FOOD ANALYSIS : FAT METHODS
Solid, dry food in a thimble
Solvent cycles again and again
Liquid milk in a flask
Ammonia frees the fat, then solvent pulls it out
Figure 1: Both methods end the same way, by weighing the fat after the solvent is gone. They differ in how the fat is freed first.
Introduction
The Soxhlet method and the Mojonnier method both find fat by pulling it out with a solvent and weighing what is left after the solvent is gone. Soxhlet is the method for solid foods: a dry, ground sample sits in a thimble while a solvent cycles through it again and again until all the fat is washed out. Mojonnier is the method for milk and liquid dairy foods: the fat in milk is held inside a protein shell and an emulsion, so ammonia is used first to dissolve the protein and free the fat, and then ether pulls the fat out in a Mojonnier flask. The Mojonnier method is the Rose-Gottlieb method carried out in the Mojonnier flask, and it is the reference method for fat in milk.
Quick comparison
Table 1: Soxhlet method compared with Mojonnier method
| Aspect | Soxhlet method | Mojonnier method |
|---|---|---|
| Foods it suits | Solid foods: oilseeds, biscuits, meat products, snack foods | Liquid milk and milk products: milk, skim milk, cream, condensed milk, milk powder |
| Sample | Dry, ground sample in a porous thimble | About 10 g of liquid milk in a Mojonnier fat extraction flask |
| How the fat is freed | Repeated washing with hot solvent | Ammonia dissolves the protein shell, ethanol helps the proteins settle, then ether extracts the fat |
| Solvent | Usually petroleum ether or diethyl ether | Diethyl ether and petroleum ether, boiling range 40 to 60 °C, with ethyl alcohol |
| Apparatus | Soxhlet extractor, flask, heater | Mojonnier fat extraction flask, flat bottom flask or dish, oven, desiccator |
| End point | Extraction runs until the last cycles remove nothing more | Drying at 102 plus or minus 2 °C until two successive weighings differ by no more than 1 mg |
| Result | Crude fat, weighed after the solvent is removed | Fat percent by weight, the reference value for milk |
| Blank | Not a routine part of the test | Reagent blank is subtracted: if the blank is more than 0.5 mg, purify or replace the reagents |
| Repeat check | Run until no more fat comes out | Duplicate results by the same analyst must not differ by more than 0.03 g fat per 100 g product |
| Reference | Standard method for crude fat in solid foods | AOAC Official Method 905.02, Fat in milk; ISO 1211:2010 |
Scientific explanation
Definition
Soxhlet method: a method that washes fat out of a dry sample with a solvent that cycles again and again, then weighs the fat. Mojonnier method: the Rose-Gottlieb gravimetric method for fat in milk, in which ammonia frees the fat from its protein shell and ether extracts it in a Mojonnier flask, and the fat is weighed after drying.
Working principle
Soxhlet: the dried, ground sample is placed in a porous thimble inside a Soxhlet extractor. A solvent, usually petroleum ether or diethyl ether, is boiled in a flask below, and its vapour rises, condenses, and drips into the thimble, washing the fat out of the sample. When the thimble fills, the solvent siphons back into the flask, carrying the fat with it. The cycle repeats many times, until all the fat has been washed out. The solvent is then removed by evaporation, and the fat left in the flask is weighed. Mojonnier: the milk sample is treated with ammonia and ethyl alcohol: the ammonia dissolves the protein and the alcohol helps the proteins settle. Fat is extracted with diethyl ether and petroleum ether. The mixed ethers are evaporated and the residue weighed. The fat is extracted twice, three times for high fat contents, and the extract is dried in an air oven at 102 plus or minus 2 °C until its weight stops changing.
Apparatus
Soxhlet: Soxhlet extractor with thimble, boiling flask, heater, and balance. Mojonnier: Mojonnier fat extraction flask or Rohrig tube, cork or synthetic rubber stopper that the solvents do not attack, 100 mL flat bottom flask or stainless steel or aluminium dish, air oven, and desiccator.
Measurement procedure
Soxhlet: dry and grind the sample, place it in the thimble, run the solvent cycles until extraction is complete, evaporate the solvent, and weigh the fat. The sample must be dry before extraction, because water blocks the solvent. Mojonnier: weigh about 10 g of milk into the extraction flask, add 1.25 mL of ammonia and mix, add 10 mL of ethyl alcohol and mix, add 25 mL of diethyl ether and shake vigorously for about one minute, add 25 mL of petroleum ether and shake again, let the upper ether layer separate until it is clear, and decant it off. Repeat the extraction twice with 15 mL of each solvent, evaporate the solvent completely, dry the flask at 102 plus or minus 2 °C for two hours, cool in a desiccator and weigh, then heat again for 30 minutes and weigh again until the difference between two successive weights does not exceed 1 mg. Wash the fat out of the flask with petroleum ether, dry and weigh the flask again: the difference in weights is the weight of fat extracted. Correct the weight by the blank determination on the reagents.
Factors affecting readings
Soxhlet: a wet sample blocks the solvent and reads low, and stopping the cycles early leaves fat behind and reads low. Ether is flammable and its vapour is heavier than air, so the work is done away from flames and in a ventilated space. Mojonnier: the ammonia must dissolve the protein fully, or fat stays trapped and the result reads low. Old or emulsified samples may need a little alcohol to help the layers separate. The reagents must be pure, because the blank is subtracted and a blank above 0.5 mg means the reagents must be purified or replaced.
Formulas and calculations
Soxhlet, crude fat percent = weight of extracted fat divided by sample weight, times 100. Mojonnier, fat percent by weight = weight of extracted fat divided by weight of milk, times 100.
Worked example from the FTN Food Analysis notes, Soxhlet: a 5.000 g sample of ground biscuit yields 1.000 g of extracted fat after drying. Crude fat = 1.000 divided by 5.000, times 100 = 20.0 percent. If the biscuit held 4.0 percent moisture, the fat on a dry basis is 20.0 divided by 0.96 = 20.8 percent.
Worked example for Mojonnier, using the milk manual procedure: a 10 g milk sample gives 0.350 g of extracted fat after drying to constant weight and blank correction. Fat = 0.350 divided by 10, times 100 = 3.5 percent. The milk manual notes that milk holds about 3.5 percent fat, so this result fits the expected value.
Industrial applications
- Soxhlet measures fat in biscuits, oilseeds, meat products, and snack foods, where the fat is locked inside particles.
- Mojonnier is the reference test for fat in milk, skim milk, cream, and milk powder, and it settles payment and trade disputes.
- Dairy laboratories use the Mojonnier result to check fast routine tests such as the Gerber method.
- Food labels for solid foods carry the crude fat value from solvent extraction, and milk labels carry the Mojonnier reference value.
Advantages and limitations
Soxhlet method: it handles solid foods that no milk method can test, it needs simple glassware, and the repeated cycling pulls out nearly all the fat. Its limits are time, flammable solvent, and the need for a dry sample: using it on milk would fail, because milk is mostly water and water blocks the solvent.
Mojonnier method: it is the reference method for milk fat, it frees fat that solvent alone cannot reach, and its blank and duplicate rules keep it honest. Its limits are time and care: strict attention to detail is essential for reliable results, the solvents are flammable, and the method suits liquid dairy foods only.
FAQ
Question: Why can Soxhlet not test milk?
Because milk is mostly water, and water blocks the solvent. The fat in milk is also held inside a protein shell and an emulsion, so solvent washing alone cannot reach it.
Question: Why does the Mojonnier method use ammonia?
Ammonia dissolves the protein that encloses the fat in milk, so the ether can then reach the fat and pull it out. Without it, the fat would stay trapped and the result would read low.
Question: What does constant weight mean in the Mojonnier test?
Heating and weighing is repeated until two successive weights differ by no more than 1 mg. Only then is the fat taken as fully dry.
Question: Why is the blank checked in the Mojonnier method?
Because the reagents themselves can leave a small residue. The blank is subtracted from the result, and if the blank is more than 0.5 mg the reagents must be purified or replaced.
References and further reading
- FTN Food Analysis, Unit 4: Protein and fat analysis (notes on disk): Soxhlet method, worked example 20.0 percent crude fat, Gerber compared with Soxhlet.
- Manual for Analysis of Milk and Milk Products, FSSAI, 2012 (book extract on disk, milk-analysis-manual.txt): Rose-Gottlieb / Mojonnier method, sections 1.3.4.2 to 1.3.4.2.3, AOAC Official Method 905.02, duplicate limit 0.03 g per 100 g.
- ISO 1211:2010, Milk: Determination of fat content, gravimetric method (reference method), cited in the milk manual and in the Buchi Mojonnier application note.
- Nielsen, S. S., Food Analysis Laboratory Manual, Springer: fat analysis methods (consulted through Google Books).
