4 o 11 he selec i i y o he p oduced u u al ne e exceeded 80%, and only a small amoun o humin was de ec ed in he eac ion essel. In he opposi e case, when he eac ion was pe o med a 160 ◦ C, he u u al o ma ion was accele a ed be o e 20 min, and he yield con inued o inc ease slowly un il i eached 69% a e 60 min. A 180 ◦ C, o a sho pe iod comp ised be ween 10 and 20 min, u u al was p oduced wi h a high yield o 71%, bu beyond his ime, he u u al concen a ion s a ed o decline due o i s esini ica ion, mo e e ec i e a his empe a u e. Thus, he op imized eac ion ime and empe a u e we e clea ly de ined. In o de o dec ease he eac ion ime, he empe a u e was ixed a 170 ◦C o 40 min unde mic owa e i adia ion. Molecules 2016, 21, 1102 4 o 11 only a small amoun o humin was de ec ed in he eac ion essel. In he opposi e case, when he eac ion was pe o med a 160 °C, he u u al o ma ion was accele a ed be o e 20 min, and he yield con inued o inc ease slowly un il i eached 69% a e 60 min. A 180 °C, o a sho pe iod comp ised be ween 10 and 20 min, u u al was p oduced wi h a high yield o 71%, bu beyond his ime, he u u al concen a ion s a ed o decline due o i s esini ica ion, mo e e ec i e a his empe a u e. Thus, he op imized eac ion ime and empe a u e we e clea ly de ined. In o de o dec ease he eac ion ime, he empe a u e was ixed a 170 °C o 40 min unde mic owa e i adia ion. Figu e 2. E ec o eac ion ime and empe a u e on (a) u u al yield and (b) xylose con en s. Reac ion condi ions: Na ion NR50 (2 pelle s), NaCl (95 mg), D-xylose (1.0 mmol), wa e (1 mL), CPME (3 mL) and MW. 2.4. E ec o he Ra io Wa e -CPME on he Fu u al Yield In o de o e alua e he e ec o he a io wa e -CPME on D-xylose con e sion and u u al yield, i e a ios o wa e -CPME (4:0, 3:1, 1:1, 1:3, 0:4; / ) we e s udied (Figu e 3). When he eac ion was pe o med in pu e wa e , as expec ed, he yield o he p oduced u u al ne e exceeded 38%, and he selec i i y was mode a e wi h a alue o 57%. Besides, he con e sion o D-xylose was no comple e since he Na ion pelle s deac i a ed du ing he p ocess because o he deposi ion o a la ge amoun o humins on he ca alys su ace. By inc easing he wa e -CPME a io, he selec i i y o 0 10 20 30 40 50 60 70 80 90 100 020 40 60 Fu u al yield (%) Reac ion ime (min) (a) 160℃ 170℃ 180℃ 0 10 20 30 40 50 60 70 80 90 100 020 40 60 Xylose con en s (%) Reac ion ime (min) (b) 160℃ 170℃ 180℃ Figu e 2. E ec o eac ion ime and empe a u e on ( a ) u u al yield and ( b ) xylose con en s. Reac ion condi ions: Na ion NR50 (2 pelle s), NaCl (95 mg), D-xylose (1.0 mmol), wa e (1 mL), CPME (3 mL) and MW. 2.4. E ec o he Ra io Wa e -CPME on he Fu u al Yield In o de o e alua e he e ec o he a io wa e -CPME on D-xylose con e sion and u u al yield, i e a ios o wa e -CPME (4:0, 3:1, 1:1, 1:3, 0:4; / ) we e s udied (Figu e 3). When he eac ion was pe o med in pu e wa e , as expec ed, he yield o he p oduced u u al ne e exceeded 38%, and he selec i i y was mode a e wi h a alue o 57%. Besides, he con e sion o D-xylose was no comple e since he Na ion pelle s deac i a ed du ing he p ocess because o he deposi ion o a la ge Molecules 2016,21, 1102 5 o 11 amoun o humins on he ca alys su ace. By inc easing he wa e -CPME a io, he selec i i y o u u al inc eased wi h alues o 56%, 71% and 79% o wa e -CPME olume ic a ios o 3:1, 1:1 and 1:3, espec i ely. Fu he mo e, he coe icien pa i ion ound in he di e en wa e -CPME a ios abo e was also de e mined as ollow. A 170 ◦ C, 1.0 mmol o u u al was pa i ioned be ween he wo phases in he p esence o he ca aly ic sys em, and he alue was calcula ed using his o mula: Kp = [ u u al]wa e /[ u u al]CPME . Acco ding he HPLC measu emen , excep a sligh dec ease o u u al concen a ion in bo h laye s, by inc easing he olume o CPME, he coe icien emained almos cons an un il i eached a inal alue o 4.1 o he wa e -CPME (1:3, / ) olume a io. When he eac ion was pe o med in 4 mL o pu e CPME, he selec i i y o u u al emained blocked a 2% due o he lack o he solubili y o D-xylose in his o ganic sol en . These esul s con i med he impo ance o a minimum o 1 mL o wa e as he eac ion sol en . A 170 ◦ C, upon i adia ion in a closed sys em, wa e emained pola enough compa ed o CPME, and D-xylose is easily in con ac wi h HCl. O e all, wa e is he e he lowe phase, and 1 mL seems o be enough despi e he di icul y o con ol he inne hea ing empe a u e o he essel agg a a ed by he g ea numbe o ions p esen in solu ion due o he dissocia ion o NaCl [29]. Molecules 2016, 21, 1102 5 o 11 u u al inc eased wi h alues o 56%, 71% and 79% o wa e -CPME olume ic a ios o 3:1, 1:1 and 1:3, espec i ely. Fu he mo e, he coe icien pa i ion ound in he di e en wa e -CPME a ios abo e was also de e mined as ollow. A 170 °C, 1.0 mmol o u u al was pa i ioned be ween he wo phases in he p esence o he ca aly ic sys em, and he alue was calcula ed using his o mula: Kp = [ u u al]wa e /[ u u al]CPME. Acco ding he HPLC measu emen , excep a sligh dec ease o u u al concen a ion in bo h laye s, by inc easing he olume o CPME, he coe icien emained almos cons an un il i eached a inal alue o 4.1 o he wa e -CPME (1:3, / ) olume a io. When he eac ion was pe o med in 4 mL o pu e CPME, he selec i i y o u u al emained blocked a 2% due o he lack o he solubili y o D-xylose in his o ganic sol en . These esul s con i med he impo ance o a minimum o 1 mL o wa e as he eac ion sol en . A 170 °C, upon i adia ion in a closed sys em, wa e emained pola enough compa ed o CPME, and D-xylose is easily in con ac wi h HCl. O e all, wa e is he e he lowe phase, and 1 mL seems o be enough despi e he di icul y o con ol he inne hea ing empe a u e o he essel agg a a ed by he g ea numbe o ions p esen in solu ion due o he dissocia ion o NaCl [29]. Figu e 3. E ec o he wa e -CPME olume ic a io on dehyd a ion o D-xylose o u u al. Reac ion condi ions: Na ion NR50 (2 pelle s), NaCl (95 mg), D-xylose (1.0 mmol), wa e -CPME, MW, 170 °C and 40 min. 2.5. In luence o he Ra io Na ion NR50-NaCl on he Fu u al Yield To he bes o ou knowledge, he inne s uc u e o he Na ion NR50 pelle s emains unclea , bu all sul onic acid unc ions we e supposed o be accessible. In ou p ocess, when NaCl was added o he aqueous phase, a ca ion exchange be ween he Na+ o he sal and he H+ o he sul onic acid g oups o Na ion occu ed, and HCl was eleased in he aqueous phase. Besides, he sal o med a coa ing on he pelle s and seemed o p o ec hem om he deposi ion o humins and, hus, om explosion. The impac o he a io o Na ion NR50-NaCl on he dehyd a ion o D-xylose p ocess was in es iga ed using he op imized me hod desc ibed abo e: Na ion NR50 ( wo pelle s), NaCl, D-xylose (1.0 mmol), wa e (1 mL), CPME (3 mL), MW, 170 °C and 40 min. As epo ed in Figu e 4, he mass o NaCl was p og essi ely inc eased un il a maximum o selec i i y was ob ained wi h 95 mg o NaCl. Howe e , wi h only 50 mg o NaCl, he eac ions s a ed o u nish be e selec i i ies (78%). This esul co obo a es a ecen s udy ha demons a es he impac o inc easing he concen a ion o NaCl on he p oduced u u al yield [30]. The same p o ocol was ealized using only one pelle o Na ion NR50 (45 mg), bu u u al was ob ained in a lowe yield (72%) (Figu e S2, ESI). 0 10 20 30 40 50 60 70 80 90 100 4:0 3:1 1:1 1:3 0:4 （％） Wa e -CPME a io ( / ) Fu u al yield Xylose con e sion Fu u al selec i i y Figu e 3. E ec o he wa e -CPME olume ic a io on dehyd a ion o D-xylose o u u al. Reac ion condi ions: Na ion NR50 (2 pelle s), NaCl (95 mg), D-xylose (1.0 mmol), wa e -CPME, MW, 170 ◦C and 40 min. 2.5. In luence o he Ra io Na ion NR50-NaCl on he Fu u al Yield To he bes o ou knowledge, he inne s uc u e o he Na ion NR50 pelle s emains unclea , bu all sul onic acid unc ions we e supposed o be accessible. In ou p ocess, when NaCl was added o he aqueous phase, a ca ion exchange be ween he Na + o he sal and he H + o he sul onic acid g oups o Na ion occu ed, and HCl was eleased in he aqueous phase. Besides, he sal o med a coa ing on he pelle s and seemed o p o ec hem om he deposi ion o humins and, hus, om explosion. The impac o he a io o Na ion NR50-NaCl on he dehyd a ion o D-xylose p ocess was in es iga ed using he op imized me hod desc ibed abo e: Na ion NR50 ( wo pelle s), NaCl, D-xylose (1.0 mmol), wa e (1 mL), CPME (3 mL), MW, 170 ◦ C and 40 min. As epo ed in Figu e 4, he mass o NaCl was p og essi ely inc eased un il a maximum o selec i i y was ob ained wi h 95 mg o NaCl. Howe e , wi h only 50 mg o NaCl, he eac ions s a ed o u nish be e selec i i ies (78%). This esul co obo a es a ecen s udy ha demons a es he impac o inc easing he concen a ion o NaCl on he p oduced u u al yield [ 30 ]. The same p o ocol was ealized using only one pelle o Na ion NR50 (45 mg), bu u u al was ob ained in a lowe yield (72%) (Figu e S2, ESI). Molecules 2016,21, 1102 6 o 11 Molecules 2016, 21, 1102 6 o 11 Figu e 4. E ec o he amoun o NaCl on he dehyd a ion o D-xylose o u u al. Reac ion condi ions: Na ion NR50 (2 pelle s), NaCl, D-xylose (1.0 mmol), wa e (1 mL), CPME (3 mL), MW, 170 °C and 40 min. To comple e ou s udy, NaCl was eplaced by o he ino ganic sal s, such as KCl o LiCl. In ela ion o he ca ion size and hei ela i e mobili y inside he Na ion pelle s, a compe i ion be ween wa e and he posi i ely-cha ged species was expec ed o ha e an in luence on he elease o he p o ons in he eac ion. Ne e heless, by using he same sal mola a io, no a ia ion o pH was eco ded, and he yield o p oduced u u al was cons an wi h a alue o 80%. Na ion NR50 has a capaci y o 0.8 mmol o H+ pe g am. Fo wo pelle s and an excess o NaCl, i co esponded o a pH o 1.1 in he ial i he o ali y o he p o ons was eleased in he medium. The pH o ou eac ion media eached a alue o 1.3 on a e age, which means ha a pa o he sul onic g oups g a ed on he esin emained p o ona ed. To e i y i he D-xylose con e sion was only ca alyzed by he HCl eleased in he medium o also by he esin, a eac ion wi h an HCl solu ion a a pH o 1.3 (0.05 mol·L−1) was pe o med o 40 min in he p esence o 90 mg o NaCl a 170 °C in wa e -CPME,1:3, / . A lowe u u al yield o 73% was ob ained ins ead o he 80% eached wi h he mix u e o Na ion and NaCl. All o hese obse a ions pe mi ed es ablishing ha a new acid ca aly ic sys em was ob ained ha ing a syne ge ic e ec be ween Na ion NR50 and NaCl. 2.6. Reusabili y o he Na ion NR50 Se e al consecu i e eac ions we e pe o med wi h he same Na ion NR50 pelle s. A e each eac ion, he esin pelle s we e eco e ed om he eac ion medium and egene a ed in an acidic HCl ba h o e nigh . The eac ions we e pe o med o 40 min a 170 °C in wa e -CPME, 1:3, / . The ac i i y o he Na ion NR50 pelle s was kep cons an o h ee consecu i e cycles (Figu e 5). A e he ou h cycle, he u u al yield began o dec ease. This obse a ion can be explained by he g adual deac i a ion o he pelle s by humin deposi ion. 0 10 20 30 40 50 60 70 80 90 100 10 20 30 50 70 95 （％） Mass o NaCl (mg) Fu u al yield Xylose con e sion Fu u al selec i i y Figu e 4. E ec o he amoun o NaCl on he dehyd a ion o D-xylose o u u al. Reac ion condi ions: Na ion NR50 (2 pelle s), NaCl, D-xylose (1.0 mmol), wa e (1 mL), CPME (3 mL), MW, 170 ◦ C and 40 min. To comple e ou s udy, NaCl was eplaced by o he ino ganic sal s, such as KCl o LiCl. In ela ion o he ca ion size and hei ela i e mobili y inside he Na ion pelle s, a compe i ion be ween wa e and he posi i ely-cha ged species was expec ed o ha e an in luence on he elease o he p o ons in he eac ion. Ne e heless, by using he same sal mola a io, no a ia ion o pH was eco ded, and he yield o p oduced u u al was cons an wi h a alue o 80%. Na ion NR50 has a capaci y o 0.8 mmol o H + pe g am. Fo wo pelle s and an excess o NaCl, i co esponded o a pH o 1.1 in he ial i he o ali y o he p o ons was eleased in he medium. The pH o ou eac ion media eached a alue o 1.3 on a e age, which means ha a pa o he sul onic g oups g a ed on he esin emained p o ona ed. To e i y i he D-xylose con e sion was only ca alyzed by he HCl eleased in he medium o also by he esin, a eac ion wi h an HCl solu ion a a pH o 1.3 (0.05 mol · L −1 ) was pe o med o 40 min in he p esence o 90 mg o NaCl a 170 ◦ C in wa e -CPME,1:3, / . A lowe u u al yield o 73% was ob ained ins ead o he 80% eached wi h he mix u e o Na ion and NaCl. All o hese obse a ions pe mi ed es ablishing ha a new acid ca aly ic sys em was ob ained ha ing a syne ge ic e ec be ween Na ion NR50 and NaCl. 2.6. Reusabili y o he Na ion NR50 Se e al consecu i e eac ions we e pe o med wi h he same Na ion NR50 pelle s. A e each eac ion, he esin pelle s we e eco e ed om he eac ion medium and egene a ed in an acidic HCl ba h o e nigh . The eac ions we e pe o med o 40 min a 170 ◦ C in wa e -CPME, 1:3, / . The ac i i y o he Na ion NR50 pelle s was kep cons an o h ee consecu i e cycles (Figu e 5). A e he ou h cycle, he u u al yield began o dec ease. This obse a ion can be explained by he g adual deac i a ion o he pelle s by humin deposi ion. Molecules 2016,21, 1102 7 o 11 Molecules 2016, 21, 1102 7 o 11 Figu e 5. Reusabili y o he aqueous phase con aining Na ion NR50 and NaCl o he D-xylose dehyd a ion o u u al. Reac ion condi ions: Na ion NR50 (2 pelle s), NaCl (95 mg), D-xylose (1.0 mmol), wa e (1 mL), CPME (3 mL), MW, 170 °C and 40 min. 2.7. SEM Obse a ions EDX and Analysis o Na ion NR50 Pelle s Fo each s age o ou s udy, he mo phology o he Na ion esins was obse ed by SEM. The pho og aphs o hese obse a ions a e p o ided (Figu e 6) in associa ion wi h a se ies o EDX analysis spec a (Figu e S3, ESI). Figu e 6a shows he ine po osi y o he na i e Na ion NR50 su ace. The EDX spec um S3a ga e he main cons i uen s o Na ion: luo ine, ca bon, oxygen and sul u . The pelle analyzed in Figu e 6b was used in a eac ion wi hou NaCl. The su ace o he deg aded ca alys was cha ac e ized by a ne wo k o c acks o a ious sizes and by he deposi ion o an o ganic esidue on he su ace. Fo his sample, he EDX spec um S3b showed an en ichmen o he ca alys wi h ca bon and oxygen, e idence o he humin deposi ion. When he eac ion was conduc ed in a NaCl aqueous solu ion, he eco e ed Na ion NR50 pelle s (Figu e 6c) appea ed o emain in ac . Howe e , he su ace o he ca alys was en i ely coa ed by NaCl c ys als, an obse a ion con i med by he EDX analysis S3c. When hese pelle s we e used wi hou ea men in an aqueous HCl solu ion, a u he ca aly ic un in he p esence o D-xylose ga e only u u al in a 21% yield. Indeed, because o he pa ial exchange o p o ons H+ wi h Na+ ca ions, he ca alys was pa ially deac i a ed. Finally, he Na ion pelle can be egene a ed in an acidic ba h o HCl o eco e i s ac i i y. As we can see in he image (Figu e 6d), he ino ganic sal coa ing was comple ely emo ed by his ea men , and i was p o en by he supp ession o bo h he Na and Cl peaks on he co esponding EDX spec um S3d. Figu e 6. FE-SEM images o he Na ion NR50 su ace mo phology ob ained om: (a) p is ine Na ion; (b) he eac ion wi hou sal ; (c) wi h sal a e one ca aly ic un; (d) a e egene a ion in concen a ed HCl solu ion (scale ba = 50 µm). 0 20 40 60 80 100 1 2 3 4 Fu u al yield (%) Cycle Figu e 5. Reusabili y o he aqueous phase con aining Na ion NR50 and NaCl o he D-xylose dehyd a ion o u u al. Reac ion condi ions: Na ion NR50 (2 pelle s), NaCl (95 mg), D-xylose (1.0 mmol), wa e (1 mL), CPME (3 mL), MW, 170 ◦C and 40 min. 2.7. SEM Obse a ions EDX and Analysis o Na ion NR50 Pelle s Fo each s age o ou s udy, he mo phology o he Na ion esins was obse ed by SEM. The pho og aphs o hese obse a ions a e p o ided (Figu e 6) in associa ion wi h a se ies o EDX analysis spec a (Figu e S3, ESI). Figu e 6a shows he ine po osi y o he na i e Na ion NR50 su ace. The EDX spec um S3a ga e he main cons i uen s o Na ion: luo ine, ca bon, oxygen and sul u . The elle analyzed in Figu e 6b was used in a eac ion wi hou NaCl. The su ace o he deg aded ca alys was cha ac e ized by a ne wo k o c acks o a ious sizes and by he deposi ion o an o ganic esidue on he su ace. Fo his sample, he EDX spec um S3b showed an en ichmen o he ca alys wi h ca bon and oxygen, e idence o he humin deposi ion. When he eac ion was conduc ed in a NaCl aqueous solu ion, he eco e ed Na ion NR50 pelle s (Figu e 6c) appea ed o emain in ac . Howe e , he su ace o he ca alys was en i ely coa ed by NaCl c ys als, an obse a ion con i med by he EDX analysis S3c. When hese pelle s we e used wi hou ea men in an aqueous HCl solu ion, a u he ca aly ic un in he p esence o D-xylose ga e only u u al in a 21% yield. Indeed, because o he pa ial exchange o p o ons H + wi h Na + ca ions, he ca alys was pa ially deac i a ed. Finally, he Na ion pelle can be egene a ed in an acidic ba h o HCl o eco e i s ac i i y. As we can see in he image (Figu e 6d), he ino ganic sal coa ing was comple ely emo ed by his ea men , and i was p o en by he supp ession o bo h he Na and Cl peaks on he co esponding EDX spec um S3d. Molecules 2016, 21, 1102 7 o 11 Figu e 5. Reusabili y o he aqueous phase con aining Na ion NR50 and NaCl o he D-xylose dehyd a ion o u u al. Reac ion condi ions: Na ion NR50 (2 pelle s), NaCl (95 mg), D-xylose (1.0 mmol), wa e (1 mL), CPME (3 mL), MW, 170 °C and 40 min. 2.7. SEM Obse a ions EDX and Analysis o Na ion NR50 Pelle s Fo each s age o ou s udy, he mo phology o he Na ion esins was obse ed by SEM. The pho og aphs o hese obse a ions a e p o ided (Figu e 6) in associa ion wi h a se ies o EDX analysis spec a (Figu e S3, ESI). Figu e 6a shows he ine po osi y o he na i e Na ion NR50 su ace. The EDX spec um S3a ga e he main cons i uen s o Na ion: luo ine, ca bon, oxygen and sul u . The pelle analyzed in Figu e 6b was used in a eac ion wi hou NaCl. The su ace o he deg aded ca alys was cha ac e ized by a ne wo k o c acks o a ious sizes and by he deposi ion o an o ganic esidue on he su ace. Fo his sample, he EDX spec um S3b showed an en ichmen o he ca alys wi h ca bon and oxygen, e idence o he humin deposi ion. When he eac ion was conduc ed in a NaCl aqueous solu ion, he eco e ed Na ion NR50 pelle s (Figu e 6c) appea ed o emain in ac . Howe e , he su ace o he ca alys was en i ely coa ed by NaCl c ys als, an obse a ion con i med by he EDX analysis S3c. When hese pelle s we e used wi hou ea men in an aqueous HCl solu ion, a u he ca aly ic un in he p esence o D-xylose ga e only u u al in a 21% yield. Indeed, because o he pa ial exchange o p o ons H+ wi h Na+ ca ions, he ca alys was pa ially deac i a ed. Finally, he Na ion pelle can be egene a ed in an acidic ba h o HCl o eco e i s ac i i y. As we can see in he image (Figu e 6d), he ino ganic sal coa ing was comple ely emo ed by his ea men , and i was p o en by he supp ession o bo h he Na and Cl peaks on he co esponding EDX spec um S3d. Figu e 6. FE-SEM images o he Na ion NR50 su ace mo phology ob ained om: (a) p is ine Na ion; (b) he eac ion wi hou sal ; (c) wi h sal a e one ca aly ic un; (d) a e egene a ion in concen a ed HCl solu ion (scale ba = 50 µm). 0 20 40 60 80 100 1 2 3 4 Fu u al yield (%) Cycle Figu e 6. FE-SEM images o he Na ion NR50 su ace mo phology ob ained om: (a) p is ine Na ion; ( b ) he eac ion wi hou sal ; ( c ) wi h sal a e one ca aly ic un; ( d ) a e egene a ion in concen a ed HCl solu ion (scale ba = 50 µm). Molecules 2016,21, 1102 8 o 11 2.8. Fu u al P oduc ion om Xylan The p oduc ion o u u al om xylan in ol es a wo-s ep eac ion: a pseudo- i s o de i e e sible depolyme iza ion and a pseudo- i s o de dehyd a ion. In ou condi ions op imized o he dehyd a ion o D-xylose (Na ion NR50 (2 pelle s), NaCl (95 mg), wa e (1 mL), CPME (3 mL), MW, 170 ◦ C and 40 min), he xylan oligome u nished he a ge u u al in a 19% yield. Thus, an inc ease o he ime pe iod (60 min s. 40 min) and he a ia ion o he empe a u e (130, 140, 150, 160, 170, 180 and 190 ◦ C) we e ealized (Figu e 7). As empe a u e ose om 130 ◦ C o 160 ◦ C, hyd olysis o xylan in o D-xylose was accele a ed, and he amoun o suga eleased in he media inc eased o each a maximum o 42%. Then, om 140 ◦ C, D-xylose was dehyd a ed in o u u al, in a ange o empe a u es om his empe a u e o 170 ◦ C, bu he u u al concen a ion emained unde he concen a ion o he eleased xylose. The yield inc eased om 30% o 55% wi h an inc easing empe a u e om 170 ◦ C o 190 ◦ C. In ega ds o ou esul s, he op imized condi ions o u u al o ma ion om xylan we e iden i ied: Na ion NR50 (2 pelle s), NaCl (95 mg), wa e (1 mL), CPME (3 mL), MW, 190 ◦C and 60 min. Molecules 2016, 21, 1102 8 o 11 2.8. Fu u al P oduc ion om Xylan The p oduc ion o u u al om xylan in ol es a wo-s ep eac ion: a pseudo- i s o de i e e sible depolyme iza ion and a pseudo- i s o de dehyd a ion. In ou condi ions op imized o he dehyd a ion o D-xylose (Na ion NR50 (2 pelle s), NaCl (95 mg), wa e (1 mL), CPME (3 mL), MW, 170 °C and 40 min), he xylan oligome u nished he a ge u u al in a 19% yield. Thus, an inc ease o he ime pe iod (60 min s. 40 min) and he a ia ion o he empe a u e (130, 140, 150, 160, 170, 180 and 190 °C) we e ealized (Figu e 7). As empe a u e ose om 130 °C o 160 °C, hyd olysis o xylan in o D-xylose was accele a ed, and he amoun o suga eleased in he media inc eased o each a maximum o 42%. Then, om 140 °C, D-xylose was dehyd a ed in o u u al, in a ange o empe a u es om his empe a u e o 170 °C, bu he u u al concen a ion emained unde he concen a ion o he eleased xylose. The yield inc eased om 30% o 55% wi h an inc easing empe a u e om 170 °C o 190 °C. In ega ds o ou esul s, he op imized condi ions o u u al o ma ion om xylan we e iden i ied: Na ion NR50 (2 pelle s), NaCl (95 mg), wa e (1 mL), CPME (3 mL), MW, 190 °C and 60 min. Figu e 7. Fu u al yield om xylan as a unc ion o empe a u e o 60 min. Reac ion condi ions: Na ion NR50 (2 pelle s), NaCl (95 mg), xylan (1.0 mmol), wa e (1 mL), CPME (3 mL), MW, 170 °C and 60 min. 3. Expe imen al Sec ion 3.1. Gene al In o ma ion Subs a es we e pu chased om Ac os O ganic (D-xylose ≥ 99%, L-a abinose ≥99%), xylan om beech wood ≥90% om Sigma Ald ich (Sain -Quen in Falla ie , F ance). Ca alys (Na ion NR50) was pu chased om Sigma Ald ich. Sol en s we e pu chased om Ac os (cyclopen yl me hyl e he , Illki ch, F ance) and Fishe Scien i ic (ace oni ile, Illki ch, F ance). The s anda d ( u u al 99%) was ob ained om Ac os. All ma e ials we e used wi hou u he pu i ica ion. The wa e used in all expe imen s was Millipo e Milli-Q g ade. Each sample was analyzed sepa a ely by means o a Shimadzu P ominence HPLC. Pen oses we e de ec ed wi h a low empe a u e e apo a i e ligh sca e ing de ec o (ELSD-LTII, Shimadzu, Ma ne_la-Vallée, F ance), and u u al was de ec ed wi h a UV-VIS de ec o (SPD-M20A, Shimadzu, Ma ne-la-Vallée, F ance) a a wa eleng h o 275 nm. The column used was a G ace P e ail C18 column (250 × 4,6 mm, 5 µm). The mobile phase was a MeOH-H2O (9:1) solu ion lowing a a a e o 0.5 mL·min−1. The column o en was se a 40 °C. D-Xylose con e sion (X), u u al yield (Y) and u u al selec i i y (S) we e calcula ed by he ollowing equa ions: 0 10 20 30 40 50 60 70 80 90 100 130 150 170 190 Fu u al yield and xylose con en s (%) Tempe a u e (°C) Fu u al om xylose Xylose eleased om xylan Figu e 7. Fu u al yield om xylan as a unc ion o empe a u e o 60 min. Reac ion condi ions: Na ion NR50 (2 pelle s), NaCl (95 mg), xylan (1.0 mmol), wa e (1 mL), CPME (3 mL), MW, 170 ◦ C and 60 min. 3. Expe imen al Sec ion 3.1. Gene al In o ma ion Subs a es we e pu chased om Ac os O ganic (D-xylose ≥ 99%, L-a abinose ≥ 99%), xylan om beech wood ≥ 90% om Sigma Ald ich (Sain -Quen in Falla ie , F ance). Ca alys (Na ion NR50) was pu chased om Sigma Ald ich. Sol en s we e pu chased om Ac os (cyclopen yl me hyl e he , Illki ch, F ance) and Fishe Scien i ic (ace oni ile, Illki ch, F ance). The s anda d ( u u al 99%) was ob ained om Ac os. All ma e ials we e used wi hou u he pu i ica ion. The wa e used in all expe imen s was Millipo e Milli-Q g ade. Each sample was analyzed sepa a ely by means o a Shimadzu P ominence HPLC. Pen oses we e de ec ed wi h a low empe a u e e apo a i e ligh sca e ing de ec o (ELSD-LTII, Shimadzu, Ma ne_la-Vallée, F ance), and u u al was de ec ed wi h a UV-VIS de ec o (SPD-M20A, Shimadzu, Ma ne-la-Vallée, F ance) a a wa eleng h o 275 nm. The column used was a G ace P e ail C18 column (250 × 4.6 mm, 5 µ m). The mobile phase was a MeOH–H 2 O (9:1) solu ion lowing a a a e Molecules 2016,21, 1102 9 o 11 o 0.5 mL · min −1 . The column o en was se a 40 ◦ C. D-Xylose con e sion (X), u u al yield (Y) and u u al selec i i y (S) we e calcula ed by he ollowing equa ions: X=(Ini ial xylose amoun (mol)−Final xylose amoun (mol)) Ini ial xylose amoun (mol)×100 Y=Final u u al amoun (mol) Ini ial xylose amoun (mol)×100 S=Fu u al yield Xylose con e sion ×100 Regula ly, he calib a ion cu e was checked in o de o a oid e en ual expe imen al e o s associa ed wi h all measu emen s epo ed abo e. The esul s epo ed in all igu es a e a e aged alues calcula ed om h ee measu emen s. SEM (scanning elec on mic oscopy)-EDX (ene gy dispe si e X- ay di ac ion) analysis o Na ion pelle s a di e en s ages o he expe imen s was pe o med on a Quan a FEG 250 (FEI) equipped wi h a mic oanalysis de ec o o EDX (B ucke , Wissembou g, F ance). SEM mic og aphs acqui ed in seconda y elec on mode we e ob ained a low acuum, 15 kV o accele a ing ol age wi h a 10-mm wo king dis ance. EDX spec a we e collec ed a a 30 ◦ angle, a 15-kV accele a ing ol age and a 10-mm wo king dis ance. 3.2. Gene al P ocedu e o he Syn hesis o Fu u al in a Wa e -CPME Biphasic Media In a ypical expe imen , a 10-mL glass essel was cha ged wi h wa e (1 mL), CPME (3 mL), he subs a e (1.0 mmol), Na ion NR50 (2 pelle s, ≈ 95 mg) and NaCl (1.62 mmol). The essel was sealed wi h a sep um, placed in he mic owa e appa a us (An onPaa Monowa e 300) and g adually hea ed o 5 min o he desi ed empe a u e ia a esonan single mode unde magne ic s i ing (600 pm) o he desi ed ime. Tempe a u e in he essel was moni o ed by means o an IR senso . A he end o he eac ion, he essel was cooled down o 40 ◦ C using comp essed ai . Then, he wo phases we e sepa a ed. The aqueous phase was dilu ed in 200 mL o dis illed wa e and il e ed p io o analysis h ough a il e pape (10–20 µ m, VWR, Fon enay-sous-Bois, F ance). The o ganic phase was dilu ed in 200 mL o ace oni ile and il e ed p io o analysis h ough a sy inge il e (PTFE, 0.45 µ m, VWR). All expe imen s we e epea ed a leas h ee imes, and he de ia ion was lowe han 5%. 4. Conclusions In summa y, his wo k epo s a new e icien p ocess o he p oduc ion o u u al in an 80% yield. The me hod used a mix u e o Na ion NR50 and NaCl in a wa e -CPME biphasic sys em a 170 ◦ C unde mic owa e i adia ion. The associa ion o Na ion NR50 and NaCl gene a ed an unusual s abili y o he esin a 170 ◦ C and a syne ge ic e ec in acid ca alysis. Rega ding hese p omising esul s, he ansposi ion o his me hodology o cellulose o s a ch in o de o p oduce 5-hyd oxyme hyl u u al (HMF) will be de eloped in due cou se. Supplemen a y Ma e ials: The Supplemen a y Ma e ials a e a ailable online a : h p://www.mdpi.com/ 1420-3049/21/8/1102/s1. Acknowledgmen s: S.L.G. would like o hank he Minis è e de l’Educa ion Na ionale e de la Reche che o he inancial suppo . This wo k was pe o med in pa ne ship wi h he COST Ac ion FP 1306 Valo isa ion o ligno-cellulosic biomass s eams o sus ainable p oduc ion o chemicals, ma e ials and uels using low en i onmen al impac echnologies. Au ho Con ibu ions: F ede ic Delbecq and Ch is ophe Len concei ed and designed he expe imen s; Sa ah Le Guenic and Da id Ge gela pe o med he expe imen s; Sa ah Le Guenic, Clai e Ceballos and F ede ic Delbecq analyzed he da a; Ch is ophe Len con ibu ed eagen s/ma e ials/analysis ools; F ede ic Delbecq and Ch is ophe Len w o e he pape . Con lic s o In e es : The au ho s decla e no con lic o in e es .