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

Class Agenda How are hemicelluloses separated from cellulose and lignin? How are individual hemicelluloses separated? How is the composition of individual hemicelluloses determined? How are the linkages determined? Lignin analysis PSE 406 - Lecture 17

How are hemicelluloses separated from cellulose and lignin? Generate Holocelulose Remember….in this procedure lignin is removed through the action of sodium chlorite PSE 406 - Lecture 17

Cellulose Isolation A 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% PSE 406 - Lecture 17

How are the hemicelluloses separated from cellulose? Cellulose is not soluble in almost any solvents. What are hemicelluloses soluble in? NaOH or KOH!!!!! PSE 406 - Lecture 17

Isolation Scheme: Softwoods HClO2 Holocellulose KOH Soluble Insoluble Hemicellulose Mixture Residue PSE 406 - Lecture 17

What is in the residue? Cellulose It is not soluble in much of anything Galactoglucomannan (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 solublize this material PSE 406 - Lecture 17

Isolation Scheme: Softwoods HClO2 Holocellulose KOH Soluble Insoluble Hemicellulose Mixture Residue There have been many different schemes developed to separate hemicelluloses. These schemes are typically based on solubility differences. The scheme above which we will use as an example was developed by Timell in 1961. We will cover in much more depth systems used to separate individual wood components in a later lecture. Prior to this schematic, the wood is first extracted with organic solvents to remove extractive compounds. Therefore, the wood used is extractive free. The first step of the above process involves treatment of the wood with HClO2 (chlorite). Lignin reacts with the chlorite and is removed leaving a mixture of cellulose and hemicelluloses which is termed holocellulose. These carbohydrates are little affected by the chlorite treatment. The different carbohydrates have different solubilities in KOH (as well as NaOH) and thus these chemicals can be used to separate the mixture into soluble and insoluble fractions. The insoluble mixture contains cellulose and glucomannans (alkali soluble galactoglucomannans). A mixture of NaOH and sodium borate will solublize the glucomanns leaving relatively pure insoluble cellulose. The crude glucomannan mixture can be further purified through an extraction process with barium hydroxide (Ba(OH)2). Barium ions will form an insoluble complex with mannose because of the configuration of the hydroxyl groups on C2 and C3 in mannose. NaOH/Borate Insoluble Soluble Text Cellulose Crude Glucomannan Ref: Timell: TAPPI 44, 88-96 1961 PSE 406 - Lecture 17

What makes up the rest of the hemicellulose mixture? Xylans Galactoglucomannans (water soluble) Maybe some pectins, a little glucans, and who know what else We are mainly concerned with the top two…. How do we separate the xylans from the galactoglucomannans? PSE 406 - Lecture 17

Barium Because of the orientation of the C2 and C3 hydroxyl groups in mannose, it will form an insoluble complex with barium ions. Therefore the addition of Ba(OH)2 will cause glucomannans to precipitate out of solution PSE 406 - Lecture 17

Isolation Scheme:Softwoods Hemicellulose Mixture Ba(OH)2 Soluble Insoluble Mixture Crude Galactoglucomannan Ba(OH)2 Ba(OH)2 Soluble Insoluble Soluble Insoluble The soluble fraction from the previous schematic contains a mixture of arabinoglucuronoxylan and galactoglucomanan. Barium hydroxide can once again be used to separate these two components. The xylan components are soluble in Ba(OH)2 solutions while most of the galactoglucomanns will precipitate. In this scheme, therefore, It takes two successive Ba(OH)2 extractions to accomplish the separation. As you can see, this procedure doesn’t take into account the presence of minor hemicelluloses such as galactans, arabinans, etc. Arabino Glucuronoxylan Galactoglucomann Discard Galactoglucomannan Text Ref: Timell: TAPPI 44, 88-96 1961 PSE 406 - Lecture 17

How is the composition of individual hemicelluloses determined? How can hemicelluloses be broken down into individual sugars. Acid hydrolysis of glycosidic linkages. Enzymatic hydrolysis PSE 406 - Lecture 17

Hemicellulose Analysis The individual sugars are quantified using gas or liquid chromatography. Often the individual components require derivitization before analysis. Other analytical techniques are used to positively identify components PSE 406 - Lecture 17

Chromatography Chromatography 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. Sample UV, RI FID,MS, Etc. Column gas Detector Packing material liquid Compounds separate through: adsorption, size exclusion, boiling points Time PSE 406 - Lecture 17

Derivitization Gas Chromatography - Chemicals to be analyzed must be volatile: Sugars and uronic acids are not volatile. Blocking hydroxyl groups will make chemicals volatile. Derivitization procedures: Methylation Acetylation Silylation Derivatization are employed for 2 reasons: to permit the analysis of compounds with inadequate volatility or stability, or to improve chromatographic behavior or detectebility. GC we convert polar N-H or O-H or S-H into thermally stable, non-polar groups. TMS is used for silylation reaction (Trimethyl silyl)-TMS CH3-O CH3-CO Si PSE 406 - Lecture 17

Chromatography Once the components are separated, they are detected by a number of different types of systems. A chromatograph is produced in which the components are seen as peaks. Quantification is accomplished by measuring the peak area Poorly reproduced figure from Supelco PSE 406 - Lecture 17

Determination of Linkages How is it possible to determine how the individual sugars are linked? Methylation of the free hydoxyl groups Acid Hydrolysis Chromatographic determination of products PSE 406 - Lecture 17

Pyranose? Furanose? Mild hydrolysis of hemicellulose results in the presence of monomers, dimers, trimers, etc. of the hemicelluloses. These materials can be separated by chromatography and compared to known dimers. PSE 406 - Lecture 17

Lignin-Important Questions How much lignin is in a sample? Wood Plant Material Pulp PSE 406 - Lecture 17

Quantification of Lignin Wood and non-woody materials Acid Insoluble lignin (along with acid soluble lig) Pulp Kappa number Other non woody materials (or I don’t have a large sample to work with) Acetyl bromide PSE 406 - Lecture 17

Acid Insoluble Lignin (Klason) Goal 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 PSE 406 - Lecture 17

Klason Procedure Wood meal (or pulp) is treated with 72% H2SO4 for 2 hours. The material is then diluted to 3% H2SO4 and then boiled for 4 hours. These two steps dissolves the carbohydrates leaving chunks of lignin floating in the acid The lignin is filtered, washed and weighed. PSE 406 - Lecture 17

Acid Soluble Lignin A 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. The 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. PSE 406 - Lecture 17

Lignin Content of Pulp Pulps contain only small amounts of lignin so a different (and quicker) method is used: the kappa number. This procedure is based upon the fact that lignin reacts very quickly with KMnO4 while carbohydrates (mostly) react very slowly. PSE 406 - Lecture 17

Kappa Number Procedure Pulp is dissolved in water and reacted with a KMnO4 solution for 10 minutes under very controlled conditions. The goal is to consume 50% of the KMnO4 in this time. Excess KMnO4 is consumed with potassium iodide forming I2 (iodine). The iodine is titrated with sodium thiosulfate to a starch endpoint. PSE 406 - Lecture 17

Kappa Number Information This method is typically used with pulp containing low amounts of lignin (chemical unbleached pulp). It was found about 15 years ago that hexenuronic acids formed during kraft pulping from uronic acids consume KMnO4 thus giving false kappa numbers (if based only on lignin). A typical kappa number for an unbleached kraft pulp is around 20. PSE 406 - Lecture 17

Kappa to Klason Correction factors have been developed to convert kappa numbers to Klason lignin. These factors are different for different processes and species. Kraft pulps: % Klason = kappa number * 0.15 Sulfite pulps: % Klason = kappa number * 0.167 (or 0.187 depending on who did the work). Kappa number 20 = ~3% lignin PSE 406 - Lecture 17

Acetyl Bromide Procedure This procedure was developed to measure lignin content in small samples. Samples are dissolved by reaction with acetyl bromide (with a little perchloric acid) in acetic acid. The solution is analyzed by UV (remember lignin absorbs, carbohydrates do not). The amount of lignin in the sample is determined by comparison against standards. Every material seems to have a different standard number. PSE 406 - Lecture 17