Supporting Information

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1 Supporting Information Copyright Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 2012 An Integrated Approach for the Production and Isolation of 5-Hydroxymethylfurfural from Carbohydrates Svilen P. Simeonov, [a, b, c] Jaime A. S. Coelho, [a, b] [a, b] and Carlos A. M. Afonso* cssc_ _sm_miscellaneous_information.pdf

2 Supplementary Information Integrated Simple Approach for the Production and Isolation of 5-Hydroxymethylfurfural (HMF) from Carbohydrates Svilen P. Simeonov 1,2 Jaime A. S. Coelho 1 Carlos A. M. Afonso 1,3 Page Experimental Details 4 General procedure for the transformation of fructose to HMF using dimethylsulfoxide (DMSO) based on reported procedure 4 General procedure for the transformation of fructose to HMF and isolation using tolylsulfoxide 5 General procedure for the transformation of fructose (2 g scale) to HMF and isolation using ammonium salts as reaction media (Tables S1 and S2) 5 Procedure for the transformation of fructose (20 g scale) to HMF and isolation using tetraethylammonium bromide (TEAB) as reaction media and reaction media reuse (Table S3) 7 Procedure for the transformation of fructose (2 g scale) to HMF in 1:10 fructose/et 4 NBr ratio (w/w) and reaction media reuse 8 Procedure for the transformation of fructose (10 g scale) to HMF in 1:10 fructose/et 4 NBr ratio (w/w) 9 General procedure for the continuous transformation of fructose (1-5 g scale) to HMF using TEAB as reaction media 10 Procedure for the transformation of glucose to HMF using TEAB as reaction media 11 Procedure for the transformation of sucrose to HMF using TEAB as reaction media 12 Procedure for the transformation of inulin to HMF using TEAB as reaction media 13 Table S1. Selection of experiments for the transformation of fructose to HMF using ammonium salts as reaction media without catalyst 5 Table S2. Selection of experiments for the transformation of fructose to HMF catalysed by Amberlyst-15 using ammonium salts as reaction media 6 Table S3. Transformation of fructose (5 to 20 g scale) to HMF catalysed by Amberlyst-15 using TEAB as reaction media 8 Table S4. Catalyst and reaction media reuse for the preparation of HMF from fructose 10 Table S5. Continuous preparation of HMF from fructose using TEAB as reaction media 11 Table S6. Preparation of HMF from glucose using TEAB as reaction media 12 Figure 1. 1 H NMR spectra of HMF purified by column chromatography (98 % purity by HPLC) 13 1 imed.ul, Faculdade de Farmácia da Universidade de Lisboa, Av. Prof. Gama Pinto Lisboa Portugal. 2 Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Acad. G.Bonchev str., bl.9, 1113 Sofia, Bulgaria 3 CQFM, Centro de Química-Física Molecular and IN-Institute of Nanosciences and Nanotechnology, Instituto Superior Técnico, Lisboa, Portugal To whom correspondence should be addressed. carlosafonso@ff.ul.pt 1

3 Figure C NMR spectra of HMF purified by column chromatography (98 % purity by HPLC) 14 Figure 3. 1 H NMR spectra of commercial HMF (Aldrich Ref. H40807) (96% purity by HPLC) 14 Figure 4. Example of 1 H NMR spectra of HMF obtained from fructose in 2 g scale (Table S2, entry 18, 98% purity by HPLC) 15 Figure 5. 1 H NMR spectra of HMF obtained from fructose in 20 g scale (1 st cycle, 99% purity by HPLC) 16 Figure 6. 1 H NMR spectra of HMF obtained from fructose in 20 g scale (2 nd cycle, 97% purity by HPLC) 17 Figure 7. Example of 1 H NMR spectra of final crude reaction 18 Figure 8. Example of 1 H NMR spectra of HMF after precipitation of the TEAB 19 Figure 9. Example of 1 H NMR spectra of HMF after precipitation of the TEAB and filtration through silica 20 Figure 10. Example of 1 H NMR spectra of the TEAB after precipitation 21 Figure H NMR spectra of HMF obtained from fructose in 2 g scale ratio 1:10 (1st cycle, 100% purity by HPLC) 22 Figure H NMR spectra of HMF obtained from fructose in 2 g scale ratio 1:10 (2nd cycle, 98.7% purity by HPLC) 23 Figure H NMR spectra of HMF obtained from fructose in 2 g scale ratio 1:10 (3rd cycle, 99.5% purity by HPLC) 24 Figure H NMR spectra of HMF obtained from fructose in 2 g scale ratio 1:10 (6th cycle, 96.3% purity by HPLC) 25 Figure H NMR spectra of HMF solution before filtration through a pad of silica gel obtained from fructose in 2 g scale ratio 1:10 (6th cycle, 96.3% purity by HPLC). Here the crystallization is much better and almost no ammonium salt is presented 26 Figure H NMR spectra of HMF obtained from fructose in 10 g scale ratio 1:10 (99% purity by HPLC) 26 Figure 17. HPLC chromatogram of HMF purified by column chromatography using Kromasil 100, C18, 250x4.6mm (98% purity by HPLC) 27 Figure 18. Example of HPLC chromatogram of HMF obtained from fructose in 2 g scale using HICHROM C18, 250x4.6mm column (Table S2, entry 18, 98% purity by HPLC) 28 Figure 19. HPLC chromatogram of HMF obtained from fructose in 20 g scale using HICHROM C18, 250x4.6mm column (1 st cycle, 99% purity by HPLC) 29 Figure 20. HPLC chromatogram of HMF obtained from fructose in 2 g scale ratio 1:10 (1st cycle, 100% purity by HPLC) 29 Figure 21. HPLC chromatogram of HMF obtained from fructose in 2 g scale ratio 1:10 (2nd cycle, 98.7% purity by HPLC) 30 Figure 22. HPLC chromatogram of HMF obtained from fructose in 2 g scale ratio 1:10 (3rd cycle, 99.5% purity by HPLC) 31 Figure 23. HPLC chromatogram of HMF obtained from fructose in 2 g scale ratio 1:10 (4th cycle, 99.8% purity by HPLC) 32 Figure 24. HPLC chromatogram of HMF obtained from fructose in 2 g scale ratio 1:10 (5th cycle, 99.8% purity by HPLC) 32 Figure 25. HPLC chromatogram of HMF obtained from fructose in 2 g scale ratio 1:10 (6th cycle, 96.3% purity by HPLC) 33 Figure 26. HPLC chromatogram of HMF obtained from fructose in 2 g scale ratio 1:10 (7th cycle, 94.9% purity by HPLC) 34 Figure 27. HPLC chromatogram of HMF obtained from fructose in 2 g scale ratio 1:10 (8th cycle, 94 purity by HPLC) 34 2

4 Figure 28. HPLC chromatogram of HMF obtained from fructose in 10 g scale ratio 1:10 (99% purity by HPLC) 35 Figure 29. Photographs of the continuous apparatus 37 3

5 Experimental Details General: All reagents were purchased from Sigma-Aldrich, Alfa Aesar and Merck and have been used without further purification. Et 4 NBr Sigma cat kg (water content 1 % w/w), old (>15 years) and wet (average water content of 14 % w/w) Et 4 NBr from Laboratorios Azevedos Sociedade Industrial Farmaceutica Lisboa, Pr 4 NCl Alfa Aesar cat , Pr 4 NBr Alfa Aesar cat. A11522, Me 4 NBr Alfa Aesar cat. A13368, Me 4 NBr Alfa Aesar cat. A13368, Bu 4 NBr Aldrich cat , CholineCl Sigma cat. C g, Amberlyst-15 (wet) cat G, Fructose Merck extra pure and commercial grade from supermarket. HPLC analysis have been performed on Dionex P680 pump, Dionex UVD 340S diode array detector, detection at 275nm, manual injector with 20µl loop, column HICHROM C18, 250x4.6mm, R t (HMF) = 8.7 min or Kromasil 100, C18, 250x4.6mm. R t (HMF) = 10.7 min. Mobile phase gradient from 1:99 to 50:50 for 40 min acetonitrile:water, flow 1 ml/min, The purity of HMF was determined by comparing the obtained integration area of HMF with other observed minor peaks. The determination of water content in the ammonium salt was performed on Metrohm Karl Fisher coulometer (831KF coulometer Metrohm, 768KF oven Metrohm, 703TI stend Metrohm) equipped with oven: temperature for the analysis was 220 C. General procedure for the transformation of fructose to HMF using dimethylsulfoxide (DMSO) based on reported procedure: 4 To an round bottom flask equipped with an distillation apparatus (N 2 or evacuation can be also used to take out the produced water) was added 200 ml of dimethyl sulfoxide, 6 g of fructose and 0,4 g of Amberlyst-15 powders. The mixture was stirred at 120 ºC for 2.5 hours. After reaction, the catalyst was filter out and 190mL of dimethyl sulfoxide was distilled at 31ºC, 0.6 mbar (oil bath temperature: 60ºC, complete distillation led to HMF degradation) to obtain a dark orange oil which was finally purified by chromatography column to give light yellow liquid of HMF (3 g, 70%) with 98% purity by HPLC that crystalizes in freezer. General procedure for the transformation of fructose to HMF and isolation using tolylsulfoxide: To 3 g of melted tolyl sulfoxide at 120ºC was added 1 g of fructose and 0,1 g of 4 Ken-ichi Shimizu, Rie Uozumi, Atsushi Satsuma, Catalysis Communications, 2009, 10,

6 Amberlyst-15 powders. The mixture was stirred for 2 hours. After reaction, ethyl acetate was added, filtered through a pad of silica gel and the solvent evaporated to give a yellow solid. This solid was then recrystallized of diethyl ether/hexanes to give 2.7 g (90%) of tolyl sulfoxide as brown solid. Finally the HMF present in solution was purified by chromatography column to give brown liquid (197 mg, 28%) with 94% purity by HPLC. General procedure for the transformation of fructose (2 g scale) to HMF and isolation using ammonium salts as reaction media (Tables S1 and S2): Without catalyst: To 9.1g of Et 4 NBr (1% water content w/w) was added 0.9ml of water. The resulting mixture (10 g, 10% water content w/w) was mixed with 2g of fructose. The mixture was placed at 80 C and heat up to 100 C for 10 min. Then was stirred at 100 C for 15 min (no conversion) the reaction time was extended to 1.5h. The mixture was cooled down to r.t. and the water was evaporated. The resulting solid was washed with EtOAc (50 ml). The solvent was decanted and the solid was dissolved in hot EtOH (2 ml) then under vigorous stirring was added EtOAc (200ml). The resulting precipitated was filtered out and the combined solutions was filtered through a pad of silica gel (10 g) and evaporated to give brown liquid of crude HMF (0.7g, 50%) with 96% purity by HPLC. Table S1. Selection of experiments for the transformation of fructose to HMF using ammonium salts as reaction media without catalyst. a Entry initial water Pre-heated Time Pre-heated Final heated Time of final heated Yield Purity Reaction media (rm) content % (w/w) temp (ºC) temp (min) temp (ºC) temp (min/h) (%) b (%) 1 Pr 4N + Cl - c h 0 2 Me 4N + Br - c h 0 3 H 4N + Br + 1mL H 2O - c h 0 4 Et 4N + Cl -.H 2O c h 46 g 5 Choline chloride c h 51 g 6 Et 4N + Br - 14 d min 41 g 7 Et 4N + Br - 14 d min 61 g 8 Et 4N + Br - 14 d min 43 g 9 Et 4N + Br - 14 d min 79 g 10 Et 4N + Br - 14 d min 75 g 11 Et 4N + Br - e,c min h 12 i Et 4N + Br - i 10 f h h 13 Pr 4N + Br - c h h 14 Pr 4N + Br - c h h 15 Pr 4N + Br - c h h a All experiments were performed in a 1 g scale of fructose (commercial grade from supermarket) and fructose/ammonium salt ratio (w/w) of 1:5. b Isolated yield obtained by dissolution of the reaction mixture in ethanol followed by precipitation with ethyl acetate, filtration and removal of traces of ammonium salt by filtration with silica. c Used commercial sample of ammonium salt. d Old (>15 years) and wet (average water content of 14 % w/w) TEAB. e Old sample of Et 4 N + Br - (average water content of 14 % w/w w) dried under vacuum (< 1 mmhg, rt, 4-5 h). f 5

7 Determined by Karl Fisher on the commercial sample followed by addition of water. g Isolated HMF pure by TLC. h Purity of HMF determined by HPLC. i 2 g scale of fructose was used. In the presence of catalyst: To 9.1g of Et 4 NBr (1% water content w/w) was added 0.9ml of water. The resulting mixture (10 g, 10% water content w/w) was mixed with 2g of fructose and 0.2g of smashed Amberlyst-15 (10% w/w). The mixture was placed at 80 C and heated up to 100 C for 10 min. Then was stirred at 100 C for 15 min. The mixture was cooled down to r.t. and the water was evaporated. The resulting solid was washed with EtOAc (50 ml). The solvent was decanted and the solid was dissolved in hot EtOH (2 ml) then under vigorous stirring was added EtOAc (200ml). The resulting precipitated was filtered out and the combined solutions was filtered through a pad of silica gel (10g) and evaporated to give brown liquid of HMF (1.25g, 91%) in 97% purity by HPLC. For the tested salts that did not precipitate following the previous procedures (Table S2, entry 5) the HMF was isolated by short silica gel column chromatography using EtOAc as a mobile phase. Table S2. Selection of experiments for the transformation of fructose to HMF catalysed by Amberlyst-15 using ammonium salts as reaction media. a Entry Reaction media (rm) fructose / rm ratio (w/w) water content % (w/w) Amount catalyst (% w/w) Pre-heated temp (ºC) Time Preheated temp (min) Final heated temp (ºC) Time of final heated temp (min/h) Yield Purity (%) b (%) 1 Me 4N + Cl - 1:5 c min 0 2 Me 4N + Br - 1:5 c min 0 3 Me 4N + Br ml H 2O 1:5 10 c min 26 d 4 Et 4N + Cl -.H 2O 1:5 c h 78 d 5 Bu 4N + Cl - 1:5 c min 80 h d 6 Pr 4N + Cl - 1:5 c h 28 d 7 Choline 1:5 i c min 59 d 8 Et 4N + Br - 1:5 14 e min 64 d 9 Et 4N + Br 1:5 14 e min 71 d 10 Et 4N + Br - 1:5 14 e min 79 d 11 Et 4N + Br - 1:5 14 e min 80 d 12 Et 4N + Br - 1:5 14 e min 91 d 13 Et 4NBr 1:5 g 10 f min l 14 Et 4NBr 1:5 g 10 f min l 15 Et 4NBr 1:5 g 10 f min l 16 Et 4NBr 1:4 g 10 f min l 17 Et 4NBr 1:5 g 5 f min l 18 Et 4NBr 1:5 g 15 f min l 19 Pr 4N + Br - 1:5 c min l 20 Pr 4N + Br - 1:5 k c min l 21 Et 4NBr 1:10 g 10 f min l 22 Et 4NBr 1:20 j 10 f min l 23 Et 4NBr 1:20 j 10 f min l row 16 and 21 were the same (21 deleted) 6

8 a All experiments were performed in a 1 g scale of fructose (commercial grade from supermarket) and fructose/ammonium salt ratio of 1:5 and Amberlyst-15, unless stated. b Isolated yield obtained by dissolution of the reaction mixture in ethanol followed by precipitation with ethyl acetate, filtration and removal of traces of ammonium salt by filtration with silica. c Not determined by Karl Fisher, used commercial sample of ammonium salt. d Isolated HMF pure by TLC. e Old (>15 years) and wet (average water content of 14 % w/w w) TEAB. f Determined by Karl Fisher on the commercial sample followed by addition of water. g 2 g scale of fructose was used. h Isolated yield obtained by short silica column chromatography. i 5.6 g scale of fructose was used. j 5.0 g scale of fructose was used. k Used Fructose from Merck (extra pure grade). l Purity of HMF determined by HPLC. Procedure for the transformation of fructose (20 g scale) to HMF and isolation using TEAB as reaction media and reaction media reuse (Table S3): 1 st Cycle: To 91 g of Et 4 NBr (1% water content w/w) was added 9.0 ml of water. The resulting mixture (100 g, 10% water content (w/w)) was mixed with 20g of Fructose and 2g of smashed Amberlyst-15 (10w%). The mixture was placed at 80 C and heat up to 100 C for 10 min. Then was stirred at 100 C for 15 min. The mixture was cooled down to r.t. and the water was evaporated. The resulting solid was washed with EtOAc (200 ml). The solvent was decanted and the solid was dissolved in hot EtOH (10 ml) then under vigorous stirring was added EtOAc (1000ml). The resulting precipitated was filtered out and the combined solutions was filtered through a pad of silica gel (20 g) and evaporated to give brown liquid of crude HMF (12.9g, 92%) with 99% purity by HPLC. 2 nd Cycle: The recovered Et 4 NBr was recrystallized additionally from EtOH and ethyl acetate and dried for 5h under vacuum (rotatory pump, <1 mmhg) resulting in 86g. The amount of water was determined by Karl Fisher 2%, then 7.6 ml of water was added to achieve 10% water amount and 20g of fructose was added. The mixture was placed at 80 C and heat up to 100 C for 10 min. Then was stirred at 100 C for 15 min. The mixture was cooled down to r.t. and the water was evaporated. The resulting solid was washed with EtOAc (200 ml). The solvent was decanted and the solid was dissolved in hot EtOH (10 ml) then under vigorous stirring was added EtOAc (1000ml). The resulting precipitated was filtered out and the combined solutions was filtered through a pad of silica gel (20g) and evaporated to give brown liquid of crude HMF (12 g, 86%) with 97% purity by HPLC and NMR. 3 rd Cycle: The recovered Et 4 NBr was recrystallized additionally from EtOH and ethyl acetate and dried for 5h under vacuum (rotatory pump, <1 mmhg) resulting in 68g. The amount of water was determined by Karl Fisher 3%. 5.3 ml of water was added to achieve 10% water amount and 14g of fructose was added. The mixture was placed at 80 C and heat up to 100 C for 10 min. Then was 7

9 stirred at 100 C for 15 min. The mixture was cooled down to r.t. and the water was evaporated. The resulting solid was washed with EtOAc (200 ml). The solvent was decanted and the solid was dissolved in hot EtOH (10 ml) then under vigorous stirring was added EtOAc (1000ml). The resulting precipitated was filtered out and the combined solutions was filtered through a pad of silica gel (20g) and evaporated to give brown liquid of crude HMF (9.1 g, 93%) with 93% purity by HPLC. 4 th Cycle: The recovered Et 4 NBr was recrystallized additionally from EtOH and ethyl acetate and dried for 5h under vacuum (rotatory pump, <1 mmhg) resulting in 60g. The amount of water was determined by Karl Fisher 2%, then 31g of new Et 4 NBr was added together with 8.4 ml of water to achieve 10% water amount then 20g of fructose was added. The mixture was placed at 80 C and heat up to 100 C for 10 min. Then was stirred at 100 C for 15 min. The mixture was cooled down to r.t. and the water was evaporated. The resulting solid was washed with EtOAc (200 ml). The solvent was decanted and the solid was dissolved in hot EtOH (10 ml) then under vigorous stirring was added EtOAc (1000ml). The precipitation was not good forming gum type brown solid. It was filtered out and the combined solutions was filtered through a pad of silica gel (20g) and evaporated to give brown liquid of crude HMF (9g, 64%) with 91% purity by HPLC. Table S3. Transformation of fructose (5 to 20 g scale) to HMF catalysed by Amberlyst-15 using TEAB as reaction media. a Entry Fructose (g) fructose / Et 4NBr ratio (w/w) water content % (w/w) Amount catalyst (% w/w) Pre-heated temp (ºC) Time Preheated temp (min) Final heated temp (ºC) Time of final heated temp (min) Yield Purity (%) b (%) 1 5 1:5 14 c d :5 14 c d 3 5 1:5 10 e f :5 10 e (94 g ) [92 g ] 98(97 g ) [98 g ] f :10 10 e f a All experiments were performed in a 5-20 g scale of fructose (commercial grade from supermarket) and tetraethylammonium bromide containing water. b Isolated yield obtained by dissolution of the reaction mixture in ethanol followed by precipitation with ethyl acetate, filtration and removal of traces of ammonium salt by filtration with silica. c Old (>15 years) and wet (average water content of 14 % w/w w) TEAB. d Isolated HMF pure by NMR and TLC. e Determined by Karl Fisher on the commercial sample followed by addition of water. f Purity of HMF determined by HPLC. g Repetition of the experiment by another researcher. Procedure for the transformation of fructose (2 g scale) to HMF in 1:10 fructose/et 4 NBr ratio (w/w) and reaction media reuse. 1 st to 7 th Cycles: To 18.2 g of Et 4 NBr (1% water content w/w) was added 1.8 ml of water. The resulting mixture (10 g, 10% water content w/w) was mixed with 2g of fructose and 0.2g of smashed Amberlyst-15. The mixture was placed at 80 C and heat up to 100 C for 10 min. and 8

10 then stirred at 100 C for 15 min. The reaction was cooled down to r.t. and the water was evaporated. The water from the resulting solid mixture was evaporated. Then it was dissolved in hot EtOH (10 ml) and under vigorous stirring was added EtOAc (500ml). The resulting precipitated was filtered out and the solution was filtered through a pad of silica gel (10g) and evaporated to give HMF as orange oil. The collected Et 4 NBr mixed with Amberlyst-15 was dried under vacuum (4-5 h, rotator pump, <1 mmhg) and recycled using the same conditions for 7 times. The results for each cycle are provided in Table S4, entry 7. 8 th Cycle: The recovered Et 4 NBr and Amberlyst-15 from the seventh cycle was dissolved in 300ml MeOH, and 4g of charcoal was added. The mixture was heated with stirring until it started to boil and was immediately filtered through celite. The solvent was evaporated to give 14g of purified Et 4 NBr. To the purified Et 4 NBr (14g) was added 1.6ml of water. The resulting mixture (15.6 g, 10% water content w/w) was mixed with 1.6g of fructose and 0.16g of smashed Amberlyst-15. The mixture was placed at 80 C and heat up to 100 C for 10 min. and then stirred at 100 C for 15 min. The reaction was cooled down to r.t. and the water from the resulting solid mixture was evaporated. Then it was dissolved in hot EtOH (10 ml) and under vigorous stirring was added EtOAc (500ml). The resulting precipitated was filtered out and solution was filtered through a pad of silica gel (10g) and evaporated to give HMF as orange oil (1.38g, 123%) with 94% purity by HPLC. The observed 123% yield is due to the transformation of accumulated non-reacted fructose from the previous cycle which corresponds to combined yields of 7 th and 8 th cycles of 93%. Procedure for the transformation of fructose (10 g scale) to HMF in 1:10 fructose/et 4 NBr ratio (w/w). To 91 g of Et 4 NBr (1% water content w/w) was added 9 ml of water. The resulting mixture (100 g, 10% water content w/w) was mixed with 10g of fructose and 1g of smashed Amberlyst-15 (10% w/w). The mixture was placed at 80 C and heat up to 100 C for 10 min. and then stirred at 100 C for 15 min. The reaction was cooled down to r.t. and the water from the resulting solid mixture was evaporated. Then, the mixture was dissolved in hot EtOH (30 ml) and under vigorous stirring was added EtOAc (1500ml). The resulting precipitated was filtered out and the solution was filtered 9

11 through a pad of silica gel (20g) and evaporated to give HMF as orange oil (6.8g, 97%) with 99% purity by HPLC. Table S4. Catalyst and reaction media reuse for the preparation of HMF from fructose. a Entry cycle Fructose (g) Solvent water content % (w/w) Fructose / solvent ratio (w/w) Amount catalyst (mol% ou w/w%) Preheated temp (ºC) Time Preheated temp (min) Final heated temp (ºC) Time of final heated temp (min/h) Yield Purity (%) b % e Et 4N + Br - c 1: min d 1: min 82 nd d 1: min 88 nd d 1: min 88 nd d 1: min 76 nd d 1: min Et 4N + Br - 5f 1: min ml H 2O 2 1 Et 4N + Br - 5 g 1: min ml H 2O 3 1 Et 4N + Br - 5 g 1: min ml H 2O Pr 4N + Br - h 1: h Pr 4N + Br - g 1: h 53 i Pr 4N + Br - 3 j 1: min ml H 2O 2 1 Pr 4N + Br - 3 g 1: min 71 i ml H 2O Et 4NBr 10 k 1: min Et 4NBr 10 k 1: min Et 4NBr 10 k 1: min Et 4NBr 10 k 1: min Et 4NBr 10 k 1: min Et 4NBr 10 k 1: min Et 4NBr 10 k 1: min Et 4NBr 10 k 1: min Et 4NBr 10 k 1: min Et 4NBr 10 k 1: min Et 4NBr 10 k 1: min Et 4NBr 10 k 1: min Et 4NBr 10 k 1: min l 2 Et 4NBr 10 k 1: min a All experiments were performed in a 1-20 g scale of fructose (commercial grade from supermarket) and fructose/ammonium salt ratio of 1:4, 1:5 or 1:10 and Amberlyst-15 (unless stated). b Isolated yield obtained by dissolution of the reaction mixture in ethanol followed by precipitation with ethyl acetate, filtration and removal of traces of ammonium salt by filtration with silica. c Old sample of Et 4 N + Br - (average water content of 14 % w/w w) dried under vacuum (< 1 mmhg, rt, 4-5 h). d The recovered ammonium salt was dried under vacuum (< 1 mmhg, rt, 4-5 h) before next reaction. e Purity determined by HPLC. f Old sample of Et 4 N + Br - (average water content of 14 % w/w w) dried under vacuum (< 1 mmhg, rt, 4-5 h) followed by water addition. g The recovered ammonium salt was dried under vacuum (< 1 mmhg, rt, 4-5 h) followed by water addition before next reaction. h Used commercial sample of ammonium salt. i Isolated yield obtained by short silica column chromatography. j Used commercial sample of ammonium salt followed by addition of water. k Determined by Karl Fisher on the ammonium salt followed by addition of water. l The recovered TEAB was purified and added fresh Amberlyst-15 (10 %). nd Purity not determined. General procedure for the continuous transformation of fructose (1-5 g scale) to HMF using TEAB as reaction meda (Table S5): 10

12 A mixture of Et 4 NBr containing 25% water (w/w) and fructose was passed continuously through the glass reactor of 100 mm pathway and 10 mm diameter filled with Amberlyst-15 (3.5 g) heated at 100 C inside a domestic oven (Solac) and using a peristaltic pump (Ismatec Reglo) equipped with 0.8 mm silicon tube (see details of each experiment in Table S5). The water from the collected reaction mixture was evaporated using rotavapor and then rotatory vacuum pump (<1 mmhg). Then the solid was dissolved in a minimum amount of hot EtOH and the TEAB was precipitated with the addition of EtOAc. The mixture was filtered and the filtrate was evaporated. Table S5. Continuous preparation of HMF from fructose using TEAB as reaction media. a Entry Fructose (g) Fructose / solvent ratio (w/w) Flow (ml/min) 1 2g 1: d 2g 1: e 2g 1: g 1: g 1: g 1: g 1: g 1: a All experiments were performed by passing continuously a 1-5 g scale of fructose (commercial grade from supermarket), tetraethylammonium bromide (Et 4NBr) containing 25 % (w/w) of water, through a glass reactor containing Amberlyst-15 (3.5 g) heated at 100 C. b Isolated yield obtained by dissolution of the reaction mixture in ethanol followed by precipitation with ethyl acetate, filtration and removal of traces of ammonium salt by filtration with silica. c Purity of HMF determined by HPLC. d Reused catalyst inside the glass reactor from previous experiment. e Experiment performed by passing initially through the glass reactor containing Amberlyst-15 only the solvent (Et 4NBr containing 25 % (w/w) of water) at 100 o C for conditioning purpose followed by fructose dissolved in the solvent. Yield b (%) Purity (%) c Procedure for the transformation of glucose to HMF using TEAB as reaction media: To 9.1g of Et 4 NBr was added 0.9ml of water. The resulting 10g mixture with 10% water amount was mixed with 2g of glucose and 60mg of CrCl 3.6H 2 O. The mixture was placed at 80 C and heat up to 100 C for 10 min. Then was stirred at 100 C for 15 min. The mixture was cooled down to r.t. and the water was evaporated. The resulting solid was washed with EtOAc (50 ml). The solvent was decanted and the solid was dissolved in hot EtOH (2 ml) then under vigorous stirring was added EtOAc (200ml). The resulting precipitate was filtered out and the combined solutions was filtered through a pad of silica gel (10g) and evaporated to give brown liquid of crude HMF (500mg, 35%) with 82% purity by HPLC. To 9.1g of Et 4 NBr was added 0.9ml of water. The resulting 10g mixture with 10% water amount was mixed with 2g of Glucose and 200mg phosphomolibdic acid. The mixture was placed at 80 C 11

13 and heat up to 100 C for 10 min. Then was stirred at 100 C for 15 min and more 20 min at 120 C. The mixture was cooled down to r.t. and the water was evaporated. The resulting solid was washed with EtOAc (50 ml). The solvent was decanted and the solid was dissolved in hot EtOH (2 ml) then under vigorous stirring was added EtOAc (200ml). The resulting precipitated was filtered out and the combined solutions was filtered through a pad of silica gel (10g) and evaporated to give brown liquid of crude HMF (200mg, 15%) with 87% purity by HPLC. To 9.1g of Et 4 NBr was added 0.9ml of water. The resulting 10g mixture with 10% water amount was mixed with 2g of glucose and 670mg boric acid. The mixture was placed at 80 C and heat up to 100 C for 10 min. Then was stirred at 100 C for 60 min. The mixture was cooled down to r.t. and the water was evaporated. The resulting solid was washed with EtOAc (50 ml). The solvent was decanted and the solid was dissolved in hot EtOH (2 ml) then under vigorous stirring was added EtOAc (200ml). The resulting precipitated was filtered out and the combined solutions was filtered through a pad of silica gel (10g) and evaporated to give brown liquid of crude HMF (350mg, 26%) with 85% purity by HPLC. Table S6. Preparation of HMF from glucose using TEAB as reaction media. a entry water content % (w/w) catalyst 1 10 d Phosphomo Amount of catalyst (w/w%) Pre-heated temp (ºC) Time Preheated temp (min) Final heated temp (ºC) Time of final heated temp (min/h) 15 min Yield Purity (%) b (%) c libdic acid min 2 10 d Boric acid h d CrCl min 20 nd 4 10 d CrCl 3.6H 2O min a All experiments were performed in a 2.0 g scale of glucose and glucose /TEAB ratio (w/w) of 1:5 and catalyst. b Isolated yield obtained by dissolution of the reaction mixture in ethanol followed by precipitation with ethyl acetate, filtration and removal of traces of TEAB by filtration with silica. c Purity of HMF determined by HPLC. d Determined by Karl Fisher on the commercial sample followed by addition of water. nd Purity not determined. Procedure for the transformation of sucrose to HMF using TEAB as reaction media: To 9.1g of Et 4 NBr (1% water content w/w) was added 0.9ml of water. The resulting mixture (10 g, 10% water content w/w) was mixed with 2g of sucrose and 0.2g of smashed Amberlyst-15 (10% w/w). The mixture was placed at 80 C and heat up to 100 C for 10 min. Then was stirred at 100 C for 15 min. The mixture was cooled down to r.t. and the water was evaporated. The resulting solid was washed with EtOAc (50 ml). The solvent was decanted and the solid was dissolved in hot EtOH (2 ml) then under vigorous stirring was added EtOAc (200ml). The resulting precipitated was 12

14 filtered out and the combined solutions was filtered through a pad of silica gel (10g) and evaporated to give brown liquid of HMF (0.4g, 32%) with 90% purity by HPLC. Procedure for the transformation of inulin to HMF using TEAB as reaction media: To 9.1g of Et 4 NBr (1% water content w/w) was added 0.9ml of water. The resulting mixture (10 g, 10% water content w/w) was mixed with 2g of inulin and 0.2g of smashed Amberlyst-15 (10% w/w). The mixture was placed at 80 C and heat up to 100 C for 10 min. Then was stirred at 100 C for 15 min. The mixture was cooled down to r.t. and the water was evaporated. The resulting solid was washed with EtOAc (50 ml). The solvent was decanted and the solid was dissolved in hot EtOH (2 ml) then under vigorous stirring was added EtOAc (200ml). The resulting precipitated was filtered out and the combined solutions was filtered through a pad of silica gel (10g) and evaporated to give brown liquid of HMF (0.75g, 55%) with 98% purity by HPLC f1 (ppm) Figure 1. 1 H NMR spectra of HMF purified by column chromatography (98 % purity by HPLC). 13

15 f1 (ppm) Figure C NMR spectra of HMF purified by column chromatography (98 % purity by HPLC). Figure 3. 1 H NMR spectra of commercial HMF (Aldrich Ref. H40807) (96% purity by HPLC). 14

16 _sspt f1 (ppm) Figure 4. Example of 1 H NMR spectra of HMF obtained from fructose in 2 g scale (Table S2, entry 18, 98% purity by HPLC). 15

17 _sow029hmf f1 (ppm) Figure 5. 1 H NMR spectra of HMF obtained from fructose in 20 g scale (1 st cycle, 99% purity by HPLC). 16

18 _sow030hmf f1 (ppm) Figure 6. 1 H NMR spectra of HMF obtained from fructose in 20 g scale (2 nd cycle, 97% purity by HPLC). 17

19 _sow f1 (ppm) Figure 7. Example of 1 H NMR spectra of final crude reaction. 18

20 _sow f1 (ppm) Figure 8. Example of 1 H NMR spectra of HMF after precipitation of the TEAB. 19

21 _sow029hmf f1 (ppm) Figure 9. Example of 1 H NMR spectra of HMF after precipitation of the TEAB and filtration through silica gel. 20

22 Figure 10. Example of 1 H NMR spectra of the TEAB after precipitation. No remaining HMF detected. 21

23 f1 (ppm) Figure H NMR spectra of HMF obtained from fructose in 2 g scale ratio 1:10 (1st cycle, 100% purity by HPLC). 22

24 f1 (ppm) Figure H NMR spectra of HMF obtained from fructose in 2 g scale ratio 1:10 (2nd cycle, 98.7% purity by HPLC). 23

25 f1 (ppm) Figure H NMR spectra of HMF obtained from fructose in 2 g scale ratio 1:10 (3rd cycle, 99.5% purity by HPLC). 24

26 f1 (ppm) Figure H NMR spectra of HMF obtained from fructose in 2 g scale ratio 1:10 (6th cycle, 96.3% purity by HPLC). 25

27 f1 (ppm) Figure H NMR spectra of HMF solution before filtration through a pad of silica gel obtained from fructose in 2 g scale ratio 1:10 (6th cycle, 96.3% purity by HPLC). Here the crystallization is much better and almost no ammonium salt is presented f1 (ppm)

28 Figure H NMR spectra of HMF obtained from fructose in 10 g scale ratio 1:10 (99% purity by HPLC). Figure 17. HPLC chromatogram of HMF purified by column chromatography using Kromasil 100, C18, 250x4.6mm (98% purity by HPLC). 27

29 Figure 18. Example of HPLC chromatogram of HMF obtained from fructose in 2 g scale using HICHROM C18, 250x4.6mm column (Table S2, entry 18, 97.7% purity by HPLC). Figure 19. HPLC chromatogram of HMF obtained from fructose in 20 g scale using HICHROM C18, 250x4.6mm column (1 st cycle, 99.7% purity by HPLC). 28

30 Figure 20. HPLC chromatogram of HMF obtained from fructose in 2 g scale ratio 1:10 (1st cycle, 100% purity by HPLC). Figure 21. HPLC chromatogram of HMF obtained from fructose in 2 g scale ratio 1:10 (2nd cycle, 98.7% purity by HPLC). 29

31 Figure 22. HPLC chromatogram of HMF obtained from fructose in 2 g scale ratio 1:10 (3rd cycle, 99.5% purity by HPLC). 30

32 Figure 23. HPLC chromatogram of HMF obtained from fructose in 2 g scale ratio 1:10 (4th cycle, 99.8% purity by HPLC). Figure 24. HPLC chromatogram of HMF obtained from fructose in 2 g scale ratio 1:10 (5th cycle, 99.8% purity by HPLC). 31

33 Figure 25. HPLC chromatogram of HMF obtained from fructose in 2 g scale ratio 1:10 (6th cycle, 96.3% purity by HPLC). 32

34 Figure 26. HPLC chromatogram of HMF obtained from fructose in 2 g scale ratio 1:10 (7th cycle, 94.9 % purity by HPLC). Figure 27. HPLC chromatogram of HMF obtained from fructose in 2 g scale ratio 1:10 (8th cycle, 93.8% purity by HPLC). 33

35 Figure 28. HPLC chromatogram of HMF obtained from fructose in 10 g scale ratio 1:10 (99.3% purity by HPLC). 34

36 Empty glass reactor (top) and full (bottom) with Amberlyst-15 Glass reactor placed inside the domestic oven 35

37 Operating system outside the oven (top) and on the bottom the feed flask (right) and the collected reaction mixture (left). Figure 29. Photographs of the continuous apparatus 36