Oils and Fats Major Component (%95-99) Minor Components (%1-5) Triglycerides Minor Components (%1-5) Triglyceride Derivatives Glycerol Free Fatty Acids Mono- and Diglycerides Non-Triglyceride Derivatives Phospholipids Sterols Pigments Vitamins Antioxidants Oxidation Products Trace Metals Hydrocarbons

Triglyceride Structure O C - R1 triacylglycerol = - R2 - R3 HC H2C OH glycerol HC H2C HO - C O - R1 3 fatty acids + - R3 - R2 +3H20 O R One chiral carbon with 1-3 acyl groups simpler stereochemistry than sugars - C more possible substituents acyl

Triglycerides differ from each other in regard to Number of Carbon Atoms in fatty acid chains Number of double bonds Isomerization Distribution of FA on glycerol backbone

Analytical Methods Saponification Value Iodine Value Gas Chromatographic Analysis for Fatty Acids Liquid Chromatography

Saponification Value Saponification - hydrolysis of ester under alkaline condition. The saponification value of an oil or fat is defined as the number of mg of potassium hydroxide (KOH) required to neutralize the fatty acids resulting from the complete hydrolysis of 1 g of the sample.

Saponification Value Determination Saponification # --mgs of KOH required to saponify 1 g of fat. 1. 5 g in 250 ml Erlenmeyer. 2. 50 ml KOH (0.5 N) in Erlenmeyer. 3. Boil for saponification. 4. Titrate with HCl (0.5 N) using phenolphthalein. Conduct blank determination. B - ml of HCl required by Blank. S - ml of HCl required by Sample. N- Factor of 0.5 N HCL.

Saponification Value 3 + 3 K+OH - O - R C H2C H2C OH O HC + HC KO - triacylglycerol HC H2C OH glycerol HC H2C C O - R KO - + 3 K+OH - 3 + Potassium salt Similarly; RCOOH + KOH RCOO-K+ + Glycerol MG + KOH RCOOK + Glycerol DG + 2KOH 2RCOOK + Glycerol

Saponification Value 1 mol TG 3 mol KOH required 1 g TG X mol KOH required MWKOH: 56 g = 56000 mg 1 g TG : 1 g / MWTG (g/mol) mol 1 mol TG 3x 56000 mg KOH required 1 g TG / MWTG X mg KOH required

Saponification Value What is the MWTG ? O O O - R1 - R1 C - R C H2C C

Saponification Value Which one’s MW should be taken? O O O - R1 - R1 C HC H2C O C - R1 - R2 HC H2C O C - R HC H2C

Saponification Value The Answer is the Weighted Average MW O O O - R1 HC H2C O C - R1 - R2 HC H2C O C - R HC H2C

Saponification Value Calculation of AMWTG Oil consists of only type Simple Triglyceride O C - R HC H2C

Average Moleculer Weight of FAs in Oil (AMWFA) Saponification Value Calculation of AMWTG Oil consists of Simple and Mixed type Triglyceride with two fatty acids R1 (%90 w/w) and R2 (%10 w/w) O C - R1 HC H2C O C - R1 - R2 HC H2C O C - R2 HC H2C O C - R1 - R2 HC H2C Average Moleculer Weight of FAs in Oil (AMWFA)

Saponification Value Generalized Calculation of AMWTG Oil consists of Simple and Mixed type Triglyceride with N fatty acids FA xi R1 x1 R2 x2 . . RN xN

Saponification Value Milk Fat 210-233 Coconut Oil 250-264 Cotton Seed Oil 189-198 Soybean Oil 189-195   Fat SV Lard 190-202

GC Analysis for Fatty Acids 1. Extract fat. 2. Saponify (hydrolysis under basic condition). 3. Prepare methyl ester (CH3ONa). 4. Chromatography methyl ester. 5. Determine peak areas of fatty acids. Fatty acids are identified by retention time. 6. Compare with response curve of standard.

Fatty Acids Methyl Esters: GC condition: 10% DEGS Column (from supelco) Column temperature 200C.

TRIGLYCERIDE ANALYSIS BY LIQUID CHROMATOGRAPHY Soybean Oil Solvent CH3CN/HF Column ODS- Octadesilsilan (C:18)

Iodine Number The iodine value of an oil or fat is defined as the mass of iodine absorbed by 100 g of the sample. The unsaturated fatty acid residues of the glycerides react with iodine, and thus the iodine value indicates the degree of unsaturation of the fatty acid residues of the glycerides. It is constant for a particular oil or fat, but depends on the method used. Animal fats (butter, dripping, lard) 30 - 70 Iodine Value Non-drying oils (olive, almond) 80 - 110 Iodine Value Semi-drying oils (cottonseed, sesame, soya) 80 - 140 Iodine Value Drying oils (linseed, sunflower) 120 - 200 Iodine Value The iodine value is often most useful in identifying the source of an oil. Generally, the higher iodine values indicate oils and the lower values fats. Iodine values are normally determined using Wigs or Hanus methods.

Determination of Iodine Number Iodine Value = (ml of Na2S2O3 volume for blank - ml of Na2S2O3 volume for sample)  N of Na2S2O3  0.127g/meq  100 Weight of Sample (g) Excess unreacted ICl

Theoretical Iodine Value Monoene + I2 Saturated Diene + 2*I2 Saturated Triene +3* I2 Saturated Sample: 100 g basis Assumption: Oil =TG FA xi C16:0 5 C18:0 15 C18:1 15 C18:2 40 C18:3 1 C20:0 3

Theoretical Iodine Value 1 mol C18:1 1 mol I2 (254 g) 15 g C18:1 X (g) I2 1 mol C18:2 2 mol I2 (2x254 g) 40 g C18:2 X (g) I2 Assumption: Oil =TG FA xi C16:0 5 C18:0 15 C18:1 15 C18:2 40 C18:3 1 C20:0 3

Theoretical Iodine Value 1 mol C18:3 3 mol I2 (3x254 g) 1 g C18:3 X (g) I2 Theoritical IV= IV C18:1 + IV C18:2+ IV C18:2 Real IV= 0.95xTheoricital Value