Extraction and Characterization of Lipids

There are some key factors which determine the chemical and physical properties of fats and oils include the degree of unsaturation or iodine value, the type of unsaturation (cis or trans), and the weight-average molecular weight or saponification number. Fats and oils commonly undergo processes such as hydrogenation, oxidation; which modify the physical and chemical characteristics of fats and oils by changing the iodine value and cis/trans ratio, for which specific values are often targeted. Hence monitoring changes in these values during a process is important because they define the quality of the end product. In addition, the trans content may be limited by regulations because of its association with heart disease and may be a required analysis for labeling. (Li, van de Voort, Sedman & Ismail, 1999)

Determination of Iodine Value

Iodine value is used to determine degree of unsaturation of fatty acids of an oil, fat or wax. The iodine value reflects amount of iodine, in grams, that is absorbed by 100 grams of the oil, fat or wax. Saturated oils, fats and waxes do not absorb iodine; therefore their iodine value is zero; but unsaturated oils, fats and waxes absorb iodine. The amount of iodine, absorbed by fatty acids shows higher iodine value, and the more reactive, less stable, softer and more susceptible to oxidation and randification oil, fat or wax properties. In performing tests, a known excess iodine reacts with a known weight of the oil and then the amount of iodine remaining, did not react, is determined by titration.

Hanus solution, which is dissolved in glacial acetic acid, has two advantages as a reagent in determination; stability and permanence. (Dunlap, 1906)

Sample, is weighted accurately into a 250 mL conical flask, and dissolved in chloroform. The measured volume of Hanus reagent is added and after one minute long mixing, flask is placed in the dark for 30 min. A corresponding reagent blank is simultaneously prepared and it is placed in the dark with sample.

At the end of the 30 minutes time period, the reaction between iodine and sample, is stopped by adding potassium iodide and diluting with water to prevent loss of the free iodine. The amount of iodine present is determined by titrating with sodium thiosulfate (Na₂S₂O₃) using starch indicator. Difference between absorption of iodine of sample and blank solutions can be calculated by titration. After that results are written in form of gram iodine per 100 gram of sample.

Determination of Peroxide Value

Peroxides are one of the reaction products produced in the initial stage of oxidation, and because of this, amount of peroxides gives an indication of the progress of lipid oxidation. One of the most commonly used methods to determine peroxide value is titration with potassium iodide. The lipid is dissolved in a suitable organic solvent, three volume of glacial acetic acid with two volume of chloroform and an excess of KI is added:

ROOH + KI_excess    ------>  ROH + KOH + I₂

Once the reaction has completed, the amount of ROOH that has reacted can be determined by measuring the amount of iodine formed. This is done by titration with 0.01N sodium thiosulfate and a starch indicator:

I₂ + starch + 2Na₂S₂O₃ (blue) ------> 2NaI + starch + Na₂S₄O₆(colorless)

The amount of sodium thiosulfate required to titrate the reaction shows the concentration of peroxides in the sample at the beginning. The room temperature method is recommended for the determination of peroxide values. 0.002 N Sodium thiosulfate, made up each day from 0.10 N soln. A saturated solution of potassium iodide, freshly prepared. The test tubes used should be thoroughly washed with soapy water and then rinsed with hot water and allowed to stand in chromic acid for a few hours. They should then be rinsed thoroughly in water and finally with distilled water, and should be dried in an oven before use. The test must be carried out away from windows and preferably with the aid of artificial light. (Stuffins & Weatherall, 1945)

There are several problems with determination of peroxide value as an indication of lipid oxidation. Firstly, peroxides are primary products that are broken down in the latter stages of lipid oxidation. Thus, a low value of PV may represent either the initial or final stages of oxidation. Secondly, the results of the procedure are highly sensitive to the conditions, such as light and temperature, and as a result the test must always be standardized.

Dunlap, F. L. (1906). The preparation of aldehyde-free ethyl alcohol for use in oil and fat analysis. J. Am. Chem. Soc., 28(3), 395–398.

Li, H., van de Voort, F. R., Sedman, J., & Ismail, A. A. (1999). Rapid determination of cis and trans content, iodine value, and saponification number of edible oils by fourier transform near-infrared spectroscopy. JAOCS, 76(4), 491-498.

Stuffins, C. B., & Weatherall, H. (1945). Determination of the peroxide value of oils and fats. Analyst, 70, 403-409.