Direct Conversion of Chitin Biomass to 5-Hydroxymethylfurfural In

Bioresource Technology 143 (2013) 384–390

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Bioresource Technology

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Direct conversion of chitin biomass to 5-hydroxymethylfurfural

in concentrated ZnCl2 aqueous solution ⇑ ⇑ Yingxiong Wang a, Christian Marcus Pedersen b, Tiansheng Deng a, Yan Qiao a, , Xianglin Hou a,

a State Key Laboratory of Coal Conversion, Institute of Coal Chemistry, Chinese Academy of Sciences, 27 South Taoyuan Road, Taiyuan 030001, People’s Republic of China b Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen Ø, Denmark

highlights

Concentrated ZnCl2 solution can be utilized for chitin biomass conversion to 5-HMF. D-Glucosamine and chitosan polymer can be convert into 5-HMF efﬁciently.

AlCl3 and B(OH)3 can increase 5-HMF yield from D-glucosamine slightly. In situ NMR was applied to monitor the chitin biomass conversion process. Quantitative 1H NMR was utilized for determine 5-HMF yield and substrate conversion.

article info abstract

Article history: The direct conversion of chitin biomass to 5-hydroxymethylfurfural (5-HMF) in ZnCl2 aqueous solution Received 27 February 2013 was studied systemically. D-Glucosamine (GlcNH2) was chosen as the model compound to investigate Received in revised form 4 June 2013 the reaction, and 5-HMF could be obtained in 21.9% yield with 99% conversion of GlcNH2. Optimization Accepted 8 June 2013 of the reaction parameters including the screening of 8 co-catalysts was carried out. Among them, AlCl Available online 14 June 2013 3 and B(OH)3 improved 5-HMF yield, whereas CdCl2, CuCl2 and NH4Cl had no effect. CrCl3, SnCl4 and SnCl2 showed negative effects, i.e. lower yields. Consequently, the optimal reaction conditions were found to be Keywords: 67 wt.% ZnCl aqueous solution, at 120 °C without co-catalyst. The reactions were further studied by Chitin 2 in situ NMR, and no intermediate or other byproducts, except humins, were observed. Finally, the sub- In situ NMR Biomass strate scope was expanded from GlcNH2 to N-acetyl-D-glucosamine and various chitosan polymers with 5-HMF different molecular weights, 5-HMF yield from polymers were generally lower than that from GlcNH2.

1. Introduction under high pressure in aqueous solution. Performing the reaction in ionic liquid or dimethylacetamide, however, resulted in the for- The diminishing of fossil resources and the enormous environ- mation of 3-acetamido-5-acetylfuran. Szabolcs et al. (2013) re- mental problems caused by the overuse of petrochemicals have ported that chitin biomass can be converted into LA in 37 mol%

pushed chemical researchers to search for renewable and safe re- yield under microwave irradiation at 190 °C with HCl or H2SO4 as sources, where especially biomass has gained a lot of interest dur- the catalyst. Mascal and Nikitin (2009) investigated the degrada- ing the last several years. Among the investigated biomass, tion of chitin for preparation of 5-(chloromethyl)furfural (CMF) cellulose, which is the most abundant biopolymer, has been widely in a biphasic reaction system formed by aqueous HCl and 1,2- studied (Guo et al., 2012; Lai et al., 2011; Li et al., 2011, 2012). dichloroethane, CMF was obtained in 45% yield together with However, chitin biomass (which here refers to chitin, chitosan, 29% of LA. their corresponding monomers and derivatives), are the second Chitin is the structural constituent existed in the exoskeleton or most abundant polysaccharides (after cellulose), has received less cuticles of crab and other invertebrates, as well as in the cell walls attention (Muzzarelli, 2011, 2012). Very recently, Kerton et al. of some fungi. Every year, 1 Â 1010 tons chitin is produced as (Drover et al., 2012; Omari et al., 2012a,b) reported that chitin bio- industrial waste material of ﬁsheries and in the seafood industry mass and its derivatives can be converted into the platform chem- (Kumar et al., 2004). Only a negligible amount of the chitin icals 5-hydroxymethylfurfural (5-HMF) and levulinic acid (LA) biomass generated is used in, e.g. skin moisturizers, joint-pain relievers, anti-tumoral and antimicrobial agents, as well as produc-

⇑ Corresponding authors. Tel.: +86 351 4049501; fax: +86 351 4041153. ing D-glucosamine (GlcNH2)(Rinaudo, 2006; Trombotto et al., E-mail addresses: [email protected] (Y. Qiao), [email protected] (X. Hou). 2008). Therefore, it will be of huge economic and environmental

0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.06.024 Y. Wang et al. / Bioresource Technology 143 (2013) 384–390 385 interest if these sustainable chitin biomass wastes can be trans- system developed here, is a promising strategy for the conversion formed into high value fuel and chemical products (Marzaioli et al., of chitin biomass into valuable platform chemicals. 2012). Chitin is a linear polysaccharide composed of 2-acetamido-2- 2. Methods deoxy-D-glucose (GlcNAc), while chitosan is the copolymer of GlcNAc and GlcNH2 (Fig. 1). There are strong intra- and inter- 2.1. Materials molecular hydrogen bonds between –OH, –NH2 and –NHAc groups, so highly polarized organic solvents such as dimethylformamide, Practical grade D-glucosamine hydrochloride (designated as dimethyl sulfoxide, or even high-cost ionic liquid are required to GlcNH2, white crystalline powder), N-acetyl-D-glucosamine (desig- dissolve chitin biomass. The poor solubility is the main challenge nated as GlcNAc, white powder), chitin (white crystalline powder), for the exploitation of chitin biomass, and this seriously hampers carboxylated chitosan (designated as Chitosan–COOH, flake, aver- its conversion into fuels and chemical platform molecules. Finding age molecular weight 200,000, carboxylation degree >70%), more environmentally benign, readily available, and low cost green chitosan with average molecular weight of 1000 (designated as solvent systems is of major importance for the future use of chitin Chitosan-1K, white powder, deacetylation degree P90%), 5000 as a renewable raw material for the chemical industry. (designated as Chitosan-5K, light-yellow powder, deacetylation Recently, our team utilized concentrated ZnCl2 aqueous solu- degree P90%), 50,000 (designated as Chitosan-50K, yellow pow- tion to study the degradation of lignocellulosic biomass (Deng der, deacetylation degree P90%), and 100,000 (designated as et al., 2012). This green solvent exhibits good solubility for ligno- Chitosan-100K, yellow powder, deacetylation degree P90%) were celluloses, and the dehydration of glucose, fructose, maltose, su- obtained from Golden-Shell Biochemical Co. Ltd. The origin of the crose, cellulose as well as starch was investigated in this compounds is from shrimp shell. Deuterium oxide (D2O, 99.9%) environmental friendly solvent. It was found that incompletely was supplied by Cambridge Isotope Laboratory. Zinc chloride coordinated Zn2+ ions could coordinate with the hydroxyl groups (ZnCl2) and ethanol were purchased from Beijing Chemical Reagent of carbohydrates (for example, cellulose), and thereby catalyze Company. Acetonitrile and 5-hydroxymethylfurfural (purity > 96%) their transformation into fructose and further into 5-HMF with a were purchased from J&K Chemical Co. Ltd. SnCl2Á2H2O, SnCl4Á5H2- moderate yield of 11.5 mol% of 5-HMF without any additional co- O, CrCl3Á6H2O, CdCl2ÁH2O, CuCl2ÁH2O, NH4Cl, AlCl3Á6H2O and catalyst. B(OH)3 were provided by Tianjin Chemical Reagent Factory. All re- Inspired by this work, and in the view of the molecular struc- agents utilized in this work were used without further purification, ture aspect: chitin has structural similarities with cellulose such and deionized, double distilled water was used in all experiments. as the b-(1?4) glycosidic bonds, but is different in the functional groups on the second carbon (C2), i.e. hydroxyl group (C2–OH) in 2.2. General reaction procedure cellulose and amino (C2–NH2) or acetamide (C2–NHAc) groups in 2+ chitin biomass. Considering the strong interaction between Zn In a typical experiment for the conversion of chitin biomass in and –OH, –NH and/or –NHAc (Varma et al., 2004; Wang et al., 2 ZnCl2 solution, 1.0 g of chitin biomass substrates was mixed with 2004) together with the protonation of these groups, the solubility 30 g ZnCl2 aqueous solution (33.5 or 67 wt.%) with or without addi- of chitin biomass in concentrated ZnCl2 aqueous solution is en- tional Lewis acid catalysts in a 100 mL glass flask, and heated in an hanced (the acidity is close to that of 1.6 wt.% HCl aqueous solu- oil bath under continuous stirring. Zero time was taken as soon as tion). Therefore, it is reasonable to investigate concentrated ZnCl2 the desired temperature was reached. A gradually darkening of the aqueous solution as a reaction media for chitin biomass conver- clear solution to dark slurry was usually observed under the ap- sion. Being the monomer of chitosan, GlcNH2 was chosen as the plied conditions. At certain times during the reaction period, model compound to study the reaction conditions. From the initial 0.25 mL of the reaction mixture was taken out from the reaction studies, 5-HMF was found to be the main product. Investigating flask and immediately emerged in an ice bath to quench the reac- the reaction with various co-catalysts including CrCl3, SnCl4, SnCl2, tion. The 5-HMF yield, during the reaction, was determined by CdCl2, CuCl2,NH4Cl, AlCl3 and B(OH)3 was carried out to optimize HPLC of these reaction samples, using a standard curve in order the reaction conditions. The in situ NMR was employed to study to quantify the amount. Quantitative 1H NMR spectroscopy was the reaction at the molecular level. Finally, the scope of using chitin also employed to quantify the yield of 5-HMF and the conversion biomass as the substrate was investigated by studying various chi- of monomeric substrates. 5-HMF yield was defined as: 5-HMF tin derivatives (Fig. 1). Using the chitin biomass–ZnCl2 solution yield = (moles of 5-HMF)/(moles of monomer unit) Â 100%.

OH OH OH NHCOCH3 HO O HO O HO O O O OH OH O HO HO HO O n NH2.HCl NHCOCH3 NHCOCH3 OH

(a) GlcNH2 (b) GlcNAc (c) Chitin

OH NH2 OCH2COOH NHCOCH3 HO HO O O O O O O O O O HO O HO n n NHCOCH NH2 3 OH OCH2COOH (d) Chitosan-1K (DP≈6); Chitosan-5K (DP≈31) (e) Chitosan-COOH Chitosan-50K (DP≈310); Chitosan-100K (DP≈620)

Fig. 1. Structures and abbreviation of chitin biomass investigated in this work. DP, Degree of polymerization. 386 Y. Wang et al. / Bioresource Technology 143 (2013) 384–390

2.3. Procedure for in situ NMR study main product. The structure of main product was identiﬁed by HPLC–MS and shown in Fig. S3. There is only one main peak in In situ reaction tracked by NMR with Bruker AV-III 400 for 1H HPLC spectrogram (Fig. S3a), which appeared at 6.00 min and im- and 13C frequency at 400.13 and 100.61 MHz, respectively. The ply that product is of high purity. A corresponding peak at chemical shift for 1H NMR was referenced to 4.77 ppm of residual 6.02 min in total ion currency (TIC) proﬁle (Fig. S3b) was detected.

D2O. Reaction mixture of GlcNH2 in 67 wt.% ZnCl2 D2O solution was As anticipated, the signals of m/z = 124.9, 96.9 and 94.9 detected prepared in a 5 mm heavy wall tube, with the same concentration for this main product, belongs to the fragment ions of 5-HMF as other experiments in this work. The solution was kept at room (Fig. S3c). Other peaks in Fig. S3b, such as the strong peak at temperature for 2 h to reach equilibrium, before it was transferred 3.30 min, may come from tiny amount byproduct or a contamina- into the NMR spectrometer, which was preheated to 120 °C. When tion with strong ionization properties. Moreover, the chemical the temperature inside the NMR spectrometer probe was stabilized structure and high purity of 5-HMF was further confirmed by 1H at 120 °C, 1H NMR (ns = 4) and 13C NMR (ns = 195) spectra were re- NMR (Fig. S4 and Fig. S6). Remarkably, the usually byproducts ob- corded at certain times during the measurement. served in biomass conversion, i.e. levulinic acid, formic acid and furfural, were not detected by HPLC, HPLC–MS and 1H NMR analy- 2.4. Analytic methods sis. In previous studies concerning the conversion of chitin biomass, LA has been the major product and not 5-HMF. The 2.4.1. HPLC analysis method relatively strong reaction conditions are responsible for this, e.g. The reaction mixture was taken out and injected into a glass high pressure and temperature (210 °C, and the pressure of H2O tube, followed by cooling in an ice bath to quench the reaction. at this temperature in Omari et al., 2012a), strong acidity (2 M The crude reaction mixture was kept at room temperature. HCl or 2 M H2SO4 in Szabolcs et al., 2013), (37% HCl in Mascal 0.1 mL of this reaction mixture was diluted with ethanol (95 v%) and Nikitin, 2009) were employed for chitin biomass/5-HMF con- to 5.0 mL in a volumetric flask. The solution was sonicated for version into LA. The reactions in this report are carried out at atmo- 1 min. to dissolve the sample. After centrifugation at 2500 rpm spheric pressure, 120 °C and in 67 wt.% ZnCl2 aqueous solution for 5 min. and filtration through a 0.45 lm PTFE filter, to remove (which has a pH value similar to 1.6 wt.% HCl aqueous solution, the insoluble humin polymer, the sample was analyzed using HPLC Deng et al., 2012). These improved reaction conditions are milder and the 5-HMF quantified with the calibration curve (Fig. S1). than the previously described and therefore, in the present inves- Chromatographic analysis was performed using a Venusil ODS col- tigation, the chitin biomass and/or 5-HMF are not converted into umn (5.0 lm, 250 mm Â 4.6 mm) and an UV detector. The mobile LA and 5-HMF can be isolated as the main product. This high selec- phase consisted of acetonitrile and water (10:90 v/v) with a flow tivity is one of the great advantage of applying ZnCl2 aqueous solu- rate of 0.9 mL/min. The column temperature was maintained at tion in the conversion chitin biomass to 5-HMF. 30 °C and the detection wavelength set to 280 nm. The reaction conditions were optimized using GlcNH2, GlcNAc and Chitosan-1K as model substrates. The effects of reaction tem- 2.4.2. HPLC–MS analysis method perature, time and ZnCl2 solution concentration on the substrate The HPLC–MS analysis was performed at 30 °C on a Waters Alli- conversion and 5-HMF yield were evaluated (Table 1). Both GlcNH2 ance 2695/ZQ-4000 instrument, equipped with a Waters 996 PDA (Table 1, entries 1 and 4) and GlcNAc (Table 1, entries 2 and 5) gave detector and a Venusil ODS column set. The mobile phase consisted much better results at 120 °C than at 80 °C; these temperature re- of acetonitrile and water (10/90 v/v) with a flow rate of 0.8 mL/ lated results are in agreement with previous research (Deng et al., min. The ZQ-4000 MS is equipped with an electrospray ionization 2012; Omari et al., 2012a). Therefore, the reaction temperature 2+ À source (ESI). In order to avoid the influence of the Zn and Cl on was kept at 120 °C in order to optimize the reaction conditions the MS signal, each sample was prepared by the following proce- further. dure: 1.0 mL of each reaction mixture was mixed with 3.0 mL ethyl Fig. 2 shows that 5-HMF yield from GlcNH2 increase approxi- acetate and then washed by 6.0 mL saturated Na2CO3 solution. The mately linearly at the first stage of reaction, for example, from 10 organic phase was then collected and the trace amount of water re- to 90 min. Then, 5-HMF yield stays constant or even starts to de- moved by anhydrous Na2SO4; afterwards, the organic phase was crease in time, this is probably caused by humin formation be- filtered and evaporated to remove the ethyl acetate, the remains tween 5-HMF and GlcNH2 (Stahlberg et al., 2011). For optimal 5- were dissolved in 1.5 mL deionized water and for the HPLC–MS HMF yield, the reaction should be quenched at this point, which analysis performed. can be monitored by HPLC. Under the same reaction temperature and times, comparing 2.4.3. Quantitative 1H NMR measurement GlcNH2 (Table 1, entries 1 and 6) and the polymer Chitosan-1K (Ta- 1 Quantitative H NMR was performed to measure the 5-HMF ble 1, entries 3 and 7) as substrates, higher substrate conversion for yield and the GlcNH (and GlcNAc) conversion using maleic acid 2 GlcNH2, and higher 5-HMF yield for both GlcNH2 and Chitosan-1K as internal standard substance, following the procedure described were obtained with higher ZnCl2 concentration. So higher concen- by Rundlöf et al. (2010). Maleic acid with concentration of tration of (67 wt.% ZnCl2 aqueous solution) was employed for the 1.0574 mg/mL in D2O was prepared as a standard solution, then following research (Cao et al., 1995; Deng et al., 2012). 0.1 mL of reaction mixture was mixed with 0.4 mL this standard 1 solution to give the H NMR sample. The measurements were per- 3.2. Co-catalyst screening and proposed schematic mechanism formed on a Bruker AV-III 400 at 400.13 MHz with scan times 1 1 ns = 64. A typical H NMR spectrum of this quantitative H NMR In order to know whether an extra co-catalyst would give a measurement is shown in Fig. S2. superior result, 8 co-catalysts; CrCl3, SnCl4, SnCl2, CdCl2, CuCl2, NH4Cl, AlCl3 and B(OH)3 were chosen to evaluate the effects on 3. Results and discussion 5-HMF yield and the conversion of the model compound GlcNH2. The optimized reaction conditions (67 wt.% ZnCl2 aqueous solution 3.1. Reaction parameters optimization at 120 °C) were employed. The 5-HMF yields from reaction of

GlcNH2 with and without co-catalysts were obtained by HPLC, When the concentrated ZnCl2 aqueous solution was initially ap- and plotted against the reaction time as depicted in Fig. 2. The 5- plied to chitin biomass conversion, it was necessary to identify HMF yield and substrate conversion were additionally determined Y. Wang et al. / Bioresource Technology 143 (2013) 384–390 387

Table 1 Optimization of the reaction parameters for chitin biomass conversion.a

Entry Substrate ZnCl2 concentration (wt.%) Temperature (°C) Substrate conversion (mol%) 5-HMF yield (mol%)

1 GlcNH2 67 120 99.6 21.9 2 GlcNAc 67 120 99.9 2.8 3 Chitosan-1K 67 120 ndb 10.1

4 GlcNH2 67 80 35.4 2.1 5 GlcNAc 67 80 30.8 0.2

6 GlcNH2 33.5 120 62.2 5.1 7 Chitosan-1K 33.5 120 nd 0.7

a Reaction condition: 1 g substrate in 30 g ZnCl2 solution, 90 min. b Not determined.

1 by quantitative H NMR, and the best values obtained for 5-HMF third step is the conversion of the GlcNH2–metal complex from yield; the corresponding substrate conversions are shown in its open chain form to the furanose form. This step is crucial for

Fig. 3. For easy to compare, the GlcNH2 degradation without co-catalyst was defined as control experiment. This reaction gave 30 Control 5-HMF in 21.9% yield and a GlcNH2 conversion of 99.6%. GlcNH :SnCl = 2:1 CrCl and SnCl were the first choice due to their excellent cat- 2 2 3 4 GlcNH :SnCl = 10:1 alytic ability in cellulose conversion to 5-HMF (Hu et al., 2009; 25 2 4 GlcNH :SnCl = 2:1 2 4 Zhao et al., 2007). However, CrCl3 did not promote the reaction, GlcNH :CrCl = 2:1 2 3 and the 5-HMF yield decreased to 13.0% with 70.7% GlcNH2 con- 20 version (Fig. 2a and Fig. 3, run 2). For SnCl4, 5-HMF yield decreased to only 1.7%, and 12.3% conversion when using 0.5 equivalent SnCl4 15 (Fig. 2a and Fig. 3, run 3); if the amount of co-catalyst was lowered to 0.1 equivalent SnCl added, the reaction proceeded well again 4 10 with 20.8% 5-HMF yield and 90.3% conversion of GlcNH2 (Fig. 2a and Fig. 3, run 4). Similar negative effects were observed when 5-HMF Yield (mol%) 5 SnCl2 was employed as the co-catalyst. (Fig. 2a and Fig. 3, run 5). These low 5-HMF yields are in contrast to the results when using glucose and fructose as the substrates as described in our previous 0 work (Deng et al., 2012). 30 When CdCl2, CuCl2,NH4Cl were employed as co-catalyst, the Control GlcNH :CdCl = 2:1 reaction results were comparable with control experiment 2 2 GlcNH :NH Cl = 2:1 (Fig. 2b, and runs 6–8 of Fig. 3). The addition of these three com- 25 2 4 GlcNH :CuCl = 2:1 pounds did not affect either 5-HMF yield or GlcNH2 conversion. 2 2 AlCl3, nevertheless exhibited positive effect for conversion chi- 20 tin biomass to 5-HMF (Fig. 2c and Fig. 3, run 9). As shown in Fig. 2c, 5-HMF concentration increased rapidly within the first 15 90 min, and remained stable for about 2 h; finally a yield of 23% 5-HMF was recorded, which is slightly higher than control experiment. 10 Because boron and aluminum belong to the same group in peri- 5-HMF Yield (mol%) odic table, it is interesting to try a boron based co-catalyst as well. 5

Considering that BCl3 is difﬁcult to handle and very hydroscopic, B(OH)3 was employed (Hansen et al., 2011). The 5-HMF yield 0 was increased with about 5% by the addition of B(OH)3 (Fig. 2c and Fig. 3, runs 10–11), i.e. yields of 26.5% and 27.2% 5-HMF 30 respectively were achieved within 90 min. with 0.5 equivalent or Control GlcNH :B(OH) = 2:1 2 3 1.0 equivalent B(OH)3 co-catalyst loading. Co-catalyst dosage 25 GlcNH :B(OH) = 1:1 2 3 seems less crucial for the B(OH)3–ZnCl2–chitin biomass reaction GlcNH :AlCl = 2:1 system. 2 3 The proposed mechanism for chitin biomass degradation and 5- 20

HMF formation in concentrated aqueous ZnCl2 solution is shown in Fig. S5. At the ﬁrst step, chitosan–zinc complex is formed through 15 2+ the coordination of Zn with –NH2 and 1-O groups in chitosan. This could lower the activation energy of glycosidic bond, make 10 the polymer backbone easier to hydrolyze, and the formation of monomers (GlcNH and GlcNAc) more feasible. In the second step, 5-HMF Yield (mol%) 2 5 ZnCl2 or other co-catalyst complexes with D-glucosamine (model compound) in the pyranose form, and this isomerize to the open 0 chain form. However, this isomerization step is hampered by the 0 50 100 150 200 250 300 350 400 450 500 2+ 4+ 3+ strong interaction between Sn ,Sn or Cr and GlcNH2, this Time (min) isomerization is therefore slow and only partial. This isomerization step can be favorited by Al3+ or B3+ due to their appropriate inter- Fig. 2. Conversion of GlcNH2 in 67 wt.% ZnCl2 aqueous solution at 120 °C with and action (Varma et al., 2004; Wang et al., 2004) with GlcNH2. The without co-catalyst. 388 Y. Wang et al. / Bioresource Technology 143 (2013) 384–390

Conversion 100 5-HMF Yield Measured by HPLC 5-HMF Yield Measured by NMR

Yield and Conversion 20

0 1 23 45 67 8911 10 1213 14 15 1617 18 Experiment Number

Fig. 3. Chitin biomass conversions and 5-HMF yields for investigated substrates with or without co-catalysts loading (mole ratio) in 67 wt.% ZnCl2 solution at 120 °C. (1)

GlcNH2, (2) GlcNH2:CrCl3 = 2:1, (3) GlcNH2:SnCl4 = 2:1, (4) GlcNH2:SnCl4 = 10:1, (5) GlcNH2:SnCl2 = 2:1, (6) GlcNH2:CdCl2 = 2:1, (7) GlcNH2:NH4Cl = 2:1, (8) GlcNH2:-

CuCl2 = 2:1, (9) GlcNH2:AlCl3 = 2:1, (10) GlcNH2:B(OH)3 = 2:1, (11) GlcNH2:B(OH)3 = 1:1, (12) GlcNAc, (13) Chitin, (14) Chitosan-1K, (15) Chitosan-5K, (16) Chitosan-50K, (17) Chitosan-100K and (18) Chitosan–COOH. the 5-HMF formation and hence the selectivity in the reaction (LA spectral resolution. Simultaneously, the intensity of 5-HMF signals vs. HMF). The fourth step is the deamination, where the –NH2 at 6.7, 7.6 and 9.4 ppm increased progressively. With the reaction group is removed from GlcNH2, and the enol-intermediate is proceeding, the H1 and H2 signals of GlcNH2 at 5.6, 5.2, 3.6 and formed. The proper reaction conditions, especially the acidic pH 3.3 ppm became weaker and ﬁnally negligible. Meanwhile, the environment promote this initial deamination step to occur intensity of 5-HMF signals at 6.7, 7.6 and 9.4 ppm increased con- (De et al., 2011; Hansen et al., 2011; Yang et al., 2012). Finally, 5- stantly. The reaction was also monitored by 13C NMR recorded at HMF is formed through the dehydration and keto-enol tautomer- 109 min (Fig. S7). Six signals of 5-HMF were observed, and one ization. Humins might be produced from both intermediate methylene at 57.6 ppm, four alkene peaks at 112.9, 129.9, 151.6 compounds in the fourth and ﬁnal step. In order to study the mech- and 161.6 ppm and a peak in the carbonyl region at 181.9 ppm. 119 13 anism and get more details, Sn NMR, C NMR spectra, X-ray The signal intensity of byproducts and substrate GlcNH2 are not diffraction (XRD) are currently employed. strong enough to be detected. These NMR results are in agreement

Since AlCl3 and B(OH)3 only improved the 5-HMF yield slightly, with the HPLC–MS results, and provide solid evidence that GlcNH2 the cost-beneﬁts are unfavorable, and the reaction system using mainly convert into 5-HMF and conﬁrms the high selectivity of the

ZnCl2 and without co-catalyst is therefore preferred. reaction. 3-Deoxy-glucosone and (4R,5R)-4-hydroxy-5-hydroxymethyl- 4,5-dihydrofuran-2-carbaldehyde are identiﬁed as main intermedi- 3.3. In situ NMR study ates for 5-HMF formation from glucose and fructose correspondingly (Amarasekara et al., 2008; Jadhav et al., 2011). Unfortunately, no rec- In order to get a better understanding of the reaction of chitin ognizable signals of intermediates were observed from neither 1H biomass degradation in concentrated ZnCl aqueous solutions, 2 NMR nor 13C NMR. It is probably due to the short lifetime of the inter- molecular level monitoring of the reaction was obtained by 1 mediates under the reaction conditions. in situ H NMR technology. The reaction was carried out in D2O containing 67 wt.% ZnCl2 at 120 °C. GlcNH2 was still the preferred model compound. Fig. S6 shows the time-progression stack of 1H 3.4. Substrate scope NMR spectra. According to 1H NMR spectra obtained at the initial stage of From the results described above, the following reaction param- 1 in situ reaction and H NMR spectrum of GlcNH2 at room temper- eters, 120 °C, 67 wt.% ZnCl2 aqueous solution, without co-catalyst, ature (data not shown), peaks with chemical shift of 3.6 and were employed to investigate the other chitin biomass substrates,

3.3 ppm assigned to be proton connected to C2 (H2), 5.6 and such as GlcNAc, biopolymer with different structures and molecu- 5.2 ppm were H1 of a- and b-isomers respectively. These four sig- lar weight including Chitin, Chitosan-1K, Chitosan-5K, Chitosan- nals were chosen to indicate the consumption of GlcNH2. The sig- 50K, Chitosan-100K and Chitosan–COOH. The direct preparation nals at 6.7, 7.6 and 9.4 ppm are attributed to 5-HMF, another signal of 5-HMF from these less-expensive and easily available biopoly- at 4.75 ppm is overlapped by solvent signal HDO at 4.77 ppm and mers is essential for expanding the substrate scope. can be observed by water suppression program sequence (Fig. S4). Usually, the reaction can proceed more efﬁciently if the reaction

Compared with the chemical shift of GlcNH2 and 5-HMF at room mixture is homogenous; the solubility of biopolymer of interest temperature (data not shown), the chemical shifts of GlcNH2 and was therefore investigated in 67 wt.% ZnCl2 aqueous solution prior 5-HMF are shifted downﬁeld at 120 °C. Signals belonging to dis- to studying the reactions. The pictures for chitin biomass sub- solvable humins formed during the reaction are observed as well. strates with concentration of 0.1 g/mL in 67 wt.% ZnCl2 aqueous As shown in Fig. S6, at the initial stage of reaction, i.e. within solution are shown in Fig. S8. Chitosan-1K, Chitosan-5K and Chito-

30 min, the signals of GlcNH2 decrease rapidly without losing ﬁne san–COOH can be dissolved well within 10 min, whereas swelling Y. Wang et al. / Bioresource Technology 143 (2013) 384–390 389

15 GlcNAc 4. Conclusions Chitosan-1K Chitosan-5K Chitosan-50K 5-HMF was prepared in concentrated ZnCl2 aqueous solution 12 Chitosan-100K from chitin biomass including GlcNH2 (21.9%), GlcNAc (2.8%), Chi- Chitosan-COOH Chitin tin (9.0%), Chitosan-1K (12.8%), Chitosan-5K (12.2%), Chitosan-50K 9 (8.0%), Chitosan-100K (8.6%) and Chitosan–COOH (9.2%). Eight co-catalysts were screened, but only AlCl3 and B(OH)3 improved the 5-HMF yield slightly. In situ NMR and quantitative 1HNMR 6 showed that 5-HMF is the main product, and no other byproducts like levulinic acid except humins were detectable. Consequently,

5-HMF Yield (mol%) the reaction has very high selectivity, high conversion rate and 3 moderate yield. The concentrated ZnCl2 aqueous solution, which is environmentally benign and inexpensive, is a very promising 0 media for chitin biomass conversion. 0 100 200 300 400 500 600 700 800 Time (min) Acknowledgements

Fig. 4. Time course of different chitin biomass conversion in 67 wt.% ZnCl2 aqueous The authors would like to acknowledge the financial support solution at 120 °C. Concentration: 1 g substrate in 30 g ZnCl solution. 2 from the Major State Basic Research Development Program of Chi- na (973 Program) (No. 2012CB215305). This work was also finan- and dissolution of Chitin, Chitosan-50K and Chitosan-100K were cially supported by the Natural Science Foundation of China (No. observed after 48 h due to their higher crystallinity or higher 21106172) and Science Foundation of Shanxi Province molecular weight (Kasaai, 2009). Thus, the 67 wt.% ZnCl2 aqueous (2012021009-2). solution is a suitable media for chitin biomass conversion. Surprisingly, almost all GlcNAc was consumed (Fig. 3, run 12) Appendix A. Supplementary data but the 5-HMF yield was only 2.8% (Fig. 3, run 12, and Fig. 4). This may be due to the weak interaction of –NHAc with Zn2+ arising Supplementary data associated with this article can be found, in from the steric hindrance of –Ac group (Gamage and Shahidi, the online version, at http://dx.doi.org/10.1016/j.biortech.2013.06. 2007; Guibal et al., 1995). The catalytic deamination and dehydra- 024. tion of GlcNAc to 5-HMF by Zn2+ was slow whereas humins formation is fast under these acidic hydrothermal conditions provided by References the concentrated ZnCl2 solution. Large amount water-insoluble humins were formed under the described conditions. The mass bal- Amarasekara, A.S., Williams, L.D., Ebede, C.C., 2008. Mechanism of the dehydration ance evaluation was performed by collecting and gravimetric of D-fructose to 5-hydroxymethylfurfural in dimethyl sulfoxide at 150 degrees analysis of the isolated humins. The experimental results disclosed C: an NMR study. Carbohydr. Res. 343, 3021–3024. Cao, N.J., Xu, Q., Chen, L.F., 1995. Acid-hydrolysis of cellulose in zinc-chloride that humins which is 80 wt.% of starting material was collected solution. Appl. Biochem. Biotechnol. 51–2, 21–28. from reaction mixture, and elemental analysis data showed that De, S., Dutta, S., Saha, B., 2011. Microwave assisted conversion of carbohydrates and the C/H ratio had been increased from 6.36 of GlcNAc to 10.47 of biopolymers to 5-hydroxymethylfurfural with aluminium chloride catalyst in water. Green Chem. 13, 2859–2868. humins. The exact mass lost during the humins formation is un- Deng, T.S., Cui, X.J., Qi, Y.Q., Wang, Y.X., Hou, X.L., Zhu, Y.L., 2012. Conversion of known, because the structure of humins is too complicate to be carbohydrates into 5-hydroxymethylfurfural catalyzed by ZnCl2 in water. analyzed. Therefore, it is difficult to do the evaluation of mass bal- Chem. Commun. 48, 5494–5496. ance very precisely, and the data can only provide an estimate. Drover, M.W., Omari, K.W., Murphy, J.N., Kerton, F.M., 2012. Formation of a renewable amide, 3-acetamido-5-acetylfuran, via direct conversion of N-acetyl- Higher GlcNAc conversion value but lower 5-HMF yield was ob- D-glucosamine. RSC Adv. 2, 4642–4644. tained compared with the ones obtained with GlcNH2 as the sub- Gamage, A., Shahidi, F., 2007. Use of chitosan for the removal of metal ion strate. This can also explain the results (Fig. 3, run 13, and Fig. 4) contaminants and proteins from water. Food Chem. 104, 989–996. Guibal, E., Roulph, C., Lecloirec, P., 1995. Infrared spectroscopic study of uranyl from Chitin, the polymer of GlcNAc, where the 5-HMF yield was biosorption by fungal biomass and materials of biological origin. Environ. Sci. 9.0% after 360 min. Technol. 29, 2496–2503. For Chitosan-1K, Chitosan-5K, Chitosan-50K and Chitosan- Guo, H., Qi, X., Li, L., Smith Jr., R.L., 2012. Hydrolysis of cellulose over functionalized glucose-derived carbon catalyst in ionic liquid. Bioresour. Technol. 116, 355– 100K, which are the copolymers of GlcNAc and GlcNH2, the 5- 359. HMF yield roughly decreased as the molecular weight of substrates Hansen, T.S., Mielby, J., Riisager, A., 2011. Synergy of boric acid and added salts in increased (Fig. 3, runs 14–17, and Fig. 4). As discussed in the pre- the catalytic dehydration of hexoses to 5-hydroxymethylfurfural in water. Green Chem. 13, 109–114. viously section, 5-HMF formation from carbohydrate biopolymer Hu, S.Q., Zhang, Z.F., Song, J.L., Zhou, Y.X., Han, B.X., 2009. Efficient conversion of is achieved through two steps: glycosidic bonds hydrolysis fol- glucose into 5-hydroxymethylfurfural catalyzed by a common Lewis acid SnCl4 lowed by conversion of the obtained monomers (Jiang et al., in an ionic liquid. Green Chem. 11, 1746–1749. Jadhav, H., Pedersen, C.M., Solling, T., Bols, M., 2011. 3-Deoxy-glucosone is an 2011; Omari et al., 2012a). The higher viscosity of Chitosan-50K intermediate in the formation of furfurals from D-glucose. ChemSuschem 4, and Chitosan-100K in 67 wt.% ZnCl2 aqueous solution may reduce 1049–1051. their reaction abilities and cause longer hydrolysis time. Conse- Jiang, F., Zhu, Q.J., Ma, D., Liu, X.M., Han, X.W., 2011. Direct conversion and NMR quently, the produced 5-HMF during the reaction was converted observation of cellulose to glucose and 5-hydroxymethylfurfural (HMF) catalyzed by the acidic ionic liquids. J. Mol. Catal. A Chem. 334, 8–12. into humins consequentially and decreased the yield greatly Kasaai, M.R., 2009. Various methods for determination of the degree of N- (Varum et al., 1994). acetylation of chitin and chitosan: a review. J. Agric. Food Chem. 57, 1667–1676. For the derivative Chitosan–COOH, which has a very good solu- Kumar, M., Muzzarelli, R.A.A., Muzzarelli, C., Sashiwa, H., Domb, A.J., 2004. Chitosan chemistry and pharmaceutical perspectives. Chem. Rev. 104, 6017–6084. bility in 67 wt.% ZnCl2 aqueous solution, 5-HMF yield was not as Lai, D.M., Deng, L., Li, J., Liao, B., Guo, Q.X., Fu, Y., 2011. Hydrolysis of cellulose into high as expected (Fig. 3, run 18 and Fig. 4). The –COOH group might glucose by magnetic solid acid. ChemSusChem 4, 55–58. coordinate with Zn2+ and block the following hydrolysis, deamina- Li, X., Gunawan, R., Lievens, C., Wang, Y., Mourant, D., Wang, S., Wu, H., Garcia- Perez, M., Li, C.-Z., 2011. Simultaneous catalytic esterification of carboxylic acids tion and dehydration reactions, this results in a slow reaction and a and acetalisation of aldehydes in a fast pyrolysis bio-oil from mallee biomass. low yield of 5-HMF (9.2% after 360 min.) Fuel 90, 2530–2537. 390 Y. Wang et al. / Bioresource Technology 143 (2013) 384–390

Li, X., Jiang, Y., Shuai, L., Wang, L., Meng, L., Mu, X., 2012. Sulfonated copolymers Rundlöf, T., Mathiasson, M., Bekiroglu, S., Hakkarainen, B., Bowden, T., Arvidsson, T., 1 with SO3H and COOH groups for the hydrolysis of polysaccharides. J. Mater. 2010. Survey and qualiﬁcation of internal standards for quantiﬁcation by H Chem. 22, 1283. NMR spectroscopy. J. Pharm. Biomed. Anal. 52, 645–651. Marzaioli, A.M., Bedini, E., Lanzetta, R., Perino, V., Parrilli, M., De Castro, C., 2012. Stahlberg, T., Rodriguez-Rodriguez, S., Fristrup, P., Riisager, A., 2011. Metal-free Preparation and NMR characterization of glucosamine oligomers bearing an dehydration of glucose to 5-(hydroxymethyl)furfural in ionic liquids with boric azide function using chitosan. Carbohydr. Polym. 90, 847–852. acid as a promoter. Chem. Eur. J. 17, 1456–1464. Mascal, M., Nikitin, E.B., 2009. Dramatic advancements in the saccharide to 5- Szabolcs, A., Molnar, M., Dibo, G., Mika, L.T., 2013. Microwave-assisted conversion of (chloromethyl)furfural conversion reaction. ChemSuschem 2, 859–861. carbohydrates to levulinic acid: an essential step in biomass conversion. Green Muzzarelli, R.A.A., 2011. Chitin nanostructures in living organisms. In: Gupta, N.S. Chem. 15, 439–445. (Ed.), Chitin Formation and Diagenesis, vol. 34. Springer Science, Dordrecht, pp. Trombotto, S., Ladaviere, C., Delolme, F., Domard, A., 2008. Chemical preparation 1–34. and structural characterization of a homogeneous series of chitin/chitosan Muzzarelli, R.A.A., Boudrant, J., Meyer, D., Manno, N., DeMarchis, M., Paoletti, M.G., oligomers. Biomacromolecules 9, 1731–1738. 2012. Current views on fungal chitin/chitosan, human chitinases, food Varma, A.J., Deshpande, S.V., Kennedy, J.F., 2004. Metal complexation by chitosan preservation, glucans, pectins and inulin: a tribute to Henri Braconnot, and its derivatives: a review. Carbohydr. Polym. 55, 77–93. precursor of the carbohydrate polymers science, on the chitin bicentennial. Varum, K.M., Ottoy, M.H., Smidsrod, O., 1994. Water-solubility of partially N- Carbohydr. Polym. 87, 995–1012. acetylated chitosans as a function of pH-effect of chemical-composition and Omari, K.W., Besaw, J.E., Kerton, F.M., 2012a. Hydrolysis of chitosan to yield depolymerization. Carbohydr. Polym. 25, 65–70. levulinic acid and 5-hydroxymethylfurfural in water under microwave Wang, X., Du, Y.M., Liu, H., 2004. Preparation, characterization and antimicrobial irradiation. Green Chem. 14, 1480–1487. activity of chitosan–Zn complex. Carbohydr. Polym. 56, 21–26. Omari, K.W., Dodot, L., Kerton, F.M., 2012b. A simple one-pot dehydration process to Yang, Y., Hu, C.W., Abu-Omar, M.M., 2012. Conversion of glucose into furans in the

convert N-acetyl-D-glucosamine into a nitrogen-containing compound, 3- presence of AlCl3 in an ethanol–water solvent system. Bioresour. Technol. 116, acetamido-5-acetylfuran. ChemSuschem 5, 1767–1772. 190–194. Rinaudo, M., 2006. Chitin and chitosan: properties and applications. Prog. Polym. Zhao, H.B., Holladay, J.E., Brown, H., Zhang, Z.C., 2007. Metal chlorides in ionic liquid Sci. 31, 603–632. solvents convert sugars to 5-hydroxymethylfurfural. Science 316, 1597–1600.