1. Arni Saleh Ph.D. student, xx cycle
Activity
Presentation
Arni Saleh
Ph.D. student, xx cycle
12 December 2007

2. Aromatic compounds from Sugarcane Bagasse
Title of activity presentation

3. Presentation outline Background Biomass Pretreatment
Process for extract lignin materials
Lignin extractions
Study of lignin extractions

4. Background Bagasse material
Renewable resource, economics and environment
What is lignin?

5. Background What is Bagasse?
Bagasse material
Cellulose %
Hemicellulose 19-43%
Lignin %
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.
Bagasse as all biomass materials can be fractionated into complex mixtures of cellulose, hemicellulose, lignin, and other minor components; those fractions have a complex structure from biopolymers. The percentage of bagasse generally consists of about 33-48% cellulose, 19-43% hemicelluloses and 6-32% lignin, and its composition normally depending on the region and growth conditions.
Bagasse is an agroindustrial residue in large amounts in world such as Brazil, Mexico, India, Philippines and etc. That it is a renewable resource, also economically competitive feedstocks for production of soluble materials into chemical productions, and the efficient material conversion, depending on the type of the process used, may be reduce the costs of the final product. The use of bagasse for bioproduction resolves the problem of agricultural municipal solid waste.

6. Background What is lignin?
Natural lignin composition and structure are unknown.
It is a heterogeneous polymer.
It is made up of three principal monomers.
Natural lignin composition and structure are unknown: It is not yet possible to definitely determine the complete structure of lignin molecule, because there is no standard lignin structure for reference, measurement of lignin concentration is empirical and very dependent on methodology of experiments.
lignin is obtained from Latin Word lignum that is equivalent to wood, but now is well known such as a complex polymer which combines with cellulose and hemicellulose.
Lignin is a heterogeneous polymer that structure consists of many different types inter-unit linkages, substituents, and side chains with various functional groups of phenolic polymers with a molecular weights on the order of atomic mass unit or more.
lignin is made up of three principal monomers, these polymers are derived from the hydroxycinnamyl alcohols or monolignols, p-coumaryl alcohol (C9H10O2), coniferyl alcohol (C10H12O3), and sinapyl alcohol (C11H14O4) or trans-p-coumaryl, trans-coniferyl, and trans-sinapyl alcohols.

7. Biomass Pretreatment

8. 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.

9. Processes for extract lignin materials
Hydrolysis method
Alkaline sugarcane bagasse hydrolysis
1) There are several processes can be used to remove and/or isolate and/or purify the lignin from biomass material cells. The alkaline methods are generally more effective because they are able to solubilize a greater fraction of lignin, also they are more vantage compared to other pretreatment technologies.
 2) This method breaks down the hydrogen bonds in the lignocellulosic biomass fractions by removal of the surrounding lignin structure hemicellulose and cellulose and recovering the resulting soluble monomeric.

10. Extraction of lignin from sugarcane bagasse
Hydrolysis method
Alkaline sugarcane bagasse hydrolysis
Extraction of lignin from sugarcane bagasse
Dried 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
1) The pretreated sugarcane bagasse powder was suspended in 100 ml of NaOH solution (with concentration ranging from 0.5 to 4.0 M) and autoclaved at 121°C for 1 h, using 250 ml Pyrex glass bottles with plastic caps.
2) The bagasse extract solutions obtained from alkaline hydrolysis were filtered through filters with 0.45 mm pore diameter and stored at 4°C until analyses were made.
3) Prior to the analysis, all liquid samples were acidified with H3PO4 up to pH 3.0 and filtered through membranes with 0.20 mm pore diameter then anlysae .
4) A schematic diagram of the extraction process is shown in Figure.

11. Lignin extraction

12. % of main components extracted
Lignin extraction
Bgasse (g)
alkaline (NaOH)
treatment (M)
% of main components extracted
p-coumaric acid
Ferulic acid
Syringic acid
Vanillin
0.5
79.61
16.21
1.10
3.08
1.0
76.36
17.58
1.49
4.57
2.0
74.01
19.03
1.81
5.14
3.0
75.78
18.49
1.69
4.05
4.0
76.10
18.78
1.79
3.34
76.83
15.44
3.19
4.53
67.40
18.90
3.97
9.72
69.04
19.26
3.37
8.33
67.27
21.12
3.95
7.65
78.91
15.80
2.45
2.85
83.41
14.96
0.82
0.80
82.13
16.04
0.97
0.86
79.98
17.31
1.34
1.37
80.59
17.33
1.50
0.59
78.79
18.36
1.82
1.04
76.04
19.10
3.07
73.79
20.59
2.38
3.24
5.0
67.00
24.40
3.31
5.28
71.68
21.10
2.87
4.35
71.49
20.23
3.18
5.10
The percentage of main components extracted by alkaline hydrolysis.

13. Study of lignin extractions
Experimental Techniques
The determination of pKa by Uv/Vis Spectroscopy
Study of aggregation by Uv/Vis Spectroscopy

14. Experimental Techniques
Preparing samples : preparation the stock solution by weighting 1-3 mg
pH titration: Acid-base titrations were applied by adding ml quantities of NaOH or HCL solutions.
variation in concentration: From the stock solutions, all dilutions were derived.
Measurement of absorption spectrum : The measurements were performed in 1, 2 and 5 cm quartz cuvettes, by UV/Vis spectrophotometer.
Data processing and analysis: After obtaining spectra signals, the data is transferred to standard analysis software. All spectra were baseline corrected.

15. Uv/Vis Spectroscopy It Incident light Io transmitted light
UV Spectroscopy molecules can absorb light in the ultraviolet (UV)-visible region of the electromagnetic spectrum. This stretches from the far UV zone (<200nm) to the near infrared zone ( nm). The longest visible wavelength is red and the shortest is violet.
2) Where A = absorbance, ε = molar extinction coefficient, c = molar concentration , l = path length, Io = intensity of the incident light (radiation), and It = intensity of the transmitted light after passing through the sample.
Beer-Lambert Law is A= cl = log10(Io/It)

16. 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
In all experiments carried out for pH titration, I used a 1cm pathlength cell.

17. p-Coumaric acid result data
Absorbance spectra of p-Coumaric acid, in only solvent distilled water, an environment not resistant to the effect of pH change from 2to 7.
Changes of a solution pH cause changes in the electrostatic interactions within the component and the solvent system.
Absorbance spectra of p-Coumaric acid, in only solvent distilled water.
Because changes of a solution pH cause changes in the electrostatic interactions within the component and the solvent system.
Absorbance:
2) Extinction coefficient:
3) pH titration curves of pCA and structure:

18. p-Coumaric acid result data
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 lmax(310). The reversible points indicated the p-Coumaric acid is pure.
1) Reversible range pH between 2 and 5

19. Ferulic acid result data
From the analysis of the plots Absorbance of pH Titration Curves Ferulic acid at lmax(322) and at lmax(346), the pKa1 and pKa2 values were obtained 4.72, 9.21 respectively.
Absorbance spectra of Ferulic acid, the effect of pH change from 2 to 8 and 6 to 12.
Extinction coefficient spectra of Ferulic acid, the effect of pH change from 2 to 8 and 6 to 12.
pH Titration Curves

20. Syringic acid result data
From the analysis of pH titration curves at lmax(270), and lmax(300) were obtained the
pKa1 = 4.47
pKa2 = 9.34
Absorbance spectra of Syringic acid, the effect of pH change from 2 to 7 and 7 to 12.
Extinction coefficient spectra of Syringic acid, the effect of pH change from 2 to 7 and 7 to 12.
3) pH titration curves of SA with variation of pH.
4) Structure of SA with variation of pH.

21. pH Titration Curves of Vanillin, at lmax(348),
Vanillin result data
pH Titration Curves of Vanillin, at lmax(348),
pKa = 7.25.
Absorbance spectra of Vanillin, the effect of pH change from 2 to 12.
Extinction coefficient of Vanillin, the effect of pH change from 2 to 12.
pH titration curve and structure of Vanillin with variation of pH.
Structure of Vanillin with variation of pH.

22. Study of aggregation by Uv/Vis Spectroscopy
p-Coumaric acid
Other components are under investigations.
Her present only p-Coumaric acid, other components are under investigations.

23. Absorbance spectra of a series of concentrations of p-Coumaric acid in ethanol
Absorbance spectra of a series of concentrations of p-Coumaric acid in ethanol with 1.0, 2.0 and 5.0 cm cell, graph A, B, and C respectively
Extinction coefficient and isosbestic points:
A) 1cm cell, at 224 nm (1), 245 nm (2), and 290 nm (3) respectively.
B) 2cm cell, at 228 nm (1), 242 nm (2), and 292 nm (3) respectively.
C) 5cm cell, at 227 nm (1), 241 nm (2), and 293 nm (3) respectively.
3) Deviations from Beer’s law for p-Coumaric acid, at 310 nm, (■) experimental points, (line) simulated cure.
4) Conventional Beer’s law plot for part of data over a relatively narrow concentration range.
The equilibrium constant K = 153 for the monomer m in equilibrium with its dimer d. +

24. 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.

25. The four aromatic compounds
Formula
Mol. Wt (g/mol)
Structure
pKa
p-Coumaric acid
C9H8O3
164.15
4.5, 9.35
Ferulic acid
C10H10O4
194.18
4.7, 9.2
Syringic acid
C9H10O5
198.17
4.5, 9.34
Vanillin
C8H8O3
152.14
7.3
1) Table shows the results of pKa of compounds tested.
2) 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 (in green cycle), then they have tow pKa values.

26. Thank you very much for your attention
That’s all!
Thank you very much for your attention