,
College
K
LONDON
ING S
Founded I8
University of London
Arni Saleh
Ph.D. student,
xx cycle
Activity
Presentation
e-mail: arni@unige.it
arnisaleh@hotmail.com
12 December 2007
,
College
K
LONDON
ING S
Founded I8
University of London
Aromatic compounds from
Sugarcane Bagasse
Presentation outline
•
•
•
•
•
Background
Biomass Pretreatment
Process for extract lignin materials
Lignin extractions
Study of lignin extractions
Background
• Bagasse material
• Renewable resource, economics and
environment
• What is lignin?
Background
What is Bagasse?
• Bagasse material
– Cellulose
– Hemicellulose
– Lignin
33-48%
19-43%
6-32%
• Renewable Resource, Economics and
Environment
Increasing importance is being given to biomass as a renewable and
environment friendly resource, which has motivated a great number of
economic utilization as starting materials for various bioproduction.
Background
What is lignin?
• Natural lignin composition and structure are
unknown.
• It is a heterogeneous polymer.
• It is made up of three principal monomers.
Biomass Pretreatment
Biomass Pretreatment
Bagasse was first dried in sunlight, then in oven
at 105°C, cut into small pieces and stored in
desiccator at room temperature. The dried
biomass of bagasse was ground using a
laboratory mill and screened to prepare 40 mesh
powder.
Processes for extract lignin materials
• Hydrolysis method
• Alkaline sugarcane bagasse hydrolysis
Hydrolysis method
Alkaline sugarcane bagasse hydrolysis
Dried sugarcane bagasse
Extraction of
lignin from
sugarcane
bagasse
Grounding and screening to
prepare 40 mesh powder
Treatment with NaOH in autoclave at
121°C, for 1 h
Solid residue
Waste
Extract
a) Filtration with 0.45 mm pore filters
b) Acid treatment with H3PO4 (pH 3)
c) Filtration with 0.20 mm pore filters
HPLC Analysis
Lignin extraction
Lignin extraction
Bgasse (g)
1.0
2.0
3.0
5.0
alkaline
(NaOH)
treatment (M)
0.5
1.0
2.0
3.0
4.0
0.5
1.0
2.0
3.0
4.0
0.5
1.0
2.0
3.0
4.0
0.5
1.0
2.0
3.0
4.0
% of main components extracted
p-coumaric
Ferulic
Syringic
acid
acid
acid
Vanillin
79.61
76.36
74.01
75.78
76.10
76.83
67.40
69.04
67.27
78.91
83.41
82.13
79.98
80.59
78.79
76.04
73.79
67.00
71.68
71.49
16.21
17.58
19.03
18.49
18.78
15.44
18.90
19.26
21.12
15.80
14.96
16.04
17.31
17.33
18.36
19.10
20.59
24.40
21.10
20.23
1.10
1.49
1.81
1.69
1.79
3.19
3.97
3.37
3.95
2.45
0.82
0.97
1.34
1.50
1.82
1.79
2.38
3.31
2.87
3.18
3.08
4.57
5.14
4.05
3.34
4.53
9.72
8.33
7.65
2.85
0.80
0.86
1.37
0.59
1.04
3.07
3.24
5.28
4.35
5.10
Study of lignin extractions
• Experimental Techniques
• The determination of pKa by Uv/Vis
Spectroscopy
• Study of aggregation by Uv/Vis Spectroscopy
Experimental Techniques
Uv/Vis Spectroscopy
Incident light
transmitted light
Io
It
Beer-Lambert Law is A= cl = log10(Io/It)
The determination of pKa by Uv/Vis
Spectroscopy
Results
•
•
•
•
A) p-Coumaric acid result data
B) Ferulic acid result data
C) Syringic acid result data
D) Vanillin result data
p-Coumaric acid result data
1.4
pH2.26
pH2.80
pH4.55
pH4.80
pH5.00
pH5.55
pH6.70
1.2
Absorbance spectra of p-Coumaric acid, in
only solvent distilled water, an environment
not resistant to the effect of pH change
from 2to 7.
Absorbance
1.0
0.8
0.6
0.4
0.2
0.0
200
Changes of a solution pH
cause changes in the
electrostatic interactions
within the component and
the solvent system.
220
240
260
280
300
320
340
360
1.0
1.0
0.8
Absorbance
0.6
0.4
pH7.20
pH8.60
pH9.65
pH10.27
pH11.24
0.8
0.6
Absorbance
pH2.82
pH3.07
pH3.50
pH3.90
pH4.07
pH4.30
pH4.61
pH4.67
pH5.02
pH5.42
pH6.05
pH6.26
0.2
0.4
0.2
0.0
0.0
200
250
300
350
200
225
250
275
Wavelength,  (nm)
300
325
350
O
O
OH
O
O
O
1.0
20000
5000
0
250
300
Wavelength,  (nm)
350
0.9
0.8
0.7
0
200
250
300
350
Wavelength,  (nm)
OH
0.8
O
pH =12
pH =7
p -C o u m a r ic a c id
Chi^2
R^2
= 0.00001
= 0.99927
Ab
Abh
pK
0.6713 ±0.0025
1.03417 ±0.00407
4.51195 ±0.01852
Chi^2
R^2
= 0.00066
= 0.99805
Ab
Abh
pK
0.95335 ±0.02325
0.01561 ±0.02279
9.35342 ±0.06651
0.6
0.4
0.2
400
2
4
6
8
pH
10
0.0
12
17
A(333nm)
-1
10000
OH
pH=2
A (310nm)
10000
pH7.20
pH8.60
pH9.65
pH10.27
pH11.24
 (M.cm)
15000
Extinction coefficient,
pH2.82
pH3.07
pH3.50
pH3.90
pH4.07
pH4.30
pH4.61
pH4.67
pH5.02
pH5.42
pH6.05
pH6.26
-1
Extinction coefficient,  (M.cm)
400
K a2
K a1
20000
375
Wavelength,  (nm)
1.0
200
380
Wavelength,  (nm)
p-Coumaric acid result data
1.0
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
200
225
250
275
300
325
350
375
Wavelength,  (nm)
1.00
0.95
0.90
0.85
A310
Absorbance
The effect of pH between
2 and 5, in this range
pCA is reversible and the
maximum value of
absorbance is about pH
3.5; at max(310). The
reversible points
indicated the p-Coumaric
acid is pure.
0.9
0.80
0.75
0.70
0.65
2.0
2.5
3.0
3.5
4.0
pH
4.5
5.0
5.5
6.0
pH2.06
pH2.58
pH2.81
pH3.05
pH3.51
pH3.86
pH4.91
pH5.66
pH5.10
pH4.08
pH3.64
pH3.42
pH2.72
pH2.49
pH2.37
pH2.28
pH2.22
pH2.13
Ferulic acid result data
18000
0.2
0.6
0.4
0.2
0.0
0.0
200
250
300
Wavelength,  (nm)
350
400
200
250
300
350
400
12000
10000
8000
20000
pH5.90
pH7.80
pH8.70
pH9.30
pH10.02
pH10.44
pH10.76
pH11.15
pH11.50
pH11.84
pH12.05
-1
Extinction coefficient,  (M.cm)
pH 2-8
0.4
0.8
14000
6000
4000
2000
15000
10000
5000
0
0
Wavelength,  (nm)
200
250
300
350
200
400
250
300
From the analysis of the plots
Absorbance of pH Titration
Curves Ferulic acid at max(322)
and at max(346), the pKa1 and
pKa2 values were obtained
4.72, 9.21 respectively.
350
400
Wavelength,  (nm)
Wavelength,  (nm)
1.2
1.0
Chi^2
R^2
= 0.00011
= 0.99953
Ab
Abh
pK
1.21773 ±0.00441
0.11 ±0
9.20697 ±0.01356
0.6
0.4
A346
0.8
A322
Absorbance
0.6
pH2.06
pH2.24
pH2.40
pH2.60
pH2.78
pH3.10
pH4.85
pH5.90
pH7.80
16000
pH5.90
pH7.80
pH8.70
pH9.30
pH10.02
pH10.44
pH10.76
pH11.15
pH11.50
pH11.84
pH12.05
1.0
Absorbance
pH2.06
pH2.24
pH2.40
pH2.60
pH2.78
pH3.10
pH4.85
pH5.90
pH7.80
0.8
pH 6-12
-1
1.2
Extinction coefficient,  (M.cm)
1.0
Chi^2
R^2
= 0.00004
= 0.99757
Ab
Abh
pK
0.63057 ±0.005
0.917 ±0
4.72483 ±0.04422
2
3
0.2
0.0
4
5
6
7
8
9
10
11
12
13
pH
O
O
CH3
HO
pH=2
OH
pKa1
O
O
O
CH3
HO
pH=7
Ferulic acid
pKa2
O
O
CH3
O
O
pH=12
Syringic acid result data
pH 2-7
1.5
1.0
pH 7-12
2.0
1.5
1.0
0.5
0.5
0.0
25000
20000
15000
10000
-1
-1
30000
pH1.98
pH2.23
pH2.57
pH3.02
pH3.36
pH3.82
pH4.02
pH4.44
pH4.84
pH5.43
pH6.77
Extinction coefficient,  (M.cm)
2.0
2.5
Absorbance
2.5
pH7.95
pH8.45
pH8.70
pH9.25
pH9.52
pH10.22
pH10.54
pH11.02
pH11.59
pH12.05
3.0
Extinction coefficient,  (M.cm)
pH1.98
pH2.23
pH2.57
pH3.02
pH3.36
pH3.82
pH4.02
pH4.44
pH4.84
pH5.43
pH6.77
3.0
5000
pH7.95
pH8.45
pH8.70
pH9.25
pH9.52
pH10.22
pH10.54
pH11.02
pH11.59
pH12.05
25000
20000
15000
10000
5000
0
0.0
-0.5
250
300
350
0
200
Wavelength,  (nm)
250
300
350
200
250
Wavelength,  (nm)
From the analysis of pH titration
curves at max(270), and max(300)
were obtained the
pKa1 = 4.47
pKa2 = 9.34
O
O
CH3
HO
O
CH3
pH=2
pKa1
O
CH3
HO O
CH3
pH=7
Syringic acid
200
pKa2
250
300
350
Wavelength,  (nm)
1.8
1.6
1.4
Chi^2
R^2
= 0.00169
= 0.99733
Ab
Abh
pK
1.83025 ±0.02106
0.12996 ±0.02679
9.33734 ±0.03724
1.2
1.0
0.8
0.6
0.4
0.2
O
O
350
2.0
O
OH
300
Wavelength,  (nm)
CH3
O
pH=12
Chi^2
R^2
= 0.00007
= 0.99775
Ab
Abh
pK
0.80416 ±0.00677
1.22172 ±0.004
4.46665 ±0.03111
0.0
O
O O
CH3
A(302.5 nm)
200
A(273 nm)
Absorbance
30000
3.5
3.5
0
1
2
3
4
5
6
7
pH
8
9
10
11
12
13
Vanillin result data
25000
2.5
20000
E xtinction coefficient,  (M .cm )
-1
2.0
pH 2-12
1.5
Absorbance
p H 1 .9 7
p H 2 .4 4
p H 2 .9 0
p H 3 .5 6
p H 4 .0 8
p H 4 .5 0
p H 4 .9 5
p H 6 .4 0
p H 6 .9 0
p H 7 .2 9
p H 7 .7 2
p H 7 .9 5
p H 8 .7 5
p H 9 .5 0
p H 1 0 .9 0
p H 1 1 .5 0
p H 1 2 .1 4
1.0
0.5
0.0
15000
10000
5000
0
200
250
300
350
400
200
250
Wavelength,  (nm)
300
350
400
W a v e le n g th ,  (n m )
2.5
pKa = 7.25.
2.0
A(347 nm)
pH Titration Curves of
Vanillin, at max(348),
Chi^2 = 0.01339
R^2 = 0.98958
1.5
Ab 2.39 ±0.05131
Abh 0.07 ±0.04239
pK 7.25279 ±0.06168
O
O
pKa
1.0
OCH3
OH
pH=2
0.5
O
OCH3
pH=12
Vanillin
0.0
2
4
6
8
pH
10
12
Study of aggregation by Uv/Vis Spectroscopy
• p-Coumaric acid
• Other components are under investigations.
Absorbance spectra of a series of concentrations of p-Coumaric
acid in ethanol
20000
1.0
0.5
15000
262
283
304
325
346
1 cm cell
5000
A
241
22000
10000
A
0
1 cm cell
0.0
220
Conc.1.42E4
Conc.9.45E5
Conc.6.30E5
Conc.4.20E5
Conc.2.80E5
Conc.1.87E5
Conc.1.24E5
Conc.8.30E6
Extinction coefficient ()
Absorbance
1.5
Conc.9.45E5
Conc.6.30E5
Conc.4.20E5
Conc.2.80E5
Conc.1.87E5
Conc.1.24E5
Conc.8.29E6
Extinction coefficient ()
2.0
23000
p-Coumaric acid in ethanol
25000
p-Coumaric acid in ethanol
367
3
2
1
200
225
250
Wavelength,  (nm)
275
300
325
350
Wavelengths,  (nm)
21000
Data: Emax310_E310
Model: Dim
20000
19000
Extinction coefficient () at 310 nm
20000
1.0
0.8
0.6
0.4
15000
2 cm cell
0.0
225
250
275
300
325
350
B
200
1
3
2
225
250
275
1.0
0.5
325
C
225
250
275
300
Wavelength,  (nm)
325
350
Conc.2.57E5
Conc.2.09E5
Conc.1.70E5
Conc.1.39E5
Conc.1.13E5
Conc.9.22E6
Conc.7.51E6
Conc.6.12E6
Conc.4.99E6
Conc.4.06E6
Conc.3.31E6
0.4
0.2
0
1
220
6
8
260
280
10
O
O
O
3
2
240
4
H
O
C
2
Concentration (mM)
5 cm cell
5000
200
OH
0.0
10000
0
200
0.16
350
15000
5 cm cell
0.0
0.14
0.6
300
20000
Extinction coefficient ()
1.5
0.12
0.8
p-Coumaric acid in ethanol
Conc.2.57E5
Conc.2.09E5
Conc.1.70E5
Conc.1.39E5
Conc.1.13E5
Conc.9.22E6
Conc.7.51E6
Conc.6.12E6
Conc.4.99E6
Conc.4.06E6
Conc.3.31E6
2.0
0.10
Maximum absorbance at 310 nm
Y=0.08492X, R=0.99877, SD=0.01642
Wavelengths,  (nm)
p-Coumaric acid in ethanol
0.08
Concentrations (mM)
2 cm cell
Wavelength,  (nm)
2.5
0.06
5000
0
200
0.04
10000
B
0.2
Absorbance
Absorbance
1.2
0.02
Conc.4.20E5
Conc.3.50E5
Conc.2.92E5
Conc.2.43E5
Conc.2.03E5
Conc.1.69E5
Conc.1.41E5
Conc.1.17E5
Absorbance
1.4
±83.58116
±41.79058
±11.65019
p-Coumaric acid in ethanol
p-Coumaric acid in ethanol
Extinction coefficient ()
1.6
18054.03446
9027.01712
152.53348
17000
15000
0.00
Conc.4.20E5
Conc.3.50E5
Conc.2.92E5
Conc.2.43E5
Conc.2.03E5
Conc.1.69E5
Conc.1.41E5
Conc.1.17E5
Conc.9.77E6
Conc.8.14E6
= 110203.36609
= 0.97041
Ed
Em
K
18000
16000
1.8
Chi^2
R^2
O
300
320
340
360
Wavelengths,  (nm)
HO
+
OH
OH
H
O
HO
HO
Conclusions
I am tested four aromatic compounds, p-Coumaric acid, Ferulic
acid, Syringic acid and Vanillin. All they basically have phenyl
group and the first three also have the carboxylic acid
chromophore. The first three have tow pKa values because they
have carbonyl group (>C=O) chromophore. The pKa value of a
chemical compound determines its solubility and stability.
In ethanol the p-Coumaric acid at high concentration makes
dimerisation.
The four aromatic compounds
Compound
Formula
Mol. Wt
(g/mol)
Structure
pKa
4.5, 9.35
p-Coumaric acid C9H8O3
164.15
HO
O
OH
O
O
Ferulic acid
C10H10O4
194.18
OH
H3C
4.7, 9.2
HO
O
O
OH
H3C
Syringic acid
C9H10O5
198.17
4.5, 9.34
HO
O
H3 C
O
Vanillin
C8H8O3
152.14
OCH3
OH
7.3
,
College
K
LONDON
ING S
Founded I8
University of London
That’s all!
Thank you very much for your
attention