UNIVERSITI MALAYSIA SABAH SCHOOL OF FOOD SCIENCE & NUTRITION LABORATORY REPORT NT20903 FOOD CHEMISTRY AND BIOCHEMISTRY Determination of free fatty acid(FFA) and iodine value (IV)in oil LECTURER NAME : MOHD NAZRI BIN ABDUL RAHMAN LAB SESSION : 3 OCTOBER 2011 (GROUP 4, MONDAY) Group Member Title: Determination of free fatty acid (FFA) in oil and determination of iodine value (IV) in oil Introduction Acid value or free fatty acid content is an important characteristic commonly used in quality control of fat and oil. Fats and oils are one of the large variety and important sources in the world.

It contains acidity which is the result of the degree of breakdown of the triacylglycerols by a chemical reaction known as hydrolysis or lipolysis generally. Fresh and healthy oils or fats may also have significant amounts of acidity. This fact is usually due to the errors during the biosynthesis or extraction of oils from fruits. Oils and fats that produced from low quality fruit or through a careless process will consequences in breakdown of the triacylglycerides into fatty acid. These fatty acids are called free fatty acids (FFA).

Free fatty acids can used as an immediate source of energy by many organs and can be converted by the liver into ketone bodies. Free fatty acid form during the breakdown of lipid by hydrolysis and oxidation. Thus, free fatty acid is hydrolyses from dietary lipid for absorption or utilize for lipid synthesize. Free fatty acid play important role in food system and our body system also too . Free fatty acid is same with unsaturated fatty acid, which is giving help to our body . To verify the quality of fats and oils, acid value or free fatty acid can be used.

Acid value is the mass of potassium hydroxide (KOH) in milligrams that is required to neutralize one gram of chemical substance. Acid value is normally calculated in order to determine the amount of free fatty acids. This method is applicable to all normal fat and oil. Titration with sodium hydroxide is use to determine acid value by to neutralize free fatty acid in 1g of fat. The percentage of free fatty acid is expressed in the term of acidity. While for the free fatty acid, it is calculated by a method which the molecular weight of a particular fat is used in it.

The molecular weight is vary depends on different type of fat. Type of fat or oil| Expressed as| Molecular weight (g/mol)| Coconut, palm oil kernel and any resulting fractions| Lauric acid| 200| Palm oil and any resulting fractions| Palmitic acid| 256| Other liquid oil| Oleic acid| 282| The process of breakdown of lipid by hydrolysis and oxidation generated free fatty acid. However, acid value is not possible used to differentiate between the free fatty acid formed during the process of hydrolysis and oxidation. But it still can widely used to determine the quality of fat and oil in food system.

This is an appropriate method to all fat and oil. Oil or fat that made carefully from freshly or unspoiled fruits generally will have lower acid value under 0. 5% of free fatty acid. But for extra virgin olive oil will have less than 0. 8% of free fatty acid. Based on the titration result, we can conclude the relationship between acid value and free fatty acid in the oil. The higher result of acid value we get, we can determine how higher value of free fatty acid in the oil. Therefore, the test will carry out with carefully to avoid mistaken appear in the test.

However, sometime it is hard to use acid value to differentiate between the free fatty acid formed during the hydrolysis process and oxidation process. Halogenation is one of the addition reaction. Fat or oil generally consists of double bonds between carbon atoms which inclined to undergo addition reactions. Halogen such as chlorine, bromine, iodine monochloride and iodine monobromide will across the double-bonded carbon atoms in the fat or oil during halogenation. As a result, saturation of carbon – carbon atom will occur and saturated lipid is formed. Degree of unsaturation in the ipid can also be determined by measured the amount of halogen that added to the lipid molecule. H H H H H H H H H C C = C C H + Br2 H C C C C H H H H Br Br H One application of the iodine number is the determination of the amount of unsaturation contained in fatty acids. This unsaturation is in the form of double bonds which react with iodine compounds.

The higher the iodine number, the more unsaturated fatty acid bonds are present in a fat. In a typical procedure the acid is treated with an excess of Wij’s iodine solution which a solution of iodine monochloride (ICl) in glacial acetic acid. The iodine concentration is then determined by titration with sodium thiosulfate. Iodine value is the average unsaturation measurement in fat sample. Although iodine value is expressed as the weight of iodine added to 100 g of sample, the analytical procedure used to determine the iodine normally use iodine monochloride in acetic acid solution (Wijs’ reagent) as halogenations agent.

Objective 1) To determine the free fatty acid found in oil. 2) To differentiate between the free fatty acid formed during the hydrolysis process and oxidation process. 3) To understand the relationship between acid value and free fatty acid 4) To determine the iodine value (IV) in oil 5) To measure the unsaturation of oils. 6) To study the volume of sodium thiosulphate solution used in the titration Material ; Apparatus for determination of free fatty acid in oil Conical flask 250 ml, burette 25 ml, measuring cylinder 250 ml, glass rod, glass dropper and beaker Reagents

Solvent: Mix 95% (i/i) ethanol with diethyl ether in the ration of 1:1(i/i), Neutralise with 0. 1 N sodium hydroxide before use. Sodium hydroxide: 0. 1N or 0. 5N standardized solution. Phenolphthalein Indicator Solution: 1% dilution in 95% (i/i) ethanol Sample: Cooking Oil(RBOPO) mn 256 g/mol and Margarine(Steric) mv 256 g/mol. Material and Apparatus for determination of iodine value in oil Conical flask 500 ml, measuring cylinder 100 ml, pipette ,burette 50 ml, retort stand, dropper electronic digital weight measures and beaker Reagents

Dichloromethane Potassium iodide: 10% solution Sodium thiosulphate: standardized 0. 1 N solution Starch indicator: mix 10 g of starch and 10 mg of mercury iodide in 30 ml of distilled water. Pour the mixture into 1 L of boiling distilled water. Stir for 1 minute and cool. Wijs’ reagent Sample: margarine 0. 34g, oil 0. 34 g Method for determination of free fatty acid in oil 1. Sample was weighed in conical flask and the amount of sodium hydroxide required depends on the type of fat. 2. The fat was dissolved with 150 ml of petroleum ether. 3.

The mixture was titrated with sodium hydroxide solution, 15 drops of phenolphthalein was used as indicator (the last drop has been added when the resulting pink colour lasts for at least 10 seconds). 4. The result of the titration is recorded in a table for calculation. Method for determination of iodine value in oil 1. 0. 34 g of fat was weighted in a dry 500 ml conical flask with an accuracy of 0. 001g. 2. 15 ml of dichloromethane was added to dissolve the fat . 3. 25 ml of Wijs’ reagent was pipette into a beaker and covered with aluminium foils. 4. The Wijs’ reagent was transferred to the conical flask and swirled.

Then the flask was left in a dark place for 1 hour. (leave for 2 hours for fat with iodine value more than 200 and if the fat is oxidized or polymerized). 5. 20 ml of potassium iodide solution and 150 ml of distilled water were added to the flask. 6. A burette is filled with sodium thiosulphate solution. 7. Few drops of starch indicator added into the conical flask as indicator and then titrated with the sodium thiosulphate solution. 8. Titration stopped when blue color disappears. 9. The result of the titration is recorded in a table for calculation. Precaution Step for determination of free fatty acid in oil 1.

The solvent was made sure not expired. 2. Both solution (ethanol and diethyl ether) were need to be stirred and mixed to become equivalent and made sure both layers of solution had mix together. 3. Carefully handle the chemical substances . 4. Apparatus used must be clean and dried to prevent the mixture of residue water and samples. In order to obtain accurate reading, the eyes of observer must be same level with the meniscus of the sample to reduce parallax error Precaution Steps for determination of iodine value in oil 1. Make sure that the surface of electronic balance is cleaned when weighting the samples. 2.

Make sure that the solvent is not expired. 3. Stir the solvent until all the samples dissolved completely. 4. The flask containing the indicator must be shaken well while titration. 5. Ensure the burette is titrate slowly when reaching the end point. 6. When taking the readings from burette, the eyes of observer must be level with the meniscus of the sample to reduce parallax error. Calculation for determination of free fatty acid in oil Weight of sample = w Volume of sodium hydroxide solution used (ml) = v Normality of sodium hydroxide = N

Acid fat molecular weight = M Acid value = 56. 1 Nvw Free fatty acid (FFA%) = vNM10w Note: 1. Normally when a decision has been made as ‘acidity’ and cannot be defined as oleic acid. Results Type of fat or oil sample: Cooking oil Group| 1| 2| 3| 4| 5| Mean ± SD| Weight of fat in unit gram (w)| 20| 20| 20| 20| 20| 20| * ml NAOH used(v)| 5. 6| 4. 3| Failed| 4. 5| Failed| 4. 8±0. 700| Normality of NaOH(N)| 0. 1| 0. 1| 0. 1| 0. 1| 0. 1| 0. 1| Acid fat molecular weight (M)| 200| 200| 200| 200| 200| 200| Acid value| 1. 57| 1. 21| -| 1. 6| -| 1. 35±0. 195| Free fatty acid (%)| 0. 56| 0. 43| -| 0. 45| -| 0. 48±0. 070| Acid value for group 4 = 56. 1 Nvw = 56. 1 (0. 1)(4. 5)20 = 1. 26 Average acid value = 1. 57+1. 21+1. 263 = 1. 35 Free fatty acid(%) for group 4 = vNM10w = 4. 50. 1(200)10(20) = 0. 45% Average free fatty acid=0. 56+0. 43+0. 453 = 0. 48% Type of fat or oil sample: Margarine Group| 1| 2| 3| 4| 5| Mean ± SD| Weight of fat in unit gram (w)| 10| 10| 10| 10| 10| 10| * ml NAOH used(v)| 5. 8| 1. 3| Failed| 1. 4| Failed| 2. 83±2. 570| Normality of NaOH| 0. | 0. 1| 0. 1| 0. 1| 0. 1| 0. 1| Acid fat molecular weight (M)| 200| 200| 200| 200| 200| 200| Acid value| 3. 25| 0. 73| -| 0. 79| -| 1. 59±1. 438| Free fatty acid (%)| 1. 16| 0. 26| -| 0. 28| -| 0. 57±0. 514| Acid value for group 4 = 56. 1 Nvw = 56. 1 (0. 1)(1. 4)10 = 0. 79 Average acid value = 3. 25+0. 73+0. 793 = 1. 59 Free fatty acid(%) for group 4 = vNM10w = 1. 40. 1(200)10(10) = 0. 28% Average free fatty acid=1. 16+0. 26+0. 283 = 0. 57% Calculations for determination of iodine value in oil Weight of oil or fat used= w

Volume (ml) of sodium thiosulphate solution used for sample= vs Volume (ml) of sodium thiosulphate solution used for blank= Vb Normality of sodium thiosulphate solution= N Iodine value=12. 69N (Vb- Vs)W Results Type of fat or oil sample: Cooking oil Group| 1| 2| 3| 4| 5| Mean ± SD| Weight of fat in unit gram (w)| 0. 34| 0. 34| 0. 34| 0. 34| 0. 34| 0. 34| Sample titration of Na2S2O3 used/ml(Vs)| 12. 7| 10. 6| 15. 6| 13. 0| 12. 2| 12. 82±1. 44| Blank titration of Na2S2O3 used/ml(Vb)| 0| 0| 0| 0| 0| 0| Normality of Na2S2O3(N)| 0. 1| 0. 1| 0. 1| 0. 1| 0. 1| 0. 1| Iodine value| -47. 40| -39. 56| -58. 22| -48. 52| -45. 3| -47. 85±6. 75| Iodine value for group 4 = 12. 69N (Vb- Vs)W = 12. 69(0. 1) (0- 13. 0)0. 34 = -48. 52 Average iodine value = (-47. 40)+(-39. 56)+(-58. 22)+(-48. 52)+(-45. 53)5 = -47. 846 Type of fat or oil sample: Margarine Group| 1| 2| 3| 4| 5| Mean ± SD| Weight of fat in unit gram (w)| 0. 34| 0. 34| 0. 34| 0. 34| 0. 34| 0. 34| Sample titration of Na2S2O3 used/ml(Vs)| 30. 5| 15. 6| 16. 2| 10. 2| 16. 3| 17. 76± 4. 48| Blank titration of Na2S2O3 used/ml(Vb)| 0| 0| 0| 0| 0| 0| Normality of Na2S2O3(N)| 0. 1| 0. 1| 0. 1| 0. 1| 0. 1| 0. 1| Iodine value| -113. 84| -58. 22| -60. 46| -38. 07| -60. 84| -66. 9±28. 22| Iodine value for group 4= 12. 69N (Vb- Vs)W = 12. 69(0. 1) (0- 10. 2)0. 34 = -38. 07 Average iodine value = (-113. 84)+(-58. 22)+(-60. 46)+(-38. 07)+(-60. 84)5 =-66. 29 Discussion From the experiment above, we can know that average of acid value of cooking oil of all the group is 1. 35 and the average of free fatty acid value in the cooking oil is 0. 48. Besides that, average of acid value of margarine is 1. 59 and the average of free fatty acid value of margarine is 0. 57. The average acid value of cooking oil and margarine of our group (group 4) obtained are 1. 6 and 0. 79 respectively whereas the average free fatty acid, FFA (%) of cooking oil and margarine obtained are 0. 45% and 0. 28% respectively. Based on the free fatty acids(%) and acid values calculated from data, we can observe that the free fatty acids(%) increase with the acid values. Therefore, acid value is proportional to percentage of free fatty acids. The acid value of cooking oil of group 1 and group 2 are 1. 57 and 1. 21 respectively whereas the free fatty acids (%) of group 1 and group 2 are 0. 56 and 0. 43 respectively. The acid value of margarine of group 1 and group 2 are 3. 25 and 0. 3 respectively whereas the free fatty acids (%) of group 1 and group 2 are 1. 16 and 0. 26 respectively. Meanwhile, group 3 and group 5 were failed in both of this experiment. The result of the cooking oil and margarine of acid value and the percentage of free fatty acid of our group (group 4) and group 2 are not much different to each other whereas there is a significant difference of the acid value and the percentage of free fatty acid of group 1 with our group (group 4) and group 2. The volume of phenolphthalein used were not the same among all the groups, therefore there is a difference between all the groups.

The failure of group 3 and group 5 may be due to the margarine they used was not completely dissolved in the solvent or they accidentally used the wrong solvent. An amount of 20g of refined oil is titrated slowly with NaOH. Before the titration, the color of the solution is opaque yellow. Phenolphthalein was used as an indicator. Our group (group 4) is used 15 drops of phenolphthalein before titrated with NaOH. After titrated for a while, the solution change to slight pink and the titration is stopped. The pink color lasts for at least 10 seconds and the volume of NaOH used is 4. 50ml.

Hydrolysis of cooking oil will produce free fatty acids and glycerol. The free fatty acid in percentage increased as the acid value increased. The increased of acid value will denotes an excessively high content of free fatty acids and it will undergoes hydrolysis and oxidation and the oil will turn sour or rancid. Therefore, free fatty acids increase with the increase of hydrolysis and oxidation. The factors which will trigger hydrolysis reaction in food system are temperature, presence of an enzyme and pH. The internal lipids are primarily free fatty acids. Free fatty acids are more susceptible to oxidation.

Breakdown of lipid by hydrolysis and oxidation will form free fatty acids. The iodine value, IV is used to determine the average of unsaturated of fats by adding iodine to the fats or oil. In the experiment, the iodine values of cooking oil and margarine of our group (group 4) have been determined. The iodine values of cooking oil is 48. 52 while margarine is 38. 07. The iodine value of cooking oil of group 1, group 2, group 3 and group 5 are 47. 40, 39. 56, 58. 22 and 45. 53 respectively. The iodine value of margarine of group 1, group 2, group 3 and group 5 are 118. 84, 58. 22, 60. 46 and 60. 84 respectively.

The average iodine value of cooking oil and margarine of all the group is 47. 846 and 66. 29 respectively. The difference between all the group also due to margarine not completely dilute in solvent and they don’t stir margarine and dilute it equally. There a few changes of colour that occur in the reactions. Firstly, as the dichloromethane is added into cooking oil and margarine, the solution is change to yellow colour. Then, after Wijs reagent is added, the solution becomes dark red and the solution with cooking oil inside is left in dark for an hour where the solution with margarine is left in dark place for two hour.

After that, potassium iodide and distilled water is added. Lastly, starch indicator is added, the solution becomes blue colour and the solution is titrated with sodium thiosulphate solution. The solution is titrated slowly and observed until the blue colour disappears altogether. The higher the iodine value, the more unsaturated the oil, the less stable of the oil and the higher is the potential for the oil to polymerise, oxidation and free radical production. Margarine have the higher iodine value than the cooking oil because it make up of polyunsaturated fats. The igher the temperature or the longer the exposure to heat, the greater the degree of polymerization. Therefore, the lower the iodine value, the more stable and the less inflammatory of the oil and the quality of the oil is high. Different source of oil having the different iodine value because different source of oil contain different types of fatty acid chains. These chains differ in the number of carbon atoms and the number of carbon-carbon double bonds in the chain. Questions 1) Why is high acidity considered as an unsatisfactory characteristic in the fat and oil industry?

High acidity in the fat and oil is mostly caused by incautious extraction methods, fruit fly infestation, long duration between harvesting and extraction especially for the fruit that has been bruised or spoiled during harvesting as well as fungal diseases in the fruit like apple scab, Phytophthora collar rot, gloesporium and macrophoma. High acidity means that high occurring of rancidity. It is due to high oxidation and high hydrolysis. As a result, it contributes to low quality in production of fat and oil industry.

Therefore, high acidity is considered as an unsatisfactory characteristic in the fat and oil industry. 2) Identify factors which will trigger hydrolysis reaction in food system. The factors which will trigger hydrolysis reaction in food system are temperature, presence of an enzyme and pH. Questions 1. What is the characteristic of fat does the iodine value represent? Iodine value represents the degree of unsaturated fat. Higher iodine value will have a greater degree of unsaturated fat which means have more double bonded carbon atoms in fat. 2.

Results show that the iodine value of corn oil and coconut oil is respectively 130 and 10. What is the conclusion can you make regarding the characteristic of the two oils? Corn oil will have greater degree of unsaturation than coconut oil due to it has higher iodine value. Therefore, highly unsaturated corn oil is less stable and more tend to undergo oxidation if compared to coconut oil. Therefore, corn oil is easier initiated by oxygen attacking the double bonds between carbon atoms than coconut oil. Thus, corn oil will produce highly reactive molecules which generate unpleasant and noxious odours and flavors.

So, rancidity is more likely to occur in corn oil than in coconut oil. Conclusion Based on the first experiment, we have learned the correct method to determine the free fatty acid (FFA) in oil or fat by doing this experiment. Moreover, we can also know the way to calculate the acid value of free fatty acid content. We also have learned the right method to determine the iodine value in oil by titration with sodium thiosulphate for the second experiment. Iodine value is important in determination of degree of unsaturated fat in oils and fatty acids.

This experiment is important to control the quality of fat and oil in a food system. Therefore, it is very useful in food industry in order to reduce the chances of occurring rancidity and prolong the shelf life of food(fats). Reference http://utahpests. usu. edu/plantdiseases/htm/fruit http://web. usm. my/jps/19-1-08/Article%2019-1-7. pdf http://naturalhealthychoices. weebly. com/safe-cooking-oils. html http://journeytoforever. org/biodiesel_yield. html http://www. second-opinions. co. uk/fats_and_cancer. html