tcbiomass2015
November 2-5, 2015
Bioeconomy Institute
Quantitation of Sugar in Bio-oil
Patrick A Johnston, Marjorie Rover, and Robert C. Brown
Introduction
Results and Discussion
Pyrolytic sugars from the fast pyrolysis of biomass offer an alternative route to
obtaining fermentation substrates from cellulose. We have found that the amount of
water soluble sugar is less than the hydrolyzable sugar in bio-oil. We hypothesize
that unidentified oligosaccharides and/or polysaccharides are present in the
pyrolysis oils. Identifying and quantifying these compounds have been difficult due
to their high boiling points, low vapor pressures and challenges in separating and
measuring sugars in complex media like bio-oil. In this study, high performance
liquid chromatography (HPLC) has been employed to separate and quantify
complex carbohydrates in bio-oils and determine their total sugar content. Sugar
concentrations in the bio-oil are characterized according to water solubility,
hydrolysis conversion, and molecular size exclusion. The main water soluble sugars
found in bio-oils are cellobiosan, galactose, levoglucosan, and xylose. Hydrolyzing
the oligosaccharides and polysaccharides to monomeric units such as glucose and
xylose give an estimate of the total amount of sugars present in the bio-oil. Size
exclusion chromatography (SEC) techniques such as gel filtration chromatography
(GFC) and gel permeation chromatography (GPC) allow us to determine molecular
weights and characterize unknown oligosaccharides and/or other polysaccharides.
Hydrolyzable sugar content in bio-oil pyrolytic sugar
160 6.80 SR13-M SQ Build 3984 (230974)HYPERREZ 2-26-2015 PJ H2SO4 #34 [modified by meadlab]
µRIU
1-20150218-SF2 Sug ars Sample B
HPLC C18 Column with Diode Array Detector for Small Molecules
Phenol Selective Wavelength @ 254nm
RI_1
150
2,800
13
120
UV_VIS_2
WVL:254 nm
5
lu
1-20150218SF2 Condensed Pyrolytic Sug ar
-G
Wt. %
130
1
Sugars
3,000 6-24-15 #3 [modified by MSQ]
mAU
co
se
140
110
100
Glucose
39.28
2,600
90
80
70
2,400
60
50
to
l
-S
or
2
20
3
30
2,200
bi
e
8.10
-X
yl
os
Xylose
40
2,000
10
7.73
-10
-20
27.00
1,800
23
Sorbitol
0
min
27.25
27.50
27.75
28.00
28.25
28.50
28.75
29.00
29.25
29.50
29.75
30.00
30.25
30.50
30.75
31.00
31.25
31.50
31.75
32.00
32.25
32.50
32.75
33.00
33.25
33.50
33.75
34.00
-100
0.0
2
36
24
37
44
38
39
40
41
42
19
28
30
31
32
33
34
35
43
27
25
18
-Numerous peaks indicating the presence of small molecules such as pheno
1
200
7
8
9
10
400
20
4
3
600
11 12
14
800
21
17
6
1,000
26
1,200
29
16
1,400
15
Gel Filtration Chromatography displays lower
molecular weight sugars
22
1,600
min
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0
12.0
13.0
14.0
15.0
16.0
17.0
18.0
19.0
20.0
- Numerous peaks indicating the presence of small molecules such as phenols
Objectives
Conclusions
• Identify main sugar components in bio-oil stage fractions.
• Quantify total hydrolysable sugars in bio-oil.
• Determine if high molecular weight oligosaccharides and polysaccharides are
present in distinct stage fractions.
Materials and Methods
Pyrolysis Liquid Samples
a. Heavy ends of red oak bio-oil from 8 kg/hr fluidized bed pyrolyzer with
fractionating oil recovery system [1]
II. Acid Hydrolysis of bio-oil Samples
a. Bio-oil hydrolyzed with 400mM H2SO4 at 125C for 44 min [2]
b. Thermo Scientific HyperRez - 300 mm × 7.7 mm i.d, 8 μm XP Carbohydrate
and H+ Guard column
III. Water Soluble Sugars
a. Two Bio-Rad Aminex 87P - 300 mm × 7.8 mm i.d., 9 μm and Aminex deashing guard column
IV. Gel Filtration Chromatography
a. Two Agilent PL-aquagel-OH 20 - 300 mm × 7.5 mm i.d., 5 μm and PLaquagel gaurd column
V. Diode Array Detector for Small Molecules
a. Phenomenex EVO C18 Column – 250 mm × 4.6 mm × 5 μm
VI. Instrumentation
a. Thermo Scientific Ultimate 3000 High Performance Liquid Chromatograph
(HPLC)
VII.Software
a. Thermo Scientific Chromeleon (HPLC/GPC analysis)
Gel Filtration Chromatography displays mostly
levoglucosan and cellobiosan
I.
• GFC Chromatograms of pyrolytic syrups show no evidence of oligosaccharides.
Most of the pyrolytic syrup is comprised of levoglucosan and cellobiosan.
• HPLC results on the C18 column with DAD show many small molecular weight
compounds including phenolic monomers.
• GC-FID chromatographs display phenolic compounds and other small volatile
compounds that make up a large percentage of the remaining unknown
composition of the pyrolytic syrup.
• The differences in results between the amount of water soluble sugar and
hydrolysable sugar could be due to small amounts of unaccounted sugar alcohols
in water soluble fraction.
References
1. Rover, M.R., et al., Production of clean pyrolytic sugars for fermentation.
ChemSusChem, 2014. 7(6): p. 1662-8.
- GFC Results indicate bio-oil sugars contains mostly levoglucosan
and cellobiosan.
- Larger polysaccharides and oligosacharides are not present in the biooil
2. Helle, S., et al., A kinetic model for production of glucose by hydrolysis of
levoglucosan and cellobiosan from pyrolysis oil. Carbohydr Res, 2007. 342(16): p.
2365-70.
3. Johnston, P.A. and R.C. Brown, Quantitation of sugar content in pyrolysis
liquids after acid hydrolysis using high-performance liquid chromatography
without neutralization. J Agric Food Chem, 2014. 62(32): p. 8129-33.
1