PRE-HYDROLYSIS OF THE PHENYL GLYCOSIDIC BOND IN A MODEL
COMPOUND
By
Sagar Nandkumar Deshpande
A THESIS
Submitted in Partial Fulfillment of the
Requirements for the Degree of
Master of Science
(in Chemical Engineering)
The Graduate School
The University of Maine
December, 2008
Advisory Committee:
Adriaan R. P. van Heiningen, Professor of Chemical Engineering, Co-Advisor
Martin Ocen Lawoko, FBRI's Analytical Chemist, Co-Advisor
Joseph M. Genco, Professor of Chemical Engineering
Raymond C. Fort, Jr., Professor of Chemistry
PRE-HYDROLYSIS OF THE PHENYL GLYCOSIDIC BOND IN A MODEL
COMPOUND
By Sagar Nankumar Deshpande
Thesis Advisors: Dr. A. R. P. van Heiningen and
Dr. Martin Ocen Lawoko
An Abstract of the Thesis Presented
in Partial Fulfillment of the Requirements for the
Degree of Master of Science
(in Chemical Engineering)
December, 2008
Chemical pulping of wood leads to fiber yields of only about 50 % because most
hemicelluloses and almost all the lignin end up in the spent pulping stream. The latter is
combusted for steam and electricity generation. The heating value of wood carbohydrates
is only half that of lignin. Therefore it has been proposed that a more economical use of
hemicelluloses is extraction as oligomers from wood chips prior to pulping followed by
conversion to high value-added products such as ethanol, polymers and other chemicals.
Recently, it has been shown that all lignin in soft wood is chemically linked to
carbohydrates and especially to the hemicelluloses by covalent bonds (Lawoko et. 2005).
The main LCC bonds proposed are of the ester-, a-ether-, phenyl glycosidic- and carboncarbon type. Selective cleavage of LC bonds will be essential when extracting
hemicellulose mostly free of lignin in an extraction step prior to pulping.
In this study the cleavage of the phenyl glycosidic bond of a model compound,
phenyl-B-D-glucopyranoside, was studied. The hydrolysis conditions were chosen to
simulate different
pretreatment methods such as auto hydrolysis, green liquor
pretreatment and acid hydrolysis. It was found that hydrolysis of the phenyl glucoside
model compound at near neutral aqueous conditions (pH 6) was minimal (4 %) at 170 °C.
On the other hand hydrolysis was nearly complete (95 %) at this temperature with the
addition of acetic acid at a concentration expected to be generated from deacetylation of
xylan in hardwoods (about 10 g/L). At acid conditions of pH 1.65 the temperature may be
lowered to 105 or 121 °C while still obtaining a significant (80-90 %) hydrolysis yield.
The hydrolysis experiments performed simulating green liquor extraction showed
as previously reported that the hydrolysis of phenyl-glucoside produces phenol and
levoglucosan while no glucose is formed. The result showed that hydrolysis of the phenyl
glucoside at 155 °C was essentially complete with quantitative formation of levoglucosan
and phenol rather than glucose and phenol.
Incomplete mass balances were obtained between the amount of the phenyl
glucoside reacted and phenol and glucose formed after hydrolysis at pH 1.65 at all
temperatures studied (70, 90, 105 and 121 °C). Based on the curved nature of the phenyl
glucoside calibration curve and quantification of the TOC of the starting and final
reaction mixtures, it is suggested that the alditol acetylation method does not work
properly at strong acidic conditions. No other degradation products than glucose, phenol
and levoglucosan were observed at any of the experimental conditions studied.
ACKNOWLEDGMENTS
I would like to express my deepest gratitude to my advisor Dr. Adriaan van
Heiningen for keeping faith and giving opportunity to work under OBER group. I would
like to thank Dr. Matin Lawoko who gave his constant guidance, generous support,
patience and valuable time to fulfill my work.
I would also like to take the opportunity to thank my other committee members
Dr. Joseph M. Genco, and Dr. Raymond C. Fort for spending their time and energy for
the benefit of my thesis.
I would like to thank other research group members from OBER group for
keeping friendly, healthy working environment and endless encouragement.
I would like to thank all the graduate students as well as staff of the Chemical and
Biological Engineering Department and Pilot Plant for their warm hearted helpfulness.
This research has been made possible by financial support from EPSCoR under
NSF Grant No. 0554545
Finally, I would like to dedicate this thesis to my parents, brother and fiancee.
This work would not have been possible without the everlasting support of my family
whom I owe everything.
TABLE OF CONTENTS
ACKNOWLEDGMENTS
ii
LIST OF FIGURES
vi
LIST OF TABLES
ix
Chapter
1. INTRODUCTION
1.1 Wood Pulping
1
1.2 Cellulose
2
1.3 Hemicelluloses
3
1.3.1 Galactoglucomannans
3
1.3.2 Arabinoglucuronoxylan
4
1.3.3 Glucuronoxylan
4
1.3.4 Glucomannan
5
1.4 Lignin
5
1.5 Extractives
6
2. LITERATURE REVIEW
2.1 Lignin-Carbohydrate Complexes
7
2.2 Glycosidic Type of Lignin-Carbohydrate bonds
2.2.1 Acid Catalyzed Hydrolysis of Glycosides
in
10
11
3. MATERIAL AND METHODS
3.1 Chemicals
13
3.1.1 Materials
14
3.1.2 Equipments
15
3.2 Reduction to Alditols
_
3.2.1 Reduction procedure
15
16
3.3 Acetylation of Alditols
16
3.3.1 Alditol acetylation procedure
17
3.4 Phenol extraction procedure
18
4. DESCRIPTION OF EXPIREMENTAL CONDITIONS AND SAMPLE
PREPARATION PRODURES
4.1 Hot water extraction of hardwood
20
4.1.1 Simulation of hot water extraction - Neutral Condition
22
4.1.2 Simulation of hot water extraction-Acidic Condition
22
4.2 Green liquor extraction - Alkaline Condition
23
4.3 Strong acidic treatment
26
4.4 Preparation of internal standard for phenyl-glucoside, glucose and
levoglucosan
26
4.4.1 Preparation of internal standard for phenol
27
4.5 Calibration for phenol
27
5. RESULTS AND DISCUSSION
29
5.1.1 Investigating the effect of temperature on cleavage of the
phenyl-glucosidic bond in pH 6 water
iv
30
5.1.2 Simulation of hot water extraction -Effect of temperature and
acidity
31
5.2 Green liquor extraction - Alkaline Condition
32
5.3 Strong acidic treatment
34
_
5.3.1. Strong acidic treatment at 70°C
35
5.3.2 Strong acidic treatment at 90°C
36
5.3.3. Strong acidic treatment at 105°C
37
5.3.4. Strong acidic treatment at 121°C
38
5.4 Activation energy for strong acidic treatment condition
39
5.5 Total Organic Carbon analysis
41
6. CONCLUSIONS
44
6.1 Practical Implication
45
REFERENCES
47
Appendix A Methodology Development
52
Appendix B Discussion of Product Analysis
60
Appendix C Figures and Graphs.
70
BIOGRAPHY OF AUTHOR
77
v
LIST OF FIGURES
Figure 1.1
Structure of Cellulose
2
Figure 1.2
Structure of Galactoglucomannans
4
Figure 1.3
Structure of Arabinoglucuronoxylan
4
Figure 1.4
Structure of basic building blocks of lignin
5
Figure 2.1
Proposed structure of benzyl ether bond between lignin and
hemicelluloses
Figure 2.2
8
Proposed structure of benzyl ester bonds between lignin and
hemicelluloses
9
Figure 2.3
Proposed structure of acetal bond between lignin and hemicelluloses
9
Figure 2.4
Proposed structure of glycoside bond between lignin and
.hemicelluloses
Figure 2.5
_
A-l Mechanism of acid hydrolysis of
phenyl-P-D-glucopyranoside
Figure 3.1
10
12
Molecular structure of model compound
phenyl-P-D-glucopyranoside
13
Figure 3.2
Molecular structure of internal standard inositol
14
Figure 3.3
Molecular structure of internal standard guaiacol
14
Figure 3.4
Molecular structure of glucose isomer
15
Figure 4.1
Modified Dionex ASE-100 extractor
21
Figure 4.2
Graph of temperature against time for rocking digester
24
Figure 4.3
Calibration curve for phenol in water
27
VI
Figure 5.1
Phenyl-glucoside hydrolysis in hot water in the presence of
acetic acid - constant time of 90 minutes
Figure 5.2
31
Phenyl-glucoside cleaved at simulated green liquor extraction
conditions - constant time 90 minutes
33
Figure 5.3
Phenyl-glucoside cleaved at pH 1.65 and 70 °C
35
Figure 5.4
Phenyl-glucoside cleavage at pH 1.65 and 90 °C
36
Figure 5.5
Phenyl-glucoside hydrolysis at pH 1.65 and 105 °C
37
Figure 5.6
Phenyl-glucoside cleavage atpH 1.65 and 121 °C
38
Figure 5.7
Arrhenius plot for degradation of phenyl-glucoside at pH 1.65
40
Figure 5.8
Calibration curve for phenyl-glucoside in acidic solution
43
Figure A.l
UV spectrograms..,
52
Figure A.2
HPLC Chromatograms
55
Figure A.3
Gas chromatogram for pure glucose after acetylation using
Pyridine and acetic-anhydride
Figure A.4
_
57
Gas chromatogram of pure glucose after silylation using
N-Trimethylsilylimidazole reagent
58
Figure B.l.
Graph of phenyl-glucoside cleaved at pH 1.65
60
Figure B.2
Hydrolysis of pure glucose at pH 1.65
62
Figure B.3
Hydrolysis of pure phenol at pH 1.65
63
Figure B.4
Graph plot of carbon after reaction for low acidic condition
69
Figure B.5
TOC comparison for final analysed vs. final calculated
69
Figure C.l
Graph for calibration curves for water as solvent
71
Figure C.2
Graph for calibration curves for green liquor as solvent
72
vii
Figure C.3
Percentage of phenyl-glucoside reacted on basis of glucose formed
measured by GC-MS and GC-FID
73
Figure C.4
GC-MS chromatograms
74
Figure C.5
Mechanism for formation of levoglucosan at alkaline conditions at
high temperature
76
vin
LIST OF TABLES
Table 4.1
Details of hot water extraction in presence of acetic acid
23
Table 5.1
Rate constant at pH 1.65 at different temperatures
40
Table 5.2
Carbon calculations before reaction for pure compounds at room
temperature and pH 1.65
Table 5.3
42
Carbon analysis for pure compounds at different temperature and
pH1.65
42
Table B.l
TOC for acidic hydrolysis at pH 1.65 and 70 °C
67
Table B.2
TOC for acidic hydrolysis at pH 1.65 and 90 °C
67
Table B.3
TOC for acidic hydrolysis at pH 1.65 and 105 °C
68
Table B.4
TOC for acidic hydrolysis at pH 1.65 and 121 °C
68
Table C.l
Initial concentrations of phenyl-glucoside
70
IX
Chapter 1
INTODUCTION
1.1
Wood Pulping
Wood is a heterogeneous, hygroscopic, cellular natural material. Wood is made
up of four main generic components; cellulose, hemicelluloses, lignin and a smaller
amount of extractives. The composition of wood changes depending on the type of soft
wood or hard wood. The proportion of hemicelluloses varies from 23-32 %, that of lignin
from 15-25 % and the proportion of cellulose vary from 38-50 % (Sjostrom, 1993). For
the pulp and paper industry, wood is an important source of fibers. Pulping can be done
in many ways, but mechanical and chemical pulping are the dominant processes.
In mechanical pulping, wood chips or wood logs are physically broken down into
fibers by application of mechanical force. Steam or water is used to separate and
accelerate the breaking of the fiber bonds, while only a small fraction of lignin is
removed in the process. To produce pulps with low lignin content, chemical pulping
method must be used. The wood chips are cooked with a chemical solution and then
washed and bleached. Basically, there are two different chemical pulping procedures: the
kraft or sulfate process and the sulfite process. The yield in both cases is approximately
50 % because most of the hemicelluloses and almost all the lignin ends up in the spent
pulping stream. From the spent liquid stream, the pulping chemicals are recovered and
the heat generated from combustion of the organics is used for steam and electricity
production.
The heating value of wood carbohydrates is only half that of lignin. Therefore a
more economical use of hemicelluloses is extraction as oligomers from wood chips prior
1
to pulping followed by their conversion to high value-added products such as ethanol,
polymers and other chemicals, as has been proposed by vanHeiningen (2006). However,
when the hemicelluloses pre-extraction is performed at high temperature and
acidity/alkalinity, the carbohydrates may degrade and lignin undergoes condensation
reactions. These unwanted reactions must be minimized to obtain the hemicellulose
sugars in undegraded form and avoid problems during the subsequent delignification
stage. Also it is known that the hemicelluloses are covalently bonded to lignin (Lawoko
et al, 2003). Therefore it is important to know the reactivity and stability of these
chemical bonds as a function of operating conditions used for hemicelluloses extraction.
1.2
Cellulose
Cellulose is the most abundant organic compound found in nature. It forms the
skeleton of the plant wall and has the most desired properties for making paper because
of its high molecular weight and hydrogen bonding capacity. Cellulose is a long chain of
glucose molecules typically containing as many as 10,000 P-D-glucopyranose units
linked to one another through P-(l-4)-glycosidic bonds, Because of its linear nature
cellulose adopts a partially crystalline structure, which explains its chemical stability and
lack of solubility in aqueous and organic media.
1
OH
X ^ ^ O HO -^~
HO
~~o-
v ^ ~J^~^\~v^
4
OH
\^
^
\
\
OH^i* / V -
~^^ \° \
~~s^V
OH
Figure 1.1 Structure of Cellulose (Sjostrom, 1993).
2
OH
tf
The presence of intermolecular and intramolecular hydrogen bonds contributes to
the chemical stability of cellulose. The OH-groups present at both ends of the cellulose
chain show different properties, in that the CI -OH group is a hemi-acetal and has
reducing properties, while the C4 -OH group is an alcohol and shows non-reducing
behavior (Fengel, 1984). The structure of cellulose is shown in Figure 1.1
1.3
Hemicelluloses
Hemicelluloses are low molecular weight, branched chains of xylose, arabinose,
galactose, mannose, and glucose molecules. The hemicelluloses polymers are located in
between cellulose and lignin in the plant cell wall. Hemicelluloses are chemically bonded
with lignin. The hemicelluloses are non-crystalline; provide water absorbency, elasticity,
and swelling and wet strength to the fibers (Sjostrom, 1993). A significant part of the
hemicelluloses are removed and degraded during the chemical pulping process. The
hemicelluloses in softwoods are mostly of the glucomannan type, while xylan is the
dominant type in hardwoods.
1.3.1
Galactoglucomannans
These are the principal hemicelluloses in softwood, making up of about 20% of
dry material. The backbone is a linear chain of (1-4) linked P-D-glucopyranose and P-Dmannopyranose units. The a-D galactopyranose monomer unit is (1-6) linked to a
mannopyranoside unit. Galactoglucomannans are divided into two types differing in
galactose content. The ratio of the fractions present in low galactose glucomannan
contents is 0.1:1:4 for galactose: glucose: mannose, while the galactose rich fraction has a
ratio of 1:1:3 (Gatenholm, 2002).
3
Figure 1.2 Structure of Galactoglucomannans (Sjostrom, 1993).
1.3.2
Arabinoglucuronoxylan
Arabinoglucuronoxylan represents 5 to 10 % of the dry material in softwood. It is
composed of a framework of (l-4)-(3-D-xylopyranose linked units which are partially
substituted at C-2 by 4-O-methyl-a-D-glucuronic acid groups. Another side group is a-Larabinofuranose. (Sjostrom, 1993)
Figure 1.3 Structure of Arabinoglucuronoxylan (Sjostrom, 1993).
1.3.3
Glucuronoxylan
These are the main hemicelluloses in hardwood species and represent 15 to 30%
of the dry wood material. (3-D-xylopyranose units with acetyl groups at the C2 or C3
4
positions form the back bone of these polymers. The xylan is substituted with side groups
of 4-0- methyl glucuronic acid monomer units through a link (1-2) with an average
frequency of approximately 1 uronic acid group per 10 xylose units. (Sjostrom, 1993)
1.3.4 Glucomannan
Hardwoods also contain 2 to 5% of a glucomannan composed of P-Dglucopyranose and P-D- mannopyranose linked units. The glucose: mannose ratio varies
between 1:2 and 1:1, depending on the wood species.
1.4
Lignin
Lignin (Brunow, G, 1999, Sjostrom, 1993, Watanabe, 2003, Sixta, 2006) is an
amorphous, highly complex, mainly aromatic polymer of phenyl propane units. Lignin
increases the mechanical strength properties of wood. Lignin in wood is distributed
throughout the secondary cell wall with the highest concentration in the middle lamella.
Because of the difference in the volume of the middle lamella relative to that of the
secondary cell wall, about 70% of the lignin is located in the secondary wall.
Figure 1.4 Structure of basic building blocks of lignin; [A] phenyl
propane unit, [B] guaiacyl unit and [C] syringyl unit (Sixta, 2006)
5
Lignins can be classified in several ways but they are usually identified according
to their structural elements. All plant lignin consist mainly of three basic building blocks
of phenyl propane, guaiacyl and syringyl units (see Figure 1.4) although other aromatic
type units also exist in many different types of plants. There is a wide variation of
structures within different plant species. The phenyl propane unit can be substituted at the
(3 and y carbon positions into various combinations linked together both by ether and
carbon to carbon linkages. Lignin in softwoods is mainly a polymerization product of
coniferyl alcohol and is called "guaiacyl lignin." Hardwood lignin is mainly of the
"syringyl-guaiacyl" type, i.e. a copolymer of coniferyl and sinapyl alcohols. The ratio of
these two varies in different hardwood species from a ratio of 4:1 to 1:2. Lignins found in
plants contain significant amounts of constituents other than guaiacyl- and syringylpropane units.
1.5
Extractives
Extractive covers a large number of different compounds which can be extracted
from wood by means of polar and non-polar solvents. They derive their name by being
chemicals that are removed by one of several extraction procedures. The extractives are
chemicals mainly consisting of fats, fatty acids, fatty alcohols, phenols, terpenes, steroids,
resin acids, rosin, waxes and etc (Sixta, 2006). These chemicals exist as monomers,
dimers, and polymers.
6
Chapter 2
LITERATURE REVIEW
2.1
Lignin-Carbohydrate Complexes (LCCs)
Lignin carbohydrate complexes are the class of materials in which the
hydrophobic lignin is bonded to the hydrophilic polysaccharides. These compounds are
commonly referred to as glycoconjugates. Researchers have proposed several types of
linkages between lignin and carbohydrates.
Erdmann (1866) proposed that covalent bond exists between lignin and
carbohydrate in wood based on the observation that it was difficult to separate the two
components. Braun (1952) obtained a native lignin fraction after extracting 2-3% of
wood lignin with cold ethanol. This fraction was free from carbohydrates and the degree
of wood milling did not increase the yield. Subsequently it is named 'Brauns native
lignin'. Later Traynard et al (1953) proved experimentally the existence of covalent
linkages between pentosans and lignin by hydrolysis of
poplar in buffered water
solutions at four different pH levels (range 2.2 to 4.2), and found that the percentage of
lignin dissolved to percentage of pentose dissolved was constant.
Lawoko et al (2003) reported that 50 to 90% of the residual lignin in kraft pulp
was linked to various carbohydrates in unbleached kraft pulps, and mainly to xylan and
glucomannan forming network structures. The study demonstrated that lignin is linked
through covalent bonds to all the major polysaccharides in the woody cell wall,
arabinoglucuronoxylan
(Xyl),
galactoglucomannan,
glucomannan
(GlcMan)
and
cellulose. Such a network structure may play an important mechanical role for the
"woody" properties of the secondary xylem in contrast to non-lignified plant cells as for
7
instance the seed hairs of cotton. Furthermore, the progressive degradation of lignincarbohydrate networks may be important in technical processes involving wood, such as
chemical pulping. During the end of a kraft cook and oxygen delignification, LCC
structures were observed to be degraded at different rates. The delignification rates of the
LCC types were different, with the xylan and cellulose rich LCCs being delignified faster
than the glucomannan rich LCC toward the end of a kraft cook and during oxygen
delignification.
Four types of lignin carbohydrate bonds (LC-bonds) have been proposed in the
literature. One of them is the benzyl-ether type (see Figure 2.1), where the a-hydroxyl
group of the lignin is linked to the hydroxyl group of the carbohydrate (Freudenberg and
Grion, 1959, Eriksson and Lindgren 1977, Kosikova et al 1979, Yaku et al. 1981,
Koshijima et al. 1984, Watanabe 1989). In softwood xylan, the 2-OH or 3-OH units of
the arabinose and in galactoglucomannans, the 3-OH unit of galactose are the common
linkage sites.
HO
v
^
OH CH2OH
H3CO
0
I
Figure 2.1 Proposed structure of benzyl ether bond between lignin and hemicelluloses
(Watanabe, 2003)
8
The benzyl ester type (see Figure 2.2) is formed by the linkage between the ahydroxyl group of the lignin and the carboxyl group of glucuronic acid (Freudenberg and
Harkin 1960, Yaku et al. 1976, Eriksson et al. 1980, Obst et al 1982, Lundquist et al.
1983, Watanabe and Koshijima 1988).
HOH,C—HC-HC
^
HO
OCH
OH
H O ^ )
0
C
0
H3CO
Figure 2.2 Proposed structure of benzyl ester bond between lignin and hemicelluloses
(Watanabe, 2003)
The proposed acetal type LC-bond (see Figure 2.3) shows a linkage between
lignin and two hydroxyl groups of the hemicelluloses.
CH,OH
HOH 2 C-HC-HC
H3CO
Figure 2.3 Proposed structure of acetal bond between lignin and hemicelluloses
(Watanabe, 2003)
9
In the phenyl glycoside types (see Figure 2.4), the hydroxyl groups of the lignin
are glycosylated by the reducing end of hemicelluloses (Smeltorious 1974, Kosikova et
al. 1972, Yaku et al. 1976, Joseleau and Kesraoui 1986, Kondo et al. 1990).
Figure 2.4 Proposed structure of glycoside bond between lignin and hemicelluloses
(Watanabe, 2003)
2.2
Glycosidic Type of Lignin-Carbohydrate bonds.
Research on lignin carbohydrate bonds employs different experimental methods
and use different starting materials like wood, milled wood, wood extractives, isolated
lignin, model compounds, etc. Therefore the conclusions often are speculative and thus
controversial as to the suggested type of bonds in native wood.
Kawamura and Higuchi (1952) showed that xylose was liberated from dioxane
lignin by treatment with a glycosidase, suggesting the presence of glycosidic linkages
between lignin and xylose units. Hayashi (1961) degraded wheat straw LCCs by using
glycosidase and brewer's yeast extract having P-glycosidase activity, and inferred the
presence of phenyl-|3-D-glycosidic linkages from the increase in the amount of phenolic
hydroxyl group and reducing power. Kosikova (1972) proposed the existence of phenyl-
10
glycosidic bonds by comparing the behavior of model compounds with lignin
carbohydrate complex isolated from the soluble part of methylated beech wood. The
isolated complex was investigated using UV after subjecting the wood to alkali and acid
treatment. Smelstorius (1974) showed that arabinoglucuronoxylan in the secondary cell
wall is glycosidically linked to lignin and also proposed that galactoglucomannan and
cellulose are not glycosidically linked to lignin in wood of Australian-grown Pinus
Radiata. Koshijima, Yaku, and Yamada (1976) proposed the existence of glycosidic
bonds between wood polysaccharides and lignin. Nelson and Richardson (1982)
examined the structure of LCCs by methylation analysis using GLC-MS for the
unequivocal classification of the sugar derivatives. D-glucose, D-xylose and L-rhamnose
were shown to be glycosidically linked to lignin. Some of the D-glycosyl residues carry
other (1 —> 4) linked D-glucose units, and some of the D-xylosyl residues bear other (1
—> 4) linked D-xylose unit and (1 —> 3) linked L-arabinofuranosyl group. Joseleau and
Kondo et al (1990) examined the possibility of linkages between lignin and carbohydrates
by transglycodase action of glycosidase. The result suggested the possibility of the
formation of glycosidic linkages between lignin and carbohydrate in LCC by the
participation of glycosidases during biosynthesis of lignin.
2.2.1
Acid Catalyzed Hydrolysis of Glycosides
Early work on cleavage of different type of glycosidic compounds at acidic
conditions has been reported by Moelwyn-Hughes (1928) and Veibel (1939). They
suggested that the cleavage of the glycosidic linkage follows a unimolecular mechanism.
Heidt et al (1944) determined the rate constants and activation energies of the aqueous
11
acid hydrolysis of four different types of glucopyranosides (a - methyl, p -methyl, phenyl
and benzyl). They were able to show that degradation is first-order in glucopyranoside
concentration in 0.1 N HCl at 45 to 96 °C. Overend, Rees and Sequiera, (1962) studied
rates of acidic hydrolysis for a variety of glucopyranosides including phenyl-P-Dglucopyranoside. They showed that degradation of phenyl-P-D-glucopyranoside proceeds
by unimolecular heterolysis of the conjugate acid with fission of the glucosyloxygen
bond, supporting the A-1 mechanism (see Figure 2.5). Bruyne and Wouters-Leysen
(1970) provided further supportive evidence that the degradation of phenyl-P-Dglucopyranoside proceeds by the unimolecular A-1 mechanism.
CH2OH
+
H+
=F
K
Phenyl-Glucoside
Slow
<-het
CH2OH
0
OH
OH
H20
Fast
<*?
OH
OH
Phenol
-OH
CH 2 OH
+
HO-
Figure 2.5 A-1 Mechanism of acid hydrolysis of phenyl-P-D-glucopyranoside
12
Chapter 3
MATERIAL AND METHODS
3.1
Chemicals
All chemicals used were of analytical grade. The model compound phenyl-P-D-
glucopyranoside was purchased from TCI America (purity > 99 %). The molecular
structure is shown in Figure 3.1. The molecular weight of anhydrous phenyl-P-Dglucopyranoside is 256.25 g/mol, and has the molecular formula as C12H16O6.
CH2OH
HO
f?cCv
^-"°
OH
/V>
\
/
Figure 3.1 Molecular structure of model compound phenyl-|3-D-glucopyranoside
(3-D-glucose and phenol were purchased from Fisher Scientific (both 99+ %
purity).
Inositol (Figure 3.2) was used as internal standard for the quantification of
phenyl-glycoside, glucose and levoglucosan, while guaiacol (Figure 3.3) was used as
internal standard to quantify phenol. Both chemicals were obtained from Fisher
Scientific.
Reagents used for hydrolysis of model compound phenyl-P-D-glucopyranoside
consisted of glacial acetic acid (HPLC grade), (12M) hydrochloric acid, sodium
hydroxide, sodium carbonate; sodium sulfate and anhydrous sodium sulphate; all
reagents obtained from Fisher Scientific.
13
Figure 3.2 Molecular structure of internal standard Inositol
Figure 3.3 Molecular structure of internal standard guaiacol
Chemicals used for derivatization of the sample included dichloromethane, 25 %
ammonium hydroxide, potassium borohydride solution, 1-methylimidazole, acetic
anhydride, absolute ethanol and 7.5M potassium hydroxide; obtained from Fisher
Scientific and Across Organic.
3.1.1
Materials
The materials used for the analytical methods were glass flasks,
cylinders, beakers, centrifuge tubes (50 ml), small
measuring
glass tubes (approximately 5 ml
capability) with screw lock, round bottom glass tubes (20 x 150 mm) with Teflon-lined
screw tops, pipettes (200-1000 uL, 1-5 mL) and pH paper (range 0-14).
14
3.1.2
Equipments
Hydrolyzed product were investigated using a gas chromatograph of SHIMADZU
GC2010 with mass spectrum analysis QP2010S (GC-MS). Capillary column of type RTX
225 (length 30 m, internal diameter 0.25 mm, film thickness 0.25 um). A gas
chromatograph with flame ionization detector (GC-FID) of SHIMADZU GC2010 and
capillary column of type RTX 225 and DB-225 (length 15 m, internal diameter 0.25 mm,
film thickness 0.25 um) was used for glucose analysis. A Dionex ASE-100 modified
accelerated solvent extractor, autoclave (temperature range 105 °C to 121 °C), water bath
(temperature range 40 °C to 90 °C) and rocking digester were also used for hydrolysis
experiments.
3.2
Reduction to Alditols
Derivatization using by direct acetylation of glucose yielded multiple GC peaks
because of the presence of different acetate isomers (five ring structure furanose, open
ring structure aldose and six ring structure pyranose) (Figure 3.4).
Figure 3.4 Molecular structure of glucose isomer, [A] five ring structure
furanose, [B] open ring structure aldose, [C] six ring structure pyranose
15
By performing reduction (Theander, O, 1986; Sjostrom, E 1993) prior to
acetylation, the glucose isomers were converted to the alditol form, which upon
acetylation gave one single alditol acetate peak, more specifically that of glucitol.
3.2.1
Reduction procedure
1 ml of neutralized phenyl-glucoside solution after hydrolysis was placed in a
round glass tube with screw lock. 100 uL of 25 % ammonium hydroxide solution was
added to raise the pH to 11 due to the fact that reduction must take place at alkaline
conditions. Every time it was necessary to check pH using pH paper. A known amount of
the internal standard inositol solution (prepared in 2.5 % ammonium hydroxide) was
added followed by 100 uL of reduction reagent, a potassium borohydride solution. The
potassium borohydride solution was prepared by adding 250 uL of 2.5 % ammonium
hydroxide and 750 uL of deionized water to 150 mg potassium borohydride. This
solution was kept in a water bath at a temperature of 40 °C for 1 hour during which
reduction took place. After the reduction, the excess of borohydride was reacted with
acetic acid, which was added stepwise until the pH of the mixture was slightly acidic (pH
= 4.5).
3.3
Acetylation of Alditols
The analysis in gas chromatography often involves derivatization either by
silylation or acetylation of the hydroxyl groups. The purpose of the derivatization was to
increase the volatility of the material to enable quantitative analysis. Derivatization using
alditol-acetylation was preferred over silylation and acetylation using pyridine and acetic-
16
anhydride as the reagent. This procedure was chosen for several reasons and will be
discussed later in the chapter as well as Appendix A.
3.3.1
Alditol Acetylation Procedure
(Theander, O, 1986) 0.5 mL of the neutralized reduced sample was placed in a
round glass tube (20x150 mm) with Teflon-lined screw top. This was done in duplicate to
minimize error. Methylimidazole and acetic-anhydride were used as the acetylation
reagents. Both reagents were added in quantities of 0.5 ml and 5 ml respectively, and the
glass tube was immediately screw locked. The methylimidazole functioned as catalyst
while acetic anhydride was the acetylation reagent. Mixing was done using the vortex
mixer, and then cooled in an ice bath for about 2 minutes and then kept at room
temperature for 10 minutes. 1.5 ml of ethanol was then added, the tube was screw locked
and mixture was vortex mixed and then cooled in an ice bath for 2 minutes and then the
mixture was kept at room temperature for 10 minutes so that the excess of aceticanhydride reacts with ethanol to form ethyl-acetate. 4 ml of deionized ice cold water was
added and the solution was vortex mixed, cooled in an ice bath for 2 minutes and kept at
room temperature for 5 minutes. 4 ml of freshly prepared and ice cold 7.5M potassium
hydroxide solution was then added, vortex mixed and cooled in the ice bath for 5
minutes. It was important that after addition of potassium hydroxide the glass tube was
immediately screw locked as the reaction was exothermic and otherwise would lead to
loss of material due to vaporization. Another 4 ml of 7.5M potassium hydroxide was
added in same manner. Potassium hydroxide was added in two stages to avoid excess
heating due to the exothermic reaction. The potassium hydroxide reacts with excess
17
acetic anhydride in the solution. It was added stoichiometrically so that it only reacts with
the free acetic anhydride and no de-acetylation takes place. The pH after addition of
potassium hydroxide remains constant at 6.5 starting from a pH of 4.5 after acetylation
process. After addition of 8 ml of 7.5M potassium hydroxide phase separation occurs of
the water and ethyl-acetate phases. Since the aqueous alkaline solution has a higher
density, the upper phase was ethyl acetate containing the alditol acetates. The ethylacetate phase containing the alditol acetate was transferred into a GC vial (to 1.5 ml
level) containing a pinch of anhydrous sodium sulphate which absorbed any small
amount of water remaining in the transferred phase.
The GC-MS conditions are: 1 \xL of ethyl-acetate was injected in the GC
operating in split ratio mode of 1:20. The injection temperature was maintained at 250 °C
and the detector temperature at 260 °C. The oven temperature program was as follows;
from 120 °C to 200 °C at a rate of 5 °C/min and held at 200 °C for 5 minutes, then from
200 °C to 280 °C at a rate of 4 °C/min and held at 280 °C for 10 minutes. The total
flow was maintained at 25 ml/min.
3.4
Phenol extraction procedure
Phenol formed as a result of hydrolysis of the phenyl-glucoside was isolated using
liquid-liquid extraction. Dichloromethane was used as extraction reagent (Muard, 2001).
1 ml of the neutralized solution after hydrolysis was transferred in a round glass tube with
Teflon-lined screw top. A known amount of internal standard (guaiacol solution in
dichloromethane) was added followed by 2 ml of dichloromethane. The mixture was
vortex mixed and kept at room temperature for phase separation. The density of
18
dichloromethane was higher than that of water, so the dichloromethane phase settles to
the bottom. The dichloromethane phase was transferred into a GC vial (to 1.5 ml level)
containing a pinch of anhydrous sodium sulphate which absorbed any small amount of
water present in the transferred phase.
GC-MS operating conditions: 1 uL of dichloromethane was injected in the GC
operating at a split ratio mode of 1:20. The injection temperature was maintained at 250
°C and the detector temperature at 260 °C. The oven temperature program was as
follows: the oven was maintained at 40 °C for 1 minute, then from 40 °C to 120 °C at a
rate of 5 °C/min and held for 5 minutes at 120 °C, from 120 °C to 280 °C at a rate of 4
°C/min and held at 280 °C for 10 minutes. The total flow was maintained at 27 ml/min.
19
Chapter 4
DESCRIPTION OF EXPERIMENTAL CONDITIONS AND SAMPLE
PREPARATION PROCEDURES
Pre-extraction of hemicelluloses from hard woods is being pursued at the
University of Maine to create more value from them. Knowledge of the quantity and ease
of cleavage of hemicellulose-lignin complexes is important for the design of the
subsequent
separation
and
biological
conversion
processes
of
the
extracted
hemicelluloses. In the present work, the effect of pre-extraction conditions and that of a
subsequent high temperature dilute acid treatment on the reactivity of the phenylglucosidic LCC bond was investigated using the simplest model compound, phenyl-P-Dglucopyranoside. In order to define the reaction conditions for the present model
compound study, the different extraction procedures and hydrolysis methods are
reviewed below.
4.1
Hot water extraction of hardwood
It has been shown that hydrothermal treatment of hardwoods leads to significant
removal of hemicelluloses. At the end of extraction process most of the xylan dissolves in
the form oligosaccharides, which later de-polymerizes to a smaller extent into monomeric
xylose. Arabinan and galactan are completely removed from wood while acetic acid is
released by de-acetylation of the dissolved acetylated oligosaccharides. Xylan is the
predominant component in the extract, especially at temperatures higher than 150 °C.
(Tunc and vanHeiningen, 2008)
20
Hence it was interesting to study the effect of the experimental conditions of hot
water extraction on cleavage of the phenyl-glucoside bond. The temperature conditions
investigated were 150, 160 and 170°C, while the extraction time was kept constant at 90
minutes. These experiments were performed using a Dionex ASE-100 extractor.
The ASE-100 equipment works at high pressure in the range of 11-15 MPa. When
extracting wood with water, the pressure inside the extraction cell does not remain stable.
If the pressure inside the extraction cell in the ASE-100 drops below a minimum set point
of 11 MPa, the supply pump introduces fresh water into the extraction cell. If the pressure
increases above the maximum set point of 15 MPa due to thermal expansion or
generation of gases during extraction, the relief valve (relief valve-1, Figure 4.1) opens
and releases liquid from the extraction cell.
Relief Valve - 2
-c*a—i
-t>^3-
Solven: Bottle
Extraction Cell
L-®Pump
—tsc
Static Valve
Relief Vsilve-1
Hsat
Exchanger
Three-way Valve
Expansion Tank
Needle Valve - 1
A
o
Collection Bottle-1 Collection Bottle-2
Nitrogen
11-15 MPa
Needle
Valve - 2
Waste Bottle
Nitrogen
1-1.5 MPa
Figure 4.1 Modified Dionex ASE-100 extractor (Tunc and van Heiningen, 2008)
21
In order to avoid release or addition of liquid during the extraction process, the
Dionex ASE-100 was modified by connecting a pressurized expansion tank to the
extraction cell. This allowed the ASE-100 to operate at constant volume extraction.
Moreover, a heat exchanger was added to afford condensation of any volatile products
(Tunc and vanHeiningen, 2008). The Modified ASE-100 extractor is represented
schematically in Figure 4.1.
4.1.1
Simulation of hot water extraction - Neutral Condition
A known concentration of an aqueous phenyl-glucoside solution of pH 6 is
pumped from the solvent bottle of the ASE 100 into the extraction cell. The pH of the
solution was similar to the final pH of the extract when hardwood is contacted with a
solution of "green liquor" as described in the so-called "near-neutral" extraction process
(Mao et al., 2008). The present model compound experiments were performed at
temperatures of 150, 160 and 170 °C for a constant time of 90 minutes. At the end of
each experiment, a sample taken from the collection bottle was further analyzed using the
earlier described alditol-acetylation and liquid extraction methods to quantify the amount
of remaining phenyl-glucoside and formed glucose and phenol.
4.1.2
Simulation of hot water extraction - Acidic Condition
De-acetylation of xylan occurs during hydrofhermal hemicelluloses extraction
from hard woods. The release of acetic acid creates an acidic solution. The amount of
acetic produced depends on the time and temperature of the wood extraction (Tunc and
22
vanHeiningen, 2008). Based on these results, pre-determined amounts of acetic acid were
added to the aqueous solution of the phenyl-glucoside in the ASE 100 experiments. Table
4.1 shows the amount of acetic acid added at the three different temperatures. At the end
of each of the three models compound experiments the samples collected in the ASE 100
collection bottle were neutralized using IM of sodium hydroxide solution. The
neutralized samples were analyzed using the alditol-acetylation and liquid extraction
methods for quantification of the remaining amount of phenyl-glucoside and formed
glucose and phenol.
Temperature
Acetic acid added
pH after
lMNaOH added to
(°C)
(50% by weight)
reaction
neutralize solution
150
4.42 mg/ml
3.34
1.22ml/10ml
160
6.45 mg/ml
3.29
1.25ml/10ml
170
12.76 mg/ml
3.24
1.30ml/10ml
Table 4.1 Details of hot watei* extraction in presence of acetic acid
4.2
Green liquor extraction - Alkaline Condition
Kraft pulping is the dominant technology for producing unbleached pulp. Wood
chips are pulped at high temperature and high pressure using an aqueous mixture of
caustic and sodium sulfide to produce pulps which are superior in strength compared to
that of other pulping processes. The spent liquor containing dissolved wood substance
and residual inorganic pulping chemicals are burned in the recovery boiler. The inorganic
chemicals are removed from the furnace as a smelt primarily consisting of sodium
carbonate and sodium sulfide. The molten smelt is dissolved in water to form so-called
green liquor.
23
Pre-extraction of wood with green liquor to obtain hemicelluloses for value added
products is presently being persued at the University of Maine (Mao et al, 2008), hence
the effect of green liquor on the cleavage of the phenyl-glucosidic bond as a possible
LCC was investigated in the present study.
Industrial green liquor is an alkaline solution containing 8.99 g/L sodium
hydroxide, 29.14 g/L sodium sulfide and 69.98 g/L sodium carbonate (all concentrations
expressed as sodium oxide, Na20). For green liquor pretreatment of wood, 3% (as Na20)
of green liquor is charged based on dry wood at total liquor to wood ratio of 4:1 L/kg.
For the present experiments, this composition was used to simulate the green liquor
extraction process. The concentrations used after dilution of the sodium salts are shown
in Appendix C. The present green liquor extraction experiments were performed in the
rocking digester at temperatures of 137, 146, and 155 °C for 90 minutes. Each time a
known amount of phenyl-glucoside was dissolved in a small quantity of water and then
dissolved in the green liquor solution. The pH before and after high temperature
treatment was 12.5.
180
160
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inn
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£01
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60
40
20
2U
40
60
SO
100
120
140
160
Time (minutes)
Figure 4.2 Graph of temperature against time for rocking digester
24
The rocking digester is a batch, indirectly electrically heated vessel which rotates
back and forth, thereby mixing the content of the digester. The UMaine rocking digester
has a capacity of 20 L, but for hydrolysis of the phenyl-glucoside model compound 8
small bombs of 350 ml capacity were used simultaneously. Once the sealed bombs are
properly placed inside the rocking digester, the digester was filled with water to 60 % of
its capacity and then heated to the desire temperature. This is schematically shown in
Figure 4.2. The temperature profile shown in Figure 4.2 is composed of three regions:
A. Heating period: The temperature was raised from room temperature to the desired
maximum temperature (160 °C). It takes 36 minutes to attain 160 °C temperature
from room temperature (26.1 °C)
B. Time at temperature: Time at which the desired temperature was kept constant.
C. Cooling time: Time required for cooling the digester from the high temperature to a
temperature at which the hydraulic pressure reaches atmospheric pressure
(approximately 94 °C).
After every run, the small bombs were removed from the rocking digester and
cooled under cold water. The samples were neutralized using 1ml of 12M hydrochloric
acid for 55 ml of green liquor solution. The neutralized samples were analyzed using
alditol-acetylation to quantify the remaining phenyl-glucoside and formed glucose and
using liquid extraction method to quantify the amount of formed phenol.
25
4.3
Strong acidic treatment
Acid treatment of woody biomass and pulp fibers is commonly used to dissolve
and hydrolyze the carbohydrate fraction to release and quantify the amount of lignin and
mono sugars. Also dilute acid treatment of the sugars is an important pretreatment
technique for biomass to overcome the "recalcitrance" of cellulose for production of
biofuels. Thus it was important to establish the stability of the phenyl-glucoside at strong
acidic conditions.
A strong acidity of pH 1.65 at room temperature was chosen using HC1 as acid.
Four temperatures were studied; 70 and 90 °C using a water bath, and 105 and 121 °C
using an autoclave. At the lower temperature of 70 °C the solutions in capped centrifuge
tubes were taken out of the bath every hour for up to 5 hours (i.e. 1, 2, 3, 4, 5 hours) and
after 12, 24, and 48 hours. For the temperature of 90 °C the solutions in capped vials
were taken out of the water-bath every hour for up to 4 hours.
After removing the samples from the bath or autoclave, they were immediately
cooled under cold water and neutralized using known amount of 1M of sodium hydroxide
solution. The neutralized samples were stored for further analysis using the alditolacetylation and liquid extraction method.
4.4.
Preparation
of internal standard for phenyl-glucoside, glucose and
levoglucosan
The internal standard for the quantification of phenyl-glucoside, glucose and
levoglucosan was prepared by dissolving 20 mg of Inositol in 20.0 mL solution obtained
by combining 2 mL of 25% ammonium hydroxide solution with 18 ml of deionized
water.
26
4.4.1
Preparation of internal standard for phenol
The internal standard for the quantification of phenol was prepared by dissolving
20.0 mg of guaiacol in 20.0 mL of dichloromethane.
4.5
Calibration for phenol
Five standard flasks of 15.0 mL were used for series dilution. In the first flask
15.0 mg of phenol was dissolved in 15ml with deionized water. From the first flask 5.0
mL, 4.0 mL, 3.0 mL and 2.0 mL and 1.0 mL was transferred respectively to the other
flasks and diluted to the 15.0 mL mark with deionized water. As a result, each flask had a
different phenol concentration. 1.0 ml was taken from each flask and was subjected to
dichloromethane liquid extraction. Figure 4.3 shows the GC-MS calibration curve for
phenol extracted with dichloromethane. Extraction proceeded in a similar fashion as
described in section 3.4.
Figure 4.3 Calibration curve for phenol in water
27
In a similar fashion standard solutions of phenyl-glucoside and glucose (Appendix
C, see Figure C.l) were prepared. 1ml samples were taken from each flask and then
subjected to the earlier described reduction and the alditol-acetylation procedure. For the
green liquor extraction condition, green liquor rather than water was used as dilution
solvent, and a different calibration curve (Appendix C, see Figure C.2) was obtained.
28
Chapter 5
RESULTS AND DISCUSSION
The final mass of phenyl-glucoside and phenol after the hydrolysis treatment was
determined by GC-MS, while GC-FID was used for glucose, since GC-FID gave a more
consistent result for glucose than GC-MS. Appendix C, Figure C.3 shows a comparison
of GC-MS and GC-FID data for glucose at different hydrolysis conditions.
The detector response factor, Fx, was calculated using calibration curves
(Appendix C; see Figure C.l and C.2) for each product by using mass ratio to area ratio
of the compound to the internal standard. Then the calibration curve was used to calculate
the mass of the compound after an hydrolysis experiment as shown below,
M x = (Fx) x (M1S) x (A x )
(Ax)
where M = Mass of compound (mg)
Fx = Detector response factor
A = Peak area of compound
X = Compound phenyl-glucoside / phenol / glucose or levoglucosan
IS = Internal standard inositol / guaiacol
From the data, the percentage of phenyl glucoside cleaved was calculated based
on the following three methods:
1)
On the basis of phenyl-glucoside remaining:
= (Initial mass of phenyl-glucoside - Final mass of phenyl-glucoside) x 100
(Initial mass of phenyl-glucoside)
29
2)
On the basis of glucose formed
=
(256/180) x (Final mass of glucose formed)
(Initial mass of phenyl-glucoside)
x
100
where 256 g/mol is the molar mass of phenyl-glucoside and 180
g/mol is the molar mass of glucose.
3)
On the basis of phenol formed
=
(256 / 94) x (Final mass of phenol formed)
(Initial mass of phenyl-glucoside)
x
100
where 94 g/mol is the molar mass of phenol.
Appendix C; table C.l shows the initial concentration of phenyl-glucoside for each
condition.
5.1.1
Investigating the effect of temperature on cleavage of the phenyl-glycosidic
bond in pH 6 water
The effect of pre-extraction temperature on the cleavage of the phenyl glycosidic
bond at near neutral aqueous conditions (pH of 6) was studied. It was found that there
was no significant cleavage of the phenyl-glucoside at 150 and 160 °C. At 170 °C only 4
% of the model compound was cleaved. These results show that pre-extraction at neutral
aqueous conditions has a minimal effect on the phenyl-glucoside bond at the present
selected temperatures.
30
5.1.2
Simulation of hot water extraction - Effect of temperature and acidity
Graph 5.1 shows the yield of phenyl-(3-D-glucopyranoside reacted plotted versus
hydrolysis temperature of 150, 160 and 170 °C at constant time of 90 minutes in the
presence of acetic acid. The amount of acetic acid added is plotted on right y axis.
As can be seen, the addition of acetic acid leads to a considerable increase in the
cleavage of the phenyl-glucoside bond. At 170 °C the amount of phenyl-glucoside
cleaved is in the range of 91 % to 96 % depending on the calculation basis; i.e. on the
amount of phenyl-glucoside remaining, or amount of formed glucose or phenol. The
amount of phenyl-glucoside reacted calculated using the three methods are in good
agreement, implying that a good mass balance is achieved using the present experimental
and analytical procedures.
Figure 5.1 Phenyl-glucoside hydrolysis in hot water in the presence of
acetic acid - constant time of 90 minutes
31
The results show that during hot water hemicelluloses pre-extraction of wood at
160 °C or higher for 90 minutes at a L/W ratio of about 4 a significant cleavage of
phenyl-glucoside lignin-carbohydrate bond is obtained due to release of acetic acid from
the hemicelluloses. The combined effect of temperature and acidity accomplishes the
cleavage. By increasing the temperature from 150 to 160 °C the percentage of phenylglucoside bond cleavage increases considerably from about 55 % to about 85 %.
No other products were seen in the chromatogram. It was further studied whether
any other products than glucose and phenol were formed. This study is reported in the
last part of Appendix-B. This investigation showed that there were no other products.
5.2
Green liquor extraction - Alkaline Condition
No glucose was detected at the simulated alkaline hydrolysis conditions, while
phenol was measured. This indicates that glucose is further converted to other products.
The GC-MS data revealed another significant peak that was determined by the NIST
library data base to be levoglucosan (Appendix C; see Figure C.4.C chromatograph plot
from GC-MS). To confirm this, a commercial levoglucosan was derivatised using the
alditol acetate method and then analyzed using the GC-MS system. It gave an identical
retention time and mass spectrum as the product identified in the green liquor hydrolysis
experiments. Thus, it can be concluded that at the green liquor pre-extraction conditions,
the phenyl-glucoside is cleaved to produce levoglucosan and phenol. The formation of
glucose is minimal, if any. The mechanism for the formation of levoglucosan from
phenyl-glucoside has in fact been reported in the literature (William E., 1990) and is
shown in Figure C.5.
32
The detector response factor for levoglucosan was calculated using a separate
calibration curve (Appendix C; see Figure C.2). In a similar way as before, the
percentage of phenyl glucoside cleaved was then calculated on the basis of phenylglucoside left and phenol formed and also on the basis of levoglucosan formed as shown
below:
(256 / 162) x (Final mass of levoglucosan formed)
x
100
(Initial mass of phenyl-glucoside)
Here 256 g/mol represent the molar mass of phenyl-glucoside, while 161 g/mol represent
the molar mass of levoglucosan.
100
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80
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u 60
n
a
QC40
(D
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135
140
145
150
155
160
i
Temperature (°C)
o
PG basis (GC-MS)
j
- a- Levoglucosan basis (GC-FID) j
i
- A - Phenol basis (GC-MS)
I
Figure 5.2 Phenyl-glucoside cleaved at simulated green liquor extraction
conditions - constant time 90 minutes
Figure 5.2 shows the plot of phenyl-glucoside reacted as a function of hydrolysis
temperature (137, 146 and 155 °C for 90 minutes). For the hydrolysis experiments at 137
and 146 °C, the percentage of phenyl-glucoside cleaved on the basis of levoglucosan
formed were in very good agreement with those of phenol formed and phenyl-glucoside
33
remaining. This indicates quantitative conversion of glucose to levoglucosan at these
conditions
At 155 °C however, there is no significant amount of phenyl-glucoside left (on
remaining PG basis), while the calculation on the basis of phenol and levoglucosan
formed show an 84 % cleavage. This may indicate some other degradation reaction of the
phenyl-glucoside leading to products which are not analyzed by the GC system due its
reduced volatility. Further investigation is however required to confirm this.
Based on these results it may be concluded that during wood pre-extraction at
alkaline conditions of pH 12.5 with green liquor more than 50 % cleavage of the phenylglucoside lignin-carbohydrate bonds takes place at 145 °C, and increases to
approximately 85 % at 155 °C.
5.3.
Strong acidic treatment
The LC bond cleavage was also investigated at dilute acid pretreatment
conditions. The pH of the aqueous hydrochloric acid solution used was 1.65 and the
temperatures investigated were 70, 90, 105, and 121 °C with reaction time being another
parameter. The hydrolysis of phenyl-[3-D-glucopyranoside was studied on the conversion
bases as discussed above [see equations (1), (2) and (3)]. Large mass balance
discrepancies were observed when the percent cleavage calculated based on remaining
phenyl-glycoside was compared with that based on formed phenol or glucose. This is
shown in detail in Appendix B. The discussion below will concentrate mainly on the
cleavage calculated based on the formation of glucose and phenol. However, at the end of
34
this chapter the mass balance discrepancy (shown in Appendix B) will be addressed when
discussing the carbon balance (TOC data).
5.3.1
Strong acidic treatment at 70 °C
At a low pH of 1.65 the temperature can be lowered while still achieving some
cleavage of phenyl-glucoside. Figure 5.3.A shows phenyl-glucoside cleaved at 70 °C
after 1, 2, 3, 4, 5, 24, 48, 72 hours on the basis of glucose and phenol formed.
[A]
70
40
20
60
80
Time (hours)
• Glucose basis (GC-FID)
- * - Phenol basis (GC-MS)
[B]
Figure 5.3 Phenyl-glucoside cleaved at pH 1.65 and 70 °C; [A] at 1 ,2,
3, 4, 5 , 24, 48 and 72 hours, [B] at 1 ,2 ,3, 4 and 5 hours
35
The conversion yield calculated based on the two known products formed, i.e.
phenol and glucose shows reasonable agreement with each other. The results show a
nearly linear increase in conversion up to 63 % after 72 hours.
The percentage yield of phenyl-glucoside hydrolysis on the basis of glucose
formed was somewhat higher than that on the basis of phenol formed during the first 5
hours. This was further investigated by subjecting pure glucose and pure phenol to the
same conditions of pH 1.65 at 70 °C for 1, 5 and 24 hours (Appendix B; see Figure B.2
and Figure B.3 respectively). The mass of the two components was unchanged by the
hydrolysis, clearly showing that glucose and phenol were stable at these conditions and
no other degradation products of glucose or phenol were formed.
5.3.2
Strong acidic treatment at 90 °C
Figure 5.4 shows the phenyl-glucoside hydrolysis yield at 90 °C for 1, 2, 3 and 4
hours.
]
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0
1
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2
3
Time (hours)
- Glucose basis (GC-FID)
4
5
;
- A - Phenol basis (GC-MS)
!
Figure 5.4 Phenyl-glucoside cleavage at pH 1.65 and 90 °C
36
The hydrolysis yield on the basis of phenol and glucose formed show good
agreement with each other. During the first hour, 8 % cleavage occurs and this increases
to 30% after 4 hours.
To determine whether any degradation product was formed from glucose or
phenol, the pure compounds were subjected at 90 °C for 1 and 3 hours (Appendix B;
Figure B.2 and Figure B.3 respectively). No significant conversion of either compound
was detected by gas chromatographic analysis.
5.3.3
Strong acidic treatment at 105°C
Treatment at 105 °C was performed in the autoclave. For the analysis of the data
it should be taken into account that it takes approximate 15 minutes for the reaction
mixture to reach the desired final temperature. This explains why the hydrolysis of
phenyl-glucoside at 105 °C begins after about 15 minutes as seen in Figure 5.5
90
80
70
1 60 !
S 50 !
,j?
'/
% « !
°- 30 ;
^ 20 i
10 I
/'
/
/
/
o I-*-—
0
0.5
1
1.5
2
2.5
3
Time (hours)
- o- Gluccose basis (GC-FID)
- A - Phenol basis (GC-MS)
Figure 5.5 Phenyl-glucoside hydrolysis at pH 1.65 and 105 °C
37
3.5
Only 3 % conversion is obtained at 15 minutes based on phenol and glucose
formed due to heat-up to 105 °C in this period. Figure 5.5 shows that subsequently the
cleavage increases rapidly to 61 % after 1 hour. Then the cleavage rate decreases
significantly with a yield of 78 % after 2 hours, and 85% after 3 hours. Since our data
shows no formation of degradation products from pure phenol or glucose at 105°C, the
observed reduced rate of formation of glucose and phenol may be due to depletion of the
phenyl-|3-D-glucopyranoside. However to verify this, further studies need to be
performed whereby the concentration of the phenyl-P-D-glucopyranoside is changed to
determine the reaction order of the phenyl-(3-D-glucopyranoside hydrolysis kinetics.
5.3.4
Strong acidic treatment at 121 °C
As discussed before, it takes about 15 minutes to heat up the sample to the final
autoclave temperature. This was again reflected in the initial delay time of about 15
minutes for phenyl-glucoside cleavage at 121 °C shown in Figure 5.6
100
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0
0
0.5
1
1.5
2
2.5
Time (hour)
--o--Glucose basis (GC-FID)
- A - Phenol basis (GC-MS)
Figure 5.6 Phenyl-glucoside cleavage at pH 1.65 and 121 °C
38
3.5
During the temperature rise to 121 °C about, 8% of the phenyl-glucoside was
cleaved after 15 minutes. The data shows a rapid increase in yield up to 76 % after 1 hour
and then only a small further increase to around 82 % at 2 hour and 3 hours. No
formation of degradation products from phenol or glucose was observed.
5.4
Activation energy for strong acid treatment condition
The activation energy is the amount of energy required to ensure that a reaction
happens. It can be calculated by the Arrhenius equation which is given below
k =A*exp ( - E i T T)
The Arrhenius equation can be rearranged as follows;
where k = rate percentage (hour"1)
Ea = activation energy (kJ*mor')
R = gas constant; 0.008314472 (kl^K^'mol"1)
T = Temperature (°K)
A = proportionality constant
Figure 5.7 shows a straight line Arrhenius plot of the data in Table 5.1 in the form of
log(rate) versus
jj.
39
2
j
hour1)
•
1.5 j
:
y = -6681.x + 19.38
R2 = 0.991
i !
^
I
0s
!
log [rate
v
^ \
•s.
0.5 j
0 |
0.0t>26 0.00265
-0.5
0.0027 0.00275
0.0028 0.00285
0.0029
0.00295
;
1/Temperature (°K')
Figure 5.7 Arrhenius plot of degradation of phenyl-glucoside at pH 1.65
Temperature Temperature
1/T
1
rate (k) (%.hr"')
logk
CK"
)
(°K)
(°C)
-0.052
70
343
0.00292
0.8866
0.884
90
0.00275
363
7.6715
1.769
105
378
0.00265
58.843
Table 5.1 Rate constant at pH 1.65 at different temperatures
The activation energy (Ea) was estimated from the slope of the log(k) versus -y^ curve and found
to be 128 kJ mol"1. This value is in good agreement with previous work, where the
activation energy for the cleavage of the phenyl glycoside bond at acidic conditions was
estimated to be between 125 kJ mol"1 and 135 kJ mol"1 (Overend, Rees and Sequiera,
1962)
40
5.5
Total Organic Carbon (TOC) analysis
In order to better understand the mass balance discrepancies obtained under acidic
hydrolysis conditions, the total organic carbon content of the sample was determined
using the TOC analyzer. The equations for the calculation of the theoretical amount of
organic carbon based on the GC analyses are also given below:
(CpG) Carbon from phenyl-glucoside (PG)
Mass of carbon (12x12) x (256 / 180) x (mass of glucose formed)
Molar mass of PG
(Initial mass of PG)
Please note that in the above formula the amount of PG remaining is calculated on basis
of glucose formed because of problems with the analysis of PG as will be explained later.
(CQIU)
Carbon from glucose
= Mass of carbon (6 x 12) x Mass of glucose formed
Molar mass of glucose
(Cphe) Carbon from phenol
= Mass of carbon (6 x 12) x Mass of phenol formed
Molar mass of phenol
Thus the total amount of carbon present in solution (mg/L):
(Ctotal)=
(CpQ + Cpiu + Cphe )
Total volume (ml) x 1000
At reaction time zero, i.e. when the phenyl-glucoside is dissolved in the acidic
solution at room temperature and immediately neutralized, the TOC data from the
analyzer is in excellent agreement with the calculated value (0.35% difference, see Table
5.2). When pure glucose and phenol were treated in the same way, the calculated carbon
41
showed a difference of 3% with the TOC data, still within the margin of experimental
error.
Compound
Phenyl-glucoside Glucose Phenol
Initial Carbon calculated (mg/Lit)
8.5
13.05
33.53
12.62
Carbon calculated from TOC analysis (mg/L)
8.53
34.65
% Difference
0.35
3.29
3.23
Table 5.2 Carbon calculations before reaction for the pure compounds at
room temperature and pH 1.65
Also, when pure glucose and pure phenol were treated at different temperatures
and pH 1.65, the TOC value measured and calculated from the GC analysis were in
agreement in the range of 94-97% (Table 5.3). This clearly indicated that the formation of
any degradation products from glucose or phenol is minimal at these conditions.
Compound
Temp
(°C)
Glucose (mg/L)
Carbon
from TOC
Carbon
analysis
calculated
after
before
reaction
reaction
3.452
3.543
3.543
3.491
3.543
3.406
3.543
3.402
STD
for
TOC
Phenol (mg/L)
Carbon
from TOC
Carbon
analysis
calculated
after
before
reaction
reaction
7.669
7.221
7.669
7.32
7.669
7.197
7.669
7.173
14.32
13.45
STD
for
TOC
Time
(hr)
0.068
0.001
1
0.072
0.087
2
0.032
0.039
3
0.09
0.051
4
70
0.028
1
0.01
2
10.47
9.837
0.084
90
5
14.32
13.46
0.097
0.038
1
11.14
10.94
5.31
5.01
0.07
0.024
105
3
4.04
3.81
11.84
10.98
0.022
0.051
1
121
5.99
5.86
16.33
15.86
Tabh; 5.3 Carbon analysis for pure compc>unds at c ifferent term:>erature and pH1.65
Based on the above results, the higher yield of phenyl glycoside cleaved during
acid treatment calculated based on the amount of remaining phenyl-glycoside (Appendix
42
B) is likely to be the result of an analytical problem related to the acid treatment. The
calibration curve made for this system whereby phenyl-glucoside is dissolved in the
acidic solution and then immediately neutralized, then preparing standards as discussed in
section 4.4, is shown in Figure 5.8. The lack of linearity of the calibration curve, which is
typical for the other calibration curves, is another indication of an analytical problem.
This may be either an incomplete acetylation of the acid treated phenyl-glucoside or an
incomplete extraction of the derivatized product to the ethyl acetate phase. Both problems
p
bo
y = 2.961x
R2 = 0.742
o o
in ^
of Inositol
would lead to an overestimation of the hydrolysis yield of phenyl glycoside.
;
0.4 i
••-
•
o
(Mass of Glucose
p o p
^
•
+ ^^
c/> 0 . 5
03
^
S*
0.05
0.1
0.15
0.2
0.25
0.3
(Area PG / Area of Inositol)
Figure 5.8 Calibration curve for phenyl-glucoside in acidic solution (pH 1.65)
43
Chapter 6
CONCLUSIONS
The combined effect of temperature and acidity on the hydrolysis of
phenyl-P-D-glucopyranoside was studied experimentally. The hydrolysis yield was
quantified using GC/MS and GC/FID analysis after alditol acetylation derivatization of
the products and starting material. The hydrolysis conditions were chosen to simulate
different pretreatment methods such as auto hydrolysis, green liquor pretreatment and
acid hydrolysis. It was found that hydrolysis of the phenyl glucoside model compound at
near neutral aqueous conditions (pH 6) was minimal (4 %) at 170 °C. On the other hand
hydrolysis was nearly complete (95 %) at this temperature with the addition of acetic acid
at a concentration expected to be generated from deacetylation of xylan in hardwoods
(about 10 g/L). At acid conditions of pH 1.65 the temperature may be lowered to 105 or
121 °C while still obtaining a significant (80-90 %) hydrolysis yield.
The hydrolysis experiments performed simulating green liquor extraction
showed as previously reported that the hydrolysis of phenyl-glucoside produces phenol
and levoglucosan while no glucose is formed. The result showed that hydrolysis of the
phenyl glucoside at 155 °C was essentially complete with quantitative formation of
levoglucosan and phenol rather than glucose and phenol.
Incomplete mass balances were obtained between the amount of the phenyl
glucoside reacted and phenol and glucose formed after hydrolysis at pH 1.65 at all
temperatures studied (70, 90, 105 and 121 °C). Based on the curved nature of the phenyl
glucoside calibration curve and quantification of the TOC of the starting and final
reaction mixtures, it is suggested that the alditol acetylation method does not work
44
properly at strong acidic conditions. No other degradation products than glucose, phenol
and levoglucosan were observed at any of the experimental conditions studied.
6.1 Practical Implications of this Study
The model compound experiments represent a homogeneous system while wood
pre-extraction is a heterogeneous process. Also the effect of the chemical environment in
close proximity to the phenylglycosidic bond in wood may affect the bond cleavage. For
instance, the presence of the electron donating methoxyl group in the C3-position of the
aromatic ring would enhance the cleavage. Similarly, the presence of other reactive
species in solution may affect the kinetics. However, the present LCC model compound
study provides a good understanding of how reactive the LC bond is at different
conditions during real wood pre-extraction processes. Cleavage of the LC bond is
important since it may enhance the dissolution of hemicelluloses. Furthermore, if wood is
further processed to pulp by kraft cooking, cleavage of the phenylglycosidic bond would
increase the phenolic content of lignin and thus enhance lignin reactions leading to
delignification.
During practical hot water pre-extraction of wood acetic acid is produced by
deacetylation of xylan. Other organic acids e.g. formic acid and lactic acid are also
formed, albeit in small amounts. The present model compound data suggests that at 160
°C and 90 minutes reaction time only 18-15 % of the phenyl glycosidic bond will be left,
while at 170 °C complete cleavage is achieved.
It is common practice to convert polysaccharides to monosugars by acid
hydrolysis using 3 % or 4 % sulfuric acid in an autoclave at 121 °C. The monosugars
45
may be converted to other products e.g. ethanol, or simply be analyzed for composition
determination. Based on the present study which was performed at milder conditions than
the traditional acid hydrolysis discussed above (pH 1.65 versus pH 0), it is reasonable to
assume that at the traditional acid hydrolysis conditions, the phenylglycosidic bond is
completely cleaved.
Extraction of wood with green liquor is performed to neutralize the organic acids
that would otherwise be produced if only hot water was used. Thus, a milder extraction is
achieved as is reflected in the final near-neutral pH of about 6. The near neutral pH is
reached relatively quickly as can be inferred from the rapid pressurization of the digester
due to release of CO2 from decomposition of Na2CC>3 shortly after temperature is
reached. In the present model compound study however, the green liquor used was not
neutralized as was reflected in the same pH of 12.5 before and after treatment. On the
other hand the present study showed that phenylglycoside hydrolysis was minimal even
at 170 °C at neutral aqueous conditions. Therefore the present data suggest that the LCC
phenylglycosidic bond remains intact during "near-neutral" green liquor pre-extraction of
hardwoods.
46
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•
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•
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•
Bruyne and Wouters-Leysen. The influence of acids on the hydrolysis of
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•
Ebringerova, A; Hromadkova, Z. Xylans of industrial and biomedical importance
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Eriksson, O. and Lindgren, BO. About the linkage between lignin and
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•
Eriksson O., Goring D.A.I, and Lindgren B.O. Structural studies on the chemical
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•
Fengel D.; Wegener, G. Wood: Chemistry, ultrastructure, reactions. Walter de
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•
Freudenberg, K. and Grion, G. Contribution to the mechanism of formation of
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•
Gatenholm, P.; Tenkanen, M. Hemicelluloses: Science and technology. American
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47
•
Gellerstedt, G. Gel permeastion chromotography. In methods in lignin chemistry.
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•
Hayashi A. Studies on the chemical combination
between lignin and
carbohydrate. Part X. The existence of phenyl-P-D-glucosidic bond. Journal of
the Agricultural. Chemical Society of Japan. (1961). 35, 83-86.
•
Heidt, Lawrence J.; Purves, Clifford B. Thermal rates and activation energies for
the aqueous acid hydrolysis of a- and P- methyl, phenyl and benzyl Dglucopyranosides, a- and P- methyl and P- benzyl D-fructopyranosides and dimethyl D-fructofuranoside. Journal of the American Chemical Society. (1944). 66,
1385-9.
•
Jean-Paul Joseleau and Rachid Kesraoui. Glycosidic bonds between lignin and
carbohydrates. Holzforschung. (1986). 40, 163-168.
•
Kawamura I; Higuchi T. Relation between lignin and carbohydrates in wood. V.
Methylation of carbohydrates contained in dioxane lignin. Journal soc. Textile
cellulose (Japan). (1952). 8, 335-337.
•
Kondo R; Sako T; Iimori T; Imamura H. Formation of glycosidic LCC in the
enzymatic dehydrogenative polymeraization of coniferyl alchol. Mokuzai
Gakkaishi. (1990). 36(4), 332-338,
•
Kosikova B; Joniak D. and Skamla J. Lignin-Carbohydrate bond in beech wood.
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•
Kosikova, B., Joniak, D. and Kosikova, L. The properties of benzyl ether bonds in
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48
•
Koshijima, T., Watanabe, T. and Azuma, J. Existence of benzylated carbohydrate
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•
Lawoko. M, Henriksson G. and Gellerstedt G.. New method for quantitative
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•
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•
Mao H, Genco J.M, Yoon S.H, A. van Heiningen and Pendse H, Technical
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•
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•
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•
Moelwyn-Hughes, E. A. Kinetics of the hydrolysis of certain glucosides (salicin,
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•
Neilson M. J. and Richards G. N. Chemical structure in a LCC isolated from the
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49
•
Overend, W.G., Rees, C.W and Sequiera, J.S. Reaction at position 1 of
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•
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•
Sjostrom. E. Wood Chemistry Fundamentals and Applications 2nd Edition,
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•
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•
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•
Theander, O and Westerlund, E, A.
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34, 330-336.
•
Veibel, Stig. and Kem. Maanedsblad. Enzymic and acid hydrolysis of glucosides.
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•
Watanabe, T. and Koshijima, T. Evidence for an ester linkage between lignin and
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•
Watanabe, T.; Koshijima, T. Association between Lignin and Carbohydrates in
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50
•
William E.M High
Temperature
Alkaline
Degradation
of Phenyl-P-D-
Glucopyranoside, Journal of Wood Chemistry and Technology. (1990). 10(2), 209215.
•
Yaku, F., Yamada, Y. and Koshijima, T. Lignin-carbohydrate complex. Part
II.Enzymatic
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of acidic polysaccharide
in Bjorkman LCC.
Holzforschung. (1976). 30, 148-156.
•
Yaku, F., Tanaka, R. and Koshijima, T. Lignin carbohydrate complex. Part IV.
Lignin as side chain of the carbohydrate in Bjorkman LCC. Holzforschung.
(1981). 35(4), 177-181.
•
Brunow, G. http://www.helsinki.fi/~orgkm_ww/lignin_structure.htmI. (1999).
51
APPENDIX A
METHODOLOGY DEVELOPMENT
This chapter includes methods tried to quantify and analyze products after
hydrolysis of phenyl-glucoside.
UV Visible Spectroscopy
UV-Visibility method was favored for being a non-sophisticated and easy
method.
Pretreatment
of
sample
was
not
necessary.
An
HP8453
UV-VIS
Spectrophotometer was used. Figure A. 1 shows a typical UV spectrogram.
IA]
;;
' •
•'.}
i
[Bl
•:
?
i A
>
/
:;,;.
Figure A. 1 UV spectrograms of [A] Phenyl-glucoside at acidic conditions [B] Phenylglucoside at alkaline conditions
52
Figure A. 1 Continued [C] Phenol at acidic conditions [D] Phenol at alkaline conditions
[E] Guaiacol at acidic conditions
53
[F]
Figure A. 1 Continued [F] Guaiacol at alkaline conditions
A shortcoming of UV spectroscopy is that glucose has no UV response without
use of dyes; hence it is difficult to use for glucose. Another problem is that the UV
adsorption of phenyl-glucoside and phenol overlap i.e. phenyl-glucoside - 266nm,
272nm (figure A.l.A and A.l.B) and phenol 270nm, 275nm (figure A.l.C and A.l.D).
Finally the UV adsorption of guaiacol (internal standard) occurred at different
wavelength depending on the pH; at acidic conditions at 223nm, 274nm (figure A.l.E)
and at alkaline nature at 240nm, 290nm (figure A. 1 .F).
54
HPLC Analysis
Figure A.2 HPLC chromatograms [A] pure phenyl-glucoside- UV
detector [B] Pure phenol -UV detector [C] phenyl-glucoside
hydrolysed in presence of HCl - 90°C- 3hours
55
HPLC with UV and RI detector was used for analysis of glucose, phenylglucoside and phenol. An HPX-87p type of detector was used (300mm x 7.8mm), oven
temperature 60°C, eluent HPLC grade water (0.6 ml/min), total run time 50inutes, UV
wavelength 272nm. Sample was neutralized, filtered and then analyzed.
The UV peak for phenyl-glucoside and phenol are broad and overlap (phenylglucoside 29 to 35 minutes and phenol 21 to 31minutes). Also the RI detector was not
effective for low concentrations glucose; hence a new approach was necessary.
56
GC-MS Analysis -Derivatization using Acetylation
RTX-225 and DB-225 columns (50% cyanopropylmethy 1/50% phenylmethyl
polysiloxane) were preferred for analysis. Since complete water removal was necessary,
rotary vacuum evaporation was preferred over freeze drying because of time required
(vacuum evaporator - 15 minutes and freeze drying - 18 hours). Derivatization was
necessary to make the products volatile, and acetylation was considered because of its
universal acceptance.
ity
200000
w
i
150000
solute Int
c
100000
<
50000
Glucose
/
0
—
16 17
I
A
:
18
19
20
21
22
23
24
25
Time (min)
Figure A.3 Gas chromatogram of pure glucose after acetylation using
pyridine: acetic-anhydride (1:1 v/v)
After
removing water from
the sample, pyridine and
acetic-anhydride
(Gellerstedt, 1992) were mixed in a ratio of 1:1 by volume and kept overnight to
complete the reaction; Un-reacted acetic-anhydride was removed by washing using
methanol in an ice bath. Toluene was used to remove pyridine and then the mixture was
subjected to rotary evaporation (water bath temperature maintained at 35°C). The dry
57
products were dissolved in ethyl-acetate which was used as solvent for GC-MS analysis.
Figure A.3 shows the gas chromatogram for pure glucose after acetylation.
GC-MS Analysis -Derivatization using Silylation
Derivatization using silylation was preferred over acetylation because of its short
preparation time.
After
removing
water
from
the
sample,
1.5
ml
of
silylation
reagent N-
trimethylsilylimidazole (Tri-Sil-Z) was added and the solution was kept in a water bath
maintained at 65 °C to achieve complete dissolution. The sample was filtered and was
then ready for gas chromatographic analysis. The silylation reagent Tri-Sil-Z was bought
from Pierce-Biotechnology. The reagent is a mixture trimethylsilylimidazole (TMSI) in
dry pyridine (1.5 mEq/ml = 1 part TMSI: 4 parts pyridine)
6000000
jnsifr
^ 5000000
4000000
2til
3000000
ibsolut
Glucose
2000000
«i
1000000
Glucose
Ml
13
14
15
16
17
18
19
20
Time (min)
Figure A.4 Gas chromatogram of pure glucose after silylation
using N-Trimethylsilylimidazole reagent
Removal of water may be done either by freeze drying or by rotary vacuum
evaporation. However both methods must deal with a salt residue after neutralizing the
58
sample. More over in the rotary vacuum evaporator there is a possibility of losing volatile
products with water at high vacuum. Other major problem is the presence of more than
one peak for glucose, which makes quantification more difficult.
Since reduction using potassium hydroxide prior to acetylation converts all
isomers of glucose into one alditol form, only one peak in seen in the chromatogram after
alditol-acetylation. Acetylation using methylimidazol as reagent removes water from the
acetyl phase, and hence rotary evaporation and freeze drying are unnecessary.
59
APPENDIX B
DISCUSSION OF PRODUCT ANALYSIS
Problems with phenyl-glucoside analysis at acidic conditions (pH 1.65)
At acidic hydrolysis conditions of pH 1.65, the yield of phenyl-glucoside cleaved
calculated on the basis of un-reacted phenyl-glucoside was significantly larger than that
on the basis of the two products, glucose and phenol. Figure B.l shows the percentage of
phenyl-glucoside cleaved calculated using the three methods at the four temperatures.
100
[A]
[B]
Time (hours)
o PG basis (GC-MS)
- A -Phenol basis (GC-MS)
--Q--Glucose basis (GC-FID)
Figure B.l. Phenyl-glucoside cleaved at pH 1.65; [A] 70 °C; [B] 90 °C
60
[C]
100
. * •
/
0
0
1
2
3
4
Time (hours)
o PG basis (GC-MS)
- Q- Gluccose basis (GC-FID)
- A - P h e n o l basis (GC-MS)
[D]
-B-
-B
Figure B.l. Continued; [C] 105 °C; [D] 121 °C
To investigate this disagreement, the following experiment was performed; phenylglucoside was treated at pH 1.65 and room temperature rather than at the higher
temperatures, and the mixture was analyzed by alditol-acetylation and dichloromethane
liquid extraction. Again it was observed that 35% of the phenyl-glucoside had
disappeared while there was no formation of glucose and phenol. The possibility of
degradation of glucose and phenol was eliminated by testing the pure components (see
Figures B2 and B3). This suggests that the treatment of phenyl-glucoside with
61
hydrochloric acid at pH of 1.65 leads to a systematically lower analysis for the phenyl
glucoside remaining.
2.5
[A]
2.4
2.2
2.1
2
25
[B]
Figure B.2 Hydrolysis of pure glucose at pH 1.65. [A] 70 °C; [B] 90°C
62
1.4
1.38
1.36
1.34
1.32
1.3
1.28
1.26
1.24
1.22
1.2
[A]
30
[B]
Time (hours)
•Initial mass
-a-Final calculated mass
Figure B.3 Hydrolysis of pure phenol at pH 1.65. [A] 70 °C; [B] 90°C
63
Sample composition after sodium borohydride reduction
Final samples prepared at acidic conditions (pH 1.65, 70 °C - 2, 5 and 24 hours
and 121 °C - 1 hour) and at high temperature in the presence of acetic (150, 160 and 170
°C) were subjected to the potassium borohydride reduction as usual, but then the
solutions were extracted with dichloromethane to verify whether any other phenolic
products than phenol were formed.
The liquid-liquid extraction after reduction was done in the same way as used for
the analysis of phenol as discussed in chapter-3, except that no internal standard was
used. The dichloromethane phase was analyzed using GC-MS. No significant other
compound than phenol were seen.
Analysis of the water phase formed after acetylation
Samples prepared at acidic conditions (pH 1.65, 70 °C - 2, 5 and 24 hours and
121 °C - 1 hour) and at high temperature in the presence of acetic (150, 160 and 170 °C)
were used. A water phase is formed after alditol-acetylation of these samples. To verify
whether any PG was remaining in the water phase, the pH of the water phase was
reduced to 1 from pH 6.5 using hydrochloric acid, and then exposed to 121 °C for 1 hour.
This condition was selected since all phenyl-glucoside would hydrolyze after this
treatment. Subsequently the sample was directly acetylated using pyridine and aceticanhydride as reagents (see Appendix-A). No compound was detected by GC-MS, except
for traces of levoglucosan.
64
Determination of the presence of gluconic acid in the reaction mixture
This investigation was undertaken based on two assumptions:
1) Gluconic acid is formed after hydrolysis of phenyl-glucoside, and
2) Gluconic acid is not derivatized using the alditol acetylation method.
Pure gluconic acid was dissolved in deionized water, and subjected to reduction
and alditol acetylation as discussed in Chapter-3. GC-MS didn't show any gluconic acid
peak, proving that the alditol-acetylation method was inadequate to derivatize gluconic
acid.
Hence a new method was developed to derivatize gluconic acid using an ion
exchange resin. A DOWEX 1x8, 200-400 mesh ion exchange resin was pre-treated to
convert it to the sodium form. Thus, a known amount of resin was washed three times
with 6ml water followed by three times washing with 5ml 3M of sodium acetate. Each
time before washing the resin was kept at room temperature for 10 minutes in the sodium
acetate solution. Subsequently the resin was kept for 10 minutes in 5ml of 0.5M acetic
acid before washing with 0.05M of acetic acid to convert the resin to the acetate form.
The freshly prepared resin in the acetate form was mixed with a gluconic acid
solution for 1 hour and then the resin was removed by filtration. The filtrate was set
aside, and the resin was first washed with water and then with 2.5ml of 1M hydrochloric
acid to remove adsorbed gluconic acid. The hydrochloric acid filtrate was evaporated to
dryness and kept overnight in a desiccator.
65
2.5 mL of a buffer solution of pH 8 prepared using 0.025M sodium-tetraborate
and 0.1M hydrochloric acid was added to the dry residue. The pH was checked regularly
to make sure that the pH does not exceed 6.5.
Reduction and acetylation was then performed on the above prepared solution as
discussed in chapter-3, except that potassium borohydride was dissolved in 0.5ml of the
buffer solution and no any internal standard was used. The gluconic acid peak was clearly
seen in the GC-MS chromatogram; showing that the above method derivatizes gluconic
acid and allows its detection by GC-MS.
Subsequently two neutralized samples after acidic hydrolysis (pH 1.65, 70 °C - 5
hours and 121 °C - 1 hour) and one green liquor treatment sample (155°C) were
subjected to the above described resin assisted reduction acetylation method.
No gluconic acid peak was seen in the GC-MS chromatograms of all the above
samples, proving that gluconic acid is not formed during hydrolysis of the phenylglucoside.
66
TOC measurement
The total organic carbon (TOC) content of the starting phenyl-glucoside solution
and final hydrolyzed solution can be calculated if the concentrations of all organics are
known. Also the TOC may be determined experimentally both before and after
hydrolysis of the phenyl-glucoside solution. Thus, if the experimentally determined TOC
is higher than the TOC calculated based on the measured concentrations of the organics,
then not all the organics are identified in the mixture or their measured concentrations are
inaccurate. The TOC calculation was done using the equations described in chapter 5.4.
Note again that in the amount of PG after hydrolysis is calculated on basis of glucose
formed because of problems with the analysis of PG.
Time (hour)
2
4
1
3
TOC calculated before reaction
5.34
5.34
(mg/L)
5.34
7.88
Final TOC measured after
5.02
reaction (mg/L)
7.416
4.908
5.059
Final TOC calculated after
reaction (mg/L)
7.498
4.735
5.229
5.576
STD of TOC measured after
reaction (mg/L)
0.072
0.091
0.081
0.077
Table B.l TOC for acidic hyc rolysis at p H1.65an< i 7 0 ° C
Time (hour)
1
TOC calculated before reaction
(mg/L)
8.644
Final TOC measured after
reaction (mg/L)
8.064
Final TOC calculated after
reaction (mg/L)
7.989
STD of TOC measured after
reaction (mg/L)
0.03
Table B.2 TOC for acidic hydrolysis
67
5
5.34
4.95
5.709
0.098
2
3
4
8.644
8.644
8.644
8.175
7.959
8.121
7.981
7.148
8.372
0.072
0.067
0.102
at pH 1.65 and 90 °C
Time (hour)
1
3
TOC calculated before reaction (mg/L)
9.48
8.2
TOC measured after reaction (mg/L)
8.96
8.175
TOC calculated after reaction (mg/L)
8.821
8.754
STD of TOC measured after reaction (mg/L)
0.081
0.05
Table B.3 TOC for acidic hydrolysis at pH 1.65 and 105 °C
Time (hour)
1
3
TOC calculated before reaction (mg/L)
9.04
5.99
TOC measured after reaction (mg/L)
8.85
5.86
TOC calculated after reaction (mg/L)
8.98
5.81
STD of TOC measured after reaction (mg/L)
0.015
0.088
Table B.4 TOC for acidic hydrolysis at pH 1.65 anc 1121°C
The measured TOC values after hydrolysis are compared to those calculated from
the known starting phenyl-glucoside concentrations in Figure B.4 for all four hydrolysis
temperature experiments. It can be seen that the measured value is somewhat smaller than
the calculated value. This difference may be attributed to experimental error and a
slightly too low TOC calibration. The measured TOC values after reaction are plotted
against the TOC values calculated from the organic concentrations in Figure B.5. A
straight line through the origin is obtained with a slope of 0.98 indicating small fraction
of carbon is unaccounted for by the (measured) concentrations of phenyl-glucoside,
glucose and phenol.
68
Initial carbon calculated before
reaction (mg/L)
10
9
8
y = 1.053x
R2 = 0.986
7
6
5
4
3
2
1
0
10
Final carbon from TOC analysis after reaction (mg/L)
Figure B.4 TOC comparison for all pHl.65 hydrolysis experiments. Initial vs. final
y = 0.989x
R2 = 0.935
10
Final carbon calculated after reaction (mg/L)
Figure B.5 TOC comparison for all pHl.65 hydrolysis experiments. Final analysed vs.
final calculated
69
APPENDIX C
FIGURES AND GRAPHS
Initial concentration of phenyl-glucoside
Condition
Concentration (mg/mL)
Effect of temperature and acidity
0.949
150 ° C - 90 minutes
160 ° C - 90 minutes
0.92
170 ° C - 9 0 minutes
0.93
Green liquor extraction
137 ° C - 90 minutes
0.986
146 ° C - 90 minutes
1.146
155 ° C - 9 0 minutes
1.132
Strong acidic condition
70 °C - 1 hour
0.935
70 °C - 2 hour
0.599
70 °C - 3 hour
0.602
70 °C - 4 hour
0.603
0.599
70 °C - 5 hour
0.682
70 ° C - 1 2 hour
70 °C - 24 hour
0.602
70 °C - 48 hour
0.599
90 °C 90 °C 90 °C 90 °C -
1 hour
2 hour
3 hour
4 hour
0.824
0.822
0.824
0.824
1 0 5 ° C - 1 hour
105 ° C - 2 hour
105 ° C - 2 hour
0.27
0.317
0.268
121 ° C - l h o u r
1.076
121 ° C - 2 h o u r
0.916
121 ° C - 3 h o u r
0.98
Table C.l Initial concentrations of phenyl-glucoside
70
Calibration curves for phenyl-glucoside and glucose in water
Figure C.l Calibration curves for water as solvent [A] Phenylglucoside - GC-MS [B] Glucose - GC-FID
71
Calibration curves for green liquor as solvent
[A]
1
2
3
4
(Area of PG) / (Area of Inositol)
[B]
->
10
re —»
8°
2. o
roc
[C]
y = 0.566x
R2 = 0.976
c
% o
is
re
E
o
10
15
20
(Area of levoglucosan) / (Area of Inositol)
Figure C.2 Calibration curves for green liquor as solvent [A]
Phenyl-glucoside - GC-MS [B] Phenol - GC-MS [C]
Levoglucosan - GC-FID
72
Comparison of phenyl-glucoside yield on basis of glucose formed using GC-MS and
GC-FID analysis.
[A]
155
160
165
170
80
175
[B]
o
20
60
40
80
[C]
35
Figure C.3 Percentage of phenyl-glucoside reacted on basis of glucose formed
measured by GC-MS and GC-FID [A] Simulation of hot water extraction;
[B] pH 1.65 - 70 °C; [C] pH 1.65 - 90 °C
73
25000
[A]
20000
Glucose
33
18
800000
[B]
Phenol
v
10
15
20
25
Time (min)
Figure C.4 GC-MS chromatograms; [A] Alditol acetylation - pH 1.65, 90
°C , 4hours [B] Phenol extraction - pH 1.65, 90 °C , 4 hours
74
60000
[C]
40000
Inositol
Phenylglucoside
(I.S)
12
18
32
17
23
28
33
Time (min)
Figure C.4 GC-MS chromatograms; [C] Alditol acetylation - green liquor,
137 °C - 90 minutes [D] Alditol acetylation - pH 1.65, 105 °C , 3hours
75
Mechanism for formation levoglucosan from phenyl-glucoside
• OH
OH
HO'* 5 ' HO
HO
®o
^o>
Figure C.5 Mechanism for formation of levoglucosan at alkaline conditions and
high temperature. (1) phenyl-glucoside, (2) phenolate ion, (3) 1, 2- anhydro a-Dglucopyranose (4) levoglucosan
(William E., 1990)
76
BIOGRAPHY OF THE AUTHOR
Sagar Nandkumar Deshpande was born in Aurangabad, India on March 26th,
1982. He was raised in Mumbai, the capital metropolitan city of India. He graduated with
a Bachelor of Science degree from the Chemical Engineering Department of Mumbai
University in June 2005. Upon graduating, Sagar enrolled in University of Maine for
graduate studies in January 2006 where he pursued his Master of Science in Chemical
Engineering. He is a candidate for the Master of Science degree in Chemical Engineering
from The University of Maine in December, 2008.
77