1. PSE 406 - Lecture 171 Wood Chemistry PSE 406 Lecture 17 Chemical Isolation and Analysis II Hemicelluloses and Lignin Analysis

2. PSE 406 - Lecture 172 Class Agenda lHow are hemicelluloses separated from cellulose and lignin? lHow are individual hemicelluloses separated? lHow is the composition of individual hemicelluloses determined? lHow are the linkages determined? lLignin analysis

3. PSE 406 - Lecture 173 How are hemicelluloses separated from cellulose and lignin? lGenerate Holocelulose lRemember….in this procedure lignin is removed through the action of sodium chlorite

4. PSE 406 - Lecture 174 Cellulose Isolation lA Tappi Standard procedure for cellulose isolation from holocellulose is as follows: »Extract holocellulose with 5% and then 24% KOH to remove hemicelluloses. The remaining material is termed alpha-cellulose –This results in cellulose of reduced molecular weight and some yield loss. Typical recoveries are 40-60%

5. PSE 406 - Lecture 175 How are the hemicelluloses separated from cellulose? lCellulose is not soluble in almost any solvents. lWhat are hemicelluloses soluble in? »NaOH or KOH!!!!!

6. PSE 406 - Lecture 176 Isolation Scheme: Softwoods Wood HClO 2 Holocellulose KOH Hemicellulose MixtureResidue SolubleInsoluble

7. PSE 406 - Lecture 177 What is in the residue? lCellulose »It is not soluble in much of anything lGalactoglucomannan (not the water soluble) »It turns out that this hemicellulose is not all that alkali soluble at this level of KOH »It takes the addition of NaOH and borate to solubilize this material

8. PSE 406 - Lecture 178 Isolation Scheme: Softwoods Wood HClO 2 Holocellulose KOH Hemicellulose MixtureResidue Cellulose Crude Glucomannan SolubleInsoluble Ref: Timell: TAPPI 44, 88-96 1961 NaOH/Borate SolubleInsoluble Text

9. PSE 406 - Lecture 179 What makes up the rest of the hemicellulose mixture? lXylans lGalactoglucomannans (water soluble) lMaybe some pectins, a little glucans, and who know what else lWe are mainly concerned with the top two…. »How do we separate the xylans from the galactoglucomannans?

10. PSE 406 - Lecture 1710 Barium lBecause of the orientation of the C2 and C3 hydroxyl groups in mannose, it will form an insoluble complex with barium ions. lTherefore the addition of Ba(OH) 2 will cause glucomannans to precipitate out of solution.

11. PSE 406 - Lecture 1711 Isolation Scheme:Softwoods Hemicellulose Mixture Ba(OH) 2 SolubleInsoluble Mixture Crude Galactoglucomannan Ba(OH) 2 Galactoglucomann SolubleInsoluble Ba(OH) 2 Galactoglucomannan Discard SolubleInsoluble Ref: Timell: TAPPI 44, 88-96 1961 Arabino Glucuronoxylan Text

12. PSE 406 - Lecture 1712 How is the composition of individual hemicelluloses determined? lHow can hemicelluloses be broken down into individual sugars. »Acid hydrolysis of glycosidic linkages. »Enzymatic hydrolysis

13. PSE 406 - Lecture 1713 Chromatography gas liquid Sample Column Packing material Detector UV, RI FID,MS, Etc. Compounds separate through: adsorption, size exclusion, boiling points Time lThe individual sugars are quantified using gas or liquid chromatography. lChromatography is the process in which chemicals are transported by liquid or gas past a stationary phase. The individual components are attracted to different degrees to the stationary phase and thus travel at different speeds and are separated.

14. PSE 406 - Lecture 1714 Chromatography lOnce the components are separated, they are detected by a number of different types of systems. lA chromatograph is produced in which the components are seen as peaks. lQuantification is accomplished by measuring the peak area 1. Maltotriose 2. Cellobiose 3. Glucose 4. Mannose 5. Arabinose 6. Adonitol 7. Arabitol 8. Xylitol

15. PSE 406 - Lecture 1715 Lignin-Important Questions lHow much lignin is in a sample? »Wood »Plant Material »Pulp

16. PSE 406 - Lecture 1716 Quantification of Lignin lWood and non-woody materials »Acid Insoluble lignin (along with acid soluble lig) lPulp »Kappa number lOther non woody materials (or I don’t have a large sample to work with) »Acetyl bromide

17. PSE 406 - Lecture 1717 Acid Insoluble Lignin (Klason) lGoal is separate carbohydrates from lignin »Lignin condenses (reacts with lignin) to become very water insoluble (it becomes very large) »Acid cleaves glycosidic linkages in carbohydrates forming individual sugars. »Sugars dissolve in water (acid) and lignin does not

18. PSE 406 - Lecture 1718 Klason Procedure lWood meal (or pulp) is treated with 72% H 2 SO 4 for 2 hours. The material is then diluted to 3% H 2 SO 4 and then boiled for 4 hours. »These two steps dissolves the carbohydrates leaving chunks of lignin floating in the acid lThe lignin is filtered, washed and weighed.

19. PSE 406 - Lecture 1719 Acid Soluble Lignin lA certain percentage of the lignin is soluble in the Klason lignin procedure »This amount is very small with softwoods but higher >5% in hardwoods and grasses. lThe filtrate from the Klason procedure is collected and the UV absorbance is checked. »Lignin absorbs UV light, sugars do not »The amount of lignin that is soluble is determined through comparing the UV absorbance to a standard.

20. PSE 406 - Lecture 1720 Lignin Content of Pulp lPulps contain only small amounts of lignin so a different (and quicker) method is used: the kappa number. lThis procedure is based upon the fact that lignin reacts very quickly with KMnO 4 while carbohydrates (mostly) react very slowly.

21. PSE 406 - Lecture 1721 Kappa Number Procedure lPulp is dissolved in water and reacted with a KMnO 4 solution for 10 minutes under very controlled conditions. »The goal is to consume 50% of the KMnO 4 in this time. lExcess KMnO 4 is consumed with potassium iodide forming I 2 (iodine). lThe iodine is titrated with sodium thiosulfate to a starch endpoint.

22. PSE 406 - Lecture 1722 Kappa Number Information lThis method is typically used with pulp containing low amounts of lignin (chemical unbleached pulp). lIt was found about 15 years ago that hexenuronic acids formed during kraft pulping from uronic acids consume KMnO 4 thus giving false kappa numbers (if based only on lignin). lA typical kappa number for an unbleached kraft pulp is around 20.

23. PSE 406 - Lecture 1723 Kappa to Klason lCorrection factors have been developed to convert kappa numbers to Klason lignin. These factors are different for different processes and species. lKraft pulps: % Klason = kappa number * 0.15 lSulfite pulps: % Klason = kappa number * 0.167 (or 0.187 depending on who did the work). lKappa number 20 = ~3% lignin

24. PSE 406 - Lecture 1724 Acetyl Bromide Procedure lThis procedure was developed to measure lignin content in small samples. lSamples are dissolved by reaction with acetyl bromide (with a little perchloric acid) in acetic acid. lThe solution is analyzed by UV (remember lignin absorbs, carbohydrates do not). lThe amount of lignin in the sample is determined by comparison against standards. »Every material seems to have a different standard number.