Analysis of oil and Fat(Unit 7)

It prepared for Undergraduate students teaching purpose

1. Unit 7A: Analysis of Oil and Fat 7.1 Unsaturation-Iodine value 7.2 Saponification - Free fatty acid, Sap. Value 7.3 Melting Behaviour – Solid Fat content Low temperatureproperties 7.4 Oxidation – peroxide value, Anisidine value, Stability, Shelf life, Stability Trials, Taste Panels 7.5 Gas Chromatography 4/30/2020 Dr. Mohammed Danish/ Oil and Fat Technology/ UniKL MICET 1

2. Unsaturation in oil and Fat (Iodine vale) The unsaturated oils and fat (PUFA)can be identified through the iodine number. The iodine number equals the number of mg of iodine required to saturate the fatty acids present in 100 mg of the oil or fat. Oils rich in saturated fatty acids have low iodine numbers. 4/30/2020 Dr. Mohammed Danish/ Oil and Fat Technology/UniKL MICET 2

3. Procedure for determining Iodine value A fixed volume of oil or fat dissolved in chloroform, then Huebl’e iodine is added to it. As long as double bonds are available, the colour of iodine does not appear in the solution as the iodine is absorbed by the double bonds. When all the double bonds are saturated, the colour of iodine appears in the solution 4/30/2020 Dr. Mohammed Danish/ Oil and Fat Technology/UniKL MICET 3

4. The iodine value can be calculated from the volume of the oil taken, specific gravity of the oil and the volume of the Huebl’s iodine required to impart colour in the oil solution. Huebl’s iodine : it is prepared by dissolving separately 26 g iodine in 30 g mercuric chloride in about 250 mL of ethanol each, mix the two, and make it to 1 L with ethanol). Control : five mL of chloroform is taken in a dry porcelain dish and 2-3 drops of Huebl’s iodine are added. This serves as a control for colour comparison. Oil testing : 5 mL of chloroform is taken in dry porcelain dish, 0.5 mL of oil (e.g. olive oil) is added and dissolved by gentle swirling, Huebl’s iodine is added slowly from the burette until the colour of iodine (matching with the control) appears in the solution. The burette reading should be noted. 4/30/2020 Dr. Mohammed Danish/ Oil and Fat Technology/UniKL MICET 4

5. Iodine number calculation The specific gravity of olive oil is 0.915, so 0.5 mL of the oil weighs 450 mg. Suppose that x mL of Huebl’s iodine is used to saturate 0.5 mL of the olive oil. 1 mL of the Huebl’s iodine contains 26 mg of iodine, x mL of the Huebl’s iodine contains 26x mg of iodine Since, 450 mg of oil takes 26x mg of iodine Therefore, 1 mg of oil takes 26x/450 mg of iodine Therefore, 100 mg of oil takes (26x/45).100 4/30/2020 Dr. Mohammed Danish/ Oil and Fat Technology/UniKL MICET 5

6. Saponification Saponification value : It is the amount (mg) of alkali (potassium hydroxide) required to totally neutralized the fatty acid part of the oil and fat resulting from the complete hydrolysis of 1 g of the sample. A soap is formed during saponification. For example, 4/30/2020 Dr. Mohammed Danish/ Oil and Fat Technology/UniKL MICET 6

7. Lower molecular weight fatty acids and esters requires more alkali for saponification, so the saponification value is inversely proportional to the mean of the molecular weights of the FA and esters. The saponification value is not, in general, so useful for identification purposes. It is useful for detecting the presence of oil and fats which contain a high proportion of lower fatty acids. 4/30/2020 Dr. Mohammed Danish/ Oil and Fat Technology/UniKL MICET 7

8. Procedure for determining Saponification value Take 0.2 to 0.5 g of oil or fat in a 50-100 mL Erlenmeyer flask. Add 10 mL of 0.5 N ethanolic potassium hydroxide solution and mix with oil sample (already taken in Erlenmeyer flask). Heat at 80-85 °C in water bath for 30 min. Cool it at temperature between 30-40 C, then titrate with 0.5N HCl standard solution (Add 2-3 drops of indicators phenolphthalein or methylene blue) Carry out blank test also without oil. 4/30/2020 Dr. Mohammed Danish/ Oil and Fat Technology/UniKL MICET 8

9. Calculation of saponification value Saponification value (mg/g)   4/30/2020 Dr. Mohammed Danish/ Oil and Fat Technology/UniKL MICET 9 Where, S = oil weight A = titration volume of blank (mL) B = Titration volume of sample (mL) F = Factor of 0.5 N HCl standard solution 28.05 = is the half of the molecular weight of KOH Preparation of 0.5 N ethanolic KOH standard solution: take 35 g of KOH, dissolve in 20 mL of water, then make up to 1000 mL with 95% (v/v) ethanol or absolute alcohol.

10. Melting behaviour of oil and fat The melting point data, solid fat index, and other solidification procedures are all used to predict the consistency of the finished oil and fat products. For edible fat and oils products, the liquid/solids level at various temperature in relation to body temperature can give good indications of the mouth feel, gumminess, workability, and overall general behaviour to cool, ambient, and elevated temperature. 4/30/2020 Dr. Mohammed Danish/ Oil and Fat Technology/UniKL MICET 10

11. Melting point Analysis Melting point is usually defined as the point at which a material changes from a solid to a liquid. It must be noted, natural fats do not have a true melting point. Pure compound have sharp and well-defined melting points, fat and oils are complex mixture of compounds, so normally it have a range of melting points. Fat and oils melting point procedure have standardized by the AOCS (American Oil Chemists Society) and other associations. 4/30/2020 Dr. Mohammed Danish/ Oil and Fat Technology/UniKL MICET 11

12. The melting methods vary considerably in the endpoint determination, conditioning of the sample, amount of automation, time requirements, attention required, degree of melt, and so on. The following methods can be used to determine the melting point of the oil and fat: Capillary melting point Softening point Slipping point Wiley melting point Mettler dropping point 4/30/2020 Dr. Mohammed Danish/ Oil and Fat Technology/UniKL MICET 12

13. Methods of melting point measurement Capillary melting point (AOCS method Cc1-25) : in this method, capillary tubes (1 milli meter inside diameter) are filled to height of 10 milli meters with melted fat. The end of the capillary sealed and tempered for 16 h at 4 to 10 °C. After tempering the tubes are heated in water bath at the rate of 0.5 °C starting 8-10 °C below the expected melting point until fat became completely clear. 4/30/2020 Dr. Mohammed Danish/ Oil and Fat Technology/UniKL MICET 13

14. Wiley melting point (AOCS method Cc 2-38) : A solidified fat disc is solidified and chilled in a metal form for 2 hours or more. The disc is then suspended in an alcohol-water bath and slowly heated while being stirred with a rotating thermometer. The Wiley melting point occurs when the fat disc becomes completely spherical. The subjective interpretation of the end pint is again a major disadvantage, and slight variation in sample tempering or heating rate interferes with reproducibility of the results. Another disadvantages is the constant attention required to determine the endpoint. 4/30/2020 Dr. Mohammed Danish/ Oil and Fat Technology/UniKL MICET 14

15. Mettler dropping point (AOCS method Cc18-80): it utilized the Mettler instrument which equipped with control unit and the dropping furnace. A sample cup designed for the furnace is filled with 17 drops of the melted fat samples and tempered in a freezer for 15 minutes. The cold solidified sample is warmed in the temperature-programmed furnace until it becomes fluid enough to flow. When the sample flows, it trips a photoelectric circuit, which records the temperature on a digital read out. This method has become standard in many oil and fat laboratory. This method offer fully automatic end point determination. Good correlation of results with Wiley melting point results. Time required to test melting point is less than an hour. This method can be used to analyse products with low melting points 4/30/2020 Dr. Mohammed Danish/ Oil and Fat Technology/UniKL MICET 15

16. Solid fat content Solid fat index (SFI): solid fat index is an empirical measure of the solid fat content. It is calculated from the specific volume at various temperatures utilizing a dilatometric scale measured in units of milliliter times 1000. SFI results are arbitrary because assumption are made and liberties are taken for precise measurement. AOCS Method Cd 10-57, dilatometry is the basis for solid fat measurement in oil and fat industry. The dilatometric solid fat index has been used extensively for product development, process control, and quality specification for oils and fat. 4/30/2020 Dr. Mohammed Danish/ Oil and Fat Technology/UniKL MICET 16

17. SFI assumptions Use of volumetric instead of gravimetric measurements. Use of solution other than mercury as the confining liquid. Assumption that the slopes of the liquid and solid lines are parallel. Assumption that the slope of the liquid line is the same for all fats. Assumption that the melting dilation is 0.1 Some of the assumption are made out of necessity and other made for convenience. Even with these liberties and assumptions the results have relative significance. 4/30/2020 Dr. Mohammed Danish/ Oil and Fat Technology/UniKL MICET 17

18. Dilatometer A dilatometer is a scientific instrument that measures volume changes caused by a physical or chemical process. A familiar application of a dilatometer is the mercury-in-glass thermometer, in which the change in volume of the liquid column is read from a graduated scale. Dr. Mohammed Danish/ Oil and Fat Technology/UniKL MICET 4/30/2020 18

19. SFC measurement through NMR AOCS method Cd 16-81, Allow to determines the quantity of solid glycerides in a fat by measuring the pulsed NMR signal of the liquid fat. The hydrogen atom in a solid crystal does not give a NMR signal, but hydrogen atom in a liquid environment does. Thus, NMR measures the actual amount of liquid in sample. The Dilatometer and NMR analysis may be correlated, but the method do not provide identical results. The most significant difference between the two techniques is experienced with the fats containing high solid contains at 10.0 and 21.1 °C (50 and 70 °F). 4/30/2020 Dr. Mohammed Danish/ Oil and Fat Technology/UniKL MICET 19

20. Difference between SFC and SFI 4/30/2020 Dr. Mohammed Danish/ Oil and Fat Technology/UniKL MICET 20

21. Low temperature properties Measurement of the heat of fusion of fat rather than its thermal expansion appeared to offer promise as a mean of determining fat solid contents. Differential scanning calorimetry (DSC) was developed for measurement of the solid contents of vegetable and animal fats. It is less time-consuming method for determining fat solid content. In brief, the DSC measured the heat of fusion of a frozen or completely solid fat sample. The area under the melting curve is measured, then selected partial areas are measured as a percentage of the whole. The temperature selected for partial area measurement are normally those used for the SFI by dilatometry. 4/30/2020 Dr. Mohammed Danish/ Oil and Fat Technology/UniKL MICET 21

22. Advantages of DSC analysis of fat The thermal history determined can provide clues to the tempering and storage condition for the fat sample; The fat melt curve can help distinguish between two fat products with identical SFI value; A fat solid range from -10 °C for soft oil to 70 °C for hard hardfats would be possible. Results should be available in approximately 1 hour Correlation with SFI results appear good. Sample required for analysis 5 to 10 milligrams. 4/30/2020 Dr. Mohammed Danish/ Oil and Fat Technology/UniKL MICET 22

23. Disadvantages of DSC analysis Accurate weighing of small quantity of sample poses the problem of representative sampling. Variation of fat solids is not constant and is greater at low temperatures. Precision has been found to be poorer than with the standard SFI dilatometric techniques. DSC has never been accepted as a quality control tool to replace SFI. Because too much information from temperature programmed and non-precise results are major obstacle in it. DSC can be useful research tool but not used in routine analysis for quality control tool. 4/30/2020 Dr. Mohammed Danish/ Oil and Fat Technology/UniKL MICET 23

24. Oxidation of oil and fats Oxidation and hydrolysis of oil and fat caused rancidity, which give sharp pungent smell mixed with stale and musty odour. To measure the rancidity in oil and fat following test can be conducted: Peroxide value Anisidine vales Stability Analysis 4/30/2020 Dr. Mohammed Danish/ Oil and Fat Technology/UniKL MICET 24

25. Peroxide value Oxidation of lipid is a major cause of deterioration, and hydroperoxides formed by the reaction between oxygen and the unsaturated fatty acids are the primary products of this reaction. The hydroperoxide have no flavor or odor but break down rapidly to form aldehydes, which strong, disagreeable flavor and odor . The peroxide concentration, usually expressed as peroxide value, is a measure of oxidation or rancidity in its early stages. Peroxide value (PV) measures the concentration of substance responsible for oxidation in terms of milliequivalents of peroxide per 1000 gm of oil or fat samples. 4/30/2020 Dr. Mohammed Danish/ Oil and Fat Technology/UniKL MICET 25

26. Determination method of peroxide values Oil samples equivalent to 0.2 g were mixed with 9.8 mL chloroform/ methanol (7:3 ratio). Mix it well in a screw-capped vials on a vortex mixer for 5 sec. Then, 100 µL of 10 mM xylenol orange was mixed and vortexed for 5 sec. Then 50 µL of 36 mM iron (II) solution was added and the sample was mixed on a vortex mixer for 5 sec. After 5 min of incubation at room temperature, the absorbance of the sample can be determined at 560 nm using UV-Vis spectrophotometer. To plot standard calibration curve for Fe(III), stock solution of ferric chloride (10 µg/mL) can be used and the dilution were made by chloroform/methanol (7:3 ratio).the standard Fe(III) concentrations can be prepared in the range of 5-30 µg/L. The peroxide values were calculated as the mequivalent of Fe(III) per Kg of oil used. 4/30/2020 Dr. Mohammed Danish/ Oil and Fat Technology/UniKL MICET 26

27. The calibration curve of Fe(III) chloride is represented in figure. The mole of Fe(II) react with one mole of ROOH form oxidized product Fe(III) as represented in the chemical equation. 4/30/2020 Dr. Mohammed Danish/ Oil and Fat Technology/UniKL MICET 27

28. Iodine method for peroxide value 5 g of oil or fat dissolve in 10 mL chloroform in the Erlenmeyer flask and mixed with 15 mL glacial acetic acid and 1 mL saturated KI. The mixture were left in the dark at room temperature for 5 minutes, Then 75 mL of distilled water was add and the mixture was shaken vigorously. Finally 1 mL of 1% starch solution was added and the resulting solution was titrated with 0.002 N sodiumthiosulphate until the colour become clear. The same procedure should be applied to a blank solution (without fat or oil). 4/30/2020 Dr. Mohammed Danish/ Oil and Fat Technology/UniKL MICET 28

29. Calculation The peroxide value Where, V1 = is the amount of Na 2 S 2 O 3 used for titration (mL) V0 = amount of Na 2 S 2 O 3 used for the blank (mL) N = the normality of Na 2 S 2 O 3 M = amount of fat or oil sample (g)   4/30/2020 Dr. Mohammed Danish/ Oil and Fat Technology/UniKL MICET 29

30. Anisidine Value Anisidine value measures the amount of alpha- and beta- unsaturated aldehydes present in the oil. This method is based on the fact that in the presence of acetic acid, p-anisidine reacts with the aldehydic compounds in an oil, producing yellowish reaction products. The colour intensity depends not only on the amount of aldehydic compounds present but also on their structure. 4/30/2020 Dr. Mohammed Danish/ Oil and Fat Technology/UniKL MICET 30

31. Anisidine value determination methods 1 g of oil or fat sample should dissolve in 25 mL of isooctane, label as A1 solution. Then 5 mL of A1 solution mixed with 1 mL p-anisidine (25% in glacial acetic acid) solution, label as A2. Prepare reference solution by adding 1.0 mL of 2.5 g/L solution of p-anisidine in glacial acetic acid in 5.0 mL of isooctane, the solution shaken to mix it well. The absorbance of both test solution and reference solution were measured at 350 nm. 4/30/2020 Dr. Mohammed Danish/ Oil and Fat Technology/UniKL MICET 31

32. Calculations The p-anisidine value was calculate as: Where, A2 = absorbance of test solution A2 A1 = absorbance of test solution A1 m = mass of the oil or fat to be examined in test solution A1, in grams.   4/30/2020 Dr. Mohammed Danish/ Oil and Fat Technology/UniKL MICET 32

33. The slides end here! Thanks for your patience! 4/30/2020 Dr. Mohammed Danish/ Oil and Fat Technology/UniKL MICET 33

34. Project tiles Margarin products Mayonnaise products Baking shotenings Frying Sortenings Emusifiers Dairy Analog Shortening Biodiesel production from edible oil Lubricants production from oil and fat Soap and Detergents from oil and fat 4/30/2020 Dr. Mohammed Danish/ Oil and Fat Technology/UniKL MICET 34