Preparation Sulfuric Acid 72%

(Specific gravity 1.6338 at 20oC)

Materials

Bequer (1.5L), graduate cylinder, hydrometer, thermometer, concentrated H2SO4 (~96%).

Procedure

Measure 665ml of concentrated H2SO4 (~96%) and slowly pour into 300ml of DI water keeping the bequer immerse in cold water. After the solution cool down make up the final volume to approximately 1L, measure the specific gravity, at 20oC, with the hydrometer. The sp gr MUST be 1.6338. Analysis of Extractives (Prepare of Extractive-free Samples)

Scopes

Extractives include chlorophyll, waxes, or other minor components that can be removed by benzene- ethanol extraction. This procedure covers the determination of extractives in a biomass sample. Extractives percentages are measured and used to convert compositions from an extractives-free basis to and as-received basis. The results are reported, on a dry weight basis, as a weight percentage of the biomass. Also, this procedure serves as a method to remove extractives from biomass prior to analysis to prevent interference with later analytical steps (such as lignin and structural carbohydrates).

Apparatus

1. Analytical balance, accurate to 1 mg or 0.1 mg;

2. Convection oven set to 105 + 5 °C for glassware drying;

3. Vacuum oven set to 45 + 2 °C;

4. Soxhlet extraction apparatus;

5. Pyrex glass extraction thimbles (C type);

6. Boiling flasks and glass beads or boiling stone;

Procedures

o 1. Dry thimbles in a convection drying oven @ 105 ± 3 C for 4 hour; record the weight of thimble (wT) to the nearest 0.1 mg.

2. Add 5 gram of AD sample to a tared extraction thimble. The height of the biomass in the thimble must not exceed the height of the Soxhlet siphon tube; place a piece of Whatman #1 filter paper (pre-cut to fit the size of thimble) on top of sample; label the top edge of the thimble with a pencil.

3. Dry thimble + air dry sample in a vacuum oven @ 45 ± 2oC overnight, record the oven-dried thimble + sample weight (wOD).

4. Assemble the Soxhlet apparatus. Insert the thimble into the Soxhlet tube.

5. Add 225 mL toluene-ethanol mixture (2:1 v/v) to receiving flask. Place the receiving flask on the Soxhlet apparatus. Adjust the heating mantles to provide a minimum of 4-6 siphon cycles per hour.

6. Reflux for 24 hours;

7. When reflux time is complete, turn off the heating mantles and allow the glassware to cool to room temperature;

8. Remove the thimble from Soxhlet tube, keep in hood and allow the solids to dry; 9. Put thimble + extracted sample in a vacuum oven @ 45 ± 2oC overnight, record the oven-dried thimble + extracted sample weight (wE);

10. The amount of extractives removed is calculated as shown in equation 3:

11. Run in triplicate and report extractives as an average with standard deviation;

12. The amount of recovered extractives can be measured by evaporating to dryness the benzene- ethanol mixture in a rotary evaporator. The benzene-ethanol mixture can be used in further extractions.

13. Keep extractive-free samples in zipper bags for future analysis. Klason Lignin Content

VI. Determination of Acid Insoluble (Klason) Lignin and Acid Soluble Lignin

Scope:

This procedure is suitable for samples that do not contain extractives. This procedure uses a two-step acid hydrolysis to fractionate the biomass into forms that are more easily quantified. The lignin fractionates into acid insoluble material (AISL, klason lignin) and acid soluble material (ASL). The acid insoluble material may also include ash and protein, which must be accounted for during gravimetric analysis. The acid soluble lignin is measured by UV-Vis spectroscopy.

Apparatus and Reagents

1. Analytical balance, accurate to 0.1 mg;

2. Convection drying oven, with temperature control of 105 ± 3oC;

4. Autoclave, suitable for autoclaving liquids, set to 121 + 3 °C;

5. Filtration setup, equipped with a vacuum source and vacuum adaptors for crucibles;

6. Desiccator containing desiccant;

7. Filtering crucibles, 30 mL, medium porosity;

8. Erlenmeyer flasks, 500 mL;

9. Serum bottles, 100 mL;

10. Sulfuric acid, 72% w/w (specific gravity 1.6338 at 20oC)

Procedures

1. Prepare extractive-free samples

2. Clean and dried crucibles. Weigh the crucibles to the nearest 0.1 mg and record this weight (w C);

3. Weigh 300.0 + 10.0 mg of the extractive-free oven-dried sample into a small beaker, 10ml;

4. Add 3.00 + 0.01 mL (or 4.92 + 0.01 g) of 72% sulfuric acid to each beaker; thoroughly mix sample with acid using a glass rod, keep the glass rod in each beaker;

5. Place the beakers at room temperature and incubate the sample for 120 minutes. Using the stir rod, stir the sample every five to ten minutes. Stirring is essential to ensure even acid to particle contact and uniform hydrolysis; 6. Carefully transfer samples from beaker to serum bottle by washing with DI water; a total volume of 84 mL DI water is required to dilute the acid to a 4% concentration;

3. Weigh 300.0 + 10.0 mg of the sample directly into serum bottle;

4. Add 3.00 + 0.01 mL (or 4.92 + 0.01 g) of 72% sulfuric acid to bottle; thoroughly mix sample with acid using a glass rod, keep the glass rod in each bottle;

5. Place the serum bottles in a water bath set at 30 + 3 °C and incubate the sample for 60 + 5 minutes. Using the stir rod, stir the sample every five to ten minutes without removing the sample from the bath. Stirring is essential to ensure even acid to particle contact and uniform hydrolysis.

6. Remove the bottles from the water bath. Dilute the acid to a 4% concentration by adding 84.00 + 0.04 mL deionized water;

7. Cap serum bottles with rubber stopper and seal with aluminum caps; mix the sample by inverting the tube several times to eliminate phase separation between high and low concentration acid layers;

8. Place the bottles in an autoclave safe tray, and place the tray in the autoclave; autoclave the sealed samples for one hour at 121°C, usually the liquids setting. After completion of the autoclave cycle, allow the hydrolyzates to slowly cool to near room temperature before removing the caps;

9. Vacuum filter the autoclaved hydrolysis solution through one of the previously weighed filtering crucibles; capture the filtrate in a filtering flask;

10. Transfer an aliquot, approximately 50 mL, into a sample storage bottle. This sample will be used to determine acid soluble lignin as well as carbohydrates; Acid soluble lignin determination must be done within six hours of hydrolysis. If the hydrolysis liquor must be stored, it should be stored in a refrigerator for a maximum of two weeks. It is important to collect the liquor aliquot before proceeding to step 11.

11. Use deionized water to quantatively transfer all remaining solids out of the serum bottle into the filtering crucible. Rinse the solids with a minimum of 50 mL fresh deionized water. Hot deionized water may be used in place of room temperature water to decrease the filtration time.

12. Dry the crucible and acid insoluble residue at 105 + 3 °C until a constant weight is achieved, usually a minimum of four hours.

13. Remove the samples from the oven and cool in a desiccator. Record the weight of the cricuble and dry residue to the nearest 0.1 mg (wL).

14. Place the crucibles and residue in the muffle furnace at 575 + 25 °C for 4 hours;

15. Carefully remove the crucible from the furnace directly into a desiccator and cool for 1 hour. Weigh the crucibles and ash to the nearest 0.1 mg and record the weight (wA). 16. The acid insoluble lignin (AISL) content is calculated by equation 4:

17. Analyze the sample for acid soluble lignin (ASL) as follows:

1) On a UV-Visible spectrophotometer (set to 205 nm wavelength), run a background (blank) of 10x diluted 4% sulfuric acid;

2) Dilute the sample as necessary (10x dilution is recommended) with deionized water to bring the absorbance into the range of 0.1 – 1.0, recording the dilution. Record the absorbance to three decimal places. Analyze each sample in duplicate, at minimum. Reproducibility should be + 0.05 absorbance units;

3) Calculate the amount of acid soluble lignin present using equation 5:

18. Run in duplicate and report AISL and ASL as an average with standard deviation;

Materials

Extractives free, dry wood samples (40-60mesh), dry and weighted medium pore size crucibles, flasks, erlenmeyer, reflux condenser, hot plate, stirring rods.

Procedure Extractives Removal

1- Benzene:ethanol (2:1).