Objective:  (a) To determine the total lipids in foods samples and (b) peroxide value of cooking oils 

Lipids are a group of substances that are soluble in organic solvents. A soxhelet apparatus is used to extract lipids in the food lab. In this method a dry sample (less than 10% moisture) is completely surrounded by a solvent in an extraction chamber which then siphons to a boiling flask. The process continues for several hours until all the lipid is extracted. After extraction, the solvent is removed. This can be done by using a rotary evaporator which sucks off the solvent using a vacuum. The low vacuum pressure lowers the boiling point of the solvent, allowing it to be removed at a lower temperature. The difference in weight of the extraction flask before and after extraction is the total lipids in the sample.

Further analysis can be done on the extracted lipid to determine its characteristics. For example, peroxide value tests can be carried out to determine the quality of the oil. Peroxides are primary breakdown products of lipid oxidation. They are transitory, leading to secondary breakdown products as the quality of the lipid continues to break down. Therefore, a low peroxide value could mean both the start of oxidation or advanced oxidation. Typically, you can tell if oxidation is advanced by smell. A highly rancid smell is a good indication of advanced oxidation. High-quality, freshly deodorized fats and oils will have a peroxide value of zero. Peroxide values >20 corresponds to poor quality fats and oils. For soybean oil, 1-5, 5-10, and > 10 corresponds to low, medium and high levels of oxidation respectively.

Mechanism of lipid peroxides (also called hydroperoxides and denoted by ROOH)

Lipids are oxidized by either autoxidation or photo-oxidation. Autoxidation is the most common mechanism for oxidation; initiated by factors such as light, heat and metals. In autoxidation, unsaturated fatty acids (i.e. fatty acids with one or more double bonds) loses a proton (i.e. hydrogen) from one of the double bonds. This leaves an unpaired electron. The new compound is called a free radical which is very unstable as it searches for another proton to fill the loss. To gain stability, the free radical will ‘rob’ another lipid of its proton. This will set off a chain reaction as every time a radical is formed it will snatch a proton from another lipid. Alternately, free radicals react with oxygen to form peroxides. Peroxides breakdown to other compounds that leads to rancid off-flavors and color damage in foods.

Photo oxidation occurs when UV light interferes with normal molecular oxygen (called triplet oxygen). This causes one of its electrons in its outer shell to become excited and move to a higher orbital level. This is called the singlet state. This is a high-energy and very reactive state that is capable of reacting with unsaturated fatty acids to extract a proton leading to formation of a free radical and then initiate autoxidation. Food processors add antioxidants in foods to prevent free radical chain reactions and buildup of rancid compounds and off-flavors.

The lipid peroxide test is used to determine the extent of oxidation of lipids. It is defined as the milliquivalents (mEq) of peroxide per kilogram of sample. Extracted lipid is dissolved in glacial acetic acid-chloroform solution which causes a liberation of iodine. Starch is then added to the flask to bind iodine, producing a purple color. When the starch-I2 complex is titrated against sodium thiosulphate, iodine is liberated from the starch, leaving a colorless to ‘milky’ colored solution.


(a) Total Lipids Determination

  1. Weigh and record the weight of a 250 ml round bottom flask and label the flask (in duplicate)
  2. Grind about 15 grams of dry food sample in mortar and pestle
  3. Weigh and collect 5 g (in duplicate) of the ground sample in folded filter paper prepared for soxhlet thimble
  4. Place in thimble and transfer to soxhlet extraction flask
  5. Extract lipids from sample with approximately 200 ml hexane in 250 ml flat bottom boiling flask over 12 hours
  6. Evaporate hexane from flat bottom boiling flask using roto-evaporator at 43oC
  7. Allow flat bottom boiling flask to cool and then take a final weight
  8. Calculate percentage lipids extracted

% lipids = [(Total wt of lipids extracted)/sample wt] x 100

(b) Peroxide Value Determination

  1. Use a Pasteur pipette to transfer (in duplicate) 5 g of cooking oil sample to a 250 ml Erlenmeyer flask (record the exact weight to the nearest 0.01g)
  2. Working in a fume hood, add 30 ml acetic acid:chloroform (3:2) solution and shake until the sample is completely dissolved
  3. Add 0.5 ml KI and swirl for exactly 1 minute
  4. Add 30 ml distilled water and shake vigorously to liberate the iodine from the chloroform layer
  5. Add 1 ml of starch indicator solution
  6. Titrate against sodium thiosulfate until a milky color appears
  7. Record the volume of titrant (sodium thiosulfate) used
  8. Run the peroxide value procedure again without adding any oil to the reaction flask. The titrant (ml) observed here will be your blank (B). This value will be zero if your container and reagents were not contaminated with peroxides
  9. Calculate the peroxide value

Where: S = volume of titrant (ml) for sample; S = volume of titrant (ml) for blank; N = Normality of Na2S2O3 solution (mEq/ml); 1000 = conversion of units (g/kg); W = sample mass

Lab Questions 

  1. Explain the mechanism of autoxidation and photooxidation
  2. Compare the total fats you obtained from the lipid extraction with the total fats declared on the food label. How do they compare? 
  3. What do the peroxide values that you obtained tell you about the quality of the cooking oils you tested?
  4. Discuss methods that food processors can take to minimize oxidative rancidity of food products

Reference: Nielsen, S. S. Food Analysis. (2003). New York, NY: Springer Science+Business Media, LLC.

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Courtney Simons
Courtney Simons
Dr. Simons is a food science educator. He earned his bachelor’s degree in food science, and Ph.D. in cereal science at North Dakota State University.