1. Lecture 2 Lignin Isolation and Acid Hydrolysis

2. Lignin Large macromolecule formed by various types of substructures

3. Lignin In nature is very resistant to degradation. It is bonded in complex and various types of carbohydrates.

4. LIGNIN Lignin may be defined as an amorphous, polyphenolic material arising from enzymatic dehydrogenative polymerization of three phenylpropanoid monomers, namely, coniferyl alcohol, sinapyl alcohol, and p-coumaryl alcohol. It is most commonly derived from wood, and it is an integral part of the secondary cell walls of plants and some algae.

5. STRUCTURE Lignin is a cross-linked racemic macromolecule with molecular masses in excess of 10,000 u. It is relatively hydrophobic and aromatic in nature. The degree of polymerization in nature is difficult to measure, since it is fragmented during extraction and the molecule consists of various types of substructures which appear to repeat in a haphazard manner. Different types of lignin have been described depending on the means of isolation.

6. STRUCTURE Structure of Lignin Microscopy Image

7. LIGNIN ISOLATION Several different enzymatic, chemical and mechanical methods have been developed for the isolation of lignin from wood and pulp. Some of these are; 1.Lignin Isolation by Enzymatic Hydrolysis of Pulp. 2.Lignin Isolation by Acid Hydrolysis of Pulp.

8. Lignin Isolation by Enzymatic Hydrolysis of Pulp This procedure is based on selective hydrolysis and dissolution of carbohydrates in pulp by commercial cellulolytic enzymes, leaving lignin behind as an insoluble residue. There are several drawbacks of utilizing the enzymatic hydrolysis method for isolating residual lignin.

9. Lignin Isolation by Enzymatic Hydrolysis of Pulp All residual lignin isolated with this procedure contain some carbohydrates that cannot be removed by prolonged and repetitive enzymatic treatments or by purification methods that are commonly employed for milled wood lignins.

10. Lignin Isolation by Acid Hydrolysis of Pulp This technique is frequently employed since it is relatively rapid and yields a lignin of relatively high purity, free of carbohydrate contamination.

11. Experimental Procedure for Acid Hydrolysis Isolation of Lignin 1.Pulps are washed thoroughly with deionized water and air dried prior soxhlet extraction with acetone for 24 hours. 2.A 300mL three necked round-bottom flask equipped with Friedrich’s condenser is charged with 30.0 g of dry pulp. 3.The pulps are then refluxed with 0.100 N HCl in p-dioxane at 4.00% consistency for two hours under an argon atmosphere.

12. Experimental Procedure for Acid Hydrolysis Isolation of Lignin 4. The solution is cooled and then filtered through a coarse-sintering glass Buchner funnel covered with filter paper to avoid plugging. 5. The resulting filtrate is passed through a fine sintered glass Buchner funnel packed with a celite® filter aid to remove fines.

13. Experimental Procedure for Acid Hydrolysis Isolation of Lignin 6. The lignin/water/dioxane mixture is neutralized with aqueous saturated solution of sodium bicarbonate to an apparent pH value of 5.0-5.5 and concentrated at approximately under 35oc under reduced pressure to approximately 10% of the original volume.

14. Experimental Procedure for Acid Hydrolysis Isolation of Lignin 7. Deionized water is added and the mixture is concentrated again under reduced pressure to remove the last traces of p-dioxane. 8. The resultant aqeous lignin solution is transferred to a 1L beaker, diluted with deionized water to approximately 750mL, acidified to a pH of 2.0-2.5, and transferred to a 250mL capacity centrifuge bottles.

15. Experimental Procedure for Acid Hydrolysis Isolation of Lignin 9.The centrifuge bottles are frozen, thawed, and centrifuged. 10. The supernatant is siphoned and fresh acidified water is added to wash the lignin. 11. The lignin is freeze dried for 3 to 5 days, after which it is characterized by NMR, UV and methoxyl content.

16. Experimental Procedure for Acid Hydrolysis Isolation of Lignin 12. The yield of residual lignin is calculated by determining the original pulp’s lignin content by Kappa number and acid insoluble lignin content.

17. Experimental Procedure for Acid Hydrolysis Isolation of Lignin