analysis of syringyl and guaiacyl (s/g) ratio in...
TRANSCRIPT
Presented to SACI by
Prof. Andrew Sparkfor
Mark Govender and Tammy Bush
7 December 2006
Analysis of Syringyl and Guaiacyl (S/G) ratio in lignin
Acknowledgements
• SAPPI • MONDI • CSIR• UKZN
Aim– Develop a quick and accurate method
to measure S/G ratio– Characterise and identify the acidolysis
products– Validate the new S/G ratio method
Why are S/G ratios important?
Gives a good indication of the reactivity of the lignin
Experimental designLiterature Review Establishing
Acidolysis conditions
Permanganate oxidation Lignin isolation
Characterisation and identification of
acidolysis products
Inter-lab study(Method validation)
Introduction
• Lignin is the second most abundant polymeric substance known to man
• Lignin has not been isolated in its pure form which makes both quantitative and qualitative determinations difficult
• Lignin are polymers of phenylpropane units
• Lignin is amorphous
Figure 1: Model structure of lignin (Lin and Dence,1992)
Methods of oxidising lignin into its subunits:
• Acidolysis-method is quick• Permanganate oxidation-method is slow
but it is the standard method used at present
• Nitrobenzene Oxidation, Pyrolysis, Cupric Oxidation and Thioacidolysis
Lignin can be broken down to syringyl and guaiacyl subunits:
OCH3
OH
OCH3
OH
OCH3
Guaiacyl Syringyl
Advantages of Acidolysis
• Quick sample preparation times• Reagents used for the sample
preparation are relatively cheap• Sample preparation is simple
Acidolysis• Present definition-the heating at 105 °C of
lignin or sawdust with 0.2 M HCl in dioxane-water (9:1, v/v)
• Acidolysis degradation products are formed by the selective cleavage of arylglycerol-β-aryl ethers and other weak ether linkages
Lignin
O
C O
CH O
CH2OH
OH
OOH
OO
+
0.2 M HCl105 °C
4hrs
Guaiacyl Syringyl
Experimental
Sampling and sample preparation
Lignin isolation(Klason Lignin)
Acidolysis and extraction
GC-MSanalysis
Wood Chips
S/G Ratio
Sawdust
Results• Acidolysis products were separated
using column chromatography• Characterised and identified using
GC-MS together with 1H-Nuclear Magnetic Resonance (NMR) and 13C-Nuclear Magnetic Resonance (NMR)
Column chromatography• Solvent system of 15% ethyl acetate and 85%
methylene chloride gave the best separation
• Thin layer Chromatography (TLC) was used to determine best eluent
GC ChromatogramKlason lignin
SyringylGuaiacyl
Mass Spectrum of Syringyl
Mass Spectral Analysis of Syringyl
181
153
43
Fragmentm/z
C CH3
O
OCH3CH3OOH
OCH3CH3OOH
C O
1H-Nuclear Magnetic Resonance (NMR)
AromaticCH’s
OH
2xOCH3
CH3
}
13C-Nuclear Magnetic Resonance (NMR)
C=O =C-O
=CH
} } }=C and 2xO-CH3
CO
CH3
1,2-Propanedione, 1-(4-hydroxy-3,5-dimethoxyphenyl)
MeMe
Me
C C
O O
O
OHO
Syringyl
Mass Spectrum of Guaiacyl
Mass Spectral Analysis of Guaiacyl
151
123
43
FragmentMass/charge (m/z)
CH3OOH
CH3OOH
C O
C CH3
O
1H-Nuclear Magnetic Resonance (NMR) - Guaiacyl
X
}Aromatic H’s
OH
OCH3CH3
13C-Nuclear Magnetic Resonance (NMR) - guaiacyl
} }
}C=O =C
=CH COCH3
X
1,2-Propanedione, 1-(4-hydroxy-3-methoxyphenyl)
Me
Me
C C
O O
OHO
Guaiacyl
Repeatability Study
0.052.60Grandis0.06 1.74GxT
0.012.88Smithii 0.052.50Fastigata 0.022.15 GxC 0.032.48Dunnii0.042.30Nitens
Standard deviation (4 replicates)
S/G ratioEucalyptus species
Internal standard (Butylated Hydroxy Toluene) vs S/G ratio
2
2.1
2.2
2.3
2.4
0 0.02 0.04 0.06 0.08 0.1
BUTYLATED HYDROXY TOLUENE(g)
S/G
RA
TIO
Klason lignin preparation methods
T222 om-88- preparation of Acid-insoluble lignin (Klason lignin)
• 15 ml H2SO4 (72%) added to 1 g sawdust
• 2 hours at 20oC• Boil for 4 hours• Filter off Klason lignin
T249 cm-85- carbohydrate analysis
(Isolation of Klason lignin)
• 3 ml H2SO4 (72%) added to 0.35 g sawdust
• 1 hour in water bath set at 30 oC
• 1 hour in autoclave at 121oC• Filter off Klason lignin
S/G ratios of Klason lignin from TAPPI method T222 om-88 vs. TAPPI method T249 cm-85
11.21.41.61.8
22.22.42.62.8
1.3 1.5 1.7 1.9 2.1 2.3 2.5
T249 cm-85 (S/G ratio)
T222
om
-88
(S/G
rat
io)
T222 om-88= 6 hours T249 cm-85= 3 hours
R2= 0.8401
Inter-lab. study
• Aim: To compare the Acidolysis (CSIR-FFP) method for oxidising lignin with the Nitrobenzene Oxidation method used at (Syracuse University, NY)
Comparison of lignin oxidation methods
Acidolysis Method
• Acidolysis reagent (2 ml ) is added to 0.0150 g Klason lignin
• Acidolysis mixture kept at 105 oC for 4 hours
• Acidolysis mixture is neutralised and then solvent extracted
• Acidolysis products analysed on GC to obtain S/G ratio
Nitrobenzene Oxidation
• 0.4 g of wood meal is added to a bottle containing 2.5 ml nitrobenzene and 2 M NaOH (40ml)
• Bottle placed in an oil bath (170°C ) for 2.5 hours
• 1 hour in autoclave at 121oC• Autoclave mixture is solvent
extracted and the pH is adjusted to 3
• Nitrobenzene oxidation products analysed on the GC-MS to obtain S/G ratio
Comparison of S/G ratios measured using Nitrobenzene Oxidation with Acidolysis (Klason lignin
prepared using TAPPI method T222 om-88)
0.9
1
1.1
1.2
1.31.4
1.5
1.6
1.7
1.8
1.5 1.7 1.9 2.1 2.3 2.5 2.7
Acidolysis(S/G ratio)
Nitro
benz
ene
Oxi
datio
n(S/
G ra
tio)
R2= 0.8283
Comparison of S/G ratios measured using Nitrobenzene Oxidation with Acidolysis (Klason lignin
prepared using TAPPI method T249 cm-85)
0.9
1
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
1.2 1.4 1.6 1.8 2 2.2 2.4 2.6
Acidolysis(S/G ratio)
Nit
rob
enze
ne
Oxi
dat
ion
(S/G
rat
io)
R2= 0.8847
Conclusions
Acidolysis of Klason lignin produces:• Syringyl unit 1,2-Propanedione, 1-(4-hydroxy-3,5-dimethoxyphenyl)
• Guaiacyl unit 1,2-Propanedione, 1-(4-hydroxy-3-methoxyphenyl)
• Internal standard BHT plays a major role in preventing the guaiacyl groups from polymerising
• Inter-lab. study was a success with good correlations of the S/G ratios from acidolysis and nitrobenzene oxidation of the lignin
Conclusions cont…..
• The preferred lignin isolation method is T249 cm-85 due to quicker preparation times and better correlation with nitrobenzene oxidation than T222 om-88
Conclusions cont…..