Roles of Food Lipids Lipid analysis Pham Van Hung, PhD Energy Source Carrier of Fat Soluble Vitamins Main Flavor Source of Foods Hormone and Cell Structure Nerve System Thermal Insulation of Body Mouth Feeling Texture of Foods Emulsifying Agents Mold Releasing and Anti-spattering Agent Definition Types of Lipids Any of a group of substances that in general are soluble in organic solvents, but are not soluble in water Fatty acids Neutral fats and oils Waxes Phospholipid Sterols Fat soluble vitamins lipids: Fatty Acids R Saturated Fatty Acids O C OH #1 Carbon O R C OH Acid Group Polar End - Hydrophilic End CH3 CH O CH2 CH2 CH2 CH2 CH2 C OH Octanoic Acid Non-polar End - Hydrophobic End (Fat-soluble tail) Cis and Trans Fatty Acids Unsaturated Fatty Acids CH CH CH CH CH O CH CH C OH - Octenoic Acid O CH2 CH2 CH2 CH2 CH2 C OH CH3 CH2 3, - Octadienoic Acid Short hand: H H CH3 (CH2 )7 C C (CH2 )7 10 O C OH Cis - Octadecenoic Acid (oleic) H CH3 (CH2 )7 O C C (CH2 )7 C OH H 8:1 (Δ3) Trans - Octadecenoic Acid (elaidic acid) 8:2 (Δ3,6) Polyunsaturated Fatty Acids Fatty Acids Linoleic acid: Cis, cis, 9, 12 - Octadecadienoic acid Linolenic acid: Cis, cis, cis 9, 12, 15 - Octadecatrienoic acid Arachidonic acid: Cis, cis, cis, cis 5, 8, 11, 14 - Eicosatetraenoic acid Linoleic Acid Linolenic Acid Arachidonic Acid Saturated Fatty Acids Naturally-occurring Fatty Acids Common Name O C Systematic Name Formula Common source n Cis form Not conjugated - isolated double bond Even numbered fatty acids OH Butanoic CH3(CH2)2COOH butterfat Hexanoic CH3(CH2)4COOH butterfat, coconut, palm nut oils Caprylic Octanoic CH3(CH2)6COOH Coconut, palm, nut oils, butterfat Decanoic CH3(CH2)8COOH Coconut, palm, nut oils, butterfat Dodecanoic CH3(CH2)10COOH Myristic Tetradecanoic CH3(CH2)12COOH Coconut, palm, nut oil, animal fats Palmitic CH ] CH2 Caproic Lauric CH [ CH CH CH CH Butyric Capric R Hexadecanoic CH3(CH2)14COOH practically all animal, plant fats Stearic Octadecanoic CH3(CH2)16COOH animal fat, plant fats Arachidic Eicosanoic CH3(CH2)18COOH peanut oil Coconut, palm, nut oils, butterfat Unsaturated Fatty Acids Common Name Systematic Name Formula Common source Important Fatty Acids for Nutrition A Monoethenoic Acids Oleic Cis 9-octadecenoic C17H33COOH plant and animal fats Elaidic Trans 9-Octadecenoic C17H33COOH animal fats C17H31COOH peanut, linseed, and cottonseed oils C17H29COOH linseed and other seed oils peanut seed fats • EPA (Eicosapentaenoic acid) 20:5(n-3) Omega-3, all-cis –eicosa-5,8,11,14,17 pentaenoic acid B Diethenoic Acids Linoleic 9,12-Octadecadienoic C Triethenoid Acids Linolenic 9,12,15-Octadecatrienoic Eleostearic 9,11,13-Octadecatrienoic C17H29COOH D Tetraethenoid Acids Moroctic Arachidonic 4,8,12,15Octadecatetraenoic C17H27COOH fish oils C19H31COOH 5,8,11,14Eicosatetraenoic • DHA (Docosahexaenoic acid) 22:6 (n-3) omega-3, all-cis -docosa4,7,10,13,16,19-hexaenoic acid traces in animal fats Fatty Acids Melting Points and Solubility in Water Characteristics of Fatty Acids Fatty Acids x Melting Point x Melting Point x x M.P.(C) mg/100 ml in H2O* C4 -8 - C6 -4 970 75 C8 x x z Solubility in H2O Fatty Acid Chain Length 31 C12 44 0.55 C14 54 0.18 C16 63 0.08 C18 x 16 C10 x 70 0.04 * Solubility Effects of Double Bonds on the Melting Points F A M P (C) 16:0 16:1 18:0 18:1 18:2 18:3 20:0 20:4 60 63 16 -5 -11 75 -50 FAT AND OILS M.P x x x x # Double bonds Glycerides Triacylglycerol (Triglycerides ) H2 C OH HC HC OH H2 C HO OH OH + HO HO Glycerol C O C O C O C HC (CH2 )16 CH3 H2 C O O H2 C OH O H2 C H2 C H2 C R C O O C R O H2 C R O HC R R O C Monoacylglycerol (Monoglyceride) + H2O Fatty Acids H2C O C OH O O C (CH2 )16 CH3 Diacylglycerol (Diglyceride) O H C O C (CH2 )16 CH3 O HC O C (CH2 )14 CH3 O R O O C (CH2 )16 CH3 (CH2 )16 CH3 ( C18 ) (C16 ) (C18 ) Triacylglycerol (Triglyceride) (β - palmityl distearin) Fatty Acids (%) of Fats and Oils Fats and Oils 10 12 14 16 16:1 18:0 18:1 18:2 18:3 World Supply • Vegetable oil - 68% • Animal fat - 28% • Marine oil - Fatty Acids 4% Butter 3 3 10 26 15 29 2 Melting Points of Triglycerides Triglyceride Melting Point (°C) Coconut Cottonseed 6 44 18 11 Soybean 18 53 12 24 54 Waxes Fatty acid + Long chain alcohol C6 -15 Important in fruits: C12 15 Natural protective layer in fruits, vegetables, etc C14 33 Added in some cases for appearance and protection C16 45 Beeswax (myricyl palmitate), Spermaceti (cetyl palmitate) C18 55 C18:1 (cis) -32 C18:1 (trans) O C30 H61 O C C 15 H31 O C16 H 33 O C C15 H 31 15 Phospholipid Sterols Lecithin (phosphatidyl choline) Male & female sex hormones Bile acids O O R C H2 C O C Vitamin D R Adrenal corticosteroids O CH H2 C O P Cholesterol CH O O CH2 CH 21 H 318 C H3 C + N CH3 CH Choline O_ Phosphatidic Acid 19 H3C HO Cholesterol 10 12 11 13 22 CH 20 17 14 16 CH 15 Fat Soluble Vitamins Vitamin A H3 C CH3 CH3 CH3 CH2 OH CH3 CH H 3C H 3C CH Vitamin D2 H H CH CH 3 HO Analytical Methods for The Determination of Characteristics of Fats and Oils Vitamin E R1 R2 CH3 O CH (CH2 CH2 CH CH )2 CH2 CH2CH CH(CH )2 HO R3 Acid Value Analytical Methods Number of mgs of KOH required to neutralize the Free Fatty Acids in g of fat Acid Value Saponification Value Iodine Value Gas Chromatographic Analysis for Fatty Acids Liquid Chromatography Cholesterol Determination RCOOH + KOH AV = What is the maximum acid value of the good soybean oil? - ml of KOH x N x 56 Weight of Sample Acid Value The free fatty acid content in a good soybean oil should be less than or equal to 0.05 % The average molecular weight of free fatty acids of the oil is 280 which is the molecular weight of linoleic aid ROO K + H2O + = mg of KOH Acid Value 0.05 % in 1gram is 0.5 mg fatty acid in gram of oil 56 mg of KOH reacts with 280 mg of RCOOH 280 mg of RCOOH / 56 mg of KOH = 5:1 The 0.1 mg KOH reacts with 0.5 mg RCOOH What is the content (%) of free fatty acids of a soybean oil if the acid value is 0.3? The average molecular weight of free fatty acids of the oil is 280 • What is the content (%) of free fatty acids of soybean oil if the acid value is 0.3? • Acid value 0.3 means that 0.3 mg KOH is required to react with the free fatty acid in1g (1000mg) of oil • The 56mg KOH reacts with 280 mg free fatty acid, 56 : 280 =1 : • 1mg KOH reacts with mg free fatty acid • The 0.3 mg KOH reacts with 1.5 mg of free fatty acid in gram oil • 1.5 mg free fatty acid /1000 mg oil x100(%) =0.15 % Acid Value is 0.1 Saponification Value Saponification - Hydrolysis of ester (triglycerider) under alkaline condition O H2 C O HC O C R O C R O H2 C O C R H C OH + KOH Heat HC OH + 3R O C O-K+ H C OH Definition : mgs of KOH required to saponify g of fat Saponification Value Saponification Value Definition : mgs of KOH required to saponify g of fat CH2 A Tricaprylin (MW= 450) CH CH2 CH2 B Tristearin (MW= 890) 1Gram of Oils A and B CH CH2 O O C O O C O O C O O C O O C O O C (CH2)6 CH3 (CH2)6 CH3 (CH2)6 CH3 (CH2)16 CH3 (CH2)16 CH3 (CH2)16 CH3 B A :Large molecular triacylglycerols : Small molecular triacylglycerols Sample A has large molecular weight triglyceride (e.g MW.890) • The molecular weights of tributyrin and tristearin are 300 and 900, respectively If the numbers of tributyrin in gram is 21 x 1020, what are the approximate numbers of tristearin in gram? Sample B has small molecular weight triglyceride(e.g MW.450) In one gram of sample, number of triglyceride in B is about two times more than number of triglyceride in A Less mg of KOH is needed to saponify sample A than sample B • If the saponification value is tristearin is 190, what is the approximate saponification value of tributyrin? Therefore, saponification value of A is about half of that of sample B Avogadro’s Number (N) = 6.02 x 1023 / mol Saponification Value Determination Saponification # mgs of KOH required to saponify g of fat g in 250 ml Erlenmeyer 50 ml KOH in Erlenmeyer Boil for saponification Titrate with HCl using phenolphthalein A 5.00 grams of exotic tropical oil was saponified with excess KOH The unreacted KOH was then titrated with 1.00 N HCl The blank required 40 mL of HCl and the sample required 20 mL Conduct blank determination Saponification number =[ (B – S) x N of HCl x 56] /gram of sample Please calculate the saponification value of the oil B - ml of HCl required to titrate KOH in Blank S - ml of HCl required to titrate excess KOH by Sample Saponification Values of Fats and Oils Fat Iodine Value Saponification # Milk Fat 210-233 Coconut Oil 250-264 Cotton Seed Oil Number of iodine (g) absorbed by 100 g of oil 189-198 Soybean Oil 189-195 Lard Molecular weight and iodine number can calculate the number of double bonds g of fat adsorbed 1.5 g of iodine value = 150 190-202 Iodine Value CH2 Triolein (MW= 884) CH A CH2 CH2 Trilinolein (MW= 878) CH B CH2 O O C (CH2)7 CH CH O O C (CH2)7 CH CH O O C (CH2)7 CH CH CH2 (CH2)7 CH3 (CH2)7 CH3 (CH2)7 A CH2 CH3 O O C (CH2)7 CH CH CH2 CH CH (CH2)4 CH3 O O C (CH2)7 CH CH CH2 CH CH (CH2)4 CH3 O O C (CH2)7 CH CH CH2 CH CH (CH2)4 CH3 CH CH2 B CH CH2 O O C (CH2)7 CH CH (CH2)7 CH3 O O C (CH2)7 CH CH CH2 CH CH O O C (CH2)14 CH3 O C ( C H 2) C H C H O O C (C H 2) 14 C H O O C ( C H 2) 14 C H O (CH2)4 CH ( C H 2) CH3 Iodine Value Determination CH CH CH CH Cl I + ICl Iodine chloride • The chemical equation of iodine value determination shows that one double bond requires one I2 molecule which is 254 in molecular weight 100 comes from the 100 gram of sample for the definition of iodine number Excess unreacted ICl ICl I2 + + Iodine Value = KI Na S O KCl + I2 Na S O + NaI (ml of Na2S2O3 volume for blank - ml of Na2S2O3 volume for sample) × N of Na2S2O3 × 0.127g/meq × 100 • Iodine value defines as the number of iodine in grams absorbed by 100 gram of sample Weight of Sample (g) Iodine Values of Triglycerides • Fatty acids A and B have only one double bond per molecule The molecular weights of A and B are 150 and 300, respectively The hypothetical iodine value of Compound A is 150 What is the Iodine value of compound B? • Triglycerides A and B have the very similar molecular weights of about 878 The compound A has double bonds per molecule and has iodine value is 174 The compound B has double bond per molecule What is the iodine value of the compound B? Fatty Acids # of Double-bonds Iodine # Palmitoleic Acid 95 Oleic Acid 86 Linoleic Acid 173 Linolenic Acid 261 Arachidonic Acid 320 Double Bond Determination Number of Double bonds Compound A and B have the same iodine values of 100 Compound A of molecular weight of 200 has one double bond per molecule Determine the number of double bonds of Compound B of molecular weight of 400 The unknown compound has molecular weight of 878 and iodine value of 173 Determine the number of double bonds in the unknown compound Determination of Double per Molecule GC Analysis for Fatty Acids Iodine Value x Molecule Weight Number of Double = Bonds per Molecule x 127 x 100 Extract fat Saponify (hydrolysis under basic condition) Prepare methyl ester (CH3ONa) Chromatography methyl ester Determine peak areas of fatty acids Compare with response curve of standard Fatty acids are identified by retention time Fatty Acids Methyl Esters Triacylglycerol 18:1 H 2C O C O (CH2 )16 CH3(C 18) HC O C O (CH2 )14 CH3 (C16) C Response O (CH2 )16 CH3 (C18) H 2C O 14 16 β - palmityl distearin 18 18:2 18:3 20 21:1 24 22 Time GC condition: 10% DEGS Column (from Supelco) Column temperature 200C Triglycerides in Olive Oil Triglyceride Analysis by Liquid Chromatography Soybean Oil Total Carbons : Double Bonds Fatty Acids Solvent CH3CN/HF Column 84346 (Waters Associates) RESPONSE OL2 O2L OPL O3 OSL O2P 54: 54: 52: 54: 54: 52: RETENTION TIME Cholestrol Determination Absorption Standard Curve of Cholesterol Enzymatic Methods R Cholesterol Oxidase etc + H O 2 O CH3O H2 O2 + H2N OCH3 NH2 CH3O Peroxidase HN 0-Dianisidine Oxidized 0-Dianisidine (Colorless) OCH3 NH + H2 O A at 440 nm HO m g/ml Cholesterol (Brown color) at 440 nm 10 Cholesterol Analysis by GLC Prepare cholesterol butyrate Analyze by GLC Sensitivity - 10-7 g Lipid Content Analysis 21 H 318 C H3C -H2O 19 H3 C CH3CH2CH2COOH HO 10 12 11 13 22 CH 20 17 14 16 CH 15 Gravimetric Method Gravimetric Method (1) Wet Extraction - Mojonnier (1) Wet extraction - Mojonnier (2) Dry extraction - Soxhlet Method For Milk: 1) 10 g milk + 1.25 ml NH4OH mix solubilizes protein and neutralizes Volumetric Methods (1) Babcock Method 2) + 10 ml CH3CH2OH - shake Begins extraction, prevents gelation of proteins 3) + 25 ml CH3CH2OCH2CH3 - shake and mix 4) + 25 ml petroleum ether, mix and shake Soxhlet Method Dry Extraction - Soxhlet Method Sample in thimble is continuously extracted with ether using Soxhlet condenser After the extraction of fat from the sample, evaporate ether in the flask and weigh the flask The gain of the weight of flask is the fat content 11 Volumetric Method Babcock Method Theory: Treat sample with H2SO4 or detergent Centrifuge to separate fat layer Measure the fat content using specially calibrated bottles The end! Methods: Known weight sample H2SO4 - digest protein, liquefy fat Add H2O to make fat be in graduated part of bottle Centrifuge to separate fat from other materials completely 12 ... by 100 g of oil 1 89- 198 Soybean Oil 1 89- 195 Lard Molecular weight and iodine number can calculate the number of double bonds g of fat adsorbed 1.5 g of iodine value = 150 190 -202 Iodine Value... pentaenoic acid B Diethenoic Acids Linoleic 9, 12-Octadecadienoic C Triethenoid Acids Linolenic 9, 12,15-Octadecatrienoic Eleostearic 9, 11,13-Octadecatrienoic C17H29COOH D Tetraethenoid Acids Moroctic... (Brown color) at 440 nm 10 Cholesterol Analysis by GLC Prepare cholesterol butyrate Analyze by GLC Sensitivity - 10-7 g Lipid Content Analysis 21 H 318 C H3C -H2O 19 H3 C CH3CH2CH2COOH HO 10 12 11