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Topic: Extracting fat using Soxhlet Extraction  (Read 5592 times)

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Offline Chochiana

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Extracting fat using Soxhlet Extraction
« on: April 26, 2015, 12:56:45 PM »
Hello everyone,

I want to know what are the scientific principles of extracting fat using Soxhlet Extractor? and
Couple of points as to why the yield could be low?

Thank you in advance! :)  ;D

Offline Furanone

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Re: Extracting fat using Soxhlet Extraction
« Reply #1 on: April 26, 2015, 01:44:51 PM »
The Soxhlet Apparatus is made up of three parts: the top condenser, the middle Soxhlet Extractor, and the bottom collection flask. You weigh your sample into a cellulose thimble with glass wool and place it in the middle Soxhlet Extractor. Then you select a suitable solvent (often petroleum ether due to cheap cost), and fill bottom collection flask about half full with solvent. Then in a fumehood, and on a hot plate, you gently evaporate the solvent so it travels up the side arm of the Soxhlet Extractor and it will reach top condenser where circulating cold water allows the solvent to condense and drip down onto the cellulose thimble with your sample enclosed.

As the drips collect and submerge your sample it dissolves the fat (based on like-dissolves-like) allowing passage through cellulose thimble pores, while all particulates fibre, proteins) and all small molecular weight hydrophilic components (ie. sugars, amino acids, salts, water, etc) remain inside cellulose thimble. Once the solvent reaches a certain height (where another side arm bends over to channel solvent back down to collection flask) from the condensing drips, it will flush back to collection flask taking any dissolved fats with it. Since the fats (ie. triglycerides) are non-volatile, they will remain in collection flask, while only solvent with much lower boiling point re-evaporates and condenses and dissolves more fats as the cycle repeats. It has been recommended that about 40 flushes takes place (typically 2.5-4 hours) to ensure all fat has been extracted. Now once in collection flask, the solvent can be evaporated out (recovered) and the remaining fat can be weighed to give total fat content (and used for further testing).

There are several reasons why your fat yield may be low.

1) If it was a matrix with a higher water content then channeling may have occurred where the solvent will not fully penetrate your sample extracting all the fat and instead will find channels through sample and extract only surrounding fat. The obvious solution to this problem is to dry your sample in vacuum oven at 60-70 C so that water is evaporated out, calculate the moisture/total solids content and once fat is extracted, back calculate to the final wet weight sample (using mass balance equation).

2) Another possibility is if the sample contains a high content of polar lipids (ie. phospholipids, mono & diglycerides) then a very non-polar solvent such as petroleum ether will only extract the neutral lipids and you will be underestimating your total lipids. A solution to this is to change your solvent. A common solvent is a 3:1 ratio of Chloroform:Methanol but be advised this has a higher boiling point so will require a much longer time to achieve the recommended 40 flushes. I typically wrapped Soxhlet flasks with aluminum foil to help insulate heat in, and allow the solvent evaporation & condensation to progress faster. 3:1 Chloroform:Methanol will extract all neutral lipids as well as phospholipids, mono & diglycerides so this is often performed along side Soxhlet extraction with petroleum ether to determine polar lipid content based on subtracting total lipids extracted with CH3Cl: MeOH from total lipids extracted from petroleum ether.

3) Another possible reason is you did not let extraction go to completion (ie. total 40 flushes). Each flush of solvent usually carries less sample fat with it (law of diminishing returns), but still if you did not extract long enough, then fat will remain in sample and you will be underestimating your total fat content.

4) If the fat is rancid (both hydrolytic rancidity & oxidative rancidity), then there will be a higher amount of free fatty acids (hydrolytic rancidity) in your sample which may or may not be volatile or able to be extracted (charged and thus not soluble in petroleum ether), and could cause an underestimation in your total fats (although this amount would likely be very low). For oxidative rancidity, peroxides could form at the unsaturated double bonds and then over time degrade to your typical off-flavour compounds such as propanal and hexanal. The binding of these peroxide groups to unsaturated triglycerides, and their breakdown products would be less soluble in the hydrophobic solvent, and more volatile respectively thus lost during removal of solvent from extracted fat, so this could cause underestimation in total fat results, but again this would likely be quite low unless a highly oxidized fat (and you would likely know this from smell of sample)

5) Carelessness in sample handling: If solvent was spilled (ie. splashback) before evaporation from final fat or using temperatures too high to evaporate out remaining solvent. Overloading of sample in cellulose thimble so like first point (1)) not all fat gets extracted due to channeling. Sample should be ground up well to increase surface-area-to-volume ratio to aid in fat extraction. These should be evident though from looking at your sample (final fat extract or defatted sample remaining in cellulose thimble) or from high variability (standard deviation) in your replicate results.

The above reasons for possible low fat yields are listed in order of most likely to least likely (in my opinion).

« Last Edit: April 26, 2015, 02:07:25 PM by Furanone »
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Offline jjwinkle

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Re: Extracting fat using Soxhlet Extraction
« Reply #2 on: May 05, 2015, 01:05:34 AM »
That is an outstanding answer. Another factor is how much sample is in the thimble. A common way to get the solvent to access the fat in water-containing samples is to mix anhydrous sodium sulfate with the sample before the Soxhlet extraction.

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