Lab_2_Klason_Lignin_Determination_&_Delignified_Wood_Densification

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University of British Columbia *

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373

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Chemistry

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Oct 30, 2023

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Wood 373 1 Lab 2 Klason Lignin Determination & Delignified Wood Densification Lab 2.1 Klason Lignin Determination (Week 1) Goal : During this lab, you will learn how to quantitatively determine the lignin content in wood by using the Klason Lignin method. Background : To examine the effectiveness of delignification, the lignin contents in both original and delignified wood need to be quantified. The traditional Klason method will be used to quantify the lignin content, which includes hydrolysis of wood using strong acids to remove polysaccharides to yield the insoluble residue quantified as Klason lignin (or acid-insoluble lignin). Procedures Safety requirements: Refer to the Safety Data Sheet (SDS) for all chemicals used in this experiment. In this lab, 72% sulfuric acid will be used which is highly corrosive and causes severe skin burns, can irritate the nose and throat, cause difficulties in breathing if inhaled and burn the eyes. Use extreme caution when handling, always wear safety goggles, lab coat and acid resistant gloves (double gloving recommended) and work in a fume hood. Note : The TA will provide ground original and delignified wood for the Klason lignin determination. Experimental section: The Klason lignin content will be determined in according with the published procedure (Biomacromolecules. 2016, 17, 1358-1364). Each group will perform 3 replicates for the original wood and another 3 replicates for delignified ground wood (a total of 6 samples for each group). For each repetition, accurately weigh out 0.20 g (Mo) dried original and delignified (R & L direction) ground wood and place them in individual serum bottles (6 for each team). Add 3.0 mL of 72% wt sulfuric acid (H 2 SO 4 ) to each bottle and mix thoroughly with a stirring rod for 1 minute. Let the solution stand for 2 hours, (stirring occasionally) to ensure complete hydrolysis of polysaccharides. Following the initial digestion, add 112 mL of boiled distilled water to dilute the acid concentration to 3%. Cap each bottle with a rubber septum and aluminum cap. Seal using the septa sealer. Using the label tape and Sharpie, label each bottle with your Team-sample numbers (ie Team 1: 1A-OW, 1B-OW, 1C-OW, 1A-DW, 1B-DW, 1C-DW). Place bottles in the autoclave tray provided. TA will autoclave samples at 120°C for 1 hour, allow to cool for next lab session. Tidy and clean the working area.

Wood 373 2 Lab 2.1b Klason Lignin Determination (Week 2) During the previous Lab 2.1, the acid hydrolyzed wood samples were sealed in serum bottles, autoclaved at 120°C for 1 hour and allowed to cool to room temperature. Prior to filtering the precipitate, accurately weigh and record the glass filter(s) and weigh bottle(s) on the provided sheet. Using the vacuum filtration apparatus, filter the solution and collect the precipitate onto the pre- weighed glass filter. Carefully decant the contents of the serum bottle onto the glass filter with the vacuum on. Wash the precipitate using boiled distilled water with the vacuum on. Disconnect the vacuum and transfer the precipitate on the filter paper into the pre-weighed weigh bottle (record the weigh bottle number and sample number on the provided sheet). Repeat for all 6 samples. The precipitate collected onto glass filter is oven-dried (105° ± 3°C) overnight and placed in a desiccator to cool. The students are required to return the next day to weigh and record the weight of each sample in order to calculate the Klason lignin content. The weight of the acid-insoluble lignin (m) and the original weight of the ground wood (Mo) is used to determine the Klason lignin content. Make observations of the lignin from the original wood and delignified wood. Take photos for your report. *Cleanup : All glassware will be immersed into 2% wt NaOH solution overnight and following by rinsing with distilled water by TA. Lab 2.2 Delignified Wood Densification (20 mins for each group) Goal: To get familiar with a normal way of fabricating densified wood samples. You are also expected to analyze differences between densified samples with different degrees of delignification. Procedure : Sample blocks from Lab 1 went through 4 cycles of the bleaching process (2 hours per cycle = 8 hours in total). The TA removed the samples after 1, 2, and 4 cycles, which are separated into beakers labeled 1, 2, and 4. Another beaker, labelled 8 will contain samples that were boiled for 8 hours without any chemicals to be used as a comparison. The four samples should be blotted dry with paper towels to remove the excess water on the surface in preparation for compression. All wood block samples will be compressed at the same time, sandwiched between two steel plates with a few layers of paper towels in between to absorb extra water. The wood will be pressed at 80°C with position controls: 11mm for 1 minute; 10.5 mm for 1 minute; 10.0 mm for 1 minute; 9.5 mm for 1 minute; 9 mm for 1 minute ... 2mm

Wood 373 3 for 1 minute, finally held at 80°C and 2 mm for 40 minutes. After 40 minutes, the position will be further reduced to 1.5 mm and the temperature increased to 100°C and held for another 40 minutes to obtain the densified wood. Lab 2.1c Acid Soluble Lignin Content (spectrophotometer demo) Goal: To determine the acid soluble lignin content using a UV Spectrophotometer. Procedure : After the acid hydrolysis of the ground wood in sulfuric acid, the acid insoluble lignin (Klason) is separated from the acid insoluble lignin. This fraction of acid insoluble lignin is found in the supernate. A spectrophotometer will be used for the characterization of this acid insoluble lignin. TA will demonstrate the specific procedure of using the equipment. After analysis, you will calculate the % of acid soluble lignin (ASL) in the sample provided by the TA using the following equation: ASL = UV abs (absorbance) * Volume * Dilution / ( Ɛ * ODW * Pathlength) * 100 Questions: 1. Klason lignin is the acid insoluble lignin found in wood. How much Klason lignin would you expect to find in your species of wood? 2. What would you expect if the acid hydrolysis time was less than 2 hours? 3. Why is desiccant/desiccator used when cooling the filtered Klason lignin? 4. Why should the densification process be done in incremental steps?

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