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…..