CHAPTER IX: ANALYSIS OF FATS AND OILS

Fats and oils are a heterogeneous group of predominantly hydrophobic compounds. The distinction between fats and oils does not have a chemical basis. Those fats/oils that remain liquid at normal (ambient) temperature are generally taken as oil and those that remain solid, fats. Analysis of fats and oils is carried out for various reasons, viz.: 1. Shelf life study (how long the item will remain without deterioration in quality under a given set of conditions) 2. Functional quality (e.g., suitability for use in biscuits, bakery, hydrogenation, etc.) 3. Sensory quality (e.g., rancidity) 4. Nutritional quality (e.g., melting point, polyunsaturated fatty acids) 5. As an aid in controlling production operation (e.g., control of hydrogenation, recovery of oil in mills) 6. Conformance to regulatory standards (e.g., with respect to free fatty acids, saponification value, peroxide value, moisture) 7. Detection of adulteration (e.g., contamination with mineral oil and argemone oil, adulteration of dairy ghee with vegetable ghee) 8. Advanced research (e.g., determination of fatty acid profile) Some of the routine tests carried out on fats and oils are as follows: 1. 2. 3. 4. 5. 6. 7. 8. Acid value/Free fatty acid (FFA) Saponification value, SV (also termed Saponification number) Iodine value, IV (Also termed Iodine number) Unsaponifiable matter Refractive index Melting point (for solid and semisolid items) Moisture content General tests for adulteration, such as Hexabromide test for the presence of linseed oil, Halphen test for the presence of cottonseed oil, Baudouin test for the presence of vegetable ghee in dairy ghee, Bellier turbidity test for the presence of ground nut oil, etc.

Some of the special tests used for particular fats and oils are: 1. Crismer test for rapeseed and mustard oil 2. Reichert-Meissl, Polenske and Kirschner values for dairy ghee 3. Polybromide test for linolenic oils such as linseed oil Some of the important physicochemical characteristics of common fats and oils are as follows:

467-1. are notoriously susceptible to hydrolysis.1. With prolonged storage.8 1. diglycerides. regulating bodies have set mandatory standards for edible oils. Consequently.5 1. some of them are: 1.00 The operational definitions of Acid Value and FFA are: Acid Value: Number of milligram of KOH needed to neutralize the FFA present in 1 g of oil FFA : Percentage of free fatty acids present in oil. natural fats and oils are in the triglyceride form when freshly extracted from the source. lipases (enzyme) coming from the source or contaminating microorganisms. though not a health hazard is undesirable for several reasons.469 Saponification value. elevated temperature and. monoglycerides. Such oils also bring about health problems.25 Mustard/Rapeseed oil ≤ 3. % 1. 2.5 2.468 1.5 9. Some fats/oils are relatively stable but others. most important of all. A suitable amount of oil is therefore mixed with neutralized rectified spirit to extract free fatty acids and the amount of the 105 . The oil is no longer the same as the virgin oil The oil tends to smoke during deep-frying The oil is susceptible to rancidity The product prepared from such oil turns rancid very soon Rancid oils markedly lower the esthetic value of oil. in Nepal: Mandatory standards of selected fats/oils Fat/oil FFA (as %oleic acid) Vanaspati ≤ 0. presence of excess free fatty acids is a sure indicator to unnatural state of oil. 3. In connection with the afore-mentioned points.466-1. Wij’s 120-143 92-125 103-128 110-143 Unsaponifiable matter. the triglycerides begin to break down giving rise to free fatty acids (FFA). however. DETERMINATION OF FREE FATTY ACIDS (FFA) AND ACID VALUE BACKGROUND For the most part. the neutral oil becomes a mixture of triglycerides. The presence free fatty acid in large excess. such as crude rice-bran oil.FOOD ANALYSIS Fat/oil Soybean oil Mustard seed oil Maize oil Sunflower seed oil Refractive index at 40°C 1. free fatty acids and glycerol.461-1. for example.470 1.469 1. Whichever the oil. 4. usually 282 (oleic acid) PRINCIPLE Free fatty acids are readily soluble in rectified spirit or absolute alcohol. This hydrolysis is brought about by a variety of agents: presence of moisture in the oil. mg KOH/g oil 189-195 170-184 187-195 188-194 Iodine value.50 Refined oil ≤ 0.465-1. It is calculated using an assumed average molecular weight of fatty acids.

Calculation for both acid value and FFA can be carried as follows: REQUIREMENTS • Neutral alcohol (95%. Carry out titration in triplicate CALCULATION % FFA = ml of alkali × N of alkali × 28. 7. 5. DETERMINATION OF SAPONIFICATION VALUE OF FAT/OIL BACKGROUND Saponification value of fat/oil is a very valuable test for the determination of adulteration. To facilitate extraction. of sample (g) Acid value = 9.1N NaOH using phenolphthalein indicator 106 . 6. Observe the color of the alcohol fraction for persistent pink color 8.1 Wt. 4. 2. the mixture may be warmed to about 70°C and swirled vigorously. tilt the mixture to allow separation of alcohol and fat fractions. P = percentage of unsaponifiable matter Since the oil from a given source has a remarkably constant saponification value any deviation found in the test is an indication to adulteration. Weigh out 10 g of fat/oil in a 250-ml conical flask (by difference) Add 50 ml of neutral alcohol Add a drop or two of phenolphthalein indicator Swirl the contents and place flask on the hot plate Warm the mixture to about 70°C. of sample (g) ml of alkali × N of alkali × 56. Some of the common examples of edible oils and their saponification values are: 64 Neutralize the acidity in the with 0.1N NaOH to persistent pink color In case of doubt.1N NaOH • Titration arrangement • Weighing arrangement • Hot plate PROCEDURE 1. Swirl well Titrate warm with 0.683 Saponification value Where M =Average molecular weight. alcoholic) • 0.2 (as oleic acid) Wt.FOOD ANALYSIS latter calculated by titrating with standard NaOH or KOH using phenolphthalein indicator. v/v)64 • Phenolphthalein indicator (1%. 3.0937 (100 − P ) − 12.2. The test is a rough measure of the average molecular weight of fatty acids in the oil and related thus: M= 561.

IX-1: Saponification of triglyceride The hydrolysis is limited to glycerides. Bromophenol blue changes from blue to permanent greenish yellow upon complete breakdown of the soap. giving rise to glycerol and fatty soap (Fig.5N HCl using phenolphthalein as an indicator. The alkali consumed for this is a measure of saponification value. and is defined as the number of milligram of KOH needed to saponify one gram of oil or fat.5N HCl using bromophenol blue indicator solution as the indicator. the glycerides irreversibly hydrolyze.. pigments. do not react with KOH under the above condition and they contribute to what is known as unsaponifiable matter. Sterols. phenolphthalein and bromophenol blue. PRINCIPLE When the oil is saponified with a slight excess of alcoholic KOH. A recapitulative presentation of the above-mentioned points is given in Fig. IX-2: Saponifiable.. The amount of HCl consumed is back calculated to reflect the milligrams of KOH consumed by one gram of fat/oil during the saponification. There are at least two titrimetric methods for the determination of saponification value. hydrocarbons. IX-2. CH2OCO-R1 R2-COOCH CH2OCO-R3 Mixed triglyceride + 3KOH CH2OH HOCH CH2OH Glycerol + R1-COOK R2-COOK R3-COOK Potassium soap Fig. waxes and phosphatides. viz. the reaction results in potassium soaps. A relatively easy method utilizes double indicator. IX-1). When fat is boiled with an excess of alcoholic KOH.and unsaponifiable matter in oil REQUIREMENTS • Oil sample • KOH pellets (pure) • Conical flask with condenser • Heating arrangement (hot plate or water bath) 107 . CRUDE FAT KOH Fats KOH Waxes KOH Phosphatides Non-fat Sterols Hydrocarbon Pigments KOH Potassium salts + Glycerol Potassium salts + Alcohol Potassium salts + Glycerol + K3PO4 + Amine No reaction (Unsaponifiable matter) Fig. The free KOH can be determined by titrating with 0. The KOH in the form of soap is determined by further titrating with 0. etc.FOOD ANALYSIS Soybean oil: Rapeseed oil: 189 – 195 168 – 181 Butter oil (ghee): 210 – 230 The test merits considerable attention in that successful testing is more of an art. although lipids. glycerol and unreacted KOH.

If the color does not change. If a milky color develops. Add about 30 ml rectified spirit and gently reflux the whole until the oil becomes transparent (this usually takes 25 min) 5. Neutralize the excess KOH with 0. The oil-alcohol mixture appears transparent at this stage 6. repeat the whole process using more KOH (e. Ensure that the sample is free from moisture and impurities 2. Since the degree of unsaturation is more or less characteristic to oil source. add more water (about 50ml) and mix well 9. a small amount of rectified spirit (≅ 10 ml).5N HCl consumed (the second reading.1 Wt. the test is routinely used for the determination of adulteration by other types of oils. DETERMINATION OF IODINE VALUE OF FAT/OIL BY WIJ'S METHOD BACKGROUND Probably no analytical test method in all of oleochemistry has had the universal widespread use as the measurement of unsaturation in fats and oils by iodine value determination. when determined using Wij'’ solution. fat-like globules suddenly appear. Add a drop of bromophenol blue indicator and swirl. If. Add about 500 mg KOH pellet. during titration.5N HCl. alcoholic) PROCEDURE 1. 600-800 mg) 7. Since 1898 great many innovations have been made. that is) and calculate the saponification value CALCULATION Saponification value = ml of HCl × N of HCl × 56. The test is a measure of unsaturation of a given fat or oil.3. Note the volume of 0. Melt the sample (if not already liquid) and filter warm. Iodine values of some common edible oils are: 108 . Note the reading on the burette (containing the standard HCl) and titrate till a permanent greenish-yellow color appears. the sample contains significant amounts of unsaponifiable matter or is contaminated with mineral oil 8. Weigh accurately by difference about 2 g of sample in a 250-ml conical flask 3. The first to use this concept was Von Hubl in 1884. of sample (g) 9.FOOD ANALYSIS • Standard HCl (0.5N) • Rectified spirit or absolute alcohol (aldehyde-free) • Titration arrangement • Indicators: bromophenol blue (1%.g. Slightly cool the flask and add a drop or two of phenolphthalein indicator. Add some rectified spirit (if the volume decreases) and continue refluxing till completely saponified. The pink color should just disappear 10. It should give a blue color 11. warm the flask a little and continue titration to the end point 12. and emulsify by swirling briefly 4. Iodine Value is the number of grams of iodine absorbed per 100 g of oil or fat. continuing until recently. Add a drop or two of distilled water. Intense red color indicates the presence of excess KOH. alcoholic) and phenolphthalein (1%.

Rosenmund-Kuhnhenn method. and allow to stand for 10 min. There are some difference vis-à-vis reagent preparation in Wij’s method also.20 to 0. swirl. Mix the solution thoroughly. Weigh 0. Iodine value is also used to calculate the amount of hydrogen used or wasted in vanaspati plants. It is routinely used for monitoring the degree of hydrogenation.and 5 ml (graduated) Dissolve 1g of reagent grade starch in hot water. There are several methods for measuring the iodine value of fats and oils. and then siphon off the clear liquid.1N)66 65 • Soluble starch (1%)65 • Iodine value flasks • Pipette: 25. Standardize the solution with AR grade potassium dichromate (K2Cr2O7). Transfer to a 250-ml beaker using 150ml of water. Bromine Value method. Use only fresh solution. The amount of iodine consumed is then determined by titrating the iodine released (after adding KI) with standard thiosulfate and comparing with blank in which the fat is omitted. Transfer the clear fraction into another container. PRINCIPLE Halogens add across the double bonds of unsaturated fatty acids to form addition compounds. as it is subject to microbial degradation 66 Dissolve 25g AR grade Na2S2O3. IX-3. a drop in I unit of iodine value means to the vanaspati manufacturer that 0. Some of the variations and /or equivalent methods are: Hanus method. IX-3: Reaction scheme during iodine value determination The reaction mixture is kept in dark and the titration carried out as quickly as possible since halogens are oxidized in the light. start 109 . aqueous) • Standard Na-thiosulfate (0. Add 2g of KI and mix. etc. In general. CH CH Unsaturated portion of fat + ICl Iodine monochloride CH CH I Cl Addition compound KCl + I2 Molecular iodine 2NaI + 2Na2S4O6 Na-tetrathionate ICl + KI Residual Added after titration Na2S2O3 + Na-thiosulfate I2 Fig. REQUIREMENTS • Carbon tetrachloride • Potassium iodide (10%. Iodine monochloride (ICl) is allowed to react with the fat in the dark. The reaction occurring in the test can be shown in Fig.075 kg of hydrogen has been added to every 1000 kg oil.FOOD ANALYSIS Soybean oil: Rapeseed oil: Butter oil: 120-143 94-120 26-38 The test is of tremendous value in vanaspati (hydrogenated oil) plants. allow to stand for a few days. The amount of Wij’s reagent used in this test should be more (usually by 150%) than shown by the stoichiometry.7H2O in distilled water to make 1000ml. Add 20ml of 1N HCl.23g of K2Cr2O7 (dried for 2 hrs at 105°C).

Swirl once and close the flask with the stopper. Add accurately 20 ml of Wij’s solution. Carry out a blank test upon the same quantities of reagents. Use only freshly prepared starch solution titrating with Na2S2O3 solution from the burette.2N. Add 10 ml of CCl4 and allow oil to dissolve 3. IX-4: Wij’s IV flask PRECAUTIONS 1. dry 250-ml IV flask (see Fig. Add two drops of starch solution. IX-4) 2. Weigh accurately by difference suitable quantity of oil using the formula: (20.69 Fig. as determined by titrating with Na-thiosulfate. of sample (g) Stopper × 12. Add 15 ml of 10% KI solution. should not be less than 0. of pot .1N Na2S2O3 using starch indicator towards the end of the titration (The mixture turns straw color near the end point. Continue the titration until the blue color just disappears) 7.thiosulfate × 49. adding about 80% of the required amount. in to a clean. The solution is stable for about 30 day. Stand the flask at 15-20°C for 30 min in dark 5. followed by 100 ml distilled water 6. at the same time and under the same conditions.3 ÷ expected iodine value) grams. Add 1ml of starch indicator and complete the titration to a point where the solution changes from blue-green to light green. omitting the oil.FOOD ANALYSIS • Wij’s solution 67 • Measuring cylinder. The strength of Wij’s solution. 2 sets • Electronic balance (± 1mg sensitivity) • Oil or fat sample PROCEDURE 1. The mixture immediately turns dark blue. Titrate with 0. Store the reagent in a colored bottle in dark. The reagent should be used in excess (150% of the amount absorbed by fat/oil) 3. 25 ml • Burette: 50 ml. The excess of reagent remaining for titration in the test must be 150% of the reagent absorbed CALCULATION Iodine value = ( Blank titer − Sample titer ) ml × N of Na 2S2 O3 Wt. Calculate the strength of Na-thiosulfate as follows: N of sod-thiosulfate = Wt. The stopper may be moistened with minimum of 10% KI solution 4.2 N 2.dichromate × 1000 ml of sod . The Wij’s solution should be ≅ 0. 110 .037 Add a pinch of Na2CO3 and 1ml of chloroform to preserve it from microbial degradation 67 Dissolve 8g iodine trichloride in 150ml glacial acetic acid and mix with 9g iodine dissolved in 350ml glacial acetic acid.

HCl: 2KMnO4 + 16HCl → 2KCl + 2MnCl2 + 8H2O + 5Cl2 The amounts of HCl and KMnO4 needed for chlorination are not very large. To take this into account. A slight excess of iodine does little or no harm but excess chlorine must be avoided. Take out about 25ml solution and set aside (as a reserve) in a separate flask (you will need this later) • Transfer the bulk iodine solution in a Woulfe bottle and assemble the parts as in Fig. IX-5 • Generate chlorine68 and pass through the iodine solution to form iodine monochloride • Continue passing the chlorine until the characteristic color of free iodine is discharged (solution suddenly lightens because of free chlorine) • Stop passing chlorine and test the Wij’s solution for dismantle the assembly for chlorination • Add small amounts of iodine solution (reserved earlier) until the free chlorine has been destroyed (the color again darkens). use about 5-10g of KMnO4 and 50-100ml of conc HCl. The latter method is described here. • Take 13g resublimed iodine in a 1-liter beaker • Add 200ml glacial acetic acid and dissolve by gentle heating (along with stirring). do not exceed the total volume of 1000ml. conc sulfuric acid. and heat to boiling for 10 min o Cool and add 30ml of 2% H2SO4 o Add 15ml of 15% fresh KI solution o Titrate with 0. Assemble pipettes. 68 111 . However. prepare standard sod-thiosulfate. Preparation of Wij’s solution by chlorination Before anything else. If some space is available. sufficient amounts of chlorine must be produced to force itself through the solution. and (ii) using chlorine gas and resublimed iodine.. make up the volume to 1000ml by glacial acetic acid.1± 0.1 and this can be ascertained by determining iodine content and total halogen content as follows: Iodine content: o Take 150ml of Chlorine-saturated water in a 500-ml conical flask and add some glass beads o Add 5ml of Wij’s solution o Mix. Besides. 10% KI solution and starch indicator. (i) using iodine monochloride. viz. Mix the solution well.1N sod-thiosulfate to starch end point o Note the titer (say A) Chlorine is generated in the laboratory by reacting KMnO4 and con. However some amounts of chlorine go waste during chlorination. Typically.FOOD ANALYSIS Note: Wij’s solution can be prepared by other methods also. burettes and other glassware needed for iodometric titration. Iodine dissolves very slowly and the complete dissolution can be carried out in stages by using small portions of glacial acetic acid • Transfer the dissolved portion to 1-liter volumetric flask • Add more glacial acetic acid (~ 200ml) to the undissolved iodine in the beaker and heat gently (as previously done) to affect dissolution • Transfer the dissolved portion to the volumetric flask (to pool the solution) again • Carry out this operation until iodine is completely dissolved. the iodine/chlorine ratio should be 1.

as the method is highly empirical.1N sod-thiosulfate to starch end point Note the titer (say B) Iodine/Chlorine ratio as follows: I/Cl = 2A . in which the mixture is boiled. the Wij’s solution thus prepared should consume approximately ( 3B − 2 A ) double the amount of 0. in which the liberated iodine is converted into a blue starch iodine complex.4.1N sod-thiosulfate Delivery tube Small vent to release excess pressure Conc. a variation in the iodometric method reported by Swoboda and Lea. IX-5: Preparation of Wij’s solution 9. There are numerous analytical procedures for the measurement of peroxide value. cooled water in a 500-ml conical flask Add 15ml of 15% KI solution Pipette 20ml Wij’s solution Titrate immediately with 0. DETERMINATION OF PEROXIDE VALUE BACKGROUND Peroxide value (PV) is a very sensitive indicator of the early stages of oxidative deterioration of fats and oils. and the Sully method. which measure the iodine produced from potassium iodide by the peroxides present in the oil. PV therefore provides a means of predicting the risk of the development of flavor rancidity. It has been contended that the two principal sources of error in these methods are the absorption of iodine at unsaturated bonds in the fatty acids on the one hand. the type and grade of solvent used. and the constitution and reactivity of the peroxides present in the oil. Other types of error which can arise include variation in weight of the sample. the liberation of iodine from potassium iodide by oxygen present in the solution to be titrated.FOOD ANALYSIS Total halogen content: o o o o o o Take 150ml of recently boiled. The most common methods are those based on the iodometric titration originally reported by Lea and Wheeler. HCl KMnO4 Iodine solution Chlorine bubbles Fig. and on the other. In all cases the results and accuracy of the test depend on the experimental conditions. 112 . Other methods have been recommended for peroxide value determination and these include a colorimetric method based on the oxidation of ferrous to ferric ion and the determination of the latter as ferric thiocyanate. variation in the reaction conditions such as time and temperature.

Titrate the liberated iodine with 0. milliequivalent peroxide = milliequivalent thiosulfate at the equivalence point Again. and VB = sod-thiosulfate consumed by blank (ml). when volume is in milliliter Therefore.01N or 0. Calculate Peroxide value using following equation: PV (meq/kg) = Where. Now. Keep in brown bottle 113 . The test is a volumetric one where I2. stopper the flask.1N sod-thiosulfate (see determination of Iodine Value) • Measuring cylinder: 25ml cap PROCEDURE 1. peroxides present in the fat liberate iodine. of sample (g) 2 volumes of acetic acid and 1 volume of chloroform 4 parts of pure KI in 3 parts of distilled water. N = normality of sod-thiosulfate. Appearance of blue color on addition of starch indicates presence of free iodine 5. Weigh accurately (by difference) 5g of fat or oil sample in the Iodine flask 2. Carry out a blank determination simultaneously (omitting oil) 7.VB ) × 1000 Wt. PRINCIPLE When a rancid fat or oil sample is treated with potassium iodide after dissolving in an appropriate solvent. VS = sod-thiosulfate consumed by sample (ml). formed from KI in the presence of peroxide is determined by titrating with sodium thiosulfate and conducting a blank determination.1N sod-thiosulfate until the blue color just vanishes 6. 69 70 • Iodine flasks: 250ml cap • Burette: 25-50ml cap • Pipette: 25ml cap • 0. and allow it to stand for 1min (with gentle shaking) 4. PV = milliequivalent thiosulfate / kg sample REQUIREMENTS • Oil or fat sample • Acetic acid-chloroform solvent69 • Saturated potassium iodide70 • 0.FOOD ANALYSIS Peroxide value of an oil or fat is the amount of peroxides present and expressed as milliequivalents of peroxide per 1.000g of sample.5% starch indicator (see determination of Iodine Value) • Weighing arrangement N × (VS . Add 35ml of distilled water and a few drops of starch indicator. Add 1ml of KI solution. milliequivalent = (strength × volume). Add 25ml of solvent and displace the air with CO2 3.01N and 0.

When fat is cooled rapidly the α polymorph is produced. MELTING POINT OF FAT BY OPEN-TUBE CAPILLARY METHOD BACKGROUND Fats do not melt sharply because they contain different types of fatty acids with different melting points.. β’ form gives a smooth texture whereas β form results in a very coarse granules. respectively. This facilitates faster slipping away of molecules. The melting point is therefore defined by the specific conditions of the method by which it is determined. However. melting point as such is not very reliable for establishing identity of the fat and oil. The melting points of α-. and β forms of tristearin are 55°C. Polymorphism results from different patterns of molecular packing in fat crystals. Melting point of fat increases with an increase in the degree of saturation and chain length of fatty acid. It is therefore very important to control the balance of polymorphic forms in the production of fat and fatty foods like margarine (needs β’ form). These polymorphic forms also affect the appearance and texture of fat. it is extensively used in controlling process operation (e. β’-. PRINCIPLE The temperature at which the oil or fat softens or becomes sufficiently fluid to slip or run as determined by the open-tube capillary-slip method. Three such polymorphic forms. Polymorphism in fats and oils refers to existence of more than one crystalline form of fatty acid or glyceride. viz. and 73°C.. beta prime (β’) and beta (β) have been identified. thereby resulting in low melting point. alpha (α). hydrogenation).5. ghee (needs β form). Polymorphic transformations occur from α to β’ to β and are irreversible. A typical method used in vanaspati manufacture is the open-tube capillary method. Melting point also depends on isomeric forms and polymorphism in fatty acids. which is usually quickly converted to the β’ form. Trans isomers of fatty acids (that usually form during hydrogenation process) have higher melting point because the chains are less kinked. A comparison of the three polymorphs is given below: Polymorph Alpha (α) Beta (β) Beta prime (β’) Crystal size 5 µm 20-50µm 1-2µm Melting point Lowest Highest Intermediate Stability Least stable Most stable Intermediate The stability of fat is related to the polymorphic form and the associated melting point.g. quality control. etc. Because of the reasons described above. Unsaturated bonds produce kinks in the fatty acid chain and therefore allow very loose molecular packing. REQUIREMENTS • Capillary tubes71 71 • Beaker or Thiele tube Thin-walled with uniform bore capillary glass tubes opn at both ends with following dimensions: Length = 50-60mm 114 .FOOD ANALYSIS 9. 64°C. The methods used for the determination of melting point vary considerably. and determining suitability of fat for a particular purpose.

Remove the tube and attach with a rubber band to the thermometer bulb. Make sure that the sample is absolutely dry 3. is sucked into the tube 5.1mm Outside diameter1. Introduce a capillary tube into the molten sample. so that the lower end of the capillary tube and the thermometer bulb are at the same level 8. Place the tube in a small beaker and hold it for one hour either in a refrigerator or in water maintained at 4-10°C Thermometer Capillary Rubber band Heat source Fat Theile tube Chilled water Fig.8-1.2-1. and thereafter at the rate of 0. against a piece of ice until the fat solidifies 6.5°C Inside diameter = 0.5mm 115 . Note the temperature of the water when the sample column begins to rise in the capillary tube 11.2°C sensitivity) PROCEDURE • Heat source (gas burner or spirit lamp) 1. Mix the sample thoroughly 4. Melt the sample and filter it through a filter paper to remove any impurities and last traces of moisture 2. about 10mm long. Take water at 10°C in the Thiele tube and immerse the thermometer with the capillary tube containing the sample of fat (see Fig.FOOD ANALYSIS • Thermometer (0.5°C per min 10. IX-6: Arrangement for melting point determination of fat 7. Report the average of two such separate determinations as the melting point. IX-6) 9. provided that the readings do not differ by more than 0. so that a column of the sample. till the temperature reaches 25°C. Chill the tube containing the sample immediately by touching the tube. Gradually increase the temperature by heating at the side-tube of the Thiele tube at the rate of 2°C pen min.

Reichert Meissl value can be determined by titrating the steam condensate (that contains water-soluble fatty acids) with0.1N NaOH required to neutralize the steam-volatile. REQUIREMENTS • Fat sample • Glycerol • 50% NaOH • Pumice powder: 1. and quantification by titrating again with barium hydroxide.1N NaOH. Some of these tests are described next. 9. This test measures the quantity of C8 to C14 fatty acids. TESTS FOR THE ADULATERATION OF FATS AND OILS Physicochemical properties of fats and oils are often used to identify them. this analysis is not usually done. The tests are based on the quantitative measurement of low molecular weight fatty acids (C4-C14) that are predominant in butter. Usually. and time in the lactational cycle of the cow. This test measures the quantity of C4 and C6 fatty acids. Polenske value: It is the number of milliliters of 0. PRINCIPLE Steam-volatile fatty acids can be collected by saponification and steam-distillation of oil. water-insoluble fatty acids distilled from 5g sample of fat under precise conditions specified in the method.1N NaOH.6. higher than other edible oils. DETERMINATION OF REICHERT-MEISSL. Determination of Kirschner value involves neutralization of the water-soluble fatty acids with barium hydroxide. The definitions of the terms are: Reichert-Meissl value: It is the number of milliliters of 0. In recent years. it is usually between 24 and 34. POLENSKE AND KIRSCHNER VALUES BACKGROUND These tests are widely used for identification and test of adulteration of butter. preparation of their silver salts.1N NaOH required to neutralize the steamvolatile. more than one property is measured so that the identification can be made with some assurance since natural fats and oils vary somewhat in their properties.6. separation of the water-soluble butyric acid salt by filtration.1. Polenske value can be determined by eluting the fatty acids adhering on the condenser with neutral ethanol and titrating with 0. liberation of butyric acid by acidification. Although the RM value varies for butter with season. nutrition. A few special tests are now available for the unequivocal determination of adulteration in fats and oils. water-soluble fatty acids forming watersoluble silver salts (which is the property of butyric acid). separation by steam distillation. Kirschner value: It is a measure of steam-volatile.0mm in diameter • 90% neutral ethyl alcohol (v/v) • 0.FOOD ANALYSIS 9.05 N barium hydroxide • Finely powdered silver sulfate • Titration arrangement 116 .4-2. water-soluble fatty acids distilled from 5g sample of fat under precise conditions specified in the method.

Add 20g of glycerol and 2ml of 50% NaOH solution from a burette which is protected from CO2 pick up. Add 0.5ml of NaOH 4. Pipette out 100ml of the filtrate to a dry 250ml conical flask and titrate with 0. and replace the graduated flask by a measuring cylinder to collect drippings from the condenser 12. if Kirschner value is to be determined) PROCEDURE • Weighing arrangement • Phenolphthalein indicator • Heating arrangement 1.01g of fat sample into a Polenske flask 3.1N NaOH using phenolphthalein as indicator 18. The filtrate should be free from water insoluble fatty acids 17. An old sample of oil or fat may behave similarly 7. The distillation is considered to begin when the first drop forms in the shill-head 11.1g of pumice powder and 50ml of dilute H2SO4 8. Filter through the same filter paper ensuring that all insoluble matter is transferred to the paper. Reject the first 2-3ml of the filtrate and collect the rest in a dry flask 15. Do not mix with filtrate of the distillate got in the previous step. IX-7) 9. The solution must be completely clear at this stage and pale yellow in color. or if it is darker which indicates overheating. Wash the condenser. When all the fat has saponified. cover the flask with a watch glass. Do not mix 13. still head and the 110ml graduated flask with three lots of 15ml distilled water passing each washing through the measuring cylinder. Increase the heat and distil 110ml of solution in 19 to 21 min. Wet the tip of the burette before adding alkali to free it of carbonate deposit and reject the first 0. Melt the fat sample if solid but do not heat above 50°C 2. condenser (52cm long with 30cm cooling length and 7cm entry tube) and a receiver (with graduations at 100ml and 110ml) 117 . Heat the mixture over a low flame with wire gauze until the liquid becomes clear and the fat has saponified. Stop heating soon after 110ml has distilled over. Connect to the distillation apparatus (Fig. and allow to cool 6. Weigh 5±0. Calculate RM value as follows: RM value = (Sample titer – Blank titer)ml × N of NaOH × 11 The factor 11 has been obtained as follows: 72 Apparatus consisting of flat-bottom boiling flask. 100ml flask and stopper 16. still head (10. Close the graduated flask with the stopper. Add 93ml of boiling distilled water which is free of CO2 and mix.7cm wide and 18cm high). If the solution is not clear which indicates incomplete saponification.1N NaOH (not to be used. Place the flask in a water bath at 15°C for 10 min and ensure that the 110ml graduation is below the water level 14. 4 paper. repeat the procedure with a fresh sample. Do not overheat at this stage which causes discoloration 5. Discard the filtrate. Warm the mixture until any insoluble material which may be present melts 10. Mix and filter through a 9cm Whatman No.FOOD ANALYSIS • Dilute H2SO4 (25ml H2SO4 + 1000ml H2O) • RM-Polenske-Kirschner apparatus72 • 0.

1 19.1N NaOH using phenolphthalein indicator 110ml 100ml Fig. Under such conditions.. Calculate Polenske value as follows: Polenske value = (Sample titer – Blank titer)ml ×N of NaOH ×10 RM and Polenske values are affected by low barometric pressures which occur at high altitudes. the 110ml flask with stopper and the filter paper containing the main bulk with 3 similar washings as before using 15ml portions of neutral ethanol 20.FOOD ANALYSIS Total volume collected (ml) 110 = = 11 Aliqout taken (ml)× Required N of NaOH (i. IX-7: Reichert-Meissl ditillation apparatus (not to scale) 21. Dissolve the insoluble fatty acids by three washings of the condenser.1) 100× 0.0.e. Carry our a blank determination similarly 22. Collect the alcoholic washings (45ml) in a clean dry flask and titrate with 0. correct the readings as follows: Corrected RM value = (Observed RM -10) log 760 +10 log p Observed value × (760 .45 118 Corrected Polenske value = . the measuring cylinder.45) p .

Hydrochloric acid • Furfural solution73 • Oil sample PROCEDURE 1.Tb ) ⎣ 10. Cool the distillate at 15°C for 10 min. thereby indicating the presence of vanaspati ghee. Keep in a dark place for 1hr with intermittent shaking 26. p = barometric pressure in mm of Hg at the place of determination 23.5g of finely powdered silver sulfate to the solution 25. add 0.Ta )121⎤ ⎦ Kirschner value = ( Tk . freshly distilled in ethyl alcohol 119 . Titrate 100ml of the filtrate as in RM value using 0. 4 paper 27. Filter through a dry Whatman No. The color is produced on account of reaction with sesamolin present in sesame oil.05N barium hydroxide 31.1g of pumice powder 28.000 Where. Tk and Tb = sample and blank titer respectively in Kirschner value determination Tr and Tb = sample and blank titer respectively in the RM value determination 9. mix and filter through 9cm Whatman No. and distil 110ml in 19-21 min 29. Calculate Kirschner value as follows: ⎡100 + ( Tr . BOUDOUIN TEST This test is useful in the detection of adulteration of dairy ghee with vanaspati ghee. REQUIREMENTS • Glass-stoppered test tube/measuring cylinder • Con. Take 5ml of the oil or melted fat in a 25-ml measuring cylinder (or test tube) provided with a glass stopper 73 2% furfural. Dairy ghee containing sesamolin gives a positive Baudouin test. Carry out a blank determination similarly 32.FOOD ANALYSIS Where. The test is based on the color reaction between sesamolin (a compound present in sesame oil) and furfural In Nepal. After determination of RM value. For Kirschner value. PRINCIPLE The development of pink color with furfural solution in the presence of hydrochloric acid indicates the presence of sesame oil.05N Ba(OH)2 for titration 24.2. use of sesame oil in vanaspati is mandatory. CO2-free distilled water. 10ml of dilute H2SO4 and 0. Connect to the distillation apparatus.6. Add 35ml of cold.1N NaOH with 0. proceed as in RM value determination but replace 0. 4 filter paper as before 30.

3. 100ml cap) 2. The test has some limitations. Besides. All 5ml chloroform and about 1ml of bromine drop-wise till the mixture becomes deep red in color 3. HEXABROMIDE TEST This test is of importance for detecting adulteration of edible oil with linseed oil (which is inedible). sesame oil is present. It is not suitable for test in mahua oils. hydrochloric acid Add 0. Add 10ml diethyl ether 6. Cool the test tube in an ice water-bath 4. On crystallization these glycerides exhibit a characteristic crystalline appearance when viewed under microscope. PRINCIPLE The formation of a precipitate of hexabromide when the oil in chloroform is treated with bromine and then with alcohol and ether in cold condition indicates the presence of linseed oil.4ml of furfural solution Insert the glass stopper and shake vigorously for two minutes Let is stand and allow the mixture to separate.4. Confirm by adding 5ml of water and shaking again 7. The procedure recommended by Williams Sutton for the microscopy of fat crystals have been suitably modified and given.and tetrabromides that result from oleic and linoleic acids are soluble and therefore do not interfere with this visual test.5. If the color in acid layer persists. Pipette 1ml of oil into a boiling tube (wide-mouthed. Add about 1. TEST FOR THE PRESENCE OF ANIMAL FAT BY MICROSCOPIC EXAMINATION Animal body fats such as beef tallow and lard have been shown to contain trisaturated glycerides. Add 5ml of conc. which contain polyunsaturated fatty acids.5ml of rectified spirit drop-wise while shaking the mixture until the precipitate which was first formed just dissolves 5. Appearance of precipitates indicates the presence of linseed oil The sensitivity of this test is about 1% if linseed oil in other other oils. The development of pink or red color in the lower acid layer indicates presence of sesame oil 6. 5. also give insoluble polybromide precipitate. 120 .FOOD ANALYSIS 2. Mix the contents and place the tube within the ice water-bath for 20 min 7. marine oils. 9. 3. Di. REQUIREMENTS • Boiling tubes • Chloroform • Liquid bromine • Ice water bath • Ethyl alcohol • Diethyl ether PROCEDURE 1. The test is based on the formation of insoluble polybromides when unsaturated fatty acids are brominated.6. 4. if the color disappears it is absent 9.

HCl. dropsy and sometimes total blindness due to the presence of alkaloids namely. 20% • TLC plates coated with silica gel G or precoated ready made plates cut to suitable size • UV chamber (long wave. • Glass beaker. Take about 2 g of melted fat samples in test tubes 2. Add 10 ml of diethyl ether and mix 3. PRINCIPLE The hydrochloric acid extract of the oil sample containing argemone oil when subjected to TLC for separation of alkaloid gives fluorescent spot under UV light. • Standard argemone oil extract • Pear-shaped flask • Hot water bath • Separating funnel. amount of fat taken and the time allowed for crystallization 9. 10ml cap • Aqueous NaOH.19 • Chloroform:Acetic acid:Water = 70:20:10 (v/v) • Chloroform:Acetic acid (90:10. which grows in mustard field and bears capsules full of brown black seeds. = 1. grav. In such cases separate the crystals by filtration and recrystallized in ether 4.). Hydrogenated fats deposit smaller size crystals. it is often used as an adulterant. Place the crystals on a drop of glycerin previously taken on a microscopic slide 5. 366nm) • Solvent mixture (moblile phase) • Diethyl ether • Conc. yellow poppy. Because of its resemblance with black mustard. the ends of which are more or less pointed can be seen. v/v) mixture 121 . Lard crystals are chisel-shaped. Examine the crystals under ×160 and finally ×400 magnifications. sp. Cover the crystals immediately with cover glass 6.5. In certain cases it is preferable to first crystallize with a stronger solution of fat from a mixture of ether and ethyl alcohol (1:1). TEST FOR PRESENCE OF ARGEMONE OIL Argemone (Argemone maxicana L. sanguinarine and dihydrosanguinarine. is a wild herb. 50ml cap. Plug the tubes with cotton and allow to stand for 30 min in ice water or 24 hrs at 20ºC (slow crystallization gives bigger crystals). The size and shape of the crystals depend upon the strength of solution. The oil is reported to cause glaucoma.6.FOOD ANALYSIS REQUIREMENTS • Fat sample • Ice water-bath • Ethyl alcohol • Microscope • Test tubes • Filtration unit • Glycerin PROCEDURE 1. The typical appearance of beef tallow crystallized into characteristic fan like tufts.

Spot on TLC plate with the help of spotting capillary.6. Spot side by side standard Argemone oil extract (0.1 % in ether) 7. Absorbance values below 0. Absorbance values greater than 0. Bright yellow or orange yellow fluorescent spots having Rf similar to the standard argemone oil will confirm presence of argemone oil. Allow the plate to dry 10. Allow to separate. or (b) Hexane:Acetone mixture 8. 5.0 show that the sample is highly rancid. Contents of the separatory funnel may be heated cautiously over the vent of heating water bath for some time for quick separation 3. 9. Add 5 ml conc HCL and shake vigorously for 2 – 3 minutes. adding 5ml of conc HCl and observing for pink color as the evidence of incipient rancidity. Take 10 ml sample in a separating funnel and dissolve in 15 ml Diethyl ether 2. and absorbance values around 1. The qualitative method involves vigorous mixing of 5ml of oil with 5ml of 0. 122 . The spot gives blue florescence under UV-light if plate is sprayed with 20% aqueous sodium hydroxide solution The method is very sensitive and can detect argemone oil upto 50 ppm level. KRIES TEST FOR RANCIDITY IN FATS/OILS Kries test is a very rapid test for the assessment of rancidity in fats and oils. PROCEDURE FOR QUANTITATIVE METHOD 1.FOOD ANALYSIS PROCEDURE 1.15 indicate no rancidity. 2.6. Develop the plate in (a) Butanol:Acetic acid:water.2 denote incipient rancidity. 4.1% phloroglucinol solution (in diethyl ether). Allow the solvent front to move up a distance of 10 cm 9. It can be carried out quantitatively as well as quantitatively. Place the plate under UV light in the visualization chamber 11. Shake 5ml of oil and 5 ml chloroform in a stoppered test tube Add 10ml of a 30% solution of trichloroacetic acid (in glacial acetic acid) Add 1ml of 1% solution of phloroglucinol (in glacial acetic acid) Incubate the test tube at 45ºC for 15 min Add 4ml of ethanol and immediately measure the absorbance at 545 nm. Place the beaker into a boiling water bath and evaporate till dryness 5. Transfer the acid layer to a 25 ml beaker 4. Dissolve the residue obtained after evaporation of hydrochloric acid in 1 ml of a mixture of chloroform and acetic acid (9:1) 6. 3.

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