Olive Oil Chemistry Analysis and Lab Testing & Balsamic Vinegar FAQ’s

Peroxides are primary (intermediate) oxidation products that are typically formed during oil extraction, processing and storage. The iodine produced by the addition of potassium iodide (as a reducing agent) to the oxidized sample dissolved in a chloroform/acetic acid mixture is measured by titration. The iodine is titrated with standardized sodium thiosulfate to a starch endpoint.

Free fatty acids are formed due to breakdown of the triacylglycerides in olive oils; when fatty acids are “free” when they are not bound to other molecules. Free fatty acidity is used as a marker for the quality of oil (and fruit). The free fatty acids produced chemically or enzymatically in samples dissolved in ethanol are titrated with standardized sodium hydroxide to a phenolphthalein endpoint.

During the breakdown of triacylglycerides (glycerol moiety bound to three fatty acids), diacylglycerols (glycerol moiety bound to two fatty acids and one free fatty acid) isomers are formed.  Fresh extra virgin olive oil contains high level of 1,2-diacylglycerols isomers where olive oil from unhealthy fruits and refined olive oil have an elevated level of 1,3- diacylglycerols isomers. The ratio of 1,2- and 1,3-isomers is a useful marker for assessing oil freshness and refining. Gas chromatography (GC) is used to determine the diacylglycerol profiles. Miniaturized column chromatography on a silica gel column is used to separate the isomeric diacylglycerols as the more polar fraction from the major part of other lipids. The peak areas of 1,2- and 1,3-isomers are determined after silylation by gas chromatography.

Determination of the concentration of triacylglycerols (TAGs) with equivalent carbon number 42 (ECN42HPLC) in oil by reverse phase high performance liquid. The content of triacylglycerols with ECN 42 determined by HPLC analysis and the theoretical content of triacylglycerols with ECN 42 (calculated on the basis of GLC determination of fatty acid composition) correspond within a certain limit for genuine olive oils. A difference larger than the values adopted for each type of oil points out that the oil contains seed oils.

Conjugated dienes and trienes are indicators of initial and secondary products of oxidation.  The presence of refined oil would generally increase the values of K232, K268 and ΔK. Samples are dissolved in isooctane and UV absorbance are measured spectrophotometrically at 234 nm and 268 nm (see AOCS Ch 5-91 for detailed procedures).

Upon thermal degradation of olive oil, pigments structure chlorophyll break down to pheophytins and further to pyropheophytins.  The ratio of pyropheophytins to the total pheophytins is considered to be a useful indicator to distinguish fresh oils from oils that are refined, deordorized or aged. The minor constituents are separated by high performance liquid chromatography (HPLC) with UV detection at 410 nm.

The method describes a procedure for determining the individual and total sterols and triterpene dialcohols content of olive oils and olive pomace oils. The oils are saponified with ethanolic potassium and the unsaponifiable matter is extracted with ether. The sterols and triterpene dialcohols fraction are separated from the unsaponifiable matter by silica gel chromatography. The fractions recovered from the silica gel are derivatized into trimethylsilyl ethers and analyzed by capillary column gas chromatography with a flame ionization detector.