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saponification

The document outlines a procedure for saponification (base hydrolysis) of organic materials, primarily for the analysis of total lipid extracts. It details the materials required, safety precautions, and a step-by-step procedure for extracting ester-bound lipids, including troubleshooting tips for common issues. The process involves heating samples with KOH, adjusting pH, and extracting with MTBE, with considerations for ensuring sample neutrality before further analysis.

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popescu vasilica
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17 views2 pages

saponification

The document outlines a procedure for saponification (base hydrolysis) of organic materials, primarily for the analysis of total lipid extracts. It details the materials required, safety precautions, and a step-by-step procedure for extracting ester-bound lipids, including troubleshooting tips for common issues. The process involves heating samples with KOH, adjusting pH, and extracting with MTBE, with considerations for ensuring sample neutrality before further analysis.

Uploaded by

popescu vasilica
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Sessions Biogeochemistry Lab July, 2009

Saponification (Base Hydrolysis) of Organic Materials

Introduction

Many polymeric materials can be decomposed into smaller subunits that are suitable for
GC/MS analysis by acidic or basic hydrolysis. 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. Saponification (base hydrolysis) is more
frequently employed than acid hydrolysis. The procedure is commonly applied to total lipid
extracts (TLE) to release ester-bound lipids, but can also be applied to solid materials such
as biomass or extracted residues.

Materials

• 0.5N KOH in H2O (note A) • MTBE (GC grade) • pH paper


• 4N HCl • 40 mL VOA vials • heating block (70°C)
• 5% NaCl in H2O • Teflon capliners • TurboVap

Special Hazards and Warnings

 You are working with a moderately strong 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 can sometimes burst while being heated. Keep the hood sash lowered
while samples are heating, and wear safety glasses.

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 KOH solution, cap the vial tightly using a Teflon
capliner, and heat in the heating block at 70°C for 3 hours (note B).
2. Allow to cool, then add 5mL of NaCl solution and shake thoroughly (note C).
3. Add 4N HCl dropwise from a pipette 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 D), then pipette off the organic layer (top) and collect it in a second
Sessions Biogeochemistry Lab July, 2009

VOA vial. Repeat 2 more times with fresh 10mL aliquots of MTBE, collecting a total of
~25mL of extract. Dry the extract to ~1 mL under N2 in the TurboVap.
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. When in doubt,
check the solution pH before injecting into the GC.

Notes

A. 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 2). If
you want to form methyl esters, see the method for alkaline transesterification. You can
substitute NaOH for KOH.
B. Methods from various labs differ widely in the temperature and time used for
saponification. We typically use 3 hours for saponification of total lipid extracts (TLE).
However, up to 24 hours is commonly employed for solid materials, including sediments,
biomass, and kerogen.
C. The NaCl solution is added to partition the KOH into the aqueous phase, away from the
organic phase. In a pinch, you can get away with using pure water for this step. However, a
NaCl solution is more efficient at this, so we recommend its use when possible.
D. Often the hydrolyzed products of fresh biomass produce a thick emulsion 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 more NaCl to the
aqueous layer, in an effort to ‘salt out’ the more polar materials; 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.

Troubleshooting

There is not much that can wrong with this reaction, aside from the formation of emulsions
that are difficult to extract. If the hydrolysis is incomplete, you can increase any of the
following: temperature, time, KOH concentration. If the hydrolysis is still incomplete, you
can try acid hydrolysis. The most common problem with this procedure is insufficient
removal of HCl from your sample (step 6), which leads to other problems in subsequent
steps.

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