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Adapted from Moorhead & Reynolds (1993) Am. Midl. Nat. 130:83-89.

By Jared L. DeForest

This method separates C compounds into three parts: (1) Soluble in ethanol and water, (2) sulfuric acid soluble and (3) sulfuric acid insoluble.

Equipment

Sonicating water bath, High-speed centrifuge, muffle furnace, 50-ml centrifuge tube (preweighed), pasture pipette, 15-ml centrifuge tubes, 72% H₂SO₄ (sulfuric acid), 125 ml flask, pre-weighed Pall 50mm glass fiber filter, Corning Mini-Miser Filter

Sample Preparation

1. Oven dry litter at 65掳C.
2. Grind litter sample into a fine powder.
3. Place 0.25g of sample into preweighed 50-ml centrifuge tube.

Soluble Fraction (Soluble in water and ethanol)

1. Add 25 ml of ethanol to each tube.
2. Place into 60掳C water bath for 30 m.
3. Centrifuge at 1,000 rpm for 15 minutes.
4. Suction off supernatant. (Try to keep polar lipids on top.)
5. Repeat steps 1-4 two more times.
6. Add 25 ml of water to each tube.
7. Repeat steps 1-4 two more times, but with water.
8. Oven dry samples at 60掳C for 24 h.
9. Weigh residue.

Soluble content was estimated as the difference between original and residual weight.

Hemicellulose (Acid soluble) & Lignin (Acid insoluble)

1. For the same centrifuge tube, add 2 ml of 72% sulfuric acid.
2. Incubate for 1 h at 30掳C.
3. Add 30 ml of distilled water to transfer sample to 125 ml flask.
4. Autoclave sample for 1 h at 120掳C.
5. Pass sample through a pre-weighed Millipore filter.
6. Oven-dry filter at 60掳C for 24 h.
7. Weigh filter.

Hemicellulose content calculated by difference between pre- and post acid digested dry sample weight. The residue is lignin content (acid insoluble fraction plus the mineral fraction).

Mineral Fraction (Ash)

1. Place residue from acid digested in crucible.
2. Muffle furnace at 500掳C for 24 h.
3. Weigh ash.

Subtract the ash value from the acid insoluble fraction.

Created: 10/05/06 Revised: 10/10/11