S1

Supporting Information for “Structural Analysis of Pyrolytic

Lignins Isolated from Switchgrass Fast Pyrolysis Oil”.

Michael Fortin1, Megan Mohadjer Beromi

1, Amy Lai

1, Paul C. Tarves

1, Charles A. Mullen

2,

Akwasi Boateng2, Nathan M. West

1*

1Department of Chemistry and Biochemistry, University of the Sciences, 600 South 43rd Street,

Philadelphia, Pennsylvania 19104, United States

2USDA-ARS, Eastern Regional Research Center, 600 E. Mermaid Lane, Wyndmoor, PA 19038,

United States

Table of Contents

Experimental Details ..................................................................................................................... S2

FTIR spectra of lignin samples ..................................................................................................... S4

Table S1. FTIR quantification of condensed structures................................................................ S4

Table S2. FTIR peak assignments ................................................................................................ S4

Table S3. Assignment of 13

C-1H correlation signals in the HSQC spectrum for polysaccharides

found in hemicellulose .................................................................................................................. S5

Table S4. Assignment of 13

C-1H correlation signals in the HSQC spectrum for lignin functional

groups ............................................................................................................................................ S6

DEPT 135 Spectra......................................................................................................................... S7

1H-

13C HMBC spectra................................................................................................................. S11

1H-

13C HSQC spectra .................................................................................................................. S16

GC-MS Chromatograms from [K][Mn(malonate)2(H2O)2] Oxidations ..................................... S19

Pyrolyzed Lignin Solvent Fractionation Diagram ...................................................................... S28

References ................................................................................................................................... S30

S2

Experimental Details

Production of switchgrass pyrolysis oil

The pyrolysis was conducted using the previously described reactor and methods.1-4

Carthage

switchgrass cultivar was the biomass feedstock for the pyrolysis. The biomass was ground in a

Wiley mill and sifted through minus 2-mm mesh before use. Fast-pyrolysis of switchgrass was

carried out in a bubbling fluidized bed of quartz sand at temperatures of ∼500 °C with a

residence time of <1.0 s. The system comprised a 7.5 cm (3 in.) diameter fluidized bed reactor

section, followed immediately by a cyclone to remove biochar. Pyrolysis vapors were condensed

by a series of four condensers cooled by cold water (∼4 °C); the remaining aerosols were

collected by electrostatic precipitators (ESP). The ESP fraction of the pyrolysis oil typically

contains most of the aerosols and pyrolytic lignins and hence was the fraction that was used for

isolation of the pyrolytic lignin described in this work.

Procedure for extracting lignin from biomass

The method is based on literature procedures.5-6

Ten grams of dried ball milled switchgrass was

placed in a 1 liter round bottom flask. To which was added 300 ml of dioxane: water 9:1 v/v

solution, containing 0.1 N hydrochloric acid. The flask was fixed with a reflux condenser and

refluxed under argon for 1 hour. The liquid was isolated by vacuum filtration, and neutralized

with 8 grams of sodium bicarbonate. The pH was check by litmus paper. The solution was

filtered, and concentrated to ¼ the original volume by rotary evaporation. The lignin was

precipitated from ice cold water, by adding drop-wise in 400 ml volume with rapid stirring. The

solids were filtered dried over phosphorous pentoxide, at 30 °C in a vacuum oven. Any lipids

present were extracted with by Soxhlet using hexanes as the solvent. Lignin extraction resulted

in 3.7 g, 37% efficiency.

Treatment of lignin under DFRC conditions

Based on the published procedure.7

(Step 1) Acetyl Bromide Derivatization. Acetyl bromide in acetic acid (1:9 v/v; 12.5 ml) was

added to 50 mg sample of lignin. The reaction was stirred at 50 oC for 3 h in a flask. The solvent

was then evaporated to dryness under reduced pressure.

(Step 2) Reductive Cleavage. The above residue was immediately dissolved in the acidic

reduction solvent (dioxane/acetic acid/water = 5:4:1 = v/v/v, 12.5 mL). Zinc dust (250 mg) was

added, and the mixture was stirred at room temperature for 30 min. Then reaction mixture was

then quantitatively transferred to a saturated ammonium chloride solution (50 mL) in a

separatory funnel using methylene chloride (20 mL). The aqueous layer was extracted with

methylene chloride (2 x 10 mL). The combined extracts were evaporated to dryness under

reduced pressure and placed into the desiccator, over P2O5, for 24 h. The final acetylation step of

S3

the standard acetylation step of the standard DFRC protocol was not carried out so that the

released phenols could be labeled with 31

P, and determine by NMR.

Oxidative cleavage using potassium permanganate

Based on the published procedure;8 40 mg of pyrolyzed lignin was suspended in 40 ml of tert-

butyl alcohol:water (3:1, v/v) in a 250 ml round bottom flask. Next 40 ml of 0.5 M sodium

hydroxide, 100 ml of 0.06 M sodium periodate, and 20 ml of 0.03 M potassium permanganate

were added in this order to start the reaction. The mixture was kept at a temperature of 82 oC for

6 h with vigorous stirring. The color of the reaction remained purple throughout the reaction so

no additional amounts of sodium periodate and potassium permanganate were required. The

reaction was quenched by the addition of 10 ml of ethanol added dropwise. The mixture was

allowed to stand for 20 minutes to allow the manganese dioxide, formed during oxidation, to

settle out. The solution was then filtered through a plug of silica gel to remove the manganese

dioxide, after which the plug was washed with 1% sodium bicarbonate to ensure all the lignin

oxidation products were filtered. The filtrate was extracted twice by 50 ml of diethyl ether,

which in turn was extracted with 15 ml of 1% sodium bicarbonate. The aqueous phases were

combined and adjusted to a pH of 6.5 with 9 M sulfuric acid. The solution was then evaporated

to 30 ml. The solution was then diluted with 20 ml of tert-butyl alcohol water (1:1, v/v), and 0.9

g sodium carbonate. To this solution was added 5 ml of 30% hydrogen peroxide. The solution

was then heated with stirring at 50 oC for 10 minutes. The reaction was quenched upon the

addition of 100 mg of manganese dioxide, after which the mixture was allowed to stand for 2

hours. Next the mixture was filtered through celite, and the filtrate was acidified to a pH of 2

with 9 M sulfuric acid. The solution was extracted with 50 ml of acetone:dichloromethane (1:1,

v/v) three times, and the organic layers were combined, and dried with anhydrous sodium

sulfate. The solvent was removed by rotary evaporation. The residue was suspended in

dichloromethane and derivatized with TMSCl.

Oxidative Cleavage using [K][(malonate)2(H2O)2]

A 10 ml Schlenk tube, equipped with stir-bar, was charged with 40 mg of pyrolyzed lignin and

3.53 mg (~5 mol %) [K][Mn(malonate)2(H2O)2] in 5 ml THF, or acetonitrile. The contents was

then exposed to one 1 atm of oxygen. The tube was sealed and was placed in an oil bath 80 °C

with vigorous stirring for 14h. After which the liquid was passed through a silica plug,

concentrated in vacuo. The residue was suspended in acetonitrile and injected into the GC.

S4

FTIR spectra of lignin samples

Table S1. FTIR quantification of condensed structures.

Wavenumbers F1 F2 F3 F4 Switchgrass

1510 cm-1

0.03060 0.03697 0.0136 0.345 0.4208

1595 cm-1

.03705 0.03727 0.0134 0.642 0.4238

Ratio 0.8256 0.99195 0.98529 1.00872 0.9929

Table S2. FTIR peak assignments.

Fraction 1 Fraction 2 Fraction 3 Fraction 4 Non pyrolyzed Assignments

3415 O-H stretch

3296 3898 3412 3316 3450 O-H stretch

1722 1715 1715 1713 1715 C=O stretch unconjugated ketones

1647 1612 1612 1634 1596 C=O stretch conjugated to aromatic

ring

1456 1516 1516 1456 1516 C-H deformations, asymmetric in –

CH3 and ..-CH2-

1319 1370 1370 1341 1382 Syringyl ring breathing with C-O

stretching

1228 1264 1261 1319 1261 Guaiacyl ring breathing with C-O

stretching

1120 1111 1111 1125 1099 C-H bomd deformation in syringyl

ring

1059 1067 C-O stretch sugar ring

1032 1049 1052 1041 1021 Aromatic C-H deformation plus C-O

deformation in primary alcohols

851 891 891 841 Out of plane C-H bend in aromatic

ring

A B C

S5

Table S3. Assignment of 13

C-1H correlation signals in the HSQC spectrum for polysaccharides

found in hemicellulose.

Chemical shift C/H (ppm) Assignment

58.9/3.6 -D-Xylp (C5/H5)

60.7/3.5 -D-Xylp (C4/H4)

62.6/3.4 -D-Xylp (C3/H3)

68.7/3.2 -D-Xylp (C2/H2)

76.4/3.2 -D-Xylp

79.6/4.1 -D-Xylp

99.2/5.2 4-O-MeGlcA anomeric

98.7/4.7 (1-4)--D-Glcp (C1H1)

102.7/4.3 (1-4)--D-Xylp anomeric

102.2/5.2 (1-4)--D-Araf anomeric

101.7/5.2 -D-Xylp Reducing end

114.6/5.0 -D-Xylp Reducing end

S6

Table S4. Assignment of 13

C-1H correlation signals in the HSQC spectrum for lignin functional

groups.

Chemical shift C/H (ppm) Assignment

20.7/2.1 Biaryl methylene

53.0/3.4 C-H Phenyl coumarin

53.5/3.07 C-H Resinol

55.6/3.7 OMe

62.5/3.7 (C-H) Phenyl coumarin

69.7/3.5 (C-H) Resinol

89.5/5.0 (C-H) Phenyl coumarin

104.1/6.5 (C2,6/H2,6) Syringyl

107.1/7.1 CStilbene C=O Syringyl

112.7/6.9 (C2,/H2) C=O Guaiacyl

115.5/5.8 (C-H) p- Coumaraylated ester

115/5.6 (C5/H5) in Guaiacyl

119.1/6.6 C=O C6/H6 in Guaiacyl

123.2/7.4 Phenolic ether linked ferulate ester

126.5/7.3 (CStilbene

129.4/7.3 (C2,/H2,) p-OH phenyl (cinnamyl alcohol)

129.4/6.8 (C6/H6)p-OH phenyl (cinnamyl alcohol)

129.4/5.2 (C2,6/H2,6) Coniferyl alcohol (allylic

alcohol)

132.1/7.7 (C2,6/H2,6) p-hydroxybenzoate (phenolic

OH free)

154.1/7.9 (C-H) Ferulate ester

S7

DEPT 135 Spectra

Switchgrass Lignin DEPT 135

S8

Pyrolytic Lignin DCM DEPT 135

S9

Pyrolytic Lignin EtOAc DEPT 135

S10

Pyrolytic Lignin Acetone DEPT 135

S11

1H-

13C HMBC spectra

Switchgrass Lignin HMBC

S12

Pyrolytic lignin F1 HMBC

S13

Pyrolytic lignin F4a HMBC

S14

Pyrolytic lignin F4b HMBC

S15

Pyrolytic lignin F4c HMBC

S16

HSQC Spectra

HSQC Fraction 1

S17

HSQC Fraction 2

S18

HSQC Fraction 3

S19

GC-MS Chromatograms from [K][Mn(malonate)2(H2O)2] oxidation of pyrolytic lignins.

Compound 10

S20

Compound 11

S21

Compound 12

S22

Compound 13

S23

Compound 14

S24

Compound 15

S25

Compound 16

S26

Compound 17

S27

Compound 18

S28

Extraction of pyrolyzed lignin, Part 1: Separation of crude pyrolytic lignin from bio-oil.

S29

Extraction of pyrolyzed lignin, Part 2: Separation of fractions 1-4 from hexane insoluble material

S30

References

1) Boateng, A. A.; Daugaard, D. E.; Goldberg, N. M.; Hicks, K. B. Ind. Eng. Chem. Res. 2007,

46, 1891.

2) Boateng, A. A.; Mullen, C. A.; Goldberg, N.; Hicks, K. B.; Jung, H.-J. G.; Lamb, J. F. S. Ind.

Eng. Chem. Res. 2008, 47, 4115.

3) Nsimba, R. Y.; Mullen, C. A.; West, N. M.; Boateng, A. A. ACS Sustainable Chem. Eng.

2013, 1, 260.

4) Nsimba, R. Y.; West, N.; Boateng, A. A. J. Agric. Food. Chem. 2012, 60, 12525.

5) Bjorkman, A. Nature 1954, 174, 1057.

6) Guerra, A.; Filpponen, I.; Lucia, L. A.; Saquing, C.; Baumberger, S.; Argyropoulos, D. S. J.

Agric. Food. Chem. 2006, 54, 5939.

7) Lu, F.; Ralph, J. J. Agric. Food. Chem. 1997, 45, 2590.

8) Gellerstedt, G. In Methods in Lignin Chemistry; Lin, S. Y., Dence, C. W., Eds.; Springer:

1992, p 322.