PSE 407

Wood Chemistry Laboratory

Winter 1999TTh 1:30-4:20Bloedel 209

Laboratory Project # 2

Title: Identification of Wood Species by Extractive Content

Introduction:

Your boss walks into the laboratory with three unlabeled samples of wood meal he has found in a storage cabinet; he is not a happy camper. “Doesn’t anyone in this miserable group know why we purchased labels! Am I the only one who remembers that we need to measure the holocellulose contents of these samples and we don’t even know what species they are!!!”

Since employment is an important issue in your life, you decide that you need to figure out which sample is which. You know that you ground three different woods:

  1. Western Red Cedar (Thuja plicata)
  2. Noble Fir (Abies procera)
  3. Red Alder (Alnus rubra)

Due to your extensive wood chemistry training, you know that there are big differences between the extractives of these woods. By extracting wood meal samples in a suitable you should be able to remove the majority of the extraneous materials. Further fractionation by polarity will allow you to separate the resin acids/neutrals/fatty acids from the polyphenolic compounds. A quick glance at the literature on extractives should give you a good idea of the relative amounts of these types of materials in each wood species in the above list. You (and your partner) will be working with only one of the above species so you will need to gather information from the rest of your co-workers in the laboratory to ascertain exactly what your species is. Good team play, therefore, is essential. (Welcome to the wonderful world of corporation human resources language). If you gain this information, you should be able to save your worthless job.

Background:

Extractive chemistry was covered in PSE 406 in lectures 14 and 15; you may want to pull out the dusty notes and refresh your memory. Last years TA, Andy Hanneman, wrote this short discussion on extractive chemistry that should refresh your memory.

Compounds from the various wood extractives groups are used by plants for a variety of purposes including energy storage (fats), defense compounds (resins and phenolics), and pigments (flavonoids). Some compounds are also components of the cell membrane in living cells (phytosterols). Wood extractives are generally best extracted with organic solvents (as opposed to water) and range in polarity from hexane extractable to methanol extractable. Acetone seems to be the best general solvent for simultaneous extraction of all classes of wood extractives. The wood extractives can cause significant problems in pulp and paper production. This includes paper deposits, machine deposits, foaming, and air and water pollution problems.

EQUIPMENT

  1. A Gas Chromatograph equipped with a high temperature, dimethyl polysiloxane (DB-1) megabore capillary column, and a temperature programmed on-column injection system (Perkin-Elmer Autosystem).
  2. Soxhlet extraction apparatus consisting of a thimble and extractor, a condenser, and a 250 ml. roundbottom flask.
  3. Boiling chips
  4. Spectroscopic grade acetone
  5. Glass wool
  6. Rotary evaporator
  7. Aluminum weighing pans
  8. Spectro grade hexane
  9. Sonicator
  10. Pyridine
  11. BSTFA
  12. GC autosampler vials
  13. Heating block
  14. Gas Chromatograph equipped with a, high temperature capillary column
Procedure – Wood meal Extraction
  1. Each team of students will be given a sample of unknown wood meal that has been previously freeze dried. Eliminating water through freeze drying allows for better extraction and easier quantification.
  2. Weigh out enough wood meal or pulp to fill the extraction thimble 3/4 full (lightly packed).
  3. Fill the 250 ml. flask with 150-175 ml. of spectroscopic grade acetone.
  4. Add a boiling chip to the flask and attach the flask to the soxhlet apparatus.
  5. Connect the condenser to the soxhlet and turn on the cooling water.
  6. Turn on the heaters on (notch #70). The soxhlet should cycle every 10 minutes.
  7. Allow the extraction to proceed for 2 hours at which point turn off the heat and allow the acetone to cool.
  8. Remove the thimble and spread the wood meal out in the fume hood on a paper towel to allow the remaining acetone to evaporate. This wood meal will be discarded. Label your round bottom flask of acetone and put in a stopper. This material will be worked with in your next lab.
Day 2
  1. Set the temperature on the “Rotovap” to 45. Make sure the water vessel is about 1/2 to 2/3 full.
  2. Place your round bottom flask on the stem of the Rotovap. Use a green plastic clamp to secure it in place.
  3. Turn on the rotovap vacuum, condenser water, and spinning mechanism, and evaporate off the acetone until approximately 10mls remains.
  4. Remove the flask from the heat bath and place it in the fume hood to cool.
  5. Obtain the tare weight of an aluminum pan.
  6. Pour the acetone into the aluminum pan (don't overfill it!).
  7. Using a 10 ml. graduated cylinder pour a 3 ml. aliquot of spectroscopic grade acetone into the roundbottom flask, swirl, and pour into the aluminum pan.
  8. Repeat step 15 three more times. It may be necessary to allow some acetone to evaporate between each addition.

NOTE:The acetone extractives include nonpolar resin (or lipid), as well as polyphenolics. The lipids are soluble in hexane, while most lignans, stilbenes and other acetone soluble phenolics are not. Full removal of polyphenols could require methanol, methanol/water or other polar solvent systems.

  1. Allow the acetone to evaporate to dryness.
  2. Add a 5 ml aliquot of hexane to the aluminum pan and place it in the sonicator. Be sure that there is about 1/8 inch of water in the bottom of the sonicator and turn it on for one minute.
  3. Using a pasteur pipette, mix the solution and remove most of the hexane while being careful not to pick up any insoluble material. Place the hexane in a second tared aluminum pan and allow the hexane to evaporate in the hood.
  4. Repeat the hexane extraction steps with 2 additional 5 mls aliquots. (repeating steps 18-19)
  5. After the hexane has been transferred and all of the solvents have evaporated, weigh the aluminum pans to determine the amount of each extractive fraction.
  6. You will need to set up your hexane extract sample for analysis by G.C. You will follow the same procedure as used in the standard preparation describe below.
Preparation of Standards – Work on this during extraction stage.

In order to determine if your sample contains significant levels of lipids (fatty acids, resin acids, or neutrals) or phenolics, you will need to analyze the hexane or acetone fraction using gas chromatography. You will learn how this technique works through a practice procedure described in a different handout. This section, however, contains the procedure for producing a sample containing a known chemical for use in the analysis. You are doing this so that you can make reasonable assumptions as to what chemicals you have in your extract. There will a variety of standard chemicals available to you in the laboratory. By time you get to this stage, you should have a good idea whether you are going to look for lipids or phenolics. Each team should make up a standard containing one compound.

  1. Choose compound to analyze.
  2. You will need to make up a solution containing approximately 1 mg/ml of material. For this standard, the exact weight is not needed nor do you need to be exactly at this ratio; just do not be too far off.
  3. Add 1 mg of compound to a capped glass sample jar. One mg is approximately a few (check with instructor) crystals of compound
  4. Add approximately 1 ml of pyridine to the sample jar in the hood. Gently swirl the solution until the standard dissolves.
  5. Take the sample to the instructor for addition of the silylation agent.
  6. The sample is now ready for analysis. If there is time, you can inject the sample. If not, the instructor will arrange for the sample to be injected overnight.

Report

A report format will be handed out to you this week. When this experimentation is complete, you will need to report the wood species of each one of these wood meals. You will need to back up your conclusion with experimental and literature data.

1

PSE407Winter 1999Northey/Hanneman