Introduction

Many polymeric materials can be decomposed into smaller subunits that are suitable for GC/MS analysis by acidic or basic hydrolysis. Ester and amide bonds are particularly susceptible. The products of ester hydrolysis will be alkanols plus alkanoates (at moderate to high pH) that need to be derivatized before analysis. This procedure is commonly applied to fresh biomass to collect total fatty acids, or to sediments following solvent extraction to release ester-bound lipids. Although in theory there should not be any hydrocarbons released by basic hydrolysis of kerogen, in practice we often find that there are.

Materials

• 0.5N NaOH or KOH in water (note A)
• 4N HCl
• 5% NaCl solution (in organic-free H2O)
• MTBE (or diethyl ether, etc.)
• 40mL or 60mL VOA vials
• pH paper
• heating block (70C)

Warnings

• You are working with strong acid and base. Be sure to wear gloves and safety glasses. If you don't want small holes to appear in your clothes, a lab coat is recommended.
• Capped vials have occasionally burst while being heated. Keep the heating block in the hood with the sash lowered while samples are being heated.

Procedure

1. Transfer sample to a VOA vial using an appropriate solvent (DCM, ether, etc.) and dry in the Turbovap. Add 5mL of 0.5N OH solution, cap the vial tightly, and heat in the heating block for up to 24 hours (note B).
2. Allow to cool, then add 5mL of NaCl solution and shake well. Its best to have a teflon capliner in the cap for this part.
3. Add 4N HCl dropwise to your sample to adjust pH to between 1 and 3. Check the solution pH by transferring a few drops with a clean Pasteur pipette onto wide-range pH paper. This step is necessary to ensure that organic acids are protonated and thus soluble in organic solvents.
4. Add 10mL of MTBE and shake vigorously for 2 minutes. Allow the two phases to separate (note C), then pipette off the organic layer (top) and collect it in a second VOA vial. Repeat 2 more times with fresh 10mL aliquots of MTBE, collecting a total of ~25mL of extract.
5. Depending on the next step, you may want to filter the extract through anhydrous sodium sulfate to eliminate any water that might have been collected with the sample.
6. Important! You are extracting a strongly acidic solution with a moderately polar solvent (MTBE). It is likely that your extract will be slightly acidic, especially if you used methanol as the hydrolysis solvent. If the next step is methylation of fatty acids, this is not a concern. However, if you plan on making TMS derivatives and then injecting directly into the GC/MS, this will cause problems with excessive GC column bleed. Neutralize your sample first by shaking with a small amount of 5% NaHCO3 solution, then dry over anhydrous Na2SO4. Alternatively, you can extract the aqueous solution with hexane, which will not dissolve an appreciable amount of acid.

Notes

1. Hydrolysis can be conducted in methanol, water, or any other protic solvent. Methanol is somewhat better at solubilizing polymeric organic materials, but there is a chance that some carboxyl groups will be converted to methyl esters. Adding a small amount of water to the reaction will help to limit their formation, as will shaking with acidic water (step 3). We generally use water for saponification of fresh biomass, and methanol for saponification of kerogen.
2. Methods from various labs differ widely in the temperature and time used for hydrolysis. In general, longer is better for very thick, gooey sediment extracts. For fresh biomass, a couple hours at 70C is probably sufficient.
3. Often the hydrolyzed products of fresh biomass produce a thick emulsion upon shaking that resists separation. Some tricks that may help, in order of increasing effectiveness: i) gently stir the emulsion with the tip of a long Pasteur pipette or glass stir rod; ii) add NaCl to the aqueous layer; iii) freeze the sample, then allow it to slowly thaw while gently stirring; iv) transfer the whole mess to a centrifuge tube and spin at 2000 rpm for 10 minutes. This last option always works, but is slow going.