The one

11.5.2015 MChem Advanced Project Keith Biggart
2012-13 Entry Cohort
DEPARTMENT OF CHEMISTRY
Advanced Research Project
Characterization of Cellulose Degradation Products in Pressurized Hot Water
prepared by:
Keith Biggart
Project Supervisor: Dr. Kari Hartonen
Date: 11/5/2015
Number of Words: 6489

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1. Acknowledgements
First and foremost, thanks must go to the Department of Analytical Chemistry,
University of Helsinki for inviting me to carry my Masters Research in Finland,
specifically to my project supervisor, Dr. Kari Hartonen, who helped me to plan and
carry out my research.
Thanks to Dr. John Slattery of the University of York for organizing the MChem
Year Abroad Program and Professor Brendan Keely for being my contact in York.
Finally, thanks to my family for all the support they have given me this
past year.

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Contents
1. Acknowledgements……………………………………………………………......2
2. Contents………………………………………………………………………………...3
3. Abbreviations…………………………………………………………………….......4
4. Abstract………………………………………………………………………………....6
5. Literature Review…………………………………………………………………..8
6. Introduction………………………………………………………………………....27
7. Experimental Section……………………………………………………………33
8. Results and Discussion
8.1. Residue and Precipitate Analysis………………………….45
8.2. FT-IR Analysis……………………………………………………...49
8.3. Dynamic Light Scattering Analysis………………………..55
8.4. ESI-MS Analysis……………………………………………………61
8.5. GC-MS Analysis…………………………………………………….73
9. Conclusion……………………………………………………………………………..85
10. References……………………………………………………………………………88

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3. Abbreviations
PHW – Pressurized Hot Water
PHWE - Pressurized Hot Water Extraction
FT-IR – Fourier Transform InfraRed
DLS – Dynamic Light Scattering
HPLC – High Performance Liquid Chromatography
ESI – Electrospray Ionization
ITMS – Ion Trap Mass Spectrometer
TOFMS – Time of Flight Mass Spectrometer
GC-MS – Gas Chromatography Mass Spectrometry
DMF – Dimethylfuran
MPa – Mega Pascals
VOC – Volatile Organic Compound
Kw – Ionic Product
5–HMF – 5-Hydroxymethylfurfural
𝜀 – Dielectric Constant
PTFE - Polytetrafluoroethylene
Pdi – Polydispersity Index
eV – Electron Volts
DCM - Dichloromethane
DP – Degree of Polymerization
LC-MS – Liquid Chromatography Mass Spectrometry
tR – Retention Time
MALDI – Matrix Assisted Laser Desorption Ionization

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CID – Collision Induced Dissociation
EI – Electron Ionization
DMF – Dimethylfuran
THF – Tetrahydrofuran

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4. Abstract
Lignocellulosic biomass is a potential renewable feedstock for the production of
biofuels and platform chemicals, reducing dependence on finite fossil fuels. Cellulose,
the major component of plant biomass, is a polymer with high chemical recalcitrance.
Therefore, effective treatments capable of degrading cellulose are a popular field of
research. In this thesis pressurized hot water extractions (PHWE) of cellulose were
conducted in dynamic and static mode at varying temperatures and catalytic conditions,
as an environmentally friendly method of degrading cellulose and extracting resulting
platform chemicals. FT-IR analysis, dynamic light scattering (DLS), HPLC-ESI-ITMS along
with LC-MSn, HPLC-ESI-TOFMS and GC-MS were used for qualitative characterization of
the extracts.
When extraction temperatures were increased to 250 and 300 ℃, the
percentage conversion of cellulose to PHW-soluble degradation products was 71 and 87
% respectively. Addition of metal catalysts in acidic conditions raised the percentage
conversion to over 95 %. FT-IR analysis confirmed alterations to the chemical structure
of the residual cellulose at higher temperatures. According to the DLS, the size of the
molecules within the extraction liquid decreased as the temperature increased. The
static extraction was more effective at degrading cellulose, improved further by the
addition of catalysts, notably acidic conditions coupled with metal catalysts such as
palladium on activated carbon (Pd/C). HPLC-ESI-MS identified useful glycosyl polymers
giving information on the degree of polymerization and accurate masses were
determined. The presence of platform chemicals, such as furfural, dimethylfuran (DMF)
and phenols were successfully verified via GC-MS analysis of samples processed at
higher temperatures. The methods used in this research offer an environmentally

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friendly approach to the degradation of cellulose and extraction of its PHW-soluble
degradation products.

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5. Catalytic Degradation of Cellulose into Platform Chemicals using
Pressurized Hot Water
Keith Biggart
Words - 2994

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Contents
1. Introduction
2. Pressurized Hot Water as an Extraction Solvent
3. “Green” Methods of Cellulose Treatment
4. Non-catalytic Cellulose Extraction with Pressurized Hot Water
5. Catalytic Extractions of Cellulose
5.1. Mineral Acids
5.2. Solid Acids
5.3. Carbon Dioxide
5.4. Basic Conditions
6. Platform Chemical Formation: Extraction of 5 – Hydroxymethylfurfural
7. Conclusion
8. References

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1. Introduction
The exploitation of finite fossil fuel resources to satisfy rising energy demands
will eventually result in major global energy shortages and serious environmental
damage. It is therefore imperative for the scientific community to explore potential
renewable sources of energy and valuable platform chemicals1,2. Naturally occurring
polymers, such as cellulose, make plant biomass an increasingly valuable feedstock,
showing considerable potential in reducing greenhouse gas emissions compared to
traditional sources of energy, such as coal and mineral oil3,4. Cellulose, in the form of
plant biomass, is the most abundant biopolymer on earth, with 720 billion tonnes
produced annually via photosynthesis5. Cellulose is a chemically recalcitrant
homopolymer consisting of linearly connected D-glucopyranose monomers held
covalently together via 𝛽-1,4-glycosidic bonds6, as shown here in figure 1.
However, its structure and characteristics make its use in more refined
industries, like platform chemical production, challenging. Cellulose is a homopolymer
consisting of linearly connected D-glucopyranose monomers held covalently together
via 𝛽-1,4-glycosidic bonds6. The structure is strengthened further by an extensive
network of intra- and intermolecular hydrogen bonds (figure 1) 7.
Figure 1: Structure of cellulose illustrating the array of Intra- and intermolecular
hydrogen bonds.
Glycosidic Bond

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Pressurized Hot Water (PHW) is defined as water at elevated temperatures,
from the boiling point (100 ℃) to the critical point (374 ℃, 22 MPa), whilst maintained
in its liquid state by applying suitable pressure8. Under these conditions, the dielectric
constant (strength of hydrogen bonds) decreases, lowering the polarity of the solvent.
As a result, PHW is viewed as a potential alternative to fossil fuel based VOCs9.Water in
its pressurized heated state has a significantly reduced pH due to an increase in the ion
product constant Kw. This increase in acidity has both positive and negative
connotations for PHW as an extraction solvent. Whilst aiding in the solvent’s ability to
degrade and solvate different compounds, it increases the corrosivity of the water,
damaging the equipment used10.
For the catalysis of cellulose degradation, the ability to dissolute the water
insoluble polymer is advantages. PHW at temperatures greater than 230 ℃ partially
dissolves cellulose, allowing homogeneous reaction conditions to form11,12.
Consequently, PHW and ionic liquids (many can dissolve cellulose) are fast growing,
“green” areas of research in this area. This literature review will discuss the catalytic
degradation of cellulose for the production of platform chemicals using PHW.
2. Pressurized Hot Water as an Extraction Solvent
In studies conducted by Plaza et al13 and Rababah et al14, extractions of total
phenols present from the herb rosemary were studied. Rababah’s research used
traditional methods of extraction. Using a solvent mix of commonly used polar organic
solvents (40 mL acetone:40 mL methanol:20 mL water:0.1 mL formic acid) the
rosemary was vortexed, heated (60 ℃) and finally sonicated to achieve homogeneity for
the extraction procedure. Plaza used a modern, “green” approach involving pressurized

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hot water extraction (PHWE). The extraction was completed in a closed reactor for 20
min at two different temperatures (100 and 200 ℃). For the determination of total
phenols present, both studies opted to apply the Folin-Ciocalteu assay, making the
results of the studies comparable. Rababah’s traditional extraction calculated total
phenolics at 92.5 mg/g of sample. Plaza, using the “green” extraction method calculated
156.93 mg/g of sample at 100℃. Limitations of comparison between the two studies
arise from differing extraction times and temperatures. However, it is a good example to
exhibit the potential for further development of PHW as an extraction technique.
4. Non-catalytic Cellulose Extraction with Pressurized Hot Water
PHW can act as a useful extraction medium for cellulose-derived products.
Studies reported have addressed the impact of a wide range of operational conditions,
such as reaction temperature, reaction time and reactor design. As discussed previously
in the review, the acidity of water increases as the temperature is increased with
pressure, which could catalyze the hydrolysis of the polymer. There is a large amount of
literature on noncatalytic cellulose degradation using PHW with notable contributions
coming from the groups of Minowa et al15, Adschiri et al16, and Sasaki et al17. It is difficult
to compare the results of these papers as the authors have used different reaction times
and temperatures with alternative reactor systems, i.e. static or dynamic reactors. For
example, in Minowa’s group, the extraction was conducted in a sealed autoclave over a
variety of reaction times. The study showed that the PHW successfully degraded the
cellulose into glucose with a yield of 18 %, as well as other water soluble products
(graph used unclear). The amount of glucose extracted was temperature dependent,
rising as the temperature increased before falling above 260 ℃. This result supports
that of the acid-catalyzed study discussed later; suggesting that a parasitic degradation

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pathway becomes significant above certain temperatures. The similarities seen in
results between dilute acid catalysis and PHW treatment suggests both processes
degrade cellulose by a single mechanistic pathway. In terms of the experimental
procedure, the study by Sasaki et al differs greatly from Minowa et al. As seen here in
figure 2, a flow reactor is used. This allows much shorter reaction times and a faster
heating rate as well as easy quenching, increasing the repeatability of the individual
experiments.
.
This study confirmed near complete cellulose conversion into hydrolysis products.
When comparing the different experimental setups, dynamic reactors (continuous flow)
offer advantages compared to static reactors. Superior temperature control by which
the heating unit or coil allows fast preheating to exact temperatures whilst also enabling
reactions with reduced residence times. The flow rate is controlled, increasing the
efficiency of the extraction and preventing incomplete extractions caused by solvent
Figure 2: illustrating the flow reactor Sasaki et al used in their hydrothermal
treatment of cellulose

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saturation in static systems8. The corrosion effects on the extraction cell are lessened,
lowering costs and increasing the opportunity for up scaling to industrial applications.
5. Catalytic Extractions of Cellulose
Reported studies on catalytic cellulose degradation of cellulose may be
categorized into two main groups, catalytic depolymerisation and catalytic cascade
reactions. Catalytic depolymerisation refers to the initial breakdown of the biopolymer
into simple sugars, such as glucose. These simple sugars can be fermented into
bioethanol, a renewable fuel source18. The catalytic cascade reactions are further
processes, such as hydrogenation or oxidation, creating access to a broad range of
platform chemicals19. The availability of efficient catalysts to achieve optimized cellulose
degradation is a crucial requirement for the future industrial implementations of these
processes.
5.1 Mineral Acids
Early research into the catalytic degradation of cellulose was focussed on simple
acid catalysis leading to hydrolytic cleavage of the glycosidic bond between the
anhydroglucose monomers. In 1945, Saeman et al20 confirmed the pseudo-first-order
kinetics cellulose undertakes when hydrolysed using dilute sulfuric acid. Following this
seminal research, other groups, such as Mok et al21 in 1992, studied the reaction
pathways of acid catalysed cellulose hydrolysis using filter paper as a biomass source.
Moks group used a semi-batch flow reactor that allowed sample removal as the reaction
progressed as well as complete effluent collection. The semi-batch technique used is
interesting as experimental procedures often use simple dynamic or static reactors. By
initially running water through the apparatus, Mok was able to control the temperature

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of the system before switching the feed solvent to the acid solution of known
concentration (0.05 % sulfuric acid by mass of sample). At 215 ℃, the group calculated
71 % glucose conversion. The reason for this lower-than-expected yield is discussed at
length in the paper, pointing to a hindering, parasitic reaction pathway in which the
hydrolysis of cellulose forms non-hydrolyzable oligomers as well as glucose. This
hypothesis is confirmed in a study by Malester et al22, who discovered the glucose yield
decreased when the reaction time was longer than 15 seconds. This extraction was done
in very acidic conditions (pH 0.42) so the emergence of subsequent reactions leading to
glucose degradation is unsurprising. However, in recent years, research has shifted
away from using sulfuric acid due to operating problems caused by equipment
corrosion and difficulties in catalyst regeneration from homogeneous reaction solutions.
5.2 Solid Acids
A “greener” approach to mineral acid catalysis is to use solid-state acid catalysts.
In cellulose treatment, literature is focused around activated carbons and metal
oxides/chlorides, offering Brønsted and Lewis centers as acid sites. For example, Peng et
al 23 studied the use of metal chlorides such as CrCl3 and AlCl3 for the conversion of
cellulose to levulinic acid in water with high conversion rates. Chareonlimkun et al24
studied the catalytic effects of metal oxides (TiO2 and ZrO2) on the conversion of
different biomasses in PHW. The fact that the solid acids are insoluble in the reaction
media allows for easy separation from the product mixture, however, their
heterogeneous nature may cause mass-transfer limitations. In a study conducted by
Onda et al25, the effect of sulfonated activated carbon on the hydrolysis of cellulose in
hydrothermal conditions was investigated. The particular catalyst was chosen not only
for ease of separation, but also thermal stability and surface area, important properties
to consider in hydrothermal technology. Although the paper is generally opaque, some

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very interesting findings were reported in terms of the stability of extracted glucose at
certain temperatures and the overall yield of glucose extracted via a “green” technique.
At 423 K, a total glucose yield of 40.5 % was reported. For pure activated carbon, the
yield was calculated at 6.4 % (figure 3), demonstrating the effectiveness of the added
sulfate groups on the surface of the catalyst in converting the cellulose. Furthermore, the
reporting that glucose is susceptible to degradation at temperatures exceeding 470 K in
hydrothermal conditions is significant for further research into the formation of
platform chemicals via cellulose extraction. Figure 3 illustrates the massive increase in
cellulose conversion when the activated carbon catalyst is used. The increase in glucose
production is excellent for platform chemical production and suggests the PHW alone is
ineffective in comparison.
Figure 3: Effects of different solid acid catalysts on the conversion of cellulose into
glucose and water-soluble byproducts25. (AC = Activated Carbon)

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5.3 Carbon Dioxide
A further interesting alternative to achieve acidic reaction conditions in PHW is
to supplement the water with carbon dioxide. Research in this area appears limited with
few papers available. At room temperature the solubility of CO2 in water is low,
however, as the temperature and pressure of the water increase to near-critical
conditions, the solubility markedly increases, forming an equilibrium in the water
(equation1).
The carbonic acid produced enhances the proton concentration in the water, ensuring a
catalytic effect. In a study conducted by Rogalinski et al.26 into the hydrolysis kinetics of
some biopolymers in PHW water, the effects of CO2 on the hydrolysis of cellulose is
investigated. The CO2 was added to the PHW via an HPLC pump to 100 % saturation.
The effects of the acidification of the water can be seen in figure 4.
CO2 + H2O H2CO3
H+ + HCO3
- 2H+ + CO3
2-
Equation 1: Equilibrium showing the acidification of water with CO2

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At
the lower temperature of 240 ℃ the glucose yield is increased from 7 to 12 % by the
addition of the CO2 catalyst. However, the effect of the catalyst lessens as the
temperature raises to 260 ℃ and the reaction time increases. This is reportedly because
the initial lowering of the pH by saturating the water with CO2 has good impact but with
increased temperatures, the pH of the water also decreases, numbing the effectiveness
of the catalyst. However, with increased yields at lower temperatures, CO2 in PHW is an
effective alternative in hydrolyzing cellulose into glucose and further platform
chemicals.
5.4 Basic Conditions
Treatment of cellulose under alkaline conditions is known to decrease the
crystallinity of the polymer27. This concept was investigated by Karagoz et al28 who
looked at hydrothermal treatment of woody biomass with alkaline solutions in a static
extraction system. The effects of 0.94 M solutions of Na2CO3, NaOH, KOH and K2CO3 on
Figure 4: Comparison of glucose via noncatalytic and CO2 catalysed cellulose
degradation.

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the liquefaction of the wood at 280 ℃ were studied with percentage conversion of
cellulose at 88.5, 86.0, 91.4 and 96.0 % respectively. When compared to noncatalytic
treatment, it is clear the alkaline solutions aid the degradation of the biomass. The pure
water sample converted only 58.3 % of the biomass to liquid products. Furthermore,
with increased yields in gaseous, water-soluble hydrocarbon and oil products, the
positive effects of the catalysts are clear to see. The increase in bio-oil yield is most
apparent from the data, which in some cases was 12 times greater than in neutral
conditions. Alkaline conditions were also shown to inhibit the formation of biochar,
thermally stable solid residue. This is consistent with the study by Minowa et al15 who
reported that an Na2CO3 catalyst significantly reduced the formation of char compared
to noncatalytic conditions.
In a relating study by Yin et al.29 the effects of base catalysis on the mechanistic
pathway of cellulose degradation was investigated in PHW. Its focus was on the
differences in composition of the bio-oil product depending on the initial pH of the
alkaline solvent. They documented that if the initial pH was >14, the final pH would also
be high, driving the alkaline degradation pathway with the formation of carboxylic acids.
If the initial pH was <13.5, the final pH of the extract would be acidic resulting from the
increased Kw of the water. This allows the acidic degradation pathway of cellulose to
compete with the alkaline pathway; resulting in 5-hydroxymethylfurfural (5-HMF)
production, figure 5. An alkali solution of pH 13 or 14 must be used to achieve
significant carboxylic acid production. Overall, the addition of alkali catalysts, like those
used in Karagoz’s study, are effective catalysts aiding cellulose degradation.

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6. Platform Chemicals: Extraction of 5 – Hydroxymethylfurfural
The majority of this literature review has been focused on green methods of
cellulose hydrolysis into glucose, because, to form useful platform chemicals, the
cellulose must first be hydrolyzed. In figure 6, the massive potential for the production
of platform chemicals from cellulose is portrayed.
Figure 5: Effects of alkalinity on hydrothermal conversion of cellulose into bio-oil. (A
= final pH values; B = main bio-oil components)29.

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Figure 6: possible derivations of glucose produced from cellulose into valuable platform
chemicals.66,79,80

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Some important uses of cellulose derivatives include ethylene glycol and glycerol as
polymer monomers; furan as a building block for chemical synthesis; and sorbitol, used
as a commercial sweetener and in medicinal treatments as a laxative30. Certain cellulose
derivatives are more in demand than others. The desire to be in a position to control
yields and product compositions has resulted in intense research efforts dedicated to
manipulating the cellulose degradation pathway in favour of certain platform chemicals.
5 – HMF is a versatile compound that can be synthesized from biomass.
Firstly cellulose in hydrolyzed to glucose; secondly the glucose is isomerized into the
less stable fructose isomer; and, finally dehydration of fructose to HMF31. HMF
represents a particularly attractive platform chemical as it may replace petroleum-
based fuel building blocks, and can be used as a starting chemical for biologically active
molecules based on its furan structure32.
Figure 7: 5-Hydroxymethylfurfural

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Entry Starting Biomass Solvent Catalyst Temperature (K) Time Yield (%) Reference
1 Fructose isopropanol NH4Cl 393 12 h 68.0 33
2 Fructose [BMim][BF4] Amberlyst-15 353 3 h 50.0 34
3 Fructose n/a DMSO 423 2 h 92.0 35
4 Fructose H2O HCl 458 1 min 54.0 36
5 Fructose H2O H3PO4 513 2 mins 65.3 37
6 Fructose H2O No cat 473 5 mins 21.5 38
7 Glucose DMSO Al(Otf)3 413 15 mins 60.0 39
8 Glucose THF (4.2 ml)/DMSO (1.8 ml) Sn-Mont 433 3 h 53.5 40
9 Glucose [Emim][BF4] SnCl4 373 3 h 61.0 41
10 Glucose [C4mim]Cl CrCl3 Microwave (400 W) 2 mins 91.0 42
11 Glucose H2O H3PO4 513 2 mins 30.0 37
12 Glucose H2O No cat 493 5 mins 6.2 38
13 Cellulose DMA NiCl2.H2O 393 3 h 32.0 43
14 Cellulose [C4mim]Cl CrCl3 Microwave (400 W) 2 mins 60.0 42
15 Cellulose [EMIM]Cl CuCl2 393 8 h 55.4 44
16 Cellulose H2O No cat 503 5 mins 7.0 38
17 Cellulose water-carbon dioxide binary
system (CO2 5.0 %)
CO2 523 30 mins 16.0 45
Table 1: Production of the platform chemical 5-HMF from a range of biomasses, catalysts and conditions.

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Table 1 demonstrates the wide variety of HMF synthesis options. In general
terms, fructose produces the highest yields, followed by glucose and finally cellulose.
This trend reflects the instability of the furanose ring of fructose, favouring the
dehydration to HMF. Furthermore, the need for additional isomerization and hydrolysis
steps for glucose and cellulose lower their overall HMF yields. This can be viewed by an
easy comparison between entries 10 and 14. Under the same conditions, glucose and
cellulose produce differing yields of HMF (91.0 and 60.0 % respectively). In many
studies, the temperature is kept below 500 K and the reaction time is kept short, such as
entry 5 in table 1. This is because at high temperatures and reaction times, the synthesis
of lactic acid becomes a competing pathway, reducing the yield of HMF. This has been
shown to be the case at temperatures upward of 530 K.
In many papers, ionic liquids were used as the solvent for HMF production,
utilizing its ability to solvate cellulose by disrupting the hydrogen bond network. Su et
al,44 (entry 15) dissolved metal chloride catalysts in ionic liquid prior to cellulose
treatment, collectively catalysing the transformation to HMF in excellent yield. When
taking an overall look at the solvents and catalysts used in the production of HMF, there
is an obvious focus on acidic reaction conditions with solid acid catalysts (SnCl4),
acidified ionic liquids, mineral acids, PHW and CO2 all studied. However, other solvents
used such as isopropanol and DMSO (entries 1 and 3) produce impressive HMF yields of
68.0 and 92.0 % respectively but are toxic and harmful to humans and the environment.
Shifts in future research towards “greener” solvents such as PHW are favoured.
The studies conducted by Daorattanachai et al38 (entries 6, 12 and 16) are
interesting as PHW is used as the reaction media without catalysis. The low yields of
HMF at 21.5, 6.2 and 7.0 % for fructose, glucose and cellulose feedstock respectively,
signify the importance of choosing an efficient catalyst. For example, the effectiveness of

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an acid catalyst in HMF production is illustrated on comparison between entries 4 and 6.
Upon addition of the hydrochloric acid catalyst, the HMF yield is more than doubled. The
final entry in the table can be compared to entry 16. The use of CO2 gas to increase the
acidity of the mixture and as a result increases the HMF yield compared to non-catalytic
conditions by 9.0 %. The development of the water-carbon dioxide binary system
coupled with a solid acid catalyst would potentially increase the HMF yield further.
7. Conclusion
PHW extraction of cellulose has been shown to hydrolyse cellulose with and
without a catalyst. The literature studied displays the wide array of catalytic strategies
used in current research. Unfortunately, the drawbacks currently related to the
technology may prevent PHW being utilized industrially as solvent. The added
corrosivity the preferred acidic conditions impart on the liquid makes it impractical in
terms of equipment longevity, affecting costs. Furthermore, the mass transfer
restrictions that result from the celluloses insolubility in water at low temperatures, one
reason why ionic liquids are studied extensively in this area as previously discussed.
Finally, a certain degree of technophobia will further inhibit this technology being
widespread.
Overall, this literature review has presented research demonstrating the
potential for the catalytic hydrolysis of cellulose into platform chemicals, than can
successfully exploit to most abundant biomass on the planet. Future research should be
focussed on the development of reaction conditions, extraction systems and catalysts
used to maximise the yields of valuable chemicals whilst remaining focused on the
“green” aspect of the chemistry. Proposing these alternative routes to chemical

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feedstocks are vitally important for the future reduction in the use of fossil fuels that is
so damaging to the environment.

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6. Introduction
The depletion of fossil fuel reserves and uncertainty surrounding the
petrochemical industry has increased the significance of the development of sustainable,
renewable energy sources1,2. Biomass has been identified as a potential alternative
feedstock to traditional crude oil products such as liquid fuels, organic solvents and
platform chemicals46. The most abundant biopolymer worldwide is cellulose; with
roughly 720 billion tonnes produced every year via photosynthesis5. Currently, cellulose
is mostly used to produce paper products, textile fibres and industrial grade celluose47.
However, this production only accounts for 0.5% of cellulose produced annually by
plants19. As such, the conversion of biomass resources offers a credible alternative to the
exploitation of petrochemicals.
Lignocellulose, a term encompassing the different polymers of a plants cell wall,
consists of three major components: cellulose, hemi-cellulose and lignin. In this study,
the focus was primarily on cellulose, the most abundant of these biopolymers. Cellulose
is a homopolymer consisting of linearly connected D-anhydroglucopyranose units held
covalently together by 𝛽-1,4-glycosidic bonds, as discovered by Haworth and Staudinger
in 193248,49. Each unit is rotated 180° to its adjacent monomers, forming the most stable
conformation. The repeating unit is cellobiose (figure 1).

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Figure 1: The overall structure of cellobiose, the repeating unit of cellulose. The 𝛽-1,4-
glycosidic bond is labeled.
Cellulose is distinguished by its rigid structure and high chemical recalcitrance, a result
of the strong glycosidic bonds as well as inter- and intramolecular hydrogen bonds
offering an extensive network of supramolecular support50. It is resistant to solvation in
the majority of common solvents, such as water or organic alternatives making
treatment difficult. However, cellulose is soluble in some ionic liquids, but these are
often toxic, so an environmentally friendly alternative solvent for cellulose treatment
would be a significant step forward in making the process for attractive for industrial
use51.
Green chemistry is becoming increasingly important in the design of chemical
processes, with the goal of minimizing anthropogenic damage to the environment.
Water is an alternative extraction solvent to non-environmentally friendly volatile
organic compounds (VOCs); it is non-toxic, safe, readily available and environmentally
benign.52 In this study, the effects of pressurized hot water (PHW) in the treatment of
microcrystalline cellulose were studied. PHW can be defined as water at elevated
temperatures, from above the boiling point (100 ℃) to its critical point (374 ℃), whilst
maintained in its liquid state by applying suitable pressure8,53. As the temperature is
increased, the dielectric constant of water decreases from 80 at 25 ℃ to 19 at 300 ℃
Glycosidic Bond

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(methanol, ε = 33 at 25 ℃), see figure 2. The dielectric constant is equivalent to the
polarity of the solvent.
Figure 2: Relationship between temperature and the dielectric constant of
water.54
The utilization of this property allows PHW to extract intermediate to low polarity
analytes that would not be possible at room temperature. As the temperature is raised
permittivity, surface tension and viscosity of PHW decrease, however, the diffusivity
increases8. These are desirable alterations in the characteristics of the solvent as they
allow greater penetration into the matrix, allowing an overall more efficient extraction.
Another important property of water that is altered with increased temperature
is the ionic product; Kw. Kw increases gradually with temperature (until rapid increase
when supercritical conditions are reached), causing the pH of the water to decrease,
resulting in acidic conditions. This is particularly useful for cellulose treatment as the
acid aids the initial hydrolysis of the cellulose polymer into shorter polymeric chains
and simple sugars21, see figure 3. PHW can act as the solvent, catalyst and reagent
simultaneously in acid hydrolysis cellulose degradation reactions55.
0
10
20
30
40
50
60
70
80
90
100
0 50 100 150 200 250 300
DielectricConstant(εr)
Temperature (℃)
Methanol (25 ℃)

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Figure 3: Acid hydrolysis mechanism of the cellobiose repeating unit of cellulose.
Early research into hydrothermal treatment of lignocellulosic material was
pioneered by Bobleter et al.56 in 1994, who focused on the mechanism of biopolymer
degradation in a range of conditions. This research was complemented by Sasaki17,
Adschiri16 and Minowa15 et al. who utilized the acidic properties of PHW with varying
parameters, producing high glucose conversion yields and some platform chemicals
such as 5-hydroxymethylfurfural (5-HMF). These studies convey the variety of reaction
conditions possible with varying parameters of temperature, extraction time and the
extraction system used. There are three possible methods of extraction performed by
PHW, dynamic, static (batch reactor) and semi-batch8. The dynamic extraction involves
two variables, temperature and flow rate, which can be optimized to produce an overall
more efficient extraction compared to static mode. Semi-batch extractions are useful as
they allow sampling of the extracts as the extraction is ongoing, as used by Mok et al.21
More recent studies have developed the use of catalysts to assist the degradation
of cellulose into water-soluble products. Most catalysts studied increase the acidity of
the conditions, for example, mineral acids22, metal oxides24 and CO226. Although
extractions under basic conditions are also known15,28. Pressurized hot water
extractions (PHWE) of cellulose produce a wide range of platform chemicals (figure 4)
and reaction conditions used in the surrounding literature aims to maximize the yields

31
of these chemicals. This study uses information from previous research on extraction
systems and effective catalysts to degrade cellulose into useful platform chemicals.
Figure 4: Diagram showing, after initial acid hydrolysis of cellulose polymer, derivatives
formed from glucose that have varying uses as platform chemicals.
The principle of this research has an overall focus on “green” chemistry, and so
the aims mirror this principle. The potential production of useful organic chemicals
purely from water and cellulose is a remarkable precedent. The overall aims of the
project were as follows:
 To setup apparatus suitable for PHW extractions.
 To conduct PHW extractions of microcrystalline cellulose under catalytic and
noncatalytic conditions.
 To determine the degree of degradation of the cellulose biopolymer via
analytical techniques.
 Use of dynamic light scattering (DLS), Fourier Transform Infrared Spectroscopy
(FT-IR), High Performance Liquid Chromatography-Mass Spectrometry (HPLC-

32
MS), HPLC-MS/MS and Gas Chromatography- Mass Spectrometry to obtain
qualitative information of the extracted compounds for their possible
identification.
 Compare and contrast different catalysts within the extraction method in an
attempt to optimize the extraction of particular chemicals.

33
7. Experimental
Figure 5: Overview of the entire experimental procedure. Showing the two separate
extraction techniques and the analytical techniques used to study the resulting
extraction liquids and solids.

34
PHW Extraction Apparatus
Figure 6 is a schematic of the PHWE system used in this research, consisting of a
pump (Jasco PU-980 HPLC Pump), an oven (Hewlett Packard 5890, 400 ℃ maximum), a
heating coil and a back pressure regulator (micro-metering valve)57. Stainless steel
capillary tubing (1/16 in. diameter, 0.16 cm) connected the pump to the gas
chromatograph oven. The capillary was coiled (preheater, 5.10 m length) to ensure
temperature equilibration of the solvent prior to entering the extraction cell.
Downstream of the extraction cell, a back pressure regulator was used to control the
pressure within the system and collection flask was installed for sample collection. All
connections, apart from to the extraction cell, were done by stainless steel nuts held in
place by stainless steel ferrules. Connections of the capillary to the extraction cell were
slip-free HPLC connectors, to allow convenient removal and replacement of the cell
between individual experiments.
Figure 6: PHWE apparatus flow diagram.

35
PHWE
There are two forms of PHWE: the dynamic extraction, as shown in figure 6, and
the static extraction (batch type extractions). The methods of these two PHWE
techniques differ greatly and so must be treated separately.
1. Dynamic PHWE
Non-Catalytic
Microcrystalline cellulose (0.500 g) (Alfa Aesar, Germany) was added to a
laboratory-made stainless steel extraction
cell with a volume of of 3 ml and diameter
of 1 cm with 10 𝜇𝑚 stainless steel frits at
both ends58. The cell was sealed by
screwing down the lid on top of a malleable
copper ring (see figure 7) and connected to
the PHWE capillary at both ends, and placed
into the oven. Distilled and deionized water
was used as the water source. The cell was
prefilled with water to test for leaks before setting the oven temperature. Temperatures
tested were 100, 150, 200, 225, 250 and 300 ℃ for 20 min at a flow rate of 2 ml/min and
a pressure of 200 bar. Collection of the extraction liquid started as the temperature
reached 100 ℃, however, the timer did not start until the particular extraction
temperature was reached. The extract was collected in a 50 ml volumetric flask and
removed before fresh water was pumped through the capillaries for a further 20
Figure 7: Extraction Cell58

36
minutes to clean the system. The residue (solid remaining in the extraction cell) and the
precipitate (solid suspended in the collected extraction liquid) were collected, dried
overnight, and accurately weighed.
Catalytic
A number of catalysts were used to increase the chemical activity of the PHW.
The temperatures applied for catalytic extractions were 100, 150, 200, 250 and 300 ℃.
For acidic conditions, H2SO4 (2.0 M, 10 𝜇𝑙) was added to 500 ml of distilled water (pH
4.0). The acidity of the resulting solution was dilute in order to avoid unnecessary
corrosion to the PHWE system. The method used was identical to the non-catalytic
extractions. For basic conditions, 2 solutions of different concentrations solutions were
used. To achieve a pH of 14, NaOH (1.0 M, 20 ml) was added to distilled water (380 ml).
To achieve pH 10 NaOH (0.1 M, 2 ml) was added to distilled water (500 ml). Again, the
overall method used was identical to the non-catalytic extractions.
Ecover (2Na2CO3.2H2O2), an environmentally friendly detergent was used as a
catalyst in both hetero- and homogeneous forms. At 25 ℃, the Ecover was relatively
insoluble in water, however, with sonication a solution of Ecover and water at room
temperature was formed. For the heterogeneous extraction, Ecover (5 g) was dissolved
in distilled water (1000 ml) by sonication. This solution (pH 10) was then utilized as the
extraction liquid using the same method as described previously. Extractions were
made difficult by bubbles from the H2O2 forming in the capillary connecting the water
source to the pump, causing the pumping efficiency to decrease considerably. For the
homogeneous catalysis, the Ecover (0.5 g) was loaded into the extraction cell with the
cellulose.

37
Catalytic conditions were also created by the addition of a small portion of CO2
liquid to the main water flow. The CO2 was added via a separate syringe pump (ISCO 260
D) and the two flows, water and CO2, were combined by a micovolume tee (Valco) prior
to the flow entering the oven to be heated in the coil (figure 8).
Figure 8: Directional flows of CO2 and water in the PHW - CO2 extractions.
The flow rate was split to maintain an overall flow of 2 ml/min (1.7 ml/min H2O, 0.3
ml/min CO2). The pressure was maintained at 200 bar; however, it fluctuated slightly
around this value. The method for the PHW - CO2 extractions was otherwise identical to
the one used in non-catalytic extractions. The PHW - CO2 dynamic extraction method
was also used in tandem with selected metal oxides catalysts (Fe2O3, SnO, Al2O3, RuO2,
PtO2 and Pd/C). The cellulose (0.5 g) was loaded into the cell as usual but the metal
catalyst (0.05 g, 10 %) was also loaded before mixing with a spatula.
Extraction liquids were stored at room temperature, and their colour was
recorded (see figure 9). Colours ranged from colourless to orange to brown. The
Water flow
(1.7 ml/min)
CO2
liquid flow
(0.3 ml/min)
Combined flow to
oven (2 ml/min)

38
samples had a sweet, caramel smell resulting from degradation of the cellulose into
sugars.
Figure 9: Colour change depending on extraction temperature of non-catalytic PHWE.
100 – 300 ℃ left to right.
2. Static PHWE
This method allowed the screening of a number of catalysts simultaneously.
Microcrystalline cellulose (0.01 g) was loaded in a Hastelloy reactor (corrosion resistant
Hastelloy C-22) of volume 3 ml and diameter 1 cm. Metal catalyst (0.001 g) was then
charged into the autoclave. Catalysts tested were Fe2O3 (Merck), SnO, ZnO, Al2O3, PtO2
(suppliers unknown), RuO2 (Koch-Light Industries), 10 % Pd on activated carbon and
La2O3 (Fluka AG). Distilled and ion exchanged water (0.5 ml) was then added to the
mixture and the contents of the reactor were stirred. A copper ring was placed around
the hole in the reactor and was then sealed by screwing down the lid of the Hastelloy
reactor, as shown in figure 10.

39
The reactor was then placed in the oven and set to a particular temperature (200, 250 or
300 ℃). The pressure within the cell could then be calculated using the Ideal Gas Law:
𝑃𝑉 = 𝑛𝑅𝑇 Equation 1
where: P = Pressure
V = Volume of the reactor
n = Number of moles of H2O
R = Gas Constant
T = Temperature
Table 1: Showing the pressure created inside the reactor at varying temperatures with
0.5 ml water added to the cellulose sample.
Temperature
(℃)
Pressure*
(bar)
Volume
(cm3)
Moles
(mol)
200 363 3.00 0.0277
250 401 3.00 0.0277
300 440 3.00 0.0277
* Effect of cellulose and catalyst on the volume and thus to the pressure was not taken into account which can lead to
slightly higher calculated pressure than is shown here.
Figure 10: Showing the extraction cell and copper sealing ring
prior to extraction.
Copper Ring
Hastelloy
Extraction Cell
Hastelloy Cell
Lid

40
The residual heating time of the reactor was calculated to be 30 minutes. As a result, the
extraction time used for the static experiment was 1 hour including the residual heating
time. After the extraction, the liquid extraction/reaction product (bio-oil) was removed
with a Pasteur pipette and placed into a 1 ml syringe with a polytetrafluoroethylene
(PTFE) syringe filter (VWR International) to remove any remaining cellulose and
catalyst. The samples were then placed in 1.5 ml vials and diluted by 0.5 ml distilled and
ion exchanged water ready to be analysed further. Extraction liquid colour was
recorded, however, the precipitate was discarded, as non-destructive filtration was not
possible with volumes so small. Extractions had a sweet, caramel smell. For
temperatures of 250 and 300 ℃, the initial extract was yellow for all samples, however,
when left over night, they turned orange/brown with a brown precipitate. Some
extractions produced some gases, released when the cell was open (PtO2 and RuO2 in
particular).
Analysis
Dynamic Light Scattering Analysis (DLS)
DLS analysis, using the Zetasizer Nano – S instrument (Malvern, UK), of
extraction samples was used to determine particle size within the samples with a 633
nm HeNe gas laser was used. Size range for this instrument was 0.6 nm – 6 𝜇𝑚
(Molecular Weight range 1000 – 2 x 107 Daltons). Sample (0.5-1.0 ml) was filtered by a
syringe filter to remove any remaining solid particles prior to being placed in a
disposable polystyrene cuvette for measurements. Each sample was measured with 6
repetitions (each consisting of 11 individual runs). Measurements were obtained at 25
℃ at a scattering angle of 173°. For samples with lower volumes, a micro-cuvette was

41
used. The Z-average diameter, intensity size distribution and polydispersity index (Pdi)
were calculated via the correlation function.
FT-IR Analysis
FT-IR analysis was conducted using the compact Bruker Alpha – P instrument
(Germany). A small amount of either precipitate or residue was placed on the sampling
area (attenuated total reflection, ATR, crystal) to be analyzed.
Direct Infusion ESI-ITMS
Direct sampling to ESI-MS was tested for the dynamic extracts on an ion trap
Bruker Esquire 3000+ mass spectrometer (Germany). Direct infusion of the extract into
the ESI-MS was conducted at 3 𝜇𝑙/min by a Hamilton HPLC syringe using an SP100i
syringe pump (WPI, USA). Nitrogen was used as the drying gas at 6.0 L/min at 300 ℃
and as the nebulizer gas with a pressure of 18 psi. Voltages used were: nebulizer end
plate 500 V, capillary exit 160 V and capillary 3500 V. The mass spectrometer was used
in positive ion mode with a scan range of 50 – 2000 m/z. Solvent mix was made up (80
% MeOH, 20 % H2O, 0.2 % formic acid) along with a small amount of lithium acetate.
Sample preparation: solvent mix (1 ml) was added to the extraction liquid (1 ml).
Samples were run twice, with and without the aid of the lithium acetate solution (2 𝜇𝑙).
HPLC-ESI-ITMS
No sample preparation was required other than decanting samples into vials
and placing them in the correct position of the HPLC autosampler. Hewlett-Packard
Series 1100 HPLC was used with an Atlantis dC18 (Waters, Ireland) column with 3 𝜇m

42
particle size and dimensions of 2.1 x 150 mm. Sample injection volume was 10 𝜇l with
flow rate 0.25 ml/min creating a back pressure of ~280 bar. The overall run time was
31 minutes, 27 minutes for the run and 4 minutes post run to allow the solvent ratios to
realign and to flush the column. The two solvent mixes used were MeOH with 0.1 %
acetic acid and H2O with 0.1 % acetic acid and their mixing ratios were optimized to
achieve desirable separation (figure 11).
Nitrogen was used as the drying gas at 7.5 L/min at 300 ℃ and as the nebulizer gas with
a pressure of 20 psi. Voltages used were: nebulizer end plate 500 V, capillary exit 160 V
and capillary 4000 V. The mass spectrometer was used in positive ion mode with a scan
range of 50 – 2000 m/z. HPLC-ESI-MS2 analysis was completed for the two most intense
m/z values of the original MS spectrum for each chromatographic peak.
HPLC-ESI-TOFMS
Analyses with HPLC-ESI-TOFMS completed to achieve accurate masses of
important analytes. An identical HPLC method and parameters were used with the
0
10
20
30
40
50
60
70
80
90
100
0 10 20 30 40
MixingRatiosofSolvents(%)
Time (mins)
Solvent Mixing Ratios
MeOH (%)
Solvent Mixing Ratios
H2O (%)
Figure 11: Eluent used in HPLC method.

43
TOFMS. However, a number of ESI-MS parameters were altered. The Bruker MicroTOF
(Germany) was the mass analyser. Nitrogen was used as the drying gas at 7.0 L/min at
250 ℃ and as the nebulizer gas with a pressure of 7.25 psi. Voltages used were:
nebulizer end plate 500 V, capillary exit 120 V and capillary 4200 V. For the TOFMS in
positive ion mode, the detector was calibrated for mass range 300 – 500 m/z and the
scan range used was 50 – 1000 m/z.
GC-MS
Sample preparation: sample (0.5 ml) was added to a 5 ml separating funnel. A
liquid-liquid extraction was conducted using dichloromethane (3 x 0.5 ml) (VWR
Chemicals, 99.8 %) and the organic phase (bottom phase) was recovered after each
extraction. After all three extractions were completed, the aqueous phase was discarded
and the organic phase (1.5 ml) was ready for GC-MS analysis. A Bruker Scion 436-GC
(Germany) was used with a triple quadrupole mass analyzer. The column used was
Agilent Technologies (USA) DB-1MS (100 % dimethylpolysiloxane) with parameters 30
m x 0.25 mm and a film thickness of 0.25 𝜇m. The carrier gas was helium, and the
column flow rate was 1 ml/min. The column was initially 50 ℃ for 4 min before rising to
250 ℃ at 15 ℃/min, see figure 12. The ionization technique used was electron impact
with an ionization energy of 70 eV. The triple quadrupole was operated in positive ion
mode at a scan range of 50 – 500 m/z. Sample injection volume was 1.0 𝜇l. The initial
injection was splitless at 250 ℃, however, after 2 minutes the split valve was opened (1 :
200). The spectral library used for identification was the National Institute of Standards
and Technologies (NIST).

44
Figure 12: Oven temperature programme used in the GC-MS analysis. Between minutes
4.00 and 17.33 the heating rate was 15 ℃/min.
0
50
100
150
200
250
300
0 5 10 15 20
Temperature(℃)
Time (mins)

45
8. Results and Discussion
8.1. Residue and Precipitate Mass Analysis
The cellulose conversion yield was calculated for dynamic extractions. The
amount of cellulose converted by the PHW can be calculated and comparisons made
between different catalysts. The percentage conversion was calculated using equation
217:
% 𝑐𝑜𝑛𝑣𝑒𝑟𝑠𝑖𝑜𝑛 =
𝑋0−𝑋
𝑋0
× 100 Equation 2
where: X0 = original mass of cellulose loaded into the cell
X = mass of residual cellulose
Residues were white powders up to 200 ℃ and changed from brown to black at 250 and
300 ℃ respectively as the level of charring increases. Precipitates were white at 200 ℃
and turned orange/brown at higher temperatures.

46
Table 1: The effect of the temperature and catalyst used on the % conversion and mass
of precipitate produced in dynamic PHWE.
Catalyst Temperature (℃) X0 (g) X* (g) % Conversion Precipitate Mass (g)
No Catalyst 100 0.5002 0.4866 2.7 -
150 0.5008 0.4880 2.6 -
200 0.4996 0.4731 5.3 0.0219
225 0.5016 0.4298 14.3 0.0306
250 0.4998 0.1432 71.4 0.0618
300 0.5015 0.0646 87.1 0.0790
H2SO4 100 0.5015 0.4926 1.8 -
150 0.5006 0.4835 3.4 -
200 0.5002 0.4777 4.5 0.0282
250 0.5004 0.1395 72.1 0.0585
300 0.4994 0.0694 86.1 0.0866
Dilute
NaOH
100 0.5009 0.4534 9.5 -
150 0.5008 0.4726 5.6 -
200 0.5003 0.4908 1.9 -
250 0.5006 0.1653 67.0 0.0317
300 0.5003 0.0436 91.3 0.0646
CO2 100 0.4998 0.5158 -3.20 -
150 0.5015 0.5154 -2.77 -
200 0.4994 0.4688 6.1 0.0195
250 0.5008 0.1885 62.4 0.0634
300 0.4998 0.0314 93.7 0.0754
Fe2O3 + CO2 250 0.5011 0.2343 53.2 0.0691
300 0.5002 0.0158 96.8 0.0915
SnO + CO2 250 0.5004 0.1154 76.9 0.0580
300 0.5013 0.0217 95.7 0.0530
Al2O3 + CO2 250 0.5003 0.2386 52.3 0.0521
300 0.4997 0.0237 95.3 0.0608
RuO2 + CO2 250 0.4997 0.2534 49.3 0.0415
300 0.4998 0.0000 101.2 0.0677
PtO2 + CO2 250 0.4999 0.1911 61.8 0.0388
300 0.4996 0.0000 102.1 0.0444
Pd/C + CO2 250 0.5014 0.1895 62.2 0.0176
300 0.5005 0.0002 100.0 0.0245
*For determination of X for the metal oxide and CO2 catalyzed extractions, it was assumed that all catalyst
remained in the cell after the extraction and so its mass was subtracted from the final residual mass value.

47
For non-catalytic PHWE percentage conversion of cellulose increases as the
temperature is increased. The relationship is non-linear with relatively high
temperatures (above 200 ℃) required to cause significant conversion of the biopolymer.
Figure 13: The effect of temperature on the percentage conversion of cellulose in dynamic PHWE.
(A = non-catalytic PHWE, B = catalytic PHWE). *% conversion values of < 0 were set to 0.
0
10
20
30
40
50
60
70
80
90
100
100 150 200 250 300
%Converionofcellulose
Temperature (℃)
H2SO4 Catalyst
Dilute NaOH Catalyst
CO2 Catalyst
B
0
10
20
30
40
50
60
70
80
90
100
100 150 200 250 300
%Conversionofcellulose
Temperature (℃)
A
0
10
20
30
40
50
60
70
80
90
100
100 150 200 250 300
%Converionofcellulose
Temperature (℃)
H2SO4 Catalyst
Dilute NaOH Catalyst
CO2 Catalyst
B
*

48
Between 225 ℃ and 250 ℃ the decomposition of cellulose into PHW-soluble products
increased with temperature, see figure 13. This is in concurrence with a study done by
Minowa et al.15 who reported a similar increase in cellulose conversion above 225 ℃.
The effects of temperature on the conversion of cellulose to degradation products lessen
as 100 % conversion is approached. The prevention of full cellulose conversions could
be due to the formation of char or other thermally stable products at high temperatures.
An identical trend was observed for the extractions conducted under catalytic
conditions with H2SO4, NaOH and CO2. However, it was expected that reactive conditions
created by the catalysts would increase the overall conversion of cellulose and start the
rapid decomposition at temperatures lower than 225 ℃, but these hypotheses did not
apply in these cases. At temperatures 100 and 150 ℃, the CO2 extraction showed an
increase in the mass of the residue. CO2 gas addition to the cellulose structure is unlikely
at these temperatures and the small addition to the mass is most likely an error
(variation) caused by drying.
For the CO2 PHWE with metal oxide catalysts, the percentage conversion of
cellulose appeared to increase at 300 ℃ compared to heterogeneously catalyzed (NaOH,
H2SO4, Ecover and CO2) extractions. It is possible that the acidic conditions created by
CO2 partially dissolve the metal oxides increasing their reactivity. Specifically for RuO2,
PtO2 and Pd/C, the percentage conversion of cellulose is almost 100 %. Negative residue
values resulting in over 100 % conversion could be caused by the formation of soluble
metal hydroxides causing the catalysts to be extracted out.
The precipitates were produced as the extraction liquid cooled. Trends in table
1 indicate that the mass of precipitate increases with the temperature. Precipitates
started to form in small amounts at 200 ℃ for heterogeneously catalyzed extractions
except for the dilute NaOH extraction. The values for precipitate mass were decreased in

49
the dilute NaOH extraction at 250 and 300 ℃. This evidence suggests that using NaOH as
a catalyst increases the solubility of the products in the extract. The extraction using
Pd/C and CO2 as the catalysts produced a noticeably small amount of precipitate when
compared to similar extractions (table 1).
8.2. FT-IR Analysis
FT-IR analysis was completed to determine if any structural changes occurred
to the cellulose by analyzing the residue and precipitate after PHWE. The FT-IR
spectrum of microcrystalline cellulose is shown in figure 14.
Figure 14: FT-IR spectrum of microcrystalline cellulose
The peak for the O-H stretch in the spectrum at 3332 cm-1 is a broad band that can give
information about the hydrogen bonds within the cellulose structure. The peak
Crystallinity Band
O-H stretch
C-H stretch
Absorbed
H2O
C-O stretch
Microcrystalline Cellulose spectra identification - Vmax (film)/cm-1 : 3332 (𝜈 O-H
alcohol), 2891 (𝜈 C-H), 1643 (H2O absorbed), 1427 (𝛿 CH2), 1366 (𝛿 C-H), 1313 (𝛿 O-
H), 1026 (𝜈 C-O; contains C-OH and C-O-C glycosidic bond).
C:Program FilesOPUS_65DataKeithMicrocrystalline Cellulose.2 Microcrystalline Cellulose Instrument type and / or accessory 11/02/2015
3332.15
2891.06
1643.29
1426.15
1365.83
1313.45
1158.63
1026.35
893.54
432.44
500100015002000250030003500
Wavenumber cm-1
30405060708090
Transmittance[%]
Page 1/1

50
identified at 1427 cm-1 as the –HCH and –OCH bending modes is known as the
“crystallinity band”5960. A decrease in intensity to this band signifies an overall decrease
in the crystallinity of the cellulose polymer. Finally, the peak seen at 1206 cm-1
incorporates the stretching modes of –COC and –CCO within the polymer61. Any changes
to the intensity of this peak should give an indication of rearrangements of the 𝛽-1,4-
glycosidic bonds within the polymer. Bands at 3332, 2891, 1426, 1365 and 894 cm-1 are
particularly sensitive to changes in crystallinity of the cellulose structure.
When no catalyst was used, the crystalline structure of cellulose stayed intact until 225
℃, with the only changes being peak intensities. However, for extractions done at 250
and 300 ℃, there were significant alterations to the FT-IR spectra, figures 15 and 16.
Figure 15: FT-IR of 250 ℃ no catalyst residue.
C:Program FilesOPUS_65DataKeithNo catalyst cellulose residue - 250.0 No catalyst cellulose residue - 250 Instrument type and / or accessory26/02/2015
3330.59
2903.53
1696.66
1312.35
1024.62
893.83
661.50
418.42
500100015002000250030003500
Wavenumber cm-1
7580859095
Transmittance[%]
Page 1/1
O-H stretch
C-H stretch
C=O stretch
C=C stretch
Crystallinity Band
C-O stretch
250 ℃ no catalyst residue - Vmax (film)/cm-1 : 3331 (𝜈 O-H alcohol), 2892 (𝜈 C-H),
1697 (𝜈 C=O), 1602 (𝜈 C=C aromatic), 1427 (𝛿 CH2), 1361 (𝛿 C-H), 1312 (𝛿 O-H),
1025 (𝜈 C-O; contains C-OH and C-O-C glycosidic bond).

51
C:Program FilesOPUS_65DataKeithNo catalyst cellulose residue - 300.0 No catalyst cellulose residue - 300 Instrument type and / or accessory26/02/2015
3372.11
2915.08
1697.60
1604.30
1209.44
794.36
448.51
500100015002000250030003500
Wavenumber cm-1
8284868890929496
Transmittance[%]
Page 1/1
Figures 15 and 16 show changes when compared to the microcrystalline cellulose
spectrum. The “crystallinity band” has decreased in intensity at 250 ℃ and disappeared
at 300 ℃. The O-H stretch has decreased in intensity, especially at 300 ℃, suggesting
alterations to the hydrogen-bonding network of the polymer. The two new bands
formed at 1698 and 1604 cm-1 have been assigned as C=O and C=C stretches
respectively. The resolution of these bands is poor at 250 ℃, but their intensities
increase as extraction temperature increases. Pastorova et al.62 and Sevilla et al.63
studied the FT-IR spectra of cellulose char at ranging temperatures, and found C=O and
C=C bands forming at temperatures greater than 220 ℃. This is surprising, as the
spectrum at 225 ℃ did not show these bands. The carbonyl stretch can be accounted for
by the presence carboxylic acids and ketones as a result of partial oxidation of the
300 ℃ no catalyst residue - Vmax (film)/cm-1 : 3372 (𝜈 O-H alcohol), 2926 (𝜈 C-H),
1698 (𝜈 C=O), 1604 (𝜈 C=C aromatic), 1029 (𝜈 C-O; contains C-OH and C-O-C
glycosidic bond), 794 (𝛿 C-H).
Figure 16: FT-IR of 300 ℃ no catalyst residue
C=C stretch C-O stretch
C=O stretch
O-H stretch C-H stretch

52
alcohol functional groups in cellulose, as researched by Lojewska et al.64 The band at
1209 cm-1 in both figures is still representative of C-O bonds, however, it has shifted
wavenumber at 300 ℃. Suggesting the pyranose structure still remains. Structures of
precipitates formed under non-catalytic conditions remained unchanged. This is
consistent with research by Sasaki et al.65 who studied precipitates formed in the
extracts of hydrothermally treated cellulose. They determined that the precipitates were
high degree of polymerization oligomers with a molecular structure similar to cellulose
itself.
It was hypothesized that upon the addition of an acid catalyst to the PHW
solvent, any structural changes to the cellulose would occur at lower temperatures. This
was not the case. The spectra for extractions at 200 ℃ for the H2SO4 and CO2 catalysts
were unchanged. This is most likely because relatively mild acidic conditions were used
to minimize corrosion of the apparatus. The residue at 250 ℃ with H2SO4 catalyst did
exhibit a decrease in intensity of the O-H stretch and increase in intensity to the C=O and
C=C bands, suggesting that the degradation of the polymer is more advanced at this
temperature with the aid of the H2SO4 catalyst (figure 17).

53
C:Program FilesOPUS_65DataKeith250 degrees H2SO4 residue.0 250 degrees H2SO4 residue Instrument type and / or accessory
C:Program FilesOPUS_65DataKeithNo catalyst cellulose residue - 250.0 No catalyst cellulose residue - 250 Instrument type and / or accessory
17/04/2015
26/02/2015
500100015002000250030003500
Wavenumber cm-1
7580859095
Transmittance[%]
Page 1/1
250 ℃ H2SO4 residue - Vmax (film)/cm-1 : 3332 (𝜈 O-H alcohol), 2919 (𝜈 C-H), 1698 (𝜈
C=O), 1607 (𝜈 C=C aromatic), 1021 (𝜈 C-O; contains C-OH and C-O-C glycosidic bond),
792 (𝛿 C-H).
CO2 was not able to contribute chemically to the structure of cellulose. However, for the
precipitates formed in the acidic conditions (H2SO4 and CO2), structural changes
occurred for extractions at 300 ℃ with the C=O stretch at around 1696 cm-1 displayed.
The Ecover catalyst had no particular effect on the crystalline structure of the cellulose.
When dilute NaOH was used as the catalyst, the residue at 250 ℃ was identical to that of
pure crystalline cellulose, contradictory to results for the non-catalytic extraction. This
is an anomalous result as NaOH can be used to aid the degradation of cellulose, as
proven by Karagoz et al.28 and Tan et al.66. However, at 300 ℃ significant structural
changes to cellulose were observed. The “crystallinity band” at 1427 cm-1 is not present,
as well as significant decrease in intensity of both the O-H and C-O bands at 3351 and
1201 cm-1 respectively.
O-H stretch
C-H stretch
C=O stretch
C=C stretch
C-O stretch
H2SO4 residue
No catalyst residue
Figure 17: FT-IR cellulose residues at 250 ℃. Non-catalytic and H2SO4 catalyzed
reactions.

54
For the CO2 PHWE with metal oxide, the FT-IR spectra are less reliable due to
high percentage conversion, the resulting precipitates were not separated from the
remaining catalyst and so residues were contaminated. In a number of spectra the
catalyst M-O bond stretches remained. For CO2 PHWE with metal oxide, the SnO, Fe2O3
and RuO2 catalysts were particularly effective at 300 ℃ in the degradation of the
cellulose based on a loss of intensity of the O-H stretch, see figure 18.
Figure 18: FT-IR of cellulose residue at 300 ℃ with Fe2O3 + CO2 catalysts.
C:Program FilesOPUS_65DataKeith300 degrees Fe2O3 + CO2 residue.0 300 degrees Fe2O3 + CO2 residue Instrument type and / or accessory17/04/2015
1697.24
1596.16
1225.82
522.85
435.00
500100015002000250030003500
Wavenumber cm-1
606570758085
Transmittance[%]
Page 1/1
O-H stretch C-H stretch
C=O stretch C=C stretch
M-O stretch
300 ℃ Fe2O3 + CO2 residue - Vmax (film)/cm-1 : 1697 (𝜈 C=O), 1596 (𝜈 C=C aromatic),
1226 (𝜈 C-O; contains C-OH and C-O-C glycosidic bond), 522 and 535 (𝜈 M-O).

55
0
500
1000
1500
2000
2500
100 150 200 250 300
ParticleSize(nm)
Temperature (℃ )
Figure 19: Particle size as a function of temperature for the sample extracts
processed with dynamic PHWE without catalyst. (The values used are an average of
the 6 measurements acquired per sample).
8.3. Dynamic Light Scattering Analysis
DLS analysis was performed to acquire information the degree of degradation to
the cellulose polymer. The presence of particles, such as dust, in the sample is to be
avoided as they effect the accuracy and repeatability of DLS analysis67. For the cellulose
PHW extracts, problems arose as the samples were too polydisperse (Zetasizer Nano-S
restricted to 3 different particle sizes per sample). Further difficulties were caused by
small amounts of precipitates forming in the sample after filtration. As a result, in a
number of samples the Pdi (polydispersity index) is high at around 1.00 (ideal value
< 0.50), increasing the potential error of the measurements, see figures 19 and 20.

56
Figure 20: Particle size as a function of temperature for the sample extracts
processed with dynamic PHWE with H2SO4 catalyst. (The values used are an average
of the 6 measurements acquired per sample).
0
100
200
300
400
500
600
700
800
900
1000
100 150 200 250 300
ParticleSize(nm)
Temperature (℃ )
Figure 19 shows that PHWE from 100 to 200 ℃ produced products that
decrease in particle size with temperature. At higher temperatures, range (200 to 300
℃), the particle size shows only small changes, with polymer degradation reaching its
maximum. Figure 20 shows a similar trend for the acid catalyzed PHWE. However, at
lower temperatures the particle sizes are smaller upon using the acid catalyst, reflecting
more efficient hydrolysis of the glycosidic bonds. The standard deviation for the 100
and 150 ℃ measurements are large for both processes, with high Pdi values of close to
1.00. This could be caused by the dilution effects of the dynamic PHWE method.
DLS analysis for the static PHWE products were more repeatable, with favorable
Pdi values between 0.01 – 0.5. Evidently, the static extractions produced more

57
concentrated samples. Results showed a decreased particle size with increasing
temperature as the biopolymer is degraded (table 2). The static batch-type PHWE
system allows simultaneous screening of different catalysts enabling comparisons of
particle sizes between products resulting from differing conditions. For non-catalytic
static extractions, a decrease in Z-average particle size was seen with temperature from
200 to 250 ℃. However, there was an increase in particle size from 250 to 300 ℃.
The majority of metal oxide catalyzed extractions (e.g. ZnO) lead to products that
showed consistent decrease in particle size with increased temperature was, as
displayed in table 2. The majority of extracted compounds in the ZnO samples have
diameters between 60 and 120 nm, see figure 21. A second peak becomes apparent for
extractions at 200 and 300 ℃, demonstrating the difference in product particle sizes
between the two temperatures.
Most of the particle size distributions are multimodal. The likelihood of multimodal
measurements increases with temperature, indicating the formation of a range of
degradation polymers and oligomers with varying degrees of polymerization (DP).
Figure 21: The DLS intensity as a function of size distribution of ZnO catalyzed PHWE
of cellulose at temperatures 200, 250 and 300 ℃.
200 ℃300 ℃
200, 250, 300 ℃
1

58
Comparing the particle size distribution of non-catalytic versus metal oxide catalysts
static PHWE it became apparent that the catalytic extractions were more successful in
decomposing the cellulose polymer. The Z-average values obtained at 200 ℃ were
generally much lower than in non-catalytic conditions, for example, for the SnO
catalyzed PHWE, Z-average was 125.8 nm with a low Pdi of < 0.5 compared to 748 nm in
non-catalytic conditions. At 300 ℃, under reactive conditions containing metal oxide or
Ecover catalysts, the Z-average was significantly decreased to 30 – 80 nm,
demonstrating the considerably increased reactivity for cellulose degradation.
In addition, PHWE products emerging from combined catalyst extractions such
as metal oxides coupled with acidified water (H2SO4, pH 4) were studied with DLS.
These results were compared with those obtained for pure water extractions to
determine the potential benefits of the coupled catalyst system. The results are
summarized in table 2.

59
Table 2: Changes in Z-Average particle size (nm) with varying catalysts and
temperatures in static PHWE.
Catalyst
Particle Size (nm)
200 ℃ 250 ℃ 300 ℃
No Catalyst 748.4 158.4 240.6
H2SO4 32.30 27.80 31.90
Fe2O3 85.60 17.50 33.40
Fe2O3 + H2SO4 43.60 18.00 201.7
SnO 125.8 52.40 61.90
SnO + H2SO4 39.80 26.50 34.40
Pd/C 1069 63.60 37.80
Pd/C + H2SO4 145.0 34.80 22.20
ZnO 116.5 51.90 44.60
ZnO + H2SO4 -* 41.00 45.50
PtO2 572.0 90.45 84.60
PtO2 + H2SO4 -* 30.10 16.50
*, value was not obtained for this measurement. Sample was either too polydisperse or its concentration
was not in the range of the instrument.
Clearly, the addition of H2SO4 facilitated the breakdown of cellulose for most extractions.
For example, Z-Average was significantly decreased at 300 ℃ from 84.60 to 16.50 nm
for the PtO2 extractions. Another feature of the data is the unexpected increase in Z-
Average from 250 to 300 ℃ for the Fe2O3, ZnO and SnO samples in the presence of
H2SO4. The increased product concentration of the static PHWE samples, may give rise
to multiple scattering (scattered light being re-scattered by other particles)68. Table 2
provides evidence for the effectiveness of Pd/C and PtO2 at high temperatures in
catalyzing the degradation of cellulose. When coupled with the acidified water, at 300 ℃,
the Z-Averages were only 22.20 and 16.50 nm respectively, suggesting that larger
polymer chains have been fully degraded into smaller products.

60
CO2 PHWE with Pd/C also showed promising DLS results (figure 22). The most
intense peak for this measurement is at 0.842 nm, with a second peak seen at 398 nm
(Pdi 0.228). At 0.842 nm, the cellulose polymer has been considerably degraded.
Actually, this catalyst combination showed the highest degree of degradation from the
DLS results. The increased degradation capacity of the Pd/C catalyst may result in its
partial solubility in the water/CO2 solvent.
Figure 22: Size distribution by intensity of a 300 ℃ Pd/C + CO2 catalyzed PHWE sample.

61
8.4. ESI-MS Analysis
Direct infusion ESI-MS was used to gain a more comprehensive understanding of
the degree of degradation of cellulose by changes to the overall mass range with
temperature. However, due to the large number of degradation products within each
sample, this technique proved not useful. Consequently, HPLC-ESI-MS was employed.
The product distributions obtained from HPLC-ESI-MS were similar for most of
the extractions conditions. Figure 23 shows a typical separation of the PHW-soluble
products, a result of the optimized LC-MS method. However, the ion trap mass spectra
for the chromatographic peaks obtained suffered from low-resolution, and so
identification of the compounds was difficult. Therefore, the samples were re-analyzed
by HPLC-ESI-MS/MS to obtain CID (collision-induced dissociation) spectra for the main
two fragment ions of each chromatographic peak. Increased number of scans required
for the MS2 data decreased the instrument response, limiting the amount of information
collected. In addition, HPLC-ESI-TOFMS was then used to acquire accurate masses for
selected chromatographic peaks. The response was further decreased so only the largest
peaks were detected. Without comprehensive MS2 and accurate mass data, identification
of the compounds was difficult.
The obtained MS information is summarized in table 3. Peak number 2, as seen
in the majority of chromatograms, represents a number of glucose oligomers, as is
evident from the mass difference of 162 m/Z (see figure 25). The presence of the
repeating 162 m/z mass unit is confirmed upon closer inspection of the product ions in
the MS2 spectrum of the most intense peak at 829.1 m/z, see figure 26.

62
Table 3: HPLC-ESI-MS data for dynamic extraction at 300 ℃ without catalyst.
Peak Number Retention Time (min) MS Masses (m/z)
MS/MS Masses (m/z)
(Precursor ion: Fragments)
Accurate Masses (m/z) Possible Structure
1 1.2 99.0, 162.9
99.0: 116.8, 134.7
162.9: 98.8, 176.7, 194.7
** -
2
1.5 527.1, 667.1, 689.1, 829.1, 991.2
829.1: 324.8, 486.8, 649.0, 811.0
667.4: 324.8, 486.8, 648.9
325.13025, 365.11636,
527.20955, 829.45533
Underivatised
Glycosyl Chain
(cellulose)
2.0 527.1,689.1, 829.1, 991.2 * ** -
3 4.3 1029 * ** -
4 5.0 975.1 * ** -
5 5.8 1017.2 * ** -
6 7.6 450.0 * ** -
7 7.9 645.5 * ** -
8
9.3 432.2 * **
Syringe Filter
polymer
contamination
10.1 476.2 * **
10.8 520.2 415.0, 503.1 **
11.2 564.3 459.2, 547.0 543.20521
11.7 608.3 546.9, 591.1 **
12.1 652.3 * **
12.3 578.2, 696.3 * **
12.7 622.3, 740.3 * **
13.0 666.3, 784.4 666.3: 649.1 **
13.3 710.3, 833.4 * **
9 16.1 337.1 248.9, 292.9, 334.9 ** -
10 17.2 752.4, 796.4 489.9, 584.7, 687.1, 731.0 ** -
11 22.5
512, 556, 605, 644,
688, 732, 781, 820.5
803.7: 413.1, 801.4 ** Polymer Chain
*Low sensitivity. Caused by the large increase in the number of scans thus shortening the detection time allowed for each mass in the HPLC-ESI-
MSMS.
**Low sensitivity. TOF instrument appeared to be less sensitive compared to the ion trap. Furthermore, when measuring masses more accurately,
the sensitivity will be decreased.

63
Figure 23: HPLC-ESI-MS chromatogram of the PHWE of cellulose at 300 ℃ without catalyst (zoomed)
Figure 24: PHWE of cellulose at 300 ℃ without catalyst cellulose PHWE
1
2
3 4 5
6 7
8
9
10
11

64
288.9
324.8
450.9
486.8
649.0
667.0
811.0
+MS2(829.5),1.5min#53
0.0
0.5
1.0
1.5
5x10
Intens.
200 400 600 800 1000 m/z
162 m/z
162 m/z
162 m/z
162 m/z
85.1 162.9
325.0
527.1
599.1
689.1
829.1
991.2
+MS,1.5min #150,Background Subtracted
0
2
4
6
5x10
Intens.
200 400 600 800 1000 1200 1400 1600 1800 2000m/z
Figure 25: ESI-MS spectrum of peak 2 for the PHWE sample at 300 ℃ without catalyst. (m/z values
are listed in table 6).
162 m/z
162 m/z
162 m/z
Figure 26: ESI-MS2 spectrum of peak 829 m/z. Peak masses are listed also in table 1.

65
This observed mass difference corresponds to a single glycosyl unit, monoisotopic
atomic mass 162.052822 Da (see figure 27)69, representing the
building block of the cellulose polymer, held together by the 𝛽-1,4-
glycosidic bonds. The observation of short glycosyl oligomers is
consistent with the successful hydrolysis of cellulose, following an
acid catalyzed mechanism. The mass, and consequently, the degree
of polymerization (DP) of these glucose oligomers may be
calculated using equation 370:
𝑀𝑎𝑠𝑠 𝑜𝑓 𝐶ℎ𝑎𝑖𝑛 = 𝑀𝑎𝑠𝑠 𝑜𝑓 𝐺𝑙𝑢𝑐𝑜𝑠𝑒 𝑀𝑜𝑛𝑜𝑚𝑒𝑟 + (𝑛 × 𝑀𝑎𝑠𝑠 𝑜𝑓 𝐺𝑙𝑦𝑐𝑜𝑠𝑦𝑙 𝑀𝑜𝑛𝑜𝑚𝑒𝑟)
Equation 3
Closer inspection of the MS spectrum of peak number 2 reveals that the peaks are split
into 4 signals, all of them representing the 5th degree of polymerization71. Figure 28
displays the assignment of the peaks in the sugar chain. The peak at 829.1 in the
spectrum was calculated by equation 3 using monoisotopic masses as 180.063393 + 4 x
162.052822 = 828.27467 m/z. , indicating that the observed species exists as the
Figure 27: Glycosyl unit.
811.1
829.1
847.1
851.1
867.1
869.1
871.2
+MS, 1.5min #156
0
1
2
3
4
5x10
Intens.
780 800 820 840 860 880 900 m/z
[5M2 + H]+
[M1 + 4M2 + H]+
[M1 + 4M2 + Na]+
[2M1 + 3M2 + Na]+
Figure 28: Zoomed ESI-MS spectrum of peak 2 of the PHWE sample at 300 ℃ (no
catalyst). Peaks at 811.1, 829.1, 851.1 and 869.1. M1 and M2 represent glucose and
glycosyl respectively.

66
protonated species. Using this information, the degrees of polymerization of the
oligomers, see table 4.
Table 4: The degree of polymerization and the chain composition for the most intense
fragments from the ESI-MS spectrum for peak 2.
Fragment Ion (m/z) Chain Composition Degree of Polymerization
162.9 [M2 + H]+ 1
325.0 [2M2 + H]+ 2
527.1 [M1 + 2M2 + Na]+ 3
689.1 [M1 + 3M2 + Na]+ 4
829.1 [M1 + 4M2 + H]+ 5
991.2 [M1 + 5M2 + H]+ 6
The oligomers formed have a similar molecular structure to cellulose itself. This
is consistent with results from FT-IR analysis, which identified that precipitates found in
the extract had the same structure as the original polymer. The oligomers of high
molecular weight precipitate out of solution, whilst the smaller oligomers remained
solubilized in the water, and their DP was determined from the mass spectra. In a
similar study, conducted by Sakaki et al12, DP as high as 23 was reported from MALDI-
TOFMS analysis of 295 ℃ PHWE of cellulose. The softer ionization technique used would
prevent the fragmentation of the oligomers, allowing more efficient analysis of polymers
with higher DP.
The most intense peak, number 11 as seen in the chromatogram in figure 24, is
most likely represents larger, unresolved oligomers with wide molecular weight
distributions. The MS spectrum corresponding to peak number 11 is given in figure 29.

67
The mass difference seen in this spectrum is characteristic of carbon dioxide loss (m/z
44).
Similar polymeric series of mass peaks with mass difference 44 m/z were seen
for chromatographic peak number 8 (tR = 9.3-13.3 min). After further investigation, it
was discovered that these peaks arose from contamination from the syringe filter used.
The filters are made from polytetrafluoroethylene (PTFE), so the mass difference of 44
m/z between peaks was unexpected. It was discovered that after washing the filter with
1 ml of distilled water, the contamination was avoided.
The effects of temperature on the product distribution were evident in the
chromatograms, and summarized for the static extractions in table 5. For the products
emerging from the extraction at 200 ℃, a large number of peaks were observed between
retention times 0.8 and 3.4 min. Peak number 2 is also seen in figure 30, representing
the glycosyl oligomers with DP 1-6. The 300 ℃ PHWE chromatogram has more activity.
Figure 29: MS spectrum of peak number 11.

68
Table 5: Retention times and mass spectra data for 200 and 300 ℃ static PHWE.
Peak
Number
Temperature
(℃)
Retention
Time (mins)
MS Masses (m/z) MS/MS Masses (m/z) Accurate Mass (m/z) Possible Structure
12
200
0.8 202.9, 98.9 * ** -
13 1.5 164.7, 98.9 * ** -
2 2.0
847.1, 685.0, 504.9, 364.9,
324.9, 202.8, 162.8
* 527.17525, 365.10202
Underivatized Glycosyl Chain
(cellulose)
14 2.9 194.9, 108.9 * ** -
15 3.4 578.2, 534.2 * ** -
-
1
300
1.2 99.0 * ** -
16 1.9 499.0 *
433.00021, 515.02423,
597.03405,
679.04088, 761.11757
-
17 2.0 499.0, 375.0 * ** -
18 2.5 1832.9 * ** -
19 4.1 192.9, 165.0, 91.2
192.9 : 164.8
165.0 : 146.8, 118.9, 93.0
** -
20 5.4 220.9, 160.9
220.0 : 202.7, 160.8, 122.9
160.9 : 535.2, 236.7, 194.8, 132.9,
98.9
** -
21 7.4 177.0 177.0 : 158.8, 130.9, 104.9 ** -
22 8.2 192.9, 174.9, 147.0, 91.2
192.3 : 174.8, 146.9
174.9 : 146.8, 118.9
** -
23 10.0 481.1 * ** -
9 15.9 337.1 334.8, 292.9, 248.9, 207.8, 160.7 337.20204 C19H29O5
*Low sensitivity. Caused by the large increase in the number of scans thus shortening the detection time allowed for each mass in the HPLC-ESI-MSMS.
**Low sensitivity. TOF instrument appeared to be less sensitive compared to the ion trap. Furthermore, when measuring masses more accurately, the sensitivity
will be decreased.

69
Figure 31: 300 ℃ static PHWE chromatogram
1
16
12
17
12
18
12
19
20
21 22
23
9
Figure 30: 200 ℃ static PHWE chromatogram
12
13
2
14
12
15
12

70
Three intense peaks, 18, 19 and 9 at retention times 4.1, 5.4 and 15.9 min
respectively, are not observed at the lower temperatures. However, despite good HPLC-ESI-
MS2 data, the molecular formulas for these compounds could not be assigned. For example,
peak number 19 has an intense MS peak at 165.0 m/z, further CID produced three peaks,
146.8, 118.9 and 93.0 m/z with mass losses of 18, 28 and 26 m/z respectively. The 18 m/z
is the loss of water and the 28 m/z could potentially be a loss of a carbonyl although this
analysis cannot be confirmed. The accurate mass of peak 26 was measured at 337.20204
m/z corresponding to a molecular formula of C19H29O5 according to the instrument library.
This suggested formula has a low carbon-oxygen ratio compared to cellulose, suggesting
less polar compound. This is consistent with the late elution time observed. The static
PHWE appears to have been more effective in producing a larger range of degradation
products, as displayed in figure 32. Specifically, the number of peaks present at between
retention times 1.5 – 8.0 min is significantly greater for the static extraction at 300 ℃,
suggesting the static extraction increased the degradation of cellulose into molecules with
smaller molecular weight distributions.
Dynamic PHWE
Figure 32: Overlapped HPLC-ESI-MS chromatograms of 300 ℃ PHWE using both static and dynamic
systems.
Static PHWE

71
Upon the addition of catalysts, the product distribution in the chromatograms
remained similar, however, often the peak intensities varied depending on the nature of the
catalyst used. This can clearly be seen for the PHWE, in product distribution obtained with
base catalysts, NaOH or Ecover The chromatographic peak at retention time of 1.5 min is
increased in intensity 10 fold (figure 33). This was also the case for the RuO2 catalyzed
PHWE.
The chromatographic peak represented in figure 31 was identified as a polymer with a
repeating unit of 82 m/z. The effect of the dilute acid catalysts was also evident, see figure
34. The differences to non-catalytic extractions in this chromatogram were the new peaks
27, 28 and 29. The TOFMS spectrum of peak 27, suggests the molecular formula of the
fragment 345.20574 m/z can be estimated as C21H29O4, however, no reasonable molecular
structure could be assigned. The relatively high carbon:oxygen ratio suggests a non-polar
species which is consistent with the late elution of the compound.
Figure 33: HPLC-ESI-MS chromatogram of a sample processed with static PHWE at 300 ℃ using Ecover.
tR = 1.5

72
Overall, the effect of the metal oxide catalysts on the product distribution in the static PHWE
was subtle. The composition of the extracts remained fairly constant, with minor changes in
intensity. Specifically, peaks 18 and 19 displayed changes in intensities upon the addition of
different catalysts; however, these peaks were not identified. The addition of the H2SO4 to
these reactors was similarly ineffective in changing the makeup of water-soluble
compounds from the extraction.
Sasaki and Ehara et al65,72 studied the degradation of cellulose in pressurized hot-
and supercritical water, using HPLC analysis coupled with a refractive index detector. They
identified a wide range of useful platform chemicals, such as dehydration products 5-HMF,
furfural and levoglucosan as well as different oligomers. However, Sasaki and Ehara used
specialized equipment, such as a SUGAR KS-801 HPLC column, that was not available for
this research.
Figure 34: HPLC-ESI-MS chromatogram of the dynamic PHWE at 300 ℃ with H2SO4 catalyst. Retention times are
labeled.
1
2
20
24
27
28
29
11
25
26

73
8.5. GC-MS Analysis
The extraction of polar-organic compounds using water as the solvent requires high
temperatures and pressures. The dielectric constant needs to be sufficiently lowered to
allow the extraction of less polar compounds. This was evident when conducting GC-MS
analysis of the PHWE extracts. For the extractions at 100 and 200 ℃ using the dynamic
system, no compounds were identified with the GC-MS. At these temperatures the water is
too polar. However, for extractions at 250 and 300 ℃ , a range of different
compounds/reaction products were extracted and identified at varying intensities, see table
6.
Figures 35 and 36, show that the dynamic system extracted 3 and 4 compounds at
250 and 300 ℃ respectively. The presence of furfural is promising, as confirmed by the MS
spectrum of peak 1, see figure 37. The molar mass of the compound is 96.09 g/mol, as the
molecular ion peak is displayed. The fragment at m/z 67 represents the loss of the aldehyde
functional group, 29 m/z.

74
Table 6: Compounds identified with GC-MS for PHWE samples of cellulose at 250 and 300
℃.
Peak
Number
Temperature
(℃)
Retention
Time
(min)
Probability
at 250 ℃
(Library
Match %)
Probability
at 300 ℃
(Library
Match %)
Structure
Name
Structure
1 250, 300 5.5 71.2 78.0 Furfural
2 250, 300 9.1 77.0 57.0
1,2-
benzenediol-3
methyl
3 300 9.4 - 74.0
Methyl-2-
furoate
4 250, 300 10.8 53.0 56.3
1,3,5-
Benzenetriol

75
Figure 35: GC-MS chromatogram of dynamic PHWE sample of cellulose at 250 ℃ without
catalyst. S* = solvent peak.
Figure 36: GC-MS chromatogram of dynamic PHWE sample of cellulose at 300 ℃ without
catalyst. S* = solvent peak.
S*
1
2
4
3
4
S* 1
2
4
S*
1
2 3

76
Figure 37: GC-MS (EI) spectrum of furfural. Retention time 5.0-5.5 min.
The low number of extracted polar-organics recovered with the dynamic system can
possibly be due to the loss of the volatile analytes by evaporation when collecting the hot
extract in an open flask. Furthermore, as the water cools to room temperature, the dielectric
constant increases back to normal range (𝜀 = 80). This may lead to precipitation of the less
polar analytes as their solubility in water decreases. It may be possible to avoid the loss of
organic compounds by mimicking the method applied by Hawthorne et al73. in the
extraction of organic compounds from soil. The outlet capillary was suspended in 5 ml of
CHCl3, allowing the extraction liquid (water) to bubble through the organic solvent; an
immediate liquid-liquid extraction. However, the heat from the extraction may cause the
organic solvent to evaporate. When extractions were completed using the static system, the
number of organic compounds extracted increased, table 7.

77
Table 7: Compounds identified with GC-MS in static PHWE sample processed at 300 ℃ without catalyst.
Peak
Number
Retention
Time
(min)
Probability
(Library
Match %)
Structure
Name
Structure
1 5.0 71.0 Furfural See Table 6
5 5.4 67.9 Glutaraldehyde
6 6.7 64.5
trans-2-
hexenyl
hexanoate
7 7.5 93.4
2,5-
dimethylfuran
(DMF)
8 8.2 51.3
2-cyclopenten-1-
one, 2 hydroxy, 3
methyl
9 8.6, 8.9 78.9, 89.4
Methyl phenol
(2, 3 or 4)
10 10.8 89.4 2-indanone

78
Figure 38: GC-MS chromatogram of the static PHWE sample processed at 300 ℃ without
catalyst.
More polar-organic compounds were obtained when the static system was
employed, see figure 38. Apart from furfural, a different set of cellulose degradation
products were formed. The extraction system used dictated which degradation products are
formed. There are two reasons for this. Firstly, a stainless steel reactor was used in the
dynamic extractions, as opposed to a Hastelloy C-22 reactor for the static extractions.
Research conducted by Antal et al38 determined that the corrosivness of hydrothermal
solvents can cause the metals present in Hastelloy (primarily Ni and Fe37 ) to leach into the
solvent, catalyzing the gasification of biomass. Nickel catalysts especially are known to
catalyzed cellulose degradation15. Secondly, the difference in the extraction methods
themselves; the dynamic system allows efficient removal of extracted compounds by the
flow of fresh water, whereas, extracted compounds are subjected to reactive conditions for
1
5
6 7
8
9
10

79
the entirety of the extraction in the static system. The likelihood of further degrading
extracted compounds is higher in the static method.
Some of the compounds identified from the extracts are valuable platform
chemicals. Methylphenol is often used in the pharmaceutical industry as a precursor in
syntheses and glutaraldehyde has medical applications in the treatment of resistant warts76.
Furfural and DMF (figure 39) can be used as renewable, furan-based biofuels as well as in
the pharmaceutical industry as platform chemicals.
Figure 39: GC-MS (EI) spectrum of DMF. Retention time 7.5 min.
DMF is formed via the hydrogenation of 5-HMF, a dehydration product of glucose (figure
40). It is a desirable, renewable biofuel with a suitable boiling point (366 K), low water
solubility and high energy density77. The oligomers and monomers produced by the initial
hydrolysis of cellulose undergo further dehydration, oxidation and fragmentation reactions
resulting in the formation of furfural and its derivatives. Furfural is a valuable platform
chemical and is the precursor to an array of different solvents and furan based chemicals

80
such as, tetrahydrofuran (THF), furoic acid and methylfuran, traditionally produced from
fossil fuel derivatives78.
Further investigation was conducted to determine whether the addition of varying
catalysts produced a different set of polar-organic compounds. In the dynamic extractions
under basic conditions, furanmethanol was produced in addition to the furfural. On the
addition of acidic catalysts, H2SO4 and CO2, no change to the GC-MS chromatograms
occurred, although, this is not surprising as the addition of the acid catalyzes the
degradation of cellulose via the same mechanism as PHW. However, upon the addition of
the RuO2 and Pd/C to the CO2 PHWE system, an increased number of compounds have been
extracted, see figures 41 and 42. Similar to the DLS analysis, an increase in activity of Pd/C
in the water/CO2 solvent was identified; due to its partial solubility and therefore increased
contact of the catalyst with the cellulose polymer, the reactivity of the catalyst improves.
Figure 40: Conversion of cellulose into 2,5-DMF and furfural via initial hydrolysis,
isomerization to fructose and dehydration into useful platform chemicals46.

81
Figure 41: GC-MS chromatogram of the dynamic PHWE processed at 300 ℃ with CO2
catalyst.
The PHWE coupled with metal oxide catalysts via the static system show few
changes in extracted polar-organic products when compared to the non-catalytic extraction.
Methyl cyclopropane carboxylic acid, shown figure 43, was extracted by the Al2O3 and Fe2O3
catalysts. Tin and zinc oxide produced benzoic acid and phenol respectively. Furthermore,
2 3
4
T
a
b
l
e
1
0
:
C
o
m
p
o
u
n
d
s
i
d
e
n
t
i
f
i
e
d
1
8
1
5
12
13
9
3
4
11
Figure 42: GC-MS chromatogram of the dynamic PHWE sample processed at 300 ℃ and
with CO2 + Pd/C. Peaks labeled 11, 12 and 13 are furaldehyde, hexanoic acid and sorbic
acid respectively.
2 3

82
Figure 43: Methyl
cyclopropane carboylic
acid
the PtO2 catalyst appeared to promote the synthesis of the biofuel DMF in
larger quantities as well as producing pentanoic acid.
Furfural is a useful platform chemical produced in nearly all
extractions undertaken. As the same mass of cellulose used in the extractions
was accurately weighed prior to each extraction (± 0.003 g) the amount of
furfural produced was semi-quantified by comparing peak areas (table 8).

83
In the dynamic PHWE, as the temperature was increased, the amount of furfural produced
also increased. However, for the static extraction the opposite was the case. The opposing
trends are likely due to the different conditions created by the dynamic and static
extractions, as previously discussed. The constant removal of extracted compounds in the
dynamic system prevents any parasitic reactions occurring, reducing the quantity of
Extraction
System
Catalyst Temperature (℃)
Furfural
Peak Area
Dynamic
none
250 1.894 x10
8
300 4.895 x10
8
H2SO4
250 2.963 x10
8
300 1.783 x10
9
CO2
250 3.776 x10
8
300 2.850 x10
9
NaOH
250 5.436 x10
8
300 8.361 x10
8
CO2 + Pd/C
250 5.621 x10
7
300 4.344 x10
8
Static
none
250 4.967 x10
8
300 5.864 x10
7
Ecover
250 8.334 x10
6
300 -
Fe2O3
250 5.089 x10
8
300 4.912 x10
7
SnO
250 5.850 x10
8
300 1.915 x10
8
ZnO
250 1.729 x10
8
300 3.139 x10
7
Al2O3
250 7.098 x10
9
300 2.201 x10
8
RuO2
250 5.399 x10
7
300 -
PtO2
250 3.910 x10
8
300 -
Pd/C
250 2.215 x10
8
300 -
La2O3
250 1.570 x10
8
300 4.030 x10
8
Fresh Sample
(none)
300 2.739 x10
8
Table 8: Effect of extraction system, catalyst and temperature effect on the furfural peak
area.

84
furfural produced, as seen in static PHWE. For catalysts PtO2, RuO2 and Ecover, this even
resulted in no furfural produced in static PHWE at 300 ℃.
It was observed that the acidic conditions in dynamic extractions resulted in an
increased amount of extracted furfural, i.e. 300 ℃ H2SO4 and CO2 had peak areas of 1.783
x109 and 2.850 x109 respectively. This may be due to more effective acid hydrolysis of the
original cellulose biopolymer. For the static extractions, Al2O3 was the most effective metal
oxide in terms of furfural production and Ecover was particularly poor. Overall, the
catalysts extracted similar amounts of furfural.
Samples underwent a color change from yellow to orange when left overnight. The
final entry in table 8, named fresh sample no catalyst, was an experiment to determine
whether leaving the samples over a long time, to allow other analysis techniques to be
completed, affected the products detected by GC-MS. The DCM liquid-liquid extraction for
this experiment was completed immediately after the yellow sample was removed from the
extraction cell. The value of 2.739 x108 shown in table 8 is close to the value obtained for the
orange/brown sample and so, on exposure to light, there was no clear change observed to
the furfural amount.

85
9. Conclusion
The cellulose was successfully decomposed in PHW, and its water-soluble products
were extracted. The percentage conversion of cellulose into its degradation products
remained constant and rather low at temperatures below 200 ℃, before increasing
significantly with temperature. At 250 and 300 ℃ the percentage conversion of cellulose
was 71.35 and 87.12 % respectively. Dynamic PHWE were attempted with the aid of
different catalysts, NaOH, Ecover, H2SO4 and CO2, with little effect on the percentage
conversion. The aid of metal catalysts Fe2O3, SnO, Al2O3, RuO2, PtO2 and Pd/C together with
CO2 in PHW increased the conversion of cellulose to above 95%. FT-IR analysis of the
charred residue showed alterations to the cellulose structure at temperatures 250 and 300
℃. Reduction in intensity, and even loss of the “crystallinity band”, as well as the formation
of new C=O and C=C bands proved that the structure of cellulose had been significantly
altered during PHW treatment at these temperatures. Reduction in intensity of the O-H and
C-O bands with temperature suggested both hydrogen bond network and pyranose ring
structure of cellulose had been partially dismantled. The degradation of the polymer was
further confirmed by DLS analysis. Particle sizes decreased significantly in a nonlinear
relationship with temperature from 1753 to 58.3 nm as the temperature increased from
100 to 300 ℃. The addition of various catalysts clearly aided the degradation, particularly
H2SO4 and Pd/C. This analysis also showed that the static extraction method was more
effective in breaking down the large cellulose polymer into shorter chains.
Overall, the results from the ESI-MS analyses were relatively unsuccessful,
independent of the extraction method used. However, the number of extracted compounds,
i.e. peaks in the chromatogram, clearly increased with temperature as the solvent becomes

86
more applicable for a wider range of compounds. Different catalysts extracted similar
compounds, with varying intensities. HPLC-ESI-MS results were difficult to interpret due to
a low signal to noise ratio. Although good separation was achieved, PHW-soluble
compounds were poorly characterized making identification of analytes difficult. The
chromatographic peak at retention time 1.5 min was identified as a glycosyl polymer chain,
with repeating unit 162 m/z, giving information on the degree of polymerization of the
chain. Attempts to achieve more data from HPLC-ESI-MS2 and HPLC-ESI-TOFMS on CID
patterns and accurate masses respectively were largely unsuccessful due to a loss in
response from the instruments.
GC-MS analysis was successful in the identification of useful platform chemicals at
250 and 300 ℃, when the polarity of PHW was lowered. Identified compounds such as
furfural and DMF have applications as renewable biofuels and as precursors to chemical
syntheses. Static extractions were shown to produce a wider range of polar-organic
compounds than the dynamic method. This might be attributed to additional catalytic effect
of the reactor material Hastelloy C-22 containing high percentages of nickel and chromium,
or increased contact between the cellulose and the PHW. The metal catalysts, RuO2 and
Pd/C showed increased activity when used in tandem with CO2 PHWE, extracting a larger
number of compounds compared to other dynamic extractions. In general, the addition of
different catalysts had little effect on extracted analytes.
This research could have been improved both in extraction methods and analytical
techniques. Repeating extractions would have increased the reliability of collected data.
Investigation into other variables of the dynamic extraction, such as time and flow rate,
would have enabled more comprehensive analysis. Extractions at higher temperatures, up

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