MATERIAL AND METHODS

MATERIAL AND METHOD Oil samples were analyzed for various parameters set according to the Indian Govt. norms viz. Is, FSSA/PFA and Agmark specification for particular commodity such as acid value, saponification value, rancidity, iodine value, peroxide value etc.

Rice bran oil

Various test and their methods for:

1. Oil sample

2. Oil sample A. Physical properties of oil.

2.1 Moisture content by using hot air oven method. Principle: Moisture content of oils and fats is the loss in mass of the sample on heating at 105+1C under operating conditions specified. Requirement: oil sample, hot air oven, weighing balance and dessicator. Materials and Methodology: Weighed 5-10 g of sample into the moisture disc which has been previously dried. Kept the sample in hot air oven at 105c for two hours. Put the disc in dessicator containing phosphorus pentaoxide and cooled at room temperature. Noted down the weight of the kept sample.
Heated in the oven for a further period of 1 hour, cool and weighed. Repeated this process

until change in weight between two successive observations does not exceed 1 mg.

Calculation:

Where, W= weight of material taken for the test. o W1=loss in weight of material (g) upon drying, and o W= weight of material after drying.

2.1 To determine the Refractive Index in Oils. Principle: The ratio of velocity of light in vacuum to the velocity of light on oil or fat, more generally, it expresses ratio between the sine of angle of incidence to the sine of angle of refraction when light of known wavelength passes from air into oil or fat. Requirements: Refractometer, oil sample Materials and Methodology: Taken the Liquid /melted sample. Adjusted the sample at 400C. Placed a few drop of dried sample on the lower prism, closed the prisms & allowed it to stand for 1-2 minutes. Taken the Refractive Index (RI) of sample. Applied correction factor if reading is taken at the temperature other than 400C. Temperature Corrections: R = R + K (T-T) Where, R = the reading at T0C T = the temperature at which the reading R is taken T = constant temperature i.e. 400C K = constant: 0.000365 for fats & 0.000385 for oils R = Corrected Refractive index 2.2 To determine the Specific Gravity. Principle: Specific gravity is the ratio of the density (mass of a unit volume) of a substance to the density (mass of the same unit volume) of a reference substance. The reference substance is nearly always water for liquids or air for gases.

True specific gravity, can be expressed mathematically as:

Where,

is the density of the sample and

is the density of water.

Requirements: Pycnometer fitted with a thermometer, balance, Water bath maintained at 30+0.20C. Materials and Methodology: Taken pre-cleaned and dried specific gravity bottle with stopper and thermometer already fitted in Specific gravity bottle and then weighed it. Filled the specific gravity bottle with distilled water maintained at 30 0.2 0C, stoppered it and weighed it. Drained out the water and dried the pycnometer and filled with oil at 250C. Stoppered it & immersed in the water bath maintained at 30 0.20C for 30mins. Wiped off the outer layer of Specific Gravity bottle & taken the weight. Calculation:

Where, W1 is weight of empty pycnometer, W2 is Weight of pycnometer filled with distilled water W3 is Weight of pycnometer filled with oil. B. Chemical properties of oil. 2.4 To determine the Acid Value& Free Fatty Acids in Oils. Principle: The acid value (AV) is a common parameter in the specification of fats and oils. It is defined as the weight of KOH in mg needed to neutralize the organic acids present in 1g of fat . Such reaction occurs by the action of lipase enzyme. The acid number is a measure of the amount of carboxylic acid groups in a chemical compound, such as a fatty acid, or in a mixture of compounds. In a typical procedure, a known amount of sample dissolved in organic solvent (often isopropanol), is titrated with a solution of potassium hydroxide with known concentration and with phenolphthalein as a color indicator. The measurement of free fatty acid (FFA) concentration is important in the refining of edible oils (vegetable oils) and in the manufacture of biodiesel. Free fatty acids are present in vegetable oils and are removed in the refining process. Caustic soda solution is mixed with the conditioned oil which neutralizes the acid as well as reacting with the free fatty acids in the oil and forming

soap. The soap and precipitated materials agglomerate and are separated out from the neutralised oil. Requirements: Conical flask 250ml, Burette 50ml, Balance, Standard sodium hydroxide solution 0.1N, Phenolphthalein indicator and Ethyl Alcohol neutral to phenolphthalein. Materials and Methodology: Taken 5 to 10 gm sample in 250 ml conical flask. Added 50-100 ml freshly neutralized ethyl alcohol/rectified spirit. Heated on a hot plate or water bath. When began to boil, cooled a little. Titrated as hot as possible with standard 0.1N sodium hydroxide solution using 1ml of neutralized phenolphthalein indicator until pink colour persisted for at least 10 seconds. 5)Noted down the titer value.

Calcudlation:

Where, V = Volume in ml of standard potassium hydroxide or sodium hydroxide used N = Normality of the potassium hydroxide solution or Sodium hydroxide solution W = Weight in g of the sample

The acidity is frequently expressed as free fatty acid for which calculation shall be percent by weight Acid value = Percent fatty acid x 1.99 N= 0.1

2.5 To determine the Saponification value. Principle: The saponification number is the number of milligrams of potassium hydroxide required to neutralize the fatty acids resulting from the complete hydrolysis of 1g of fat. It gives information concerning the character of the fatty acids of the fat- the longer the

carbon chain; the less acid is liberated per gram of fat hydrolyzed. It is also considered as a measure of the average molecular weight (or chain length) of all the fatty acids present.

Requirements: Alcoholic potassium hydroxide solution(3%).Dissolve 3 gm of potassium hydroxide in 100 ml of ethyl alcohol, Phenolphthalein indicator - Dissolve 1.0 g of phenolphthalein in 100 ml rectified spirit, Standard hydrochloric acid: approximately 0.5N

Materials and Methodology: Weighed 1.5-2.0 gm fat in a 250 ml flat bottom flask. Added 25 ml Ethanolic potassium hydroxide solution. Attached the flask with an air condenser and heated the contents over a water bath for30 minutes until solution became clear as indicated by absence of any oily matter. Cooled the flask, washed down the condenser with 10ml of hot alcohol neutral to phenolphthalein and titrated against 0.5M standard Hydrochloric Acid solution using 4 6 drops of phenolphthalein indicator. Noted down the titer value. Conducted the blank with all reagents. Calculation:

Where, S is Sodium thiosulphate used by the sample, B is Sodium thiosulphate used by blank, N is Normality of sodium thiosulphate used. 2.6 To determine Unsaponifiable matter. Principle: Unsaponifiable matter includes those substances frequently found dissolved in fats and oils that cannot be saponified by the usual caustic treatment, but are soluble in ordinary fat

and oil solvents. Included in this group of compounds are higher aliphatic alcohols, sterols, pigments, and hydrocarbons. Requirments: Flat bottom flask or conical flask with a ground glass joint, 250 ml capacity, Air condenser 1 meter long to fit the flask , Separating funnel, 500 ml capacity Reagents: Alcoholic potassium hydroxide solution: 6% KOH in ethanol, Ethyl alcohol: 50%, Phenolphthalein indicator solution: Dissolve one gram of phenolphthalein in 100 ml of ethyl alcohol, Petroleum ether (40-60): Analytical reagent grade.

Materials and Methodology: Weighed 5 gm oil in a 250 ml flat bottom flask. Added 50 ml of 6% alcoholic potassium hydroxide solution. Attached the flask with an air condenser and heated the contents over a water bath for not more than 1 hour to get complete saponification as indicated by absence of any oily matter and appearance of clear solution. Cooled the flask and transferred the content into a 250 ml separating funnel. Added 50 ml petroleum ether and approx 10 ml distilled water (if necessary). Shaked the Funnel. Allowed the funnel to get complete separation into two layers and decanted the lower layer into another separating funnel. Repeated this process 2 to 3 times more with the help of petroleum ether. Collected petroleum ether in a separating funnel and added 50% 50 ml alcohol. Stoppered the separating funnel and shaked for 2-3 times. Let it to stand until the layers separated. Removed lower layer and added few drops of phenolphthalein indicator. Repeated this process until pink color disappear. Collected the upper portion in weighed flask and evaporated it to dryness on a water

bath. After evaporating petroleum ether put the flask in air to dry.

Weighed until constant weight comes.

Calculation: Weight in g of the free fatty acids in the extract as oleic acid = 0.282*V*N Where, V is Volume in ml of standard sodium hydroxide solution N is Normality of standard sodium hydroxide solution

Where, A is weight in g of the residue, B is weight in g of the free fatty acids in the extract, W is weight in g of the sample 2.7 To determine the Iodine Value in Oils. Principle: The iodine value of an oil/fat is the number of grams of iodine absorbed by100g of the oil/fat, when determined by using Wijs solution. The oil/fat sample taken in carbontetrachloride is treated with a known excess of iodine monochloride solution in glacial acetic (Wijs solution). The excess of iodine monochloride is treated with potassium iodide and the liberated iodine estimated by titration with sodium thiosulfate solution. Requirements: Burette-50ml, Pipette 25ml, Measuring cylinder, Two beaker of 50ml ,Two Iodine Flask of 500ml (Erlenmeyer flask), Balance, Potassium Iodide Solution: 15% , Standard Sodium thio-sulphate solution 0.1N, Starch indicator , Wijs Iodine monochloride Solution Materials and Methodology: Taken 150-200 mg of well mixed, free from impurities & melted sample into a clean dry 500ml iodine flask. Added 10 ml Carbon tetra chloride and dissolved the content. Added 25 ml of Wijs solution and replaced the stopper after wetting with Potassium Iodide Solution. Swirled the flask and allowed standing in dark for 30mins. Taken out the flask from dark and added 10ml of Potassium Iodide Solution and 100 ml of water.

Titrated against standard Sodium thiosulphate solution using 1ml of starch indicator until the blue color formed disappeared after thorough shaking with the stopper on. Noted down the titer value.

Conducted blank test with all reagent & noted down the titer value.

Calculation:

2.8 To determine the turbidity at a certain temperature (Beliers Turbidity Temperature Test). Principle: Oils containing long chain saturated fatty acids give a precipitate at a particular temperature which is specific for the oil when their alcoholic soap solution is treated with dilute acetic acid solution and 70% ethyl alcohol. Requirements: Flat bottom flask , Water Bath (Hot and cold), Thermometer, Alcohol 70%, Alcoholic Potassium hydroxide solution 1.5N or 8.5% , Dilute acetic acid (1:2) and Phenolphthalein indicator Materials and Methodology: Taken 1 ml sample in 100 ml flat bottom flask with the aid of pipette.

Added 5 ml alcoholic Potash (8.5%) and saponified completely by heating over boiling water bath and fitting a condenser. Cooled and added 0.1 ml phenolphthalein indicator. Neutralized by adding diluted acetic acid and added an extra amount of 0.4 ml of Glacial Acetic acid. Added 50 ml of 70% alcohol and mixed. Fitted a thermometer into the flask with a velvet cork such that the bulb touches the liquid only. Heated the flask to 50OC and then cooled it to 40OC

 Then cooled the flask by immersing it in cooling bath so that the temperature drops at roughly 2OC per minute. Noted down the temperature at which distinct turbidity appears. It was allowed to cool further so that precipitate is formed. Dissolved the precipitate by gently heating the content to 50OC.

2.9 To determine the presence of Essential Oil in Oils. Principle: The pungency of mustard oil is due to the presence of allyl isothiocyanate. Allyl isothiocyanate (AITC) is the organo sulfur compound with the formula CH2CHCH2NCS. This colorless oil is responsible for the pungent taste of mustard .This pungency and the lachrymatory effect of AITC is mediated through the TRPA1 and TRPV1 ion channels. It is slightly soluble in water, but well soluble in most organic solvents. Requirements: Balance, Soxhlet Extraction Unit, Distillation Flask, Volumetric Flask, Sample, Distilled Water, Rectified Spirit, Silver Nitrate,Ammonia Solution (10%), Nitric Acid, Ferric Ammonium Sulphate Indicator and Ammonium thiocyanate. Materials and Methodology: Weighed 5gm oil in a distillation flask. Added 25ml rectified spirit and 200ml distilled water in a distillation flask. Carefully added 10ml Ammonium hydroxide and 25 ml silver nitrate to the distillation flask. Made up the final volume 200ml with steam. Left it for overnight for reaction to occurred. Then, filtered it through Whattman filter paper no 1. Taken 100 ml of it in another conical flask. Carried out the same with blank test as described above. Added 6ml nitric acid and few drops of ferric ammonium sulphate indicator. Titrated it against N/10 ammonium thiocyanate. Noted the readings of ammonium thiocyanate used in sample and blank.

Calculation:

Where, B is Titer value of blank used S is Titer value of sample used W is Weight of sample taken (g) 2.10 To determine the Peroxide Value in Oils. Principle: The peroxide value of an oil or fat is used as a measurement of the extent to which rancidity reactions have occurred during storage. Peroxide Value is one of the most widely used tests for oxidative rancidity in oils and fats, peroxide value is a measure of the concentration of peroxides and hydro peroxides formed in the initial stages of lipid oxidation. Mill equivalents of peroxide per kg of fat are measured by titration with iodide ion. Requirements: Pipette- 1 ml, Conical Flask-Glass stoppered, 250 ml, Balance, Acetic acid, Chloroform, Distilled Water, Sodium thiosulphate, Starch Solution and Potassium iodide Solution. Materials and Methodology: o Weighed 5g sample of oil/ fat in a 250ml stopper conical flask. o Added 30ml of Acetic Acid: Chloroform (3:2) solution, swirled the flask until the sample was dissolved. o Added 0.5 ml of saturated potassium iodide solution stoppered the flask. o Kept the flask in the dark for 1 to 5min with occasional shaking and added 30ml of distilled water and added 0.5ml starch indicator. o Titrated against 0.1 N sodium thiosulphate solution with constant and vigorous shaking until blue Color disappeared. o If titer value is more than 0.5ml, repeated the analysis using 0.01N Sodium Thiosulphate Solution. o Conducted blank determination of the reagents in the same way.

Calculation:

Where, S is Sodium thiosulphate used by the sample B is Sodium thiosulphate used by blank N is Normality of sodium thiosulphate

2.11 To Determine the presence of Mineral oil (By TLC Method). Principle: Being non-polar, mineral oils give faster moving spots on thin layer chromatographic plates, than the triglycerides. Materials and Methodology: Taken 1 ml oil and added 10 ml chloroform in a beaker. Spotted it on TLC plate using a capillary tube. Allowed to dried and placed the plate in a developing tank containing petroleum ether. Covered the tank and allowed the solvent to travel for 5-8 cm from the origin. Removed the plate from the tank, dried in air, sprayed with the fluorescent solution and Viewed under UV light. Interpretation: Appearance of a yellow fluorescent spot on the solvent front indicates the presence of mineral oil.

2.12 To determine the presence of argemone oil. Principle: Argemone oil is extracted from argemone seeds. It is mixed with mustard oil and sesame oil to increase their quantity. Consumption of this oil leads to health disorders among children. Argemone (Argemone maxicana L.), yellow poppy, is a wild herb, which grows in mustard field and bears capsules full of brown black seeds. Because of its resemblance with black mustard, it is often used as an adulterant. The oil is reported to cause glaucoma, dropsy and sometimes total blindness due to the presence of alkaloids namely, sangunarine and

dihydrosanguarine. 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. Requirments: 1. Apparatus: TLC plates coated with silica gel or precoated readymade plates cut to suitable size, Ultraviolet lamp, Pear-shaped flask, Hot water bath, Separating funnel, Beaker 2.Reagents: Solvent mixture (mobile phase), Butanol: Acetic acid: water 70:20:10 (v/v), Hexane or Heptane: Acetone 60:40 (v/v), Diethyl ether, Conc. Hydrochloric acid, Chloroform: Acetic acid (90:10 v/v) mixture, Standard Argemone oil extract Materials and Methodology: Taken 5ml oil and 5 ml diethyl ether. Added 5 ml conc HCl in a pear shaped flask. Shaked well for 5 minutes and put on a water bath to separate acid layer for sometimes. Transferred the whole contents in a separating funnel to separate acid layer. Draw out the acid layer in porcelain dish & evaporate it on a boiling water bath till dryness. Dissolved the residue in 1 ml chloroform & acetic Acid. Spotted the solution on a TLC plate with the help of Capillarity tube and dried it . Developed the plate in solvent mixture & allow the solvent front to move up to a distance of 10 cm. Removed the plate, dry at room temperature and viewed under UV lamp. Appearance of bright yellow fluorescent spot having Rf (Representative factor) same as standard argemone will confirm the presence of argemone oil.

Calculation: Rf Value= Distance travelled by Sample / Distance travelled by Solvent Interpretation: Standard Rf value of Adulterated Mustard Oil is 0.17 for Sangunarine and 0.31 for dihydro Sangunarine.

2.13 To determine the presence of castor oil. Principle: Castor oil is a vegetable oil obtained from the castor bean (technically castor seed as the castor plant, Ricinus communis (Euphorbiacea), is not a member of the bean family). Castor oil is a colorless to very pale yellow liquid with mild or no odor or taste. Its boiling point is 313 C (595 F) and its density is 961 kg/m3.It is a triglyceride in which approximately 90 percent of

fatty acid chains are ricinoleic acid. Oleic and linoleic acids are the other significant components Triricinolein a characteristic and predominate triglyceride component of castor oil is separated on silica gel TLC and visualized by iodine vapors. Requirements: 1. Apparatus: TLC plates, TLC developing chamber, Visualization tank (Iodine chamber), Test tube and Beaker. 2. Reagents: Chloroform and Hexane: Diethyl ether: Glacial acetic acid (60%:40%:2% (V/V)) Materials and Methodology:

Taken 1 ml oil in test tube and added 10 ml Chloroform. Shaked vigorously for one minute. Added mixture of Hexane: Diethyl ether: Glacial Acetic acid (60%:40%:2%(V/V)) into TLC chamber. Spotted few drops of above prepared sample solution on TLC plate and developed in developing tank containing Hexane: Solvent ether (1:1) up to 10 cm. Air dried the plate and put in iodine chamber. Occurrence of a spot at Rf of about 0.25 shows presence of castor oil.

2.14 Test for presence of Sesame Oil (Baudouin Test). Principle: The development of pink color with furfural solution in the presence of hydrochloric acid indicates the presence of sesame oil. The color is produced on account of reaction with sesamolin present in sesame oil. Requirements: Measuring cylinder, Hydrochloric acid (concentrated) and Furfural solution. Materials and Methodology: Taken 5ml of the oil in a 25ml test tube provided with a glass stopper. Added 5 ml of conc. hydrochloric acid and 0.4 ml of furfural solution. Inserted the glass stopper and shaked vigorously for two minutes. The development of a pink or red color in the lower acid layer indicates presence of sesame oil. Confirmed by adding 5 ml of water and shaking again. If the color in acid layer persists, sesame oil is present, if the color disappears, it is absent.

2.15 To determine the color of oil

Principle: The method determines the color of oils by comparison with Lovibond glasses of known color characteristics.The color is expressed as sum total of yellow and red slides used to match the color of oils in cells of specified size in the Lovibond tintometer. Apparatus: Lovibond tintometer, glass cells (cell size 0.25 inch, 0.5 inch, 1.0inch or 5.25 inch as required.). Materials and Methodology: Melt the sample if it is nt already liquid and filter the oil through a filter paper to remove any impurities and traces of moisture. Make sure sample is absolutely clear and free from turbidity. Clean the glass cell of desired size with carbon tetrachloride and allow it to dry. Fill it with the oil and place the cell in position in the tintometer. Match the color with yellow , red and blue colours. Report the color in terms of lovibond unit as follows: Color reading in a cell = aY+ 5bR Or Color reading in a cell =aY+10bR (for cotton seed oil) Where, a= sum total of the various yellow slides (Y) used. b= sum total of the various red slides (R) used. Y+5R are the mode of expressing the color of light colored oils and Y+10R is for colored oils. the dark

Although the yellow and the red slides required to match the color shade of oil in a tintometer are assessed separately, it is found that to a certain extent the slides are compensatory. Consequently different work may report different values for the yellow and red units of the oil exmined at different times. To obviate such personal errors a composite factor is used for cheching the color comprising the sum total of yellow(Y) units and 5 or 10 times the total of red units are specified for the oil or for the fat. 2.16 To determine the flash and smoke point of the oil. Principle: The method determines the temperature at which the sample will flash when a test flame is applied under the condition specified for the test. Apparatus: Pesky-martens closed cup flash tester, thermometer. Materials and Methodology: a) FLASH POINT: Fill the cup with the oil up to the mark in the cup. Place the lid on and properly engage the heating devices. Insert the thermometer, light the test flame. Heat the sample to that the temperature increase is about 5C to 6C per min. During the heating, turn the stirring device from 1 to 2 revolutions per second. At every 5C rise in temp. , discontinue stirring and apply the test flame by opening the device which controls the shutter and lowers and test flame into the shutter opening. Lower the test flame for .5 sec and quickly return to the raised position. As soon as the test flame has been returned to raise position

resume stirring. The flash point is the temp. indicated by the thermometer at the time of flame application that causes a distinct flash in the interior of the cup. 2.17 To determine the presence of Linseed oil (Hexabromide test). Principle: The formation of the 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. Requirements: Boiling tubes, ice water bath. Reagents: Chloroform, liquid bromine and diethyl ether. Materials and Methodology: Pipette 1ml of oil into boiling tube( wide mouthed 100ml capacity). Add 5 ml of Chloroform and add about 1ml of bromine drop wise till the mix. Becomes deep red in color and cool the test tube in an ice water bath. Add about 1.5ml of rectified sprit drop wise while shaking the mix. Until the precipitate which is formed just dissolves and then 10ml diethyl ether. Mix the contents and placed the tube in ice water bath for 30 min. Appearance of ppt indicates the presence of linseed oil. 2.18 To determine the presence of palm oil(Cloud point). Principle: The cloud point is that temp. at which under the conditions of this test a cloud is induced in the sample caused by the first stage of crystallization. Requirements: oil sample bottle, 115 ml ( 4oz), thermometer, water bath and chipped ice. Temp. of the water bath shall not be less than 2C and not more than 5C the cloud point. Materials and Methodology: Heat 60-75 grams of sample to 130C immediately before performing the test. Pour 45 ml of the heated oil into an oil sample bottle. Place the bottle in a water bath. Begin to cool the bottle in the water bath, stirring enough using the thermometer to keep the temp. uniform. When the sample has reached a temp. of 10C above the cloud point, begin stirring steadily and rapidly in a circular motion so as to prevent solidification of fat crystals on the sides or bottom of the bottle. From this point on do not remove the thermometer from the sample, since to do so may introduce air bubbles which will interfere with the test. The test bottle is maintained in such a position that the upper levels of the sample in the bottle and the water in the bath are about the same. Remove the bottle from the bath and read the temp. the cloud point is the temp. at which that portion of the thermometer immersed in the oil is no longer visible when viewed horizontally through the bottle. 2.19 To determine calcium content from oil Apparatus: Muffle furnace, volumetric flask Reagents: HCl dilute to 100ml Ammonium Hydroxide 50%

Dil. Ammonium hydroxide solution Conc. H2SO4 Standard potassium permanganate solution Procedure: Take 3g of material into a silica dish carefully & continue the ashing in a muffle furnace at a temperature not more than 450oC until the ash is white or almost so , cool the ash moisten with a few millimeters of distilled water & add 3ml of conc. HCl drop by drop. Evaporate to dryness on a water bath & continue heating for 1 hour to render silica insoluble moisten the residue with 20 ml distilled water & add about 2 to 3 ml of conc. HCl. Heat on water bath & filter through medium filter paper into a volumetric flask, wash filter paper thoroughly with hot water cools the filtrate & make it up to volume shake thoroughly. Transfer 25ml of aliquot of sodium prepared as above to a 500ml beaker, dil. to about 100ml with water and add 2 drops of methyl red. Add ammonium hydroxide solution drop wise till a brownish orange color is obtained. Add 2 drops of HCl so that the color of the solution is pink, dil. to about 150 ml, bring to boil add slowly with constant stirring, 10ml hot ammonium oxalate solution. If red color of solution changes to yellow or orange, add HCl drop wise until color again changes to pink. Leave over night and allow the ppt. to settle down. Filter the supernatant liquid through, filter paper and wash the ppt. with dil. ammonium hydroxide. Place the paper with ppt. in a beaker were the ppt. was carried and add a mixture of 125ml of water and 5 ml of conc. H2SO4. Heat to 70-90oC and titrate with standard potassium permanganate solution until the first slight pink color is obtained. Calculation:

A= Volume of KMnO4 solution in ml, required in titration. N= Normality of standard KMnO4 solution M= Moisture percentage M= Mass of material taken for test in g.

2.20 To determine phosphorus content from oil Apparatus: kjeldahl flask

Reagents: Conc.HNO3 98% pure Nitric Acid (1:1)with distill water Ammonium molybdate stock solution Nitric acid solution Potassium Nitrate solution Standard Nitric acid solution Procedure: Take a10ml aliquot of the prepared solution as taken for calcium , convert in 150 ml beaker. In a dry beaker , prepare ammonium molybdate stock solution & add 10 ml of conc. HNO3 7 whirl the beaker during addition. Pour the freshly prepared clear liquid quickly into the beaker containing the aliquot & stir. All the ppt. to stand overnight & then filter through whatman filter paper no. 42 over an ordinary funnel as far as possible only the supernatant liquid is padssed through the filter paper , retained ppt. in washed twice with dil. HNO 3 &then with potassium nitrate solution until the washing are free from acid. Acidity is checked by collecting sufficient filtrate in a test tube to which a few drops of phenolphthalein indicator solution & 1 drop of the standard sodium hydroxide solution till pink color appears. Transfer the ppt. with beaker in which ppt. was carried out. Add sufficient quantity of standard NaOH solution from a burette just sufficient to dissolve the ppt. & then 5 ml in excess. See that no yellow ppt. sticks to the filter paper. Note the total volume of standards NaOH added. Add about 10 drops of phenolphthalein indicator & titrate the excess alkali with standard nitric acid. Calculation:

A=Volume of standard NaOH solution in ml N1=Normality of standard NaOH solution B= Volume of standard nitric acid used to neutralize the excess of alkali in ml N2=Normality of standard nitric acid solution M= Moisture percentage m= Mass of material taken for test in g.

2.21 To determine fatty acid composition of oils by Gas Liquid Chromatography. Principle: The methyl esters are formed using boron trifloride or methanol and alkali and separated by gas liquid chromatography using a flame ionization detector. The pattern of methyl esters can be compared with authentic oils for identification. Requirements: 1. Gas liquid chromatography with the following characteristics a) Injection system heated to a temperature of 20 50 0 C higher than the column. b) Oven capable of heating the column to at least 220 0 C and maintaining the temperature to within 1 0 C. If temperature programming is to be employed, twin columns are recommended. c) Packed column - may be glass or stainless steel. However glass is preferred as steel may decompose polyunsaturated fatty acids having more than 3 double bonds. Some successful column packings with column length, internal diameter and operating temperature are as follows: i) 12- 15 % ethylene glycol succinate on 100 / 120 mesh gas chrom P (2m x 4 mm, at 180 0 C ) ii) 2- 10 % Apizon L on 80/ 100 mesh Chromosorb W or Celite( 2 m x 4 mm at 220 0 C ) iii) 10 % Butan-1-4 diol succinate on 80 / 100 mesh Chromosorb W or celite ( 2 m x 4 mm at 175 0 C) iv) 3 % SE 30 on 100 / 120 mesh Chromosorb G silanised ( 2m x 3mm at 190 0 C) Condition the newly prepared column by disconnecting the detector and heating the column in the oven to the normal operating temperature for 16 hours while running the carrier gas at a rate of 20 60 ml/ min. v) Detector Flame ionization detector capable of being heated to a temperature above that of the column. 2) Syringe 10 l graduated in 1/10th of a microlitre. 3) Recorder electronic with high precision with rate of response below 1.5second, width of paper 25cm, paper speed 25-150 cm / hr 4) Integrator or calculator for rapid and accurate calculations. 5) Reflux condenser 6) Graduated pipette 10 ml 7) Test tubes with ground stoppers.

8) 250 ml Separating funnels. Reagents (1)Carrier Gas Inert gas ( nitrogen , helium , argon) thoroughly dried and containing less than 10 mg / kg of oxygen. (2)Auxillary gas Hydrogen 99.9 % minimum purity. Free from organic impurities, air or oxygen. (3) Oxygen gas for the flame ionization in FID. (4)Heptane- Chromatographic quality.

For preparation of methyl esters This involves methyl esterification of the fatty acids in an alkaline medium and is suitable for neutral oils and fats with an acid value less than 2. Chemicals used:(1) Methanolic potassium hydroxide solution approx 1 N. Dissolve 5.6 gm Potassium hydroxide in 100 ml of methanol containing not more than 0.5% m/m water ( anhydrous methanol) (2) Heptane chromatographic quality. (3) Nitrogen, containing not more than 0.5 mg / Kg of oxygen.

Materials And Methodology If the oil includes fatty acids containing more than 2 double bonds, it was advisable to purge the air from the methanol and the flask by passing a stream of nitrogen into the methanol for a few minutes. Transferred about 4 gm of clear sample oil into a 100 ml round bottomed or conical flask. Add about 10 ml of n-heptane and mix it thoroughly over a vortex mixer for 4-5 times. Added 5 ml of methanolic Pot. hydroxide solution and again mix it thoroughly over a vortex for 8-9 times by constant heating it over a magnetic stirrer. The solution should become clear (5-10 minutes). The esters pass into the upper heptane layer. Separated it in a beaker and heat the ester till a dense light yellow solution is obtained. Dilute it with n-heptane and ester is ready for injection into the GLC injector port.

Work Instructions for Gas Chromatography Reader:-

1. Switch on the instrument by pressing the power switch located at the frontside of instrument. 2. Open the Nitrogen cylinder (top black) valve till the meter on the front panel of the GC shows 4kg/cm square 3. The Display panel infront of the GC displays the name of the instrument. 4. Then from the number plate first load switch is pressed, followed by pressing the previously specified number (for a specific run). After this run switch is pressed. 5. Then we wait until the GC gets to the specified temperature (usually takes anything between 25-30 mins) 6. The GC then displays READY (TEMPERATURE). 7. The hydrogen gas valve(red cylinder) is opened till it reaches 2.5 kg/cm square followed by the oxygen gas valve (grey cylinder) till it reaches 2 kg/cm square 8. From the oxygen knob in front of the panel the oxygen reading is brought to zero. 9. The FID is ignited using the electrical device provided above the GC. During this process oxygen is slowly released using the knob provided in front of the GC. It is checked whether the FiD has been ignited by using a test tube and checking the moisture. Once it is ignited the oxygen level is again brought to 2 kg/ cm square. The Software (NetWin) is opened and the run screen is loaded. 10. From The Run menu first the milli voltage is checked and is adjusted to 0.56-0.57 mv 11. Then 0.2 micro litre of heptane is injected and the run button is pressed which is at the top of the GC. This is followed by clicking the run on the software. ( This is the washing step) 12. After the washing step is over the GC is ready for sample analysis. 13. After sample analysis is over then step 12 is followed once more. 14. After this the F2 button is pressed from the number panel for starting the cooling sequence. 15. The valves of oxygen & Hydrogen cylinders are closed. 16. When the temperature of the panel in the GC shows anything less than 40 degree the nitrogen cylinders valve is closed. 17. The front panel switch is then turned off.