Food Laboratory 683 Determination of Fat content & Oil Quality

Total Fat by Soxhlet

Introduction The constituents of fat in foods are a mixture of triglycerides, phospholipids, sterols and other lipid substances. In this laboratory the analytical method for measuring total lipids is by ether extraction using a Buchi Soxhlet Fat Extraction Unit.

Procedure • Grind the dried sample prepared in the previous session with a mortar

and pestle • Wear gloves when handling the thimble and extraction cup! • Tare the thimble on the balance then weigh about 1.5 g of the ground

sample into the thimble and record the weight to four decimal places (W1) • Weigh one extraction beaker (glass beaker that the fat will collect in)

containing a glass beads (W2) to four decimal places • Attach the white plastic holder to the thimble as instructed by your

demonstrator • Place the thimble in the extraction chamber of the Buchi fat extractor as

instructed by your demonstrator. • Place your extraction beaker on the bottom hot plate as instructed by

your demonstrator. Ensure that your beaker is labeled first. • Once all the samples and extraction cups are set up your demonstrator

will show you how to lower the unit to seal the extraction cups • Your demonstrator will then show you the settings on the unit indicating

the extraction time etc • The machine can be set for the number of cycles and amount of time for

the extraction. A cycle is the filling of the extraction chamber to the white line then draining of the solvent to the extraction beaker. Based on your settings the machine will keep extracting for whichever is the longest out of the number of cycles or the extraction time.

• You can also set the time that the machine performs the rinse and drying stages.

• The cooling water flow will then be turned on by your demonstrator. This ensures that any petroleum ether vapours condense and are returned to the sample

• Add 100 mL of petroleum ether (boiling range 40-60ºC) to the top of the unit (above the distilling coils) using the funnel as instructed by your demonstrator. This ether will collect in the extraction chamber first, then after starting the machine will drain down to the extraction beaker.

• TAKE CARE ADDING THE PETROLEUM ETHER – PARTICULARLY MAKING SURE YOU DON’T PUT TOO MUCH WEIGHT ON THE FUNNEL AS IT MAY BREAK THE GLASS

• Your demonstrator will then show you how to start to machine which starts the fat extraction process

• Ensure that you observe closely what happens during the cycles and understand how the fat is extracted from your sample and how it is then able to be collected in the extraction cup

• Once the extraction cycles or extraction time has finished (based on the settings) the sample will be rinsed and dried

• Your demonstrator will raise the unit so that the extraction cups can be removed

• The extraction cups then need to be dried overnight to remove all the petroleum ether

• After drying you will need to come in to the lab to weigh the extraction cup to four decimal places (W3), which now contains only the fat content of your original sample

Calculation

% 𝐶𝑟𝑢𝑑𝑒 𝐹𝑎𝑡 =𝑊3−𝑊2

𝑊1𝑥 100

where: W1 = Weight of Sample (dry) W2 = Extraction cup weight W3 = Dried extraction cup weight Note – ensure all your weights are expressed in the same units

Quality Measures in Oil

Acidity The % free fatty acids value of an oil is calculated by determining the mg of NaOH necessary to neutralise the free acids present in 1 g of fat or oil. To express as a percentage this is usually expressed relative to oleic acid. This is usually done by titrating the oil sample with NaOH in the presence of a pH indicator such as phenolphthalein (to determine when the sample is neutral); where the greater volume of NaOH indicates greater acidity. However in this laboratory you will be using the oxitester to estimate acidity

Determination of Acidity using the Oxitester: The principle of the assay used by the oxitester is that fatty acids will react with a chromogen and when doing so form a coloured complex. The intensity of the colour produced (which is measured by absorbance at 630 nm) is proportional to the acidity of the sample.

Peroxides Unsaturated lipids are susceptible to the process of oxidative rancidity. During this process peroxides (ROOH) are formed. Although these can be broken down to secondary oxidation products (such as ketones, aldehydes, alcohols, hydrocarbons) their measurement in oils gives an indication of the progress of oxidation that the oil. Oils that have been stored for long periods of time, exposed to oxygen, light, or high temperatures will tend to have oxidise more than fresh correctly stored oils. A low peroxide value usually means that little oxidation of the lipids has occurred, however could also indicate that lipid oxidation is nearing completion and therefore the peroxides are being converted to their secondary products.

Manual Method for Determination of Peroxide Value Theory: This method determines the concentration of peroxides in the oil sample using the ability of peroxides to liberate iodine from potassium iodide. The amount of iodine liberated can then be determined in the presence of starch by titration with sodium thiosulphate as indicated in the reactions below:

𝑅𝑂𝑂𝐻 + 2𝐾𝐼 → 𝑅𝑂𝐻 + 𝐼2 + 𝐾2𝑂 2𝑁𝑎2𝑆2𝑂3 + 𝐼2 → 2𝑁𝑎𝐼 + 𝑁𝑎2𝑆4𝑂6

Reagents: The following reagents have been prepared: Acetic-chloroform (CHCl3) solvent (acetic acid:CHCl3 3:1) and is in the fume-hood Freshly prepared saturated KI solution 0.1N Na2S2O3 Procedure: Carry out the following procedure for the two oil samples you have been given Dilute the 0.1N Na2S2O3 to 0.01 N using boiled then cooled water

Weigh approximate 5 g of your oil sample into a 250 mL conical flask. Record the weight to 2 decimal places. Add 30 mL of the acetic-CHCl3 solvent to your flask in the fumehood, stopper it with aluminium foil and swirl to dissolve the oil Add 0.5 mL saturated KI solution. Let stand for 1 minute with occasional shaking, then add approximately 30 mL deionised water Add a few drops of 1% soluble starch indicator (until solution turns blue/purple). Titrate your sample with 0.01N Na2S2O3, adding the titrant drop by drop with vigorous shaking until the blue starch colour disappears Calculation: Calculate the Peroxide Value using the formula below: Peroxide Value=(S-B)xN/W x 1000 Units are mEq peroxide per kg of sample S = mL of titrant for sample B = mL of titrant for blank N = normality of the titrant 1000 = conversion of units (g/kg)

Peroxide Value using Oxitester: The peroxide value can also be determined using the oxitester. The principle of the method however is different from the manual method. Basically the peroxides oxidise the ions Fe2+ to Fe3+. The Fe3+ ions then form a red complex the intensity of which can be determined by absorbance at 505 nm. The absorbance is proportional to the peroxide concentration Determine the peroxide value for the two oil samples using the oxitester as shown by your demonstrator

Expected Peroxide Values for Oils: The fresher the oil sample the lower the peroxide value. Peroxide values >20 indicate poor quality oils that have been extensively oxidised.