The information below are a very reliable source because I translated them from my university notebooks of Fats and Oils Technology class which were edited and tought by Dr. Apostolos Kyritsakis (Msc., Ph.D. in University of Michigan) who is a well known and respected researcher and professor in Greece and worldwide. He has edited many awsum books about oils and especially about olive oil. I strongly suggest that if you are interested you should try and find them because they are truly amazing!
Determination of the phenolic substances in olive oil
A. Generally about phenols
Phenols are natural antioxidant substances that can be found in certain plant oils and mainly in olive oil. Phenols are water soluble substances and thus during the processing of the olives, a big portion migrates to the water phase. A quantity though remains in the oil and it affects its stability in oxidation. The above show that its really important to know the phenolic content of an oil.
The determination of phenols with the method described here, is based on the formation of colour using the Folin-Ciocalteau reactant agent which in the presence of sodium carbonate solution, reduces the phenols.
B. Reactant agents and solutions used
1) Methanol solution in water (60%)
2) Folin-Ciocalteau (2 N)
3) Sodium carbonate solution (35%)
4) Hexane
C. Procedure
1) In an 250 ml Erlenmeyer vial/bottle we measure 10 g of oil and we add 50 ml of hexane.
2) We add 20 ml of the methanol solution and we stir the mixture thoroughly for 2 minutes in a shaker. The process is repeated for 2 more times. Every time, after the separation of the phases, the methanolic phase is being removed and collected in a glass.
3) After the 3 extraction, the methanolic extracts are evaporized wutg a rotary evaporator at 70 C until there is only dry residue left.
4) The dry residue is dissolbed in 1 ml of methanol.
D. Calculation of the phenol content
1) 0,1 ml of the methanolic extract is added into a 10 ml round vial.
2) 5 ml of distilled water and 0,25 ml of Folin-Ciocalteau are added and the mixture is stirred thoroughly.
3) 3 min later we add 1 ml of sodium carbonate solution and we fill with distilled water.
4) After 1 hour we measure the absorbance of the blue colour that will form (if there are any phenols that is), at 725 nm. As a control is used Folin-Ciocalteau solution.
For the quantitative determination of phenols that are present in the sample, a standard curve is constructed using pure caffeic acid solutions with concentrations ranging from 0-100 μg / 10 ml of solution.
NOTE: The final content of the phenols is usually given in ppm. The result should be expressed towards the phenolic acid that is being used fot the construction of the standard curve. Except caffeic acid, olive oil contains other phenolic acids such as cumaric acid and protocatechuic acid.