Genel Lipid Analysis ( Ch 13-14) LIPID ANALYSIS(Ch.13-14) I. Quantification of lipid components a.Extractions ( wet vs dry ;solid-liq vs liq-liq.) b.Indirect methods (NMR, X rays, Conductance) II. Assays for further investigations on extracted oil a.Purity criteria: give indications of wholesomeness or adulteration (physical:n D 20 ,density; chemical: IV, SV, FAME, sterols, tocopherols compositions ) b.Quality criteria: indicates good quality or deteriorated quality b.1.MIU (Moisture, Impurities, Unsaponifiables) b.2.Rancidity : Hydrtolytic:AV and ffa Oxidative:PV,TBA,TOTOX etc b.3. Storage stability (shelf-life)Lipids: Fats and fat like compounds(not polymers) that are soluble in non-polar organic solvents(petroleum ether), but insoluble in H 2 O. Lipids have: 1. Culinary importance: Great effects in preparation of foods simnce they provide heat transfer media, carry flavours, and contribute to palatability(eating quality) of foods. 2. Nutritional importance: Provide bulk of calories in diet (1 g: 9 kcal). For healthy diets, %30 percent of total calories have to come from lipids. 3. Physiological importance: They are vital for biological functioning of cells, since they contain essential fatty acids and oil-soluble vitamins.“LIPID” 1. Fatty acids 2. Neutral fats and oils 3. Waxes 4. Phospholipid 5. Sterols 6. Fat soluble vitaminsLipids in foods occur in three different forms: 1- Simple lipids-Fats,oils are triglycerides-They are the ester of fatty acids with glycerol(all edible fats and oils); waxes are esters of fatty acids with longer chain alcohols. 2- Composite lipids: Contain components other than lipids.Ex:Phospholipids,Lipoproteins,Glycolipids: These are found in bound form with other constituents; their analyses is harder than simple lipids, because you have to hydrolyse the molecule before analysis. 3- Derived lipids: These are substances derived from lipids and are soluble in lipid-extracting solvents( i.e. fat soluble pigments, sterols, antioxidants like tocopherols and Vitamins A,D, E,K (fat-soluble vitamins).They are not true lipids but are found in lipid phase. They behave like lipids. In most analyses you will get them in the lipid fraction.Fatty Acids O R C OH #1 Carbon Acid Group O R C OH Non-polar End - Hydrophobic End (Fat-soluble tail) Polar End - Hydrophilic EndSaturated Fatty Acids CH 3 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 C OH O 1 2 4 5 6 7 83 Octanoic Acid Unsaturated Fatty Acids CH 3 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 C OH O 1 2 4 5 6 7 83 CH 3 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 C OH O 1 2 4 5 6 7 83 3 - Octenoic Acid 3, 6 - Octadienoic Acid Short hand: 8:1 (? 3) 8:2 (? 3,6) Cis 9 - Octadecenoic Acid (oleic) Trans 9 - Octadecenoic Acid (elaidic acid) O CH 3 (CH 2 ) 7 C C (CH 2 ) 7 C OH H H 9 10 O CH 3 (CH 2 ) 7 C C (CH 2 ) 7 C OH H H Cis And Trans Fatty AcidsPolyunsaturated 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 AcidNaturally-occurring fatty acids R CH 2 CH CH CH 2 CH CH CH 2 C OH O 765 43 1. Cis form 2. Not conjugated --- isolated double bond. 3. Even numbered fatty acids.FAT AND OILS Mostly Triglycerides: O H 2 C OH HC OH H 2 C OH HO C R HO C R HO C R O O O H 2 C O C R O O H 2 C O C R HC O C R + + 3 H 2 O Glycerol 3 Fatty Acids GLYCERIDES Monoglyceride (? - monostearin) Diglyceride (?, ?' - distearin) H 2 C OH HC OH H 2 C O O C (CH 2 ) 16 CH 3 H 2 C O HC OH H 2 C O O C (CH 2 ) 16 CH 3 C (CH 2 ) 16 CH 3 O Triglyceride (ß - palmityl distearin) H 2 C O HC O H 2 C O O C (CH 2 ) 16 CH 3 C (CH 2 ) 16 CH 3 O O C (CH 2 ) 14 CH 3 (C 18 ) (C 16 ) (C 18 )? - oleodipalmitin 1 - oleodipalmitin Oleic Palmitic Palmitic OPP ? - Linoleyldiolein 1 - Linoleyldiolein Oleic Linoleic Oleic LOOFATS AND OILS ARE PRIMARILY TRIGLYCERIDES (97-99%) Vegetable oil - 68% of world supply Cocoa butter - solid fat Oil seeds - liquid oil Animal fat - 28% (from Hogs and Cattle) Marine oil - 4% Whale oil cod liver oilFatty Acid Compositions (%) of Fats and Oils 43 63 826 10 3 6 12 3 44 14 10 18 1 16 26 11 4 12 16:1 7 1 18:0 15 6 3 2 18:1 29 7 18 24 1 8 : 2225 35 4 Fatty Acids Butter Coconut Cottonseed Soybean 18:3 2 8WAXES Fatty acids + Long chain alcohol Important in fruits: 1. Natural protective layer in fruits, vegetables, etc. 2. Added in some cases for appearance and protection. Beeswax (myricyl palmitate), Spermaceti (cetyl palmitate) O C 30 H 61 O C C 15 H 31 O C 16 H 33 O C C 15 H 31PHOSPHOLIPID Lecithin (phosphatidyl choline) O O H 2 C O C R O R C O CH H 2 C O P O CH 2 CH 2 N + CH 3 CH 3 CH 3 O_ Phosphatidic Acid CholineSTEROLS Male & female sex hormones Bile acids Vitamin D Adrenal corticosteroids Cholesterol Phytosterols(i.e. ?-sitosterol) HO H 3 C H 3 C H 3 CC H 3 CH 3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 21 22 19 20FAT SOLUBLE VITAMINS Vitamin A: CH 2 OH CH 3 CH 3 CH 3 CH 3 H 3 C 1 2 3 4 5 6 7 8 9Vitamin D2: Vitamin E: HO CH 2 H H H 3 C H 3 CC H 3 CH 3 CH 3 O R 1 R 2 HO R 3 CH 3 (CH 2 CH 2 CH 2 CH 2 ) 2 CH 2 CH 2 CH 2 CH(CH 3 ) 2 CH 3Fat contents of selected foods: Liquid vegetable oils ? 100% Margarine and butter ? 80% › they are emulsions of lipid in water Almonds :%55, Walnuts:%65, Cereals: 3-5% Sunflower seeds, hulled : ~60% Chocolate: ~35% , Milk : 37%, Milk powder : 27.5% Tuna fish : 4% (but there are some fatty fish like anchovies containing 15% fat)Ia. General procedure for solvent extractions: 1. Sample Preparation: Plant or animal tissues should be homogenized by grinding,blending etc, since particle size is important; it has to be ideal particle size for maximum extraction yields(i.e.0.4mm?). then it has to be dried before extraction, because if any moisture is let stay in the samples, it interferes with extraction, and the solvent's extraction capability decreases.Also hydrolysis is needed to break down any bound lipids, either by acids(HCl or H 2 SO4) or by enzymes like Takadiastase. 2. Solvent selection: You need different types of solvents for extracting different types of lipids: (Chloroform+methanol) is ideal for lipoproteins, whereas petroleum ether fractions(C6 and C5) are ideal for true lipids (triyglycerides), and ethyl ether for extracting oxidized lipids. Sometimes a ternary mixture works better (CHCl3: methanol:H2O) for extraction from animal tissues; water-saturated butanol for cereal lipids.LIPID CONTENT ANALYSES 1. Gravimetric Methods (1) Wet extraction - Roese Gottliegb & Mojonnier. (2) Dry extraction - Soxhlet Method. 2. Volumetric Methods (Babcock, Gerber Methods)1. Gravimetric Methods (1) Wet Extraction - Roese Gottlieb & Mojonnier. For Milk: 1) 10 g milk + 1.25 ml NH 4 OH mix. solubilizes protein and neutralizes. 2) + 10 ml EtOH - shake. Begins extraction, prevents gelation of proteins. 3) + 25 ml Et2O - shake and mix. 4) + 25 ml petroleum ether, mix and shake.(2) Dry Extraction - Soxhlet Method. Sample in thimble is continuously extracted with ether using Soxhlet condenser. After extraction, direct measurement of fat - evaporate ether and weigh the flask. Indirect measurement - dry thimble and weigh thimble and sample. 2. Volumetric Methods (Babcock, Gerber Methods) Theory: 1. Treat sample with H 2 SO 4 or detergent. 2. Centrifuge to separate fat layer. 3. Measure the fat content using specially calibrated bottles. Methods: 1. Known weight sample. 2. H 2 SO 4 - digest protein, liquefy fat. 3. Add H 2 O so that fat will be in graduated part of bottle. 4. centrifuge to separate fat from other materials completely. Types of Extraction: a1.. "Dry Extractions": These are basically solid- liquid type of extractions,and the final results are gravimetric. They may be either batch, multiple-batch, continuous or countercurrent extractions. There are also automated processes,like FOSSLET extractors, where the predetermined number of stages is (4-5), and cartridges are used to contain the sample. In complex matrices, digestion may be required to liberate bound lipids by hydrolysis before starting the extraction.Solvent Extraction Methods: a.1.1.Continuous(Goldfish)or semicontinuous(Soxhlet) or discontinuous(Majonnier) solvent extraction methods: Solvent from a boiling flask continuously flows over the sample held in a thimble.Fat content is measured by weight of fat removed. a.1.2.Elevated pressure/Temperature methods: SFE(Supercritical fluid extractions):ince it avoids the use of hazardous organic solvents, and uses inert, inexpensive, and nontoxic gases like CO 2 , it is gaining more popularity.a.2. “Wet nonsolvent extractions" : are preferred for liquid samples, since they involve liq-liq extraction (partitioning of material between two immiscible liquids) and yield volumetric results. Ex: Gerber and Babcock type of extraction of milk are examples of wet extraction. In milk, the fat globules are present in the form of an emulsion of “oil in water”, surrounded by a thin film of protein which has to be removed or broken by acid treatment before separating fat from water phase. Gerber Procedure: 11 ml milk and 1 ml isoamyl alcohol are mixed to improve phase separation, by increasing the water phase.Then H 2 SO4 (Sp. gr.:1.82 for breaking down the lipid-protein barrier) is added and mixture is centrifuged for 5 min, submerged in H 2 O bath (65°C, 5 min). Volumes of two respective phases gives fat-water ratio, or the upper layer gives fat content. I.b. Indirect Methods for Quantification of Lipids Especially geneticists in plant breeding research studies require “non-destructive” types of lipid analyses. For this purpose, many “instrumental systems” are developed: *NMR:Low resoolution nuclear magnetic resonance:The measured NMR value is related to the total number of protons in oil fraction of seed , independent of protons in non-oil fraction, since protons have a different magnetic resonance in oil and in water phase. It gives very good approximation of lipid levels. Calibration tables or curves are used for different H 2 0 levels(fig. 13-8). *X-ray absorbtion: Is an approved method for meat carcass samples. Lean (ya ğsız) meat absorbs more x-ray than fatty tissues (carcass-lipids)Conductance: Lean meat is 20-fold better conductor for electrical current than fat. Densitometric Methods: Density of seeds (i.e. Flaxseed) is proportional to its oil content. Likewise, density of extracting solvent changes proportional to the fat content it has extracted(i.e.perchloroethylene in FOSSLET). For meat , there is a method using a cylindrical container with a piston plate that holds meat under constant hydraulic pressure. Measured impression depth is related to fat content. Refractometry: ? D of the mixture (bromonaphtalene and fat extracted by it from chocolate) is used for approximation of fat in chocolate.II. Assays on Extracted Lipids -FAT CHARACTERISATION(Ch.14): II.a. Purity Criteria: For authenticity testing and determination of adulteration. Most analytical values of certain oils and fats are quite characteristic: a.1. Physical purity criteria are refractive index, melting point, colour, density,. By measuring these criteria, we will find values within certain accepted limits and ranges. But these alone are not enough for authenticity characterisation. Color:Either Lovibond color scale using standard red-yellow-blue filters, or determination of “photometric color index- PCI)* is required for objective description of color. Refractive Index:RI may also be used for controlling hydrogenation, since RI will decrease linearly as unsaturation decreases. Melting point, smoke, flash and fire points ) ( 4 . 56 ) ( 2 . 41 ) ( 7 . 69 ) ( 29 . 1 * 670 620 550 460 A A A A PCI - + + =Effects of Double Bonds on the Melting Points 16:0 60 16:1 1 18:0 63 18:1 16 18:2 -5 18:3 -11 20:0 75 F. A. M. P. ( 0 C) 20:4 -50 M.P. # Double bondsa.2. Chemical purity criteria: These are very characteristic for each oil and fat types: • Saponification value measuring average f.a. molecular weight and chain length •Iodine value measuring level of unsaturation • Fatty acid composition(FAME), • Individual sterol and tocopherol compositions. With an integrated evaluation of results of purity criteria analyses, we can be sure of identity and source of fats and oils (Reference methods in AOCS,IUPAC and DGF).1. Saponification Value(14.3.9) Saponification - hydrolysis of ester linkages under alkaline (KOH)condition. O C R O O C R C R O H 2 C O HC O H 2 C O KOH H H H H 2 C O HC O H 2 C O R C OK + 3 + 3Milk Fat 210-233 Coconut Oil 250-264 Cotton Seed Oil 189-198 Soybean Oil 189-195 Fat Saponification Value Lard 190-202 Saponification Value of Fats and OilsSaponification Value=-mgs of KOH required to saponify 1 g of fat. 1. 5 g in 250 ml Erlenmeyer. 2. 50 ml KOH in Erlenmeyer. 3. Boil for saponification. 4. Titrate with HCl using phenolphthalein. 5. Conduct blank determination. B - ml of HCl required by Blank. S - ml of HCl required by Sample. SP# = 56.1(B -S) x N of HCl Gram of Sample Saponification Value DeterminationSV is an index of the mean molecular weight of the triacylglycerols in sample oil or fat. The mean molecular weight of triacylglycerols when divided by three will give an approximate mean molecular weight of fatty acids present. 2. Iodine Number (14.3.8) Iodine value (IV): Halogens add to double bonds. IV is the number of grams of iodine that adds to 100 gram of oil. If 1 g of the fat adsorbs 1.5 g of iodine, then IV = 150. It is a measure of saturation-unsaturation.Iodine Value = (ml of Na 2 S 2 O 3 volume for blank - ml of Na 2 S 2 O 3 volume for sample) × N of Na 2 S 2 O 3 × 0.127g/meq × 100 Weight of Sample (g) CH CH CH CH Cl I ICl Iodine chloride + ICl KI KCl I 2 I 2 Na 2 S 2 O 3 Na 2 S 4 O 6 NaI + + 2 2 + + Excess unreacted ICl Iodine Value DeterminationIodine Numbers of Selected F.A. Palmitoleic Acid 1 95 Oleic Acid 1 86 Linoleic Acid 2 173 Linolenic Acid 3 261 Fatty Acids # of Double-bonds Iodine # Arachidonic Acid 4 320 If molecular weight and iodine number of f.a. is known, we can calculate the number of double bonds:IV/MWDuring oxidation, there will be a decline in unsatuartion and therefore in IV: The ranges of Iodine Value for olive oil, which is an index of its degree of unsaturation, is 75-94 in the Codex. For Italy this value fluctuates between 79 and 88, but for Tunisia the range is 80-97. There may be such regional or climatic variations, therefore, even though results are quite meaningful, sometimes additional tests are required for final confirmation. GC Analysis for Fatty Acids (14.6.1) 1. Extract fat. 2. Saponify (hydrolysis under basic condition). 3. Prepare “fatty acid methyl esters”or FAME (by Sodium methoxide - CH3ONa or BF3-methanol reagent). 4. Chromatography of methyl esters. 5. Determine peak areas of fatty acids. (Fatty acids are identified by their specific retention times). 6. Compare with response curve of standard. 14 18:1 18:2 20 18:3 22 21:1 24 16 18 Time ResponseFor analysing FAME: The FAME are carried in and out of the GC Column by the carrier gas and each will leave the column only after a specific retention time,Rt, which is dependant on the chain length and degree of saturation of the fatty acid. Generally, as carbon number as well as double bonds in f.atty acids increase, the retention time Rt will also increase. FAME: The fatty acid composition determined by GC (gas chromotography) is a real "Fingerprint" . It also serves for “nutritional labelling”: saturateds,monounsaturates, polyunsaturates, trans f.a. etc.For authenticity testing of butters, this analysis by itself may not sometimes be sufficient, since some margarines might have similar composition of FAME with butters. In this case,the respective sterol composition should also be determined. Sterol analysis is done by TLC followed by GC of the unsaponifiable fraction of lipids. (There are “Phytosterols” - stigmasterol, campesterol, ß-sitosterol- in the vegetable sterol fraction, but only “cholesterol” in animal fats).CHOLESTEROL DETERMINATION(14.6.4) Cholesterol by GLC:saponify extracted lipid,derivatize GCµ g/ml Cholesterol Absorption at 440 nm Spectromertic Absorption Standard Curve of Cholesterol Enzymatic Determination: Cholesterol Oxidase HO O H 2 O 2 Cholesterol Oxidase etc. + H 2 O 2 CH 3 O OCH 3 H 2 NN H 2 HN NH OCH 3 CH 3 O H 2 O Peroxidase + + 0-Dianisidine Oxidized 0-Dianisidine (Colorless) (Brown color)At 440 nm TRIGLYCERIDE ANALYSIS BY LIQUID CHROMATOGRAPHY Soybean Oil Solvent CH 3 CN/HF Column 84346 (Waters Associates) RESPONSE RETENTION TIMEOleate-containing triglycerides in olive oil OL2 54:5 44 O2L 54:4 46 OPL 52:3 46 O3 54:3 48 OSL 54:3 48 O2P 52:2 48 O2S 54:2 50 OPS 52:1 50 Fatty Acid Composition Total Acyl Carbons: Unsaturation Equivalent Carbon Number OS2 54:1 52Further Specific Identification Tests for SomeFats and Oils: ( For detection of adulteration) For butter: Reichert-Meissel index for butter is a measure of its content of volatile and water-soluble butyric acid; >24. For olive oil: Squalene (7000 mg/kg) is a very specific constituent of unsaponifiable part of olive oil, whereas in others, it varies between 30-400ppm. For cottonseed oil: Halphen test for detecting presence of cyclopropenoic acids which form a red color on heating with amyl alcohol and CS2. For sesameseed oil: Villavechia test: unsaponifiables of sesameseed oil only will form pink color with furfural. For cocobutter: Triglycerides C50 and C54 are dominant in cocobutter but not in CBE and CBS.II b. Quality Criteria IIb.1. M I U (Moisture+Insoluble Impurities) (dirt, oil seed particles in crude oils and soap in refined oils)+(unsaponifiables) : They are nonfatty constituents of oils which increase refining costs for crude oils . Therefore, every percent of increase in MIU content brings parallel price decreases in crude oil purchases. Also soap>5 ppm left in refined oils will be detrimental to oil quality . IIb.2 Fat rancidity and stability Rancidity: “Development of objectionable flavours and odours resulting from lipolysis or lipid break-down (either due to hydrolytic or to oxidative reactions)leading to lower consumer acceptance”. Stability: “Capability to remain fresh or maintain fresh taste+odour” or “resistance to spoilage during storage or “resistance of lipids to rancidity”RANCIDITY(Hydrolytic and Oxidative) A-Hydrolytic Rancidity: Involves chemical or enzymatic(lipase from microorganisms or plant cells) hydrolysis of fats into free fatty acids and mono or di-glycerides. Relevant Tests:AV and ffa a. A.V.=The number of miligrams of KOH required to neutralize 1 gram of fat or oil. T and aw b. FFA: Free fatty acids= here, acidity is expressed as percentage of weight of a specific free fatty acid in a fat or oil,thus taking into consideration the M.W. of the individual f.a.: * In olive oil, expressed as oleic acid, MW = 182 *In palm oil, expressed as palmitic acid,MW = 256 * %FFA oleic acid= 0.503 x Acid value * Acid value= 1.99 x % FFA oleic acid 100 ) 1000 ( % x Weight acids fatty MW x V FFA = AV = ml of KOH x N x 56 Weight of Sample = mg of KOHSignificance of AV and ffa: This value will estimate the amount of crude oil that will be lost during refining, so will mean higher operating costs for refining plants, and leads to price decreases in crude oil purchases. In refined oils, high values will mean either poor refining or fat breakdown in storage or use. In cases where the liberated ffa are volatile , ffa will be a measure of hydrolytic rancidity. For Virgin olive oils < 4 % oleic acid, and for refined olive oils < 0.3; but for shorter chain f.a. like butyric,capric and even lauric (C4- C12), having much lower flavour thresholds(as in chocolate), the product will be inedible after its ffa content reaches > 0.02 %. B-Oxidative Rancidity “LIPID OXIDATION “ Major flavor problems in food during storage are mainly due to the oxidation of lipid. Lipid Oxidation - free radical reactions. 1. Initiation. 2. Propagation. 3. Termination. Methods for Measuring Present Status of Lipid Oxidation: -Peroxide number: The amount of iodine liberated(Na 2 S 2 O 3 titration) from saturated KI solution by fat , expresssed in meq of peroxide O 2 /kg of fat (Lea value=expressed in millimoles of O 2 ). <10 for edible oils; measures only transient primary oxidation products like peroxides and hydroperoxides. -Thiobarbituric acid value (TBA): The pink color formed by secondary oxidation products like malonaldehydes with TBA reagent is measured at 523 nm.Correlates better with sensory analyses. -Anisidine value: Another color reaction of aldehydes(alkenals and dienals-secondary products of oxidation) with P- anisidine. -Totox value : 2 times peroxide value + anisidine value = Total oxidation products (2 is because peroxides have 2 times more oxygen than aldehydes.)In most cases, rancidity is due to products of auto- oxidation of unsaturated fatty acids by progressive addition of oxygen to lipid components. Autoxidation reactions are catalysed by presence of prooxidants like Cu, Fe, sunlight and lipoxigenase enzyme. The shelflife of fats and oils is assumed as the end of the induction period, after which the rate of formation of oxidation products (Peroxides, Oxides, Hydroperoxides) increases drastically. IP is 1.3 hours for C18:3, 19 hours for C18:2, and 82 hours for C18:1 at 40°C. Different tests have been developed to measure the extent of autoxidation, but each respective test is like a snapshot at the time tested and do not necessarily show the complete picture.Quantity of Reactants and Products Quantity of Reactants and Products TIME Induction period* Secondary oxidation products Peroxides Primary oxidation products *Induction period is the length of time before detectable rancidity, after which there is an accelerated lipid oxidationANALYSIS OF FLAVOR QUALITY & STABILITY OF OIL Peroxide Value KI CH 3 C OH HI CH 3 C OK OO ROOH HI I 2 H 2 O ROH I 2 Na 2 S 2 O 3 NaI Na 2 S 4 O 6 A. B. C. + + + + + + + 2 2 2 Peroxide Value = ml of Na2S2O3 × N × 1000 (milliequivalent peroxide/kg of sample) Grams of OilActive Oxygen Method (AOM) Determined the time required to obtain certain peroxide value under specific experimental conditions. The larger the AOM value, the better the flavor stability of the oil. TBA Test To determine the rancidity degree of meat or fish product. N N HS OH OH C CH 2 C O H O H OH OH HS N N OH SH N N CH CH CH HO H 2 O Colored Pigment + + 2Storage stability (shelf-life): Lipids vary in their susceptibility to rancidity because of inherent properties like unsaturation, presence of antioxidants, and some external factors like processing and storage conditions.Accelerated tests are used for predicting future behaviour. For example, by raising the temperature, you naturally accelerate the rate of deterioration. At the end of the induction period, you stop and perform peroxide determination analysis. End of shelf life can be defined as days at 20°C to reach P.V.=20. This point is generally the end of the induction period, and the end of the test. The longer the IP, the better the storage stability and the longer the expected shelflife. For this purpose many different “accelerated shelf-life” methods have been developed:-Schaal Oven test: Oil is kept at 63°C till rancidity is detected or till the end of incubation period(Assumption:1 day of incubation at 63°C corresponds to 10 days at 20°C.) -Active oxgyen method (AOM): O 2 is bubbled through dried and filtered oil to further accelerate autoxidation at 110°C, this is carried out till PV increases to 20 for lard and 100 for vegetable oils. The induction period is determined by discontinuous measurements of peroxide value or sensory evaluation of rancis odor. OXIDATIVE STABILITY INDEX(OSI) Oxidative Stability Index test, unlike tests such as peroxide value which determine oil quality only at the time of the test, has predictive value, thereby allowing quality decisions to be made before spoilage becomes a problem. The 0SI is an invaluable quality control tool for eliminating inferior ingredients (for example hazelnuts for chocolate manufacture), for measuring and comparing the stability of finished oil-fat products as well as low moisture snacks like patato chips, for evaluating antioxidant activity, for monitoring the deterioration of cooking oils with use, and for many other applications where oxidative rancidity is the cause of product failure. Because the OSI has predictive value, problems are detected and eliminated before they cause rejection, unlike other tests which merely indicate that problems have occurred after it is too late to correct them. -RANCIMAT Method: Rancimat value at Temperature T° is the number of hours,t, before the oil reaches the end of its induction period. This device performs shelf life analysis for at least 2 preselected high temperatures (i.e. 100 ° and 130 ° or 140°C). For this purpose, you put the sample in the device, increase the temperature to the preselected T 1 , and the products of oil oxidation under these conditions are collected by distillation into a water trap(a special collecting vessel containing deionized water). The conductivity of water is continuously measured and recorded. The abrupt change in conductivity marks the end of the induction period,t. Assumptions: Q 10 = rate at (T+10) °C/ rate at T °C = shelflife at T °C / shelflife at (T+10) °C Graphs where Conductance value(1/R) is plotted against time(t) are used for shelflife prediction. A plot of log t (time period corresponding to end of shelf- life) versus T°C throughout a range of temperatures will be a straight line. The two points corresponding to logt1 and log t2 values at temperatures T 1 and T 2 respectively will be plotted and the two points so connected as to intercept both x-axis and y-axis. The shelf life at a third temperature T 3 can then be read from graph by extrapolation. 1/R T(time)Log t (time) Temperature° C 100° 140° 20°For refined edible vegetable oils, Rancimat values c a n b e u sed i n t he g en era l eq u a t i on of l i n e t o deduce expected shelf-life: D 20 ?7.40 R 100 -7.9 (here, r =0.933) Where D 20 =days at 20°C to reach PV=20 And R 100 =Rancimat value in hours at 100 °CPhysical Methods-Thermal Methods *Differential scanning calorimeter *Thermogravimetric and pressure differential calorimetric methods *Oxidative stability-length of the incubation period-time to max weight gain and rate of weight increase-time of max enthalpy change Thermal Method- Chemiluminescence *Determine the content of effectiveness of antioxidants in oils and fats *correlates with the peroxide value and anisidine values and flavour *emitted during peroxidate breakdownIndicators of Quality of Heated Oils *Deteriotive changes-thermal oxidation,hydrolysis, polimerization *Deep fat frying potatoes with fast turnover in sunflower oil-thermoxidative changes *A dramatic rise in polar compounds; More than 25-27% polar lipids should be rejected *NIRS-determine the sum of dimer and polymer triglycerides and acid value to evaluate frying oil Changes in dielectric constantToxic Contaminants and Adulterants -Contamination by weed seeds during harvesting -Storage of safe oil in unsafe containerContamination due to Faulty Storage *Contamination of vegetable oils by mineral oils- determining the saponification values of the lubricating oil samples *Indices of Admixtures, Blends, Contaminants and Adulterants-One Fat to Another *Stable carbon isotope ratio analysis-establishing the authenticity of oils *Fourier transform infrared spectroscopy for authenticity of extra virgin olive oilAdmixture of Vegetable Oils with Other Vegetable Oils Biosynthesis of Fat -Species specific -Detection of adulteration can be based on the basis of various constituents in the fat admixtureAdmixture of Vegetable Oils with Other Vegetable Oils -Fatty Acid Composition -Triglyceride Analysis -Unsaponifiable Fraction of Oil *Sterol analysis *Tocopherol analysis *Phenolics and AlcoholsFatty Acid Composition *Without being detected by the routine physical and chemical characteristics-GLC analysis of fatty acids prove useful- presence of soybean oils in olive oil *IOOC for olive oil purity-saturated fatty acid content at position-2 *Pancreatic lipase allows the differentation between virgin and esterified olive oils- Saturated fatty acid level <1,5% in virgin olive oilFatty Acid Composition *Coconut oil -determination of saponification and IV-detect gross and simple adulteration *Analysis of methyl esters of fatty acids by GC- ratios of certain fatty acids-detection of 5% rapeseed oil in peanut oil(C22-C16) Palm kernel and coconut oils in adulterated cocoa butteras well as addition of coconut or palm kernel oil to butter (high lauric and myristic acid content). *Linolenic acid-detection of 5% grapeseed oil or <5% sunflower oil in olive oilUnsaponifiable Fraction of Oils *The presence or absence of certain individual unsaponifiable components can help in establishing the identity of each oil (authenticity or genuineness) and also in detection of admixture with another oil.Unsaponifiable Fraction of Oils Sterol Analysis *Analysis of sterols from unsaponifiable fraction -adulteration olive oil with soybean oil *Sterol composition obtained by fraction of the unsaponifiable matter is characteristic of virgin olive oils *The ratio of the content of ß-sitosterol to the sum of (stigmasterol and campesterol) is 25- 30 for virgin olive oil. Virgin olive oil has ß-sitosterol content of 94% or higherUnsaponifiable Fraction of Oil Tocopherol Analysis *Concentration of ?-tocopherols in solvent extracted cottonseed oils increased on contamination with soybean oil *Contamination of olive oil with peanut oil -detected from ingreased ß-tocopherol levels *Quantitative determination of ? and ?-tocopherols -a method for determining the adulteration of olive oil with less expensive oilsUnsaponifiable Fraction of Oil Phenolics and Alcohols *Reverse phase HPLC with UV detection and GC-MS-quantification of phenols *Olive oil press cake in olive oil can be detected on the basis of the presence of higher fatty alcohols in the formerBlends of Vegetable and Marine /Animal Fats Fatty Acid Composition *GLC analysis of fatty acids composition-detect admixture of marine oils in vegetable oils ( increased myristic, palmitoleic, C20 and C22 acid contents). *Purity and contamination of vegetable oils with lard-values of methyl esters of C8-C20 fatty acidsFatty Acid Composition *Animal fats contain tetradecanoic and hexadecanoic acids in greater amounts than does olive oil *Average molecular weight of the distilled fraction from pure olive oil has a saponification number less than 191-increases with adding animal fats Unsaponifiable Fraction *Differences in the sterol composition of animal and plant fats-an efficient method for detecting admixture *Adulterant animal fat at % 10 in vegetable oil- detected on basis of cholesterolDetection of Mixtures of Animal Fats *Detection for adulteration of beef fat by pig lard-fatty acid composition and physical characteristics *Detection fo interspecies meat adulterationis very often on the differences in fat characteristicsSensory Quality of Oils “Global” quality evaluation of virgin olive oil the IOOCproposed (1989) th following equation: O.Q.I = 2,55 + 0,91SE - 0,78AV 7,35K270 -0,066PV O.QI: Over-allquality index SE:Sensory evaluation (from3,9-9,0) AV:Acid value (from 0,1-3,3) K270 :Absorbance at 270 nm PV:Peroxide value(from 1,0-20,0)