Analysis of oils and fats

it has acid value, soaponification value, peroxide value and iodine value as per IP

1. Analysis of oils and fats Prepared by Dr. N.GOPINATHAN ASSISTANT PROFESSOR DEPARTMENT OF PHARMACEUTICAL CHEMISTRY FACULTY OF PHARMACY SRIHER [DU] CHENNAI-116 TAMILNADU

2. Saponification value Acid value Ester value Iodine value Peroxide value

3. Determination of saponification value The saponification value is the number of milligrams of KOH necessary to neutralise the free acids and to saponify the esters present in 1 g of the substance

4. Why KOH is preferred over NaOH Potassium hydroxide is also slightly smaller than sodium hydroxide; therefore, it can penetrate oil molecules faster than sodium hydroxide, thus breaking the oil’s hold on surfaces quicker. Since they are also more soluble, the oils can be rinsed away easier, especially when using hotter water or steam equipment

5. TAG + 3KOH---- 3 alkali salt + Glycerol

6. Principle- saponification value The oil sample is saponified by refluxing with a known excess of alcoholic potassium hydroxide solution. The alkali required for soaponification is determined by titration of excess KOH against the standard HCl

7. Saponification value- apparatus required Erlenmeyer flask Round bottomed flask Reflux condenser Hot water bath / hot plate / heating mantle.

8. Alcohol distillation Reflux 1.2 litre of ethanol with 10 g of KOH and 6 g of granulated aluminium or aluminium foil for 30 minutes. Distil it and collect 1 litre of ethanol after discarding first 50 ml.

9. 0.5 m ethanolic KOH 30g of KOH in one litre of distilled alcohol. K eep the temperature below 15 C while dissolving alkali. Allow to stand overnight. Decant the clear liquid and store it in a air tight container.

10. Phenolphthalein indicator Dissolve 1 g of phenolphthaein in 100 ml of rectified spirit.

11. Preparation of 0.5 M HCl Dilute 42.5 ml of concentrated HCl with water to produce 1000 ml.

12. Standardisation of o.5M HCl Weigh accurately 1.5 g of anhydrous sodium carbonate previously heated at about 270 C for one hour. Dissolve it in 100 ml of water and add 0.1 ml of methyl red solution. Add 0.5M HCl from burette with constant swirling. Until the solution becomes faintly pink.

13. Heat the solution to boiling , cool and continue the titration, Heat again to boiling and titrate further as necessary until the faint pink colour is no longer affected by continued boiling. Each ml of 0.5M HCl is equivalent to 0.02649g of sodium carbonate

14. IP procedure Weight of oil mentioned in the individual monograph otherwise Weigh 2 g of sample being examined into a 200 ml flask of borosilicate glass fitted with a reflux condenser. Add 25 ml of 0.5 M ethanolic KOH and a little pumice powder and boil under reflux on a water bath for 3 minutes.

15. IP procedure Add 1 ml of phenolphthalein solution titrate immediatley with 0.5 M HCl . Repeat the operation omitting the substance being examined

16. Calculation Saponification value = 28.05 x ( b-a )/ w W – weight of the sample in grams. B- blank A- titre value

17. Note If the oil purpose of preservation has been saturated with carbon dioxide for the purpose of preservation, gently reflux the solution of the oil in ethanol 95 % and ether for 10 minutes before titration. The oil may be freed from the CO2 by exposing it in a shallow dish in a vacuum desiccator for 24 hours before weighing the sample.

18. Significance of Saponification Value • The magnitude of saponification value gives an idea about the average molecular weight of the fat or oil. •Higher the molecular weight of the fat , the smaller is its saponification value. •Saponification Value also indicates the length of carbon chain of the acid present in that particular oil or fat. •Higher the saponification value, greater is the percentage of the short chain acids present in the glycerides of the oil or fats

19. Significance To identify the given fatty oil. To distinguish between fatty oils and mineral oils. It also help to calculate the amount of alkali required to convert a definite amount of fat or oil into soap. In detecting the adulteration of fat or oil. Detection of acids containing less than 16 carbon or more than 18 carbon.

20. Acid value

21. Introduction Free fatty acids are a source of flavors and aromas. short chain of free fatty acids tend to be water soluble and volatile with characteristic smell. long chain saturated and unsaturated fatty acids are more prone to oxidation in their free form and their breakdown products (aldehydes, ketones, alcohols, and organic acids) provide characteristic flavors and aromas Different fat samples may contain varying amount of fatty acids. In addition, the fats often become rancid during storage and this rancidity is caused by chemical and enzymatic hydrolysis of fats into free acids and glycerol

22. Acid value It is the number which expresses in milligrams the amount of KOH necessary to neutralise the free acid present in 1 g of the substance.

23. Principle RCOOH + KOH → ROO- K+ + H2O  AV is also known as neutralization  number  or acid number  or acidity The acid value is determined by directly titrating the oil/fat in an alcoholic medium against standard potassium hydroxide/sodium hydroxide solution.

24. Ip procedure Dissolve about 10 g of substance [ weight mentioned in the individual monograph ] being examined accurately weighed, in a 50 ml of mixture of equal volumes of ethanol 95 % and ether, previously neutralised with 0.1 m KOH to phenolphthalein solution. If the sample does not dissolve in the cold solvent, connect the flask with a reflux condenser and warm slowly with a frequent shaking until the sample dissolves.

25. Add 1 ml of phenolphthalein solution titrate against 0.1 M KOH until the solution remains faintly pink after shaking for 30 seconds.

26. Ethanol-ether solution Prepare a mixture of ethanol and diethyl ether (1:1, v/v). Neutralize with sodium hydroxide titrant and add 1.0 mL of phenolphthalein indicator until pink colouration is observed. Freshly prepare the solution.

27. Phenolphthalein indicator Dissolve 1 g of phenolphthaein in 100 ml of rectified spirit.

28. 0.1M KOH Dissolve about 6 g of KOH in distilled water to produce 1000 ml

29. 95% ethanol 95ml of ethanol to 100 ml with water.

30. calculation Acid value = 5.61 n/w N= the number of ml of 0.1 M KOH required. W= the weight in g of the substance.

31. Note If the oil purpose of preservation has been saturated with carbon dioxide for the purpose of preservation, gently reflux the solution of the oil in ethanol 95 % and ether for 10 minutes before titration. The oil may be freed from the CO2 by exposing it in a shallow dish in a vacuum desiccator for 24 hours before weighing the sample.

32. Significance and application of Acid value The acid value (AV) is a common parameter in the specification of fats and oils. it signifies due to attack of atmospheric oxygen, hot moist air or microorganisms. It gives how much generation of free fatty acid has taken place which leads to rancidity. It gives an indication about the age and extent of its deterioration.

33. Significance and application of Acid value Find rancid state, triglycerides are converted into fatty acids and glycerol, causing an increase in acid number. It is a measure of the free fatty acids (FFA) in a sample of oil or fat indicates hydrolysis of triglycerides. Such reaction occurs by the action of lipase enzyme it is an indicator of inadequate processing and storage conditions (i.e. high temperature, and relative humidity, tissue damage) The acid number is a measure of the amount of carboxylic acid groups in a chemical compound, such as a fatty acid, or in a mixture of compounds

34. Drawbacks of non-aqueous solvents in determination of acid value The majority of national and international standards for AV determination in fat and oils are based on the acid-base titration techniques in non-aqueous solvents. However, these techniques have a number of drawbacks : Currently used non-aqueous solvents are toxic, such as ethanol or isopropanol heated up to 60ºC or higher or diethyl ether-ethanol solvent. Incomplete solubility of test oil portion in alcohol (even under heating) caused by the formation of a dispersed system.

35. Conditions for accurate acid-base titration in hot amphoteric solvents might deteriorate due to the increase of the solvolysis constant for anion titrable weak acids with an increase in temperature. This conclusion follows from the fact that the solvent auto- protolysis constant increases, and the acid dissociation constant decreases with increased temperature. Need to previously neutralize the solvents

36. Ester value Ester value is the number of milligrams of KOH required to saponify the esters present in 1g of the substance Ester value = saponification value – Acid value

37. Iodine value

38. Iodine value The iodine value is the number which expresses in grams the quantity of halogen, calculated as iodine which is absorbed by 100 g of substance under the described condition. It may be determined by any of the following methods. It also known as   iodine adsorption value  or  iodine number  or  iodine index Saturated fatty acids will not give the halogenation reaction Oil /fat sample+ ICl or I 2 in ethanol and in presence of mercuric chloride

39. Significance The iodine value is a measure of the degree of unsaturation in an oil. It is constant for a particular oil or fat. Iodine value is a useful parameter in studying oxidative rancidity of oils since higher the unsaturation the greater the possibility of the oils to go rancid. Iodine numbers are often used to determine the amount of unsaturation in fatty acids. This unsaturation is in the form of double bonds, which react with iodine compounds.

40. Significance The higher the iodine number, the more C=C bonds are present in the fat If the iodine number is between 0-70, it will be  a fat and if the value exceeds 70  it is an oil. Starch is used as the indicator for this reaction so that the liberated iodine will react with starch to give purple coloured product and thus the endpoint can be observed.

41. Method Iodine monochloride method or Wijis method Iodine monobromide method or Hanus method Pyridine bromide method

42. Method A- iodine mono chloride method Principle The oil/fat sample taken in carbon tetrachloride is reacted with an excess of iodine monochloride solution ( Wjis solution). Unsaturated fatty acids undergo halogenation reaction resulting in the addition of an iodine atom to one carbon of the double bond. On completion of the reaction, the remaining iodine monochloride reacts with potassium iodide leading to the formation of molecular iodine. The liberated iodine is then evaluated by titration with a standard solution of sodium thiosulphate using starch as indicator. Mercuric ions are added to fasten the reaction

43. Method A- iodine mono chloride method

44. Wijis reagent Iodine monochloride is produced simply by combining the halogens in a 1:1 molar ratio, according to the equation I 2  + Cl 2  → 2 ICl When   chlorine gas is passed through iodine crystals, one observes the brown vapour of iodine mono chloride . Dark brown iodine mono chloride liquid is collected. Excess chlorine converts iodine monochloride into iodine trichloride  in a reversible reaction: ICl + Cl 2  ⇌ ICl 3

45. IP procedure Place an accurately weighed quantity of the substance (100 g)under examination in a dry 500ml iodine flask, add 10ml of carbontetrachloride and dissolve. Add 20ml of iodine monochloride solution , insert the stopper and allow to stand in the dark at a temperature between 15°C and 25°C for 30 minutes. Place 15ml of potassium iodide solution in the cup top , carefully remove the stopper , rinse the stopper and the sides of the flask with 100ml of water, shake and titrate with 0.1M sodium thiosulphate using starch solution , added towards the end of the titration , as indicator .

46. IP procedure Note the number of ml of 0.1M sodium thiosulphate (a). Repeat the operation without the substance under examination and note the number of ml required(b). (BLANK) SPECIAL SAFETY NOTE Carbon tetrachloride is a carcinogen in laboratory animals. Avoid breathing vapors or skin contact. Work in a well ventilated hood but avoid dispersing to atmosphere

47. Calculation Calculate the iodine value from the expression Iodine value = 1.269 (b-a) / w Where w= weight in grams od the substance a = the number of ml of 0.1M sodium thiosulphate b = the number of ml of 0.1M sodium thiosulphate with out substance w =weight of the substance in gram 1.269 is atomic mass of iodine

48. Iodine monobromide method or Hanus method Some of the IBr reacts with the double bonds in the unsaturated lipids, while the rest remains: R-CH=CH-R + IBr (excess) → R-CHI- CHBr -R + IBr (remaining ) The amount of IBr remaining is determined by adding excess potassium iodide to the solution to liberate iodine, and then titrating with a sodium thiosulfate (Na 2 S 2 O 3 ) solution in the presence of starch to determine the concentration of iodine released:

49. IBr (remaining) + 2KI → KBr + KI + I I 2 + starch + 2Na 2 S 2 O 3 (blue) 2NaI + starch + Na 2 S 4 O 6

50. Haus solution   Hanus solution (for iodine number). Dissolve 13.2 g of resublimed iodine in 1 L of glacial acetic acid which will pass the dichromate test for reducible matter. Add sufficient bromine to double the halogen content, determined by titration (3 mL is about the proper amount). The iodine may be dissolved by the aid of heat, but the solution should be cold when the bromine is added . Store the hanus solution in a amber bottle and away from light. The Hanus solution can be used for a year . Extremely hazardous in case of eye contact (corrosive ) and Causes severe eye burns. Extremely hazardous in case of skin contact (corrosive).

51. Procedure Unless otherwise specified, weigh accurately the quantity of substance under examination, stated in the table , place it in a dry 300ml iodine flask or which has been rinsed with glacial acetic acid unless otherwise specified in the monograph. Add 15ml of chloroform and dissolve Presumed iodine value Quantity of the substance Less than 20 1.0 20 to 60 0.25 to 0.5 61 to 100 0.15 to 0.25 More than 100 0.10 t o 0.15

52. Procedure Add slowly from a burette 25.0 ml of iodine monobromide solution , insert the stopper allow to stand the dark for 30minutes , unless otherwise specified in the monograph, shaking frequently. Add 10ml of potassium iodide solution and 100 ml of water and titrate with 0.1M sodium thiosulphate using stach solution , added towards the end od the titration, as indicator, note the number of ml required (a). Repeat the operation without the substance under examination and note the number of ml required(b). Calculate the iodine value from the expression given under method A

53. Calculation Calculate the iodine value from the expression Iodine value = 1.269 (b-a) / w Where w= weight in grams od the substance a = the number of ml of 0.1M sodium thiosulphate b = the number of ml of 0.1M sodium thiosulphate with out substance w =weight of the substance in gram 1.269 is atomic mass of iodine

54. Pyridine bromide method Place an accurately weighed quantity of the substance under examination in a dry iodine flask, add 10ml of carbon tetrachloride and dissolve. Add 25ml of pyridine bromide solution, allow to stand for 10minutes in the dark and complete the determination described under method A beginning at the words “place 15ml of….”

55. The approximate weight in gram of the substance to be taken may be calculated by dividing 12.5 by the highest expected iodine value. If more than half the available halogen is absorbed, the test must be repeated with a smaller quantity of the substance.

56. Peroxide value

57. Peroxide value It is the number of milli equivalents of active oxygen that expresses the amount of peroxide contained in 1000 g of the substance.

58. Procedure -IP Weight unless otherwise mentioned in the individual monograph . Weigh 5 g of the substance being examined, a ccurately weighed into a 250ml glass stoppered conical flask, add 30ml of a mixture of 3 volumes of glacial acetic acid and 2 volumes of chloroform swirl until dissolved and add 0.5 ml of saturated potassium iodide solution.

59. Allow to stand for exactly one minute with occassional shaking add 30 ml of water and titrate gradually with continious and vigorous shaking, with 0.01M sodium thio sulphate until yellow colour almost disappears. Add 0.5 ml of starch solution and continue the titration shaking vigorously until the blue colour just disappear [ a ml]

60. Repeat the operation omitting the substance being examined [ b ml]. The volume of 0.01M sodium thio sulphate in the blank determination must not exceed 0.1 ml

61. Calculation Peroxide value = 10 ( a-b) /w Where w = weight in g of the substance

62. Autoxidation The process of oxidation induced by air at room temperature referred to as “autoxidation”.

63. Rancidity Rancidity is a term generally used to denote unpleasant odours and flavours in foods resulting from deterioration in the fat or oil portion of a food. There are two basic types or causes of rancidity that cause and/or contribute to the degradation of stored edible oils: oxidative and hydrolytic Oxidative rancidity, known as autoxidation, occurs when oxygen is absorbed from the environment

64. Hydrolytic rancidity, also called hydrolysis or enzymatic oxidation, occurs in the absence of air, but with moisture present. Enzymes found naturally in plant oils (i.e., lipoxygenase, cyclooxygenase) and animal fats (i.e., lipase) can catalyze reactions between water and oil Microbial rancidity, in which micro-organisms such as bacteria, molds and yeast use their enzymes to break down chemical structures in the oil, producing unwanted odors and flavors. Water needs to be present for microbial growth to occur.