Chemical Structure Of Fats

Fats consist of a wide group of compounds that are generally soluble in organic solvents and generally insoluble in water. Chemically, fats are triglycerides: triesters of glycerol and any of several fatty acids. Fats may be either solid or liquid at room temperature, depending on their structure and composition. Although the words "oils", "fats", and "lipids" are all used to refer to fats, in reality, fat is a subset of lipid.[1] "Oils" is usually used to refer to fats that are liquids at normal room temperature, while "fats" is usually used to refer to fats that are solids at normal room temperature. "Lipids" is used to refer to both liquid and solid fats, along with other related substances, usually in a medical or biochemical context, which are not soluble in water. The word "oil" is also used for any substance that does not mix with water and has a greasy feel, such as petroleum (or crude oil), heating oil, and essential oils, regardless of its chemical structure. fats can be categorized into saturated fats and unsaturated fats. Unsaturated fats can be further divided into cis fats, which are the most common in nature, and trans fats, which are rare in nature but present in partially hydrogenated vegetable oils.
In biochemistry andnutrition, monounsaturatedfats or MUFA (Monounsaturated Fatty Acid) are fatty acids that have one double bond in the fatty acid chain and all of the remainder of the carbon atoms in the chain are single-bonded. By contrast, polyunsaturated fatty acids have more than one double bond.
Fatty acids are long-chained molecules having an alkyl group at one end and a carboxylic acid group at the other end. Fatty acid viscosity (thickness) and temperature increases with decreasing number of double bonds; therefore, monounsaturated fatty acids have a higher melting point than polyunsaturated fatty acids (more double bonds) and a lower melting point than saturated fatty acids (no double bonds). Monounsaturated fatty acids are liquids at room temperature and semisolid or solid when refrigerated.
Polyunsaturated fats are triglycerides in which the hydrocarbon tails constitutes polyunsaturated fatty acids (PUFA) (fatty acids possessing more than a single carbon'carbon double bond).[1][2] Polyunsaturated fat can be found mostly in nuts, seeds, fish, algae, leafy greens, and krill. "Unsaturated" refers to the fact that the molecules contain less than the maximum amount of hydrogen. These materials exist as cis or trans isomers depending on the geometry of the double bond.
Saturated fats have hydrocarbon chains which can be most readily aligned. The hydrocarbon chains in trans fats align more readily than those in cis fats, but less well than those in saturated fats. This means that, in general, the melting points of fats increase from cis to trans unsaturated and then to saturated. See the section on chemical structure of fats for more information.

Saturated fat is fat that consists of triglycerides containing only saturated fatty acids. Saturated fatty acids have no double bonds between the individual carbon atoms of the fatty acid chain. That is, the chain of carbon atoms is fully "saturated" with hydrogen atoms. There are many kinds of naturally occurring saturated fatty acids, which differ mainly in number of carbon atoms, from 3 carbons (propionic acid) to 36 (hexatriacontanoic acid).

Analysis of monounsaturated ( mustard oil ) ,saturated ( coconut oil ) ,polyunsaturated (blendid oil ) fats is done through various tests ,like:
1 ash content
2 acid value
3 free fatty acid
4 peroxide value
5 kries test
6 saponification value
7 unsaponification matter
8 protien content
9 iodine value
10 carbohydrate
11 energy value
12 moisture content
13 colour estimation
14 R I (abbe refractometer)
15 specific gravity ( picnometer )
16 flash point
17 G Cfatty acid profile
18 oil content 'solvent extraction
19 ramcidity test

Experiment: 1

Objective: Extraction of oil and to determine the oil content.
Soxhlets Extraction Unit
Crusher Flat
Glass flasks.
Petroleum Benzene ( 40??C-60??C)
Theory: The solvent used depends on its polarity. As the solvent boils the vapour passes through the condenser and the pure form of the solvent drops on the sample
1. Crush the sample in mortar.
2. Transfer and weigh 5g material to an extraction thimble, avoiding any loss.
3. Place the thimble in an extractor previously fitted.
4. Add sufficient quantity of solvent and affix the condensor and allow through it a current of cold water to flow & heat the extractor so that the action is moderate not violent.
4. Continue the extraction for 8 hours. Then remove the extraction from it's bath, take the extraction thimble, after it has drained out of the extractor & allow to evaporate from it in a current of air.
5. Remove the greater part of solvent in the flask by distillation. Heat the flask in an oven at 103??C for 1 hour to remove solvent. Cool at room temperature & weigh. Repeat the heating process & cool until weight of flask becomes constant.

Experiment: 2
Objective: Extraction of oil from Rotatory Vaccum Evaporator.
Theory: It is used in chemical laboratories for the efficient and gentle removal of solvents from samples by evaporation.
Rotary evaporation is the process of reducing the volume of a solvent by distributing it as a thin film across the interior of a vessel at elevated temperature and reduced pressure. This promotes the rapid removal of excess solvent from less volatile samples. Most rotary evaporators have four major components: heat bath, rotor, condenser, and solvent trap.
After taking out the oil from Extraction unit in flask add petroleum ether more then 1cm then the sample quantity.
Transfer the whole mixture in Rotatory Vaccum Evaporator.
Leave it for half an hour .
Now collect the pure oil free from the other unwanted materials from the flask.

Experiment: 3
Objective: To prepare blended oil i.e. polyunsaturated fats from Rice bran oil and Safflower seed oil.
Theory: Blend of Rice bran oil and Safflower seed oil in a ratio of 70:30 and so it has goodness of both the oil in same. Safflower oil is known to promote cardiovascular health and so does Rice bran oil, also Rice bran is an abundant source of anti-oxidants. The other fact I like is its lower absorption power, which is of high importance to me be it breakfast, or lunch or snack-time or dinner we definitely include so much of oil in our cooking, sometimes its just unavoidable!!
Materials Required:
Glass wares
Stirring strip
Sample oils

Take 70ml of rice bran oil in the flask
In other flask take 30ml of safflower oil.
Mix both of them gently by pouring safflower oil into rice bran.

Experiment: 4
Objective: Determine the moisture content of the given oil samples.

Need and scope: In almost all oil tests natural moisture content of the oil is to be determined. The knowledge of the natural moisture content is essential in all studies of oil mechanics. To sight a few, natural moisture content is used in determining the bearing capacity and settlement. The natural moisture content will give an idea of the state of oil in the field.
Definition: The natural water content also called the natural moisture content is the ratio of the weight of water to the weight of the solids in a given mass of oil. This ratio is usually expressed as percentage.

Materials Required:
Electric oven, maintain the temperature between 105o C to 110 oC.
Balance of sufficient sensitivity.
1.Clean the container with lid dry it and weigh it (W1).
2. Take a specimen of the sample in the container and weigh with lid (W2).
3. Keep the container in the oven with lid removed. Dry the specimen to constant weight maintaining the temperature between 105 oC to 110 oC for a period varying with the type of soil but usually 16 to 24 hours.
4. Record the final constant weight (W3) of the container with dried soil sample. Peat and other organic soils are to be dried at lower temperature (say 600 ) possibly for a longer period.

Experiment: 5
Objective: : Determination of ash content by dry ashing.
Introduction: The ash content is a measure of the total amount of minerals present within a food, whereas the mineral content is a measure of the amount of specific inorganic components present within a food, such as Ca, Na, K and Cl. Determination of the ash and mineral content of foods is important for a number of reasons:
Nutritional labeling. The concentration and type of minerals present must often be stipulated on the label of a food.
Quality. The quality of many foods depends on the concentration and type of minerals they contain, including their taste, appearance, texture and stability.
Microbiological stability. High mineral contents are sometimes used to retard the growth of certain microorganisms.
Nutrition. Some minerals are essential to a healthy diet (e.g., calcium, phosphorous, potassium and sodium) whereas others can be toxic (e.g., lead, mercury, cadmium and aluminum).
Processing. It is often important to know the mineral content of foods during processing because this affects the physicochemical properties of foods

Apparatus Required:
Muffle furnace
Weighing machine

Weight 5gm of sample in the crucible.
Keep the crucibles containing sample in the muffle furnace for atleast 5 hours.
Take out the crucibles after 5 hours and keep them on the desiccator for cooling.
Weight them on the balance and note the readings.

Experiment: 6
Objective: Determination of the fat content.
Introduction: Fat and oil content is an important measurement of nutritional value and product quality for many foodstuffs. In particular, this value is widely used to determine energy content and to calculate the proportion of other components in foods (e.g. carbohydrates). In addition, the fat and oil content may significantly affect the texture, perceived quality and flavor of products. Thus, accurate measurements of the fat and oil content enable the manufacturers to achieve higher standards in
Nutritional characterization and quality control of foodstuffs.

Apparatus Required:
Ethanol, HCL
Weighing balance
Filter paper
Water bath
Fat machine
Fat cup and other glass wares.

Weigh 2gm of sample ie oil in conical flask .
Add 2ml ethanol and 10ml 8N HCL.
Keep the flasks in water bath at about 80'C for half an hour.
Now filter it and wash with hot water until pH becomes 7.
Isolate the filter paper and keep it in the oven for drying.
Dried filter paper is inserted in the thomb of the fat machine for further processing.
Fat cups are adjusted in the fat machine to collect oil.

Experiment: 7
Objective: Colour estimation of the oil samples using Lovibond Tintometer.

Introduction: The Lovibond?? visual and automated colour measurement instruments (spectrophotometers for transmission and reflectance; colorimeters and colour comparators) are synonymous with accuracy in the analysis of liquids and solids including: edible, industrial & fuel oils, chemicals, pharmaceuticals, beverages & foodstuffs.
Operating Principle
The Lovibond?? Tintometer is a visual colorimeter designed to optimise the use of Lovibond?? glass filters. It is arranged with two adjacent fields of view, seen through the viewing tube, so that the product in the sample field and a white reflective surface in the comparison field are observed side by side, suitably illuminated. The Lovibond?? glasses are introduced into the comparison field by a simple system of sliding racks, allowing the user to compare the colour of light which is either transmitted through or reflected from the sample with that transmitted through the glasses. A series of neutral glasses in racks is also supplied; these can be introduced into the sample field to dull the colour of products which are too bright to obtain a good colour match using Lovibond?? Red, Yellow or Blue glasses. The racks are varied until a visual colour match is found for the light from the sample and its colour can then be expressed in Lovibond?? units

Apparatus Required:
Lovibond tintometer

Fill the cuvetts till the brim (mark).
Insert the cuvett one by one and note down the readings carefully.

Experiment: 8
Objective: Determination of specific gravity of sample oils using pycometer.
Introduction: Specific gravity is measured on a scale established by the American Petroleum Institute in units called API degrees (??API); the lower the number of API degrees, the higher the specific gravity of the oil.
Specific gravity is a way of relating the density of an object to the density of water to determine whether or not the object will float. The formula for specific gravity is given below:

If an object's specific gravity is less than one, then the object will float. If the object has a specific gravity of greater than one, it sinks. So dense objects sink in water and less dense objects float.
Apparatus Required:

Weight the pycometer and note it down.
Weight the pycometer completely filled with water.
Weight the pycometer completely filled with oil.
Note the reading carefully for correct calculation.

Experiment: 9
Objective: Determination of acid value for oil samples.
Introduction: In chemistry, acid value (or "neutralization number" or "acid number" or "acidity") is the mass of potassium hydroxide (KOH) in milligrams that is required to neutralize one gram of chemical substance. 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 acid number is used to quantify the amount of acid present.
Formula used:
(56.1??normality ??titre value)/(wt.of sample)

Apparatus Required:
Conical flask 200 ml
Ethyl alcohol
Phenopthelene indicator
0.1N NaOH

Take 2gm of sample in 200ml conical flask.
Add 50ml neutralized hot ethyl alcohol.
Add 3-4 drops of phenopthelene indicator.
Boil for 5 minutes.
Titer it with 0.1N NaOH.
Note down the readings carefully for each sample.

Experiment: 10
Objective: Determine the refractive index of oil samples using refractometer.
Introduction: Ernst Abbe (1840'1905), working for Carl Zeiss AG in Jena, Germany in the late 19th century, was the first to develop a laboratory refractometer. These first instruments had built-in thermometers and required circulating water to control instrument and fluid temperatures. They also had adjustments for eliminating the effects of dispersion and analog scales from which the readings were taken.
In the Abbe' refractometer the liquid sample is sandwiched into a thin layer between an illuminating prism and a refracting prism. The refracting prism is made of a glass with a high refractive index (e.g., 1.75) and the refractometer is designed to be used with samples having a refractive index smaller than that of the refracting prism. A light source is projected through the illuminating prism, the bottom surface of which is ground (i.e., roughened like a ground-glass joint), so each point on this surface can be thought of as generating light rays traveling in all directions. A detector placed on the back side of the refracting prism would show a light and a dark region.

Apparatus Required:

Spread the oil sample carefully on the refractomer.
Note down the readings carefully.

Experiment: 11
Objective: Determine the protein content in oil samples using Kjeldahl method.
Introduction: Proteins are the most amazing group of molecules in the human body. They are incredibly complex chains of smaller molecules called amino acids. These strings of amino acids are then folded into complicated shapes to create millions of critical body components. The DNA double helix is a familiar example of a protein.
The Kjeldahl method or Kjeldahl digestion in analytical chemistry is a method for the quantitative determination of nitrogen in chemical substances developed by Johan Kjeldahl in 1883.
The method consists of heating a substance with sulfuric acid, which decomposes the organic substance by oxidation to liberate the reduced nitrogen as ammonium sulfate. In this step potassium sulfate is added to increase the boiling point of the medium (from 337??C to 373??C) . Chemical decomposition of the sample is complete when the initially very dark-colored medium has become clear and colorless.
The solution is then distilled with a small quantity of sodium hydroxide, which converts the ammonium salt to ammonia. The amount of ammonia present, and thus the amount of nitrogen present in the sample, is determined by back titration. The end of the condenser is dipped into a solution of boric acid. The ammonia reacts with the acid and the remainder of the acid is then titrated with a sodium carbonate solution by way of a methyl orange pH indicator.
Degradation: Sample + H2SO4 ' (NH4)2SO4(aq) + CO2(g) + SO2(g) + H2O(g)
Liberation of ammonia: (NH4)2SO4(aq) + 2NaOH ' Na2SO4(aq) + 2H2O(l) + 2NH3(g)
Capture of ammonia: B(OH)3 + H2O + NH3 ' NH4+ + B(OH)4'
Back-titration: B(OH)3 + H2O + Na2CO3 ' NaHCO3(aq) + NaB(OH)4(aq) + CO2(g) + H2O
Apparatus Required:
Digestion tube
Selenium di oxide
Furnish chamber
Automatic distillation unit.

Add 2gm of sample in digestion tube
Add 7gm potassium sulphate
Add 5mg selenium dioxide
Keep the digestion tube in furnish chamber for 30 minutes for digestion.
Then add 50ml 35% NaOH
Fix Automatic distillation unit
Then titrate it and note down the end point.

Experiment: 12

Objective: Calculation of
Free fatty acid value of the oil samples

Introduction: Free fatty acid: In chemistry, especially biochemistry, a fatty acid is a carboxylic acid with a long aliphatic tail (chain), which is either saturated or unsaturated. Most naturally occurring fatty acids have a chain of an even number of carbon atoms, from 4 to 28. Fatty acids are usually derived from triglycerides orphospholipids. When they are not attached to other molecules, they are known as "free" fatty acids.
A carbohydrate is a large biological molecule, or macromolecule, consisting of carbon, hydrogen, and oxygen atoms, usually with a hydrogen:oxygen atom ratio of 2:1. Carbohydrates, Fats, and Oils have in common with each other because all three of them are lipids that store energy.
The name calorie is used for two units of energy.
The small calorie or gram calorie (symbol: cal) is the approximate amount of energy needed to raise the temperature of one gram of water by one degree Celsius.
The large calorie, kilogram calorie, dietary calorie, nutritionist's calorie, nutritional calorie, Calorie (capital C)] or food calorie (symbol: Cal, equiv: kcal) is approximately the amount of energy needed to raise the temperature of one kilogram of water by one degree Celsius. The large calorie is thus equal to1000 small calories or one kilocalorie (symbol: kcal).
The energy needed to increase the temperature of a given mass of water by 1 ??C depends on the atmospheric pressure and the starting temperature. Accordingly, several different precise definitions of the calorie have been used. The pressure is usually taken to be the standard atmospheric pressure (101.325 kPa).
Formulas used:
Free fatty acid value:
FFA = (Acid value)/(1.99)
CBH = 100 ' (Moisture content + Ash content +Fats + Protein) %
Energy = 4 ( CBH + P) + (9*fat) kcal/100gm

Experiment: 13
Objective: To measure the PV or a number of oil samples, and to evaluate the meaning of the results.
The peroxide value is defined as the amount of peroxide oxygen per 1 kilogram of fat or oil. Traditionally this was expressed in units of milliequivalents, although if we are using SI units then the appropriate option would be in millimoles per kilogram
The peroxide value is determined by measuring the amount of iodine which is formed by the reaction of peroxides (formed in fat or oil) with iodide ion.
2 I- + H2O + ROOH -> ROH + 2OH- + I2
Note that the base produced in this reaction is taken up by the excess of acetic acid present. The iodine liberated is titrated with sodium thiosulphate.
2S2O32- + I2 -> S4O62- + 2 I-
Reagents and solution
1. Acetic Acid - chloroform solution (7.2ml Acetic Acid and 4.8ml Chloroform).
2. Saturated Potassium Iodide solution. Store in the dark.
3. Sodium thiosulfate solution, 0.1N. Commercially available.
4. 1% Starch solution. Commercially available.
5. Distilled or deionized water.
Conduct a blank determination of the reagents.
1. Weigh 2.00 (??0.02)g of sample into a 100 ml glass stoppered Erlenmeyer flask. Record weight to the nearest 0.01g.
2. By graduated cylinder, add 12 ml of the acetic acid - chloroform solution.
3. Swirl the flask until the sample is completely dissolved (careful warming on a hot plate may be necessary).
4. Using 1 ml Mohr pipette, add 0.2 ml of saturated potassium iodide solution.
5. Stopper the flask and swirl the contents of the flask for exactly one minute.
6. Immediately add by graduated cylinder, 12 ml of either distilled or deionized water, stopper and shake vigorously to liberate the iodine from the chloroform layer.
7. Fill the burette with 0.1N sodium thiosulfate.
8. If the starting color of the solution is deep red orange, titrate slowly with mixing until the color lightens. If the solution is initially a light amber color, go to step 9.
9. Using a dispensing device, add 1 ml of starch solution as indicator.
10. Titrate until the blue gray color disappears in the aqueous (upper layer).
11. Accurately record the mls of titrant used to two decimal places.

Experiment: 14
Objective: To perform kreis kerr test, a qualitative test for the presence of aldehydes and ketones in fats and oils, created by its rancidity.
Introduction and principle:The term "rancidity" is used to describe the development of bad flavours and odours in fats and oils. It may result either from hydrolysis of the triacylglycerol present in fats and oils or from oxidation of the unsaturated fatty acids present in the triacylglycerols. The former cause may be detected by an increase in the acid value of the sample .Autooxidation at fatty acid double bonds occurs by reaction with molecular oxygen present in the atmosphere, causing the formation of labile peroxides.
The peroxides formed during autooxidation are unstable and decompose into free radicals .These initiate chain reactions which lead to eventually to decomposition of the fatty acid into various low molecular weight aldehydes and ketones.Aldehydes and ketones react with phloroglucinol developing a red colour.
Materials Required:
1% phloroglucinol. In ether solution.
concentrated hydrochloric acid.
to 1ml of sample add similar amount of conc. Hydrochloric acid.
add 2ml of phloroglucinol solution.
a red colour will develop if the oil is rancid.

Pour 1ml sample in the test tubes.
Add 1ml HCL + 1ml of phloroglucinol
Red color will develop if the oil is rancid; pink if about to rancid; no color if its fine.

Experiment: 15
Objective: To determine the iodine value of fats and oils and thus estimate the unsaturation of the fats and oils.

Principle: Fatty acids react with a halogen [ iodine] resulting in the addition of the halogen at the C=C double bond site. In this reaction, iodine monochloride reacts with the unsaturated bonds to produce a di-halogenated single bond, of which one carbon has bound an atom of iodine.

After the reaction is complete, the amount of iodine that has reacted is determined by adding a solution of potassium iodide to the reaction product.
ICl + KI ---------------> KCl + I2
This causes the remaining unreacted ICl to form molecular iodine. The liberated I2 is then titrated with a standard solution of 0..1N sodium thiosulfate.
I2 + 2 Na2S2O3 -----------------> 2 NaI + Na2S2O4
Materials Required:
' Iodine Monochloride Reagent
' Potassium Iodide
' Standardized 0.1 N Sodium thiosulphate
' 1% Starch indicator solution
' Reagent bottle
' Chloroform
' Fat sample in chloroform
' Iodination flask
' Burette and burette stand with magnetic stirrer
' Glass pipette
' Measuring cylinder
' Distilled water
Arrange all the reagent solutions prepared and the requirements on the table.
Pipette out 10ml of fat sample dissolved in chloroform to an iodination flask labeled as 'TEST".
Add 20ml of Iodine Monochloride reagent in to the flask. Mix the contents in the flask thoroughly.
Then the flask is allowed to stand for a half an hour incubation in dark.
Set up a BLANK in another iodination flask by adding 10ml Chloroform to the flask.
Add to the BLANK, 20ml of Iodine Monochloride reagent and mix the contents in the flask thoroughly.
Incubate the BLANK in dark for 30 minutes.
Mean while, Take out the TEST from incubation after 30 minutes and add 10 ml of potassium iodide solution into the flask.
Rinse the stopper and the sides of the flask using 50 ml distilled water.
Titrate the 'TEST' against standardized sodium thiosulphate solution until a pale straw colour is observed.
Add about 1ml starch indicator into the contents in the flask, a purple colour is observed.
Continue the titration until the color of the solution in the flask turns colourless.
The disappearance of the blue colour is recorded as the end point of the titration.
Similarly, the procedure is repeated for the flask labeled 'Blank'.
Record the endpoint values of the BLANK .
Calculate the iodine number using the equation below:

IV = (12.96(B-S)*Normality of thiosulphate)/(wt.of sample)

Experiment: 16
Objective: To measure oxidation stability of oils and fats using Rancimat Method.
Theory: The Rancimat method is an accelerated aging test. Air is passing through the sample in the reaction vessel at constant elevated temperature. In this process fatty acids are oxidized. At the end of the test volatile, secondary reaction products are formed, which are transported into the measuring vessel by the air stream and absorbed in the measuring solution (deionized water). The continuously recorded electrical conductivity of the measuring solution is increasing due to the absorption of the reaction products. Thus their appearance can be detected. The time until secondary reaction products are detected is called induction time. It characterizes the oxidation stability of oils and fats.
Materials Required:
Rancimat instrument
Deionized water
Glass wares
Weigh the samples in the test tubes provided with the apparatus.
Before putting the sample into the apparatus set the following parameters:
Sample size: Liquid samples: 3.0 ?? 0.1 g
Solid samples: 0.5 ' 1 g
Measuring solution 60 mL
Temperature 80 ' 160 ??C
Gas flow 20 L/h
Evaluation: Induction time
Evaluation sensitivity 1.0
Experiment: 17
Objective: To measure smoke, flash, and fire points of the fat samples.
Theory: The smoke point is the temperature at which the sample begins to smoke when tested under specified conditions.
The flash point is the temperature at which a flash appears at any point on the surface of the sample; volatile gaseous products of combustion are produced rapidly enough to permit ignition.
The fire point is the temperature at which evolution of volatiles proceeds with enough speed to support continuous combustion.
These tests reflect the volatile organic material in oils and fats, especially free fatty acids and residual extraction solvents.
Materials Required:
Cleave land open cup
Glass wares
For smoke point, fill a cleave land open cup with oil or melted fat, secure the thermometer, and place in the cabinet.
Heat the sample and note the temperature at which a thin, continuous stream of blusih smoke is given off.
Flash and fire points are performed similarly, passing a test flame over the sample at 5degree Celcius intervals.

Experiment: 18
Objective: Determination of saponification value.
Theory: The saponification value denotes the weight of potassium hydroxide, expressed as mg, required to saponify completely one gram of sample.
Materials Required:
Flat bottom flask (200 0r 250 ml)
Reflux condenser
Reagents: alcoholic KOH (approx. 0.5N solution in 95% ethyl alcohol; dissolve 35 to 40 gm of KOH pellets in alcohol in minimum quantity of water and dilute with ethyl alcohol to one litre.
HCL 0.5N
1.5-2gm sample + 25ml alc. KOH in the flask
Connect the reflux air condenser to the flask.
Heat the flask on a waterbath for not more then an hour.
Boil gently but steadily until the sample is completely saponified as indicated by absence of any oily matter and appearance of clear solution.
After the flask and condenser have cooled somewhat, wash down inside the condenser with 10ml of hot ethyl alcohol neutral to phenophthelene.
Add about 1ml of phenopthelene indicator and titrate with standard HCl 0.5N
Prepare and conduct a blank determination at the same time.

Experiment: 19
Objective: determination of unsaponifiable matter.

Theory: Unsaponifiable value is defined as the fraction of substances in oils and fats which are not saponified by caustic alkali but is soluble in ordinary fat solvents.

Materials Required:
Alcoholic KOH: Dissolve 70 to 80 gm of KOH in an equal quantity of distilled water and add sufficient aldehyde free ethyl alcohol and make up volume upto 1000ml.
Ethyl alcohol 95%
Petroleum ether
Flat bottom flask (200 0r 250 ml)
Reflux condenser

1.5-2gm sample + 25ml alc. KOH in the flask
Connect the reflux air condenser to the flask.
Heat the flask on a waterbath for not more then an hour.
Boil gently but steadily until the sample is completely saponified as indicated by absence of any oily matter and appearance of clear solution.
After the flask and condenser have cooled somewhat, wash down inside the condenser with 10ml of hot ethyl alcohol neutral to phenophthelene.
Add about 1ml of phenopthelene indicator and titrate with standard HCl 0.5N
Prepare and conduct a blank determination at the same time.

Moisture Content:: Sample Wt. of petriplate Wt. of sample Dry wt.
1. Monounsaturated 47.5196 5.1611 52.6517
2. Saturated 57.3455 5.0483 62.3784
3. Polyunsaturated 58.5033 5.0375 63.5151
Moisture content % = ((Wt.of petriplate + Wt.of sample) ' Dry wt.)/(sample wt.) * 100
Moisture content % = ((47.5196 + 5.1611) ' 52.6517)/5.1611??100 = 0.5618 %
Moisture content % = ((57.3455+5.0483)- 62.3784)/5.0483 ??100 =0.3050 %
Moisture content % = ((58.5033+5.0375)- 63.5151 )/5.0375??100=0.5101%
Ash content: Sample Petriplate wt.(P) Sample wt.(S) Final wt.(M)
1. Coconut 43.5185 5.0356 43.5943
2. Mustard black seeds 75.0037 5.0119 75.2040

Ash % = (100 (M'P))/((P+S)- M)
Ash % = (100(43.5943-43.5185))/((43.5185+5.0356)- 43.5943) = 1.5282%
Ash % = (100( 75.2040-75.0037))/((75.0037+5.0119)- 75.2040)= 4.1628%

Moisture (seeds): Sample Petriplate wt. (P) Sample wt.(S) Final wt.(M)
1. Coconut 51.6626 5.0240 56.4584
2. Mustard black seeds 40.3615 5.0275 45.0180
Table: 3
Moisture % = (100 (P+S)- M)/S
Moisture % = (100 (51.6626+5.0240)-56.4584)/5.0240=4.3431%
Moisture % = (100 ( 40.3615+5.0275)-45.0180)/5.0275=7.3794%
Fat Content: Sample Sample wt (S) Fat cup wt. (F) Final wt.(M)
1. Coconut 2.0111 79.7836 80.9170
2. Mustard seeds 2.0076 81.6225 82.3872
Table: 4
Fat % = (100 ( M-F))/S
Fat % = (100 (80.9170-79.7836))/2.0111 =56.3572%
Fat % = (100 (82.3872-81.6225))/2.0076=38.0902%
Colour Estimation: Sample Readings
1. Mustard oil (monounsaturated) 4R + 33.3Y + 1W
2. Coconut oil (saturated) 0.6Y + 0.1R
3. Blended (polyunsaturated) 1Y
Colour Estimation is Y + 5R = 1
For Mustard oil = 5(4) + 1(33.3) + 1(0) = 53.3 units
For Coconut oil = 5(0.1) + 1(0.6) = 1.1 units
For Blended oil = 1(1) = 1 unit
Specific Gravity: Sample Pycometer/ Blank wt (B) Water wt (W + B) Sample + Blank (S+B)
1. Mustard oil 22.1459 47.4963 45.6133
2. Coconut oil 22.3072 46.6123 44.5089
3. Blended oil 21.8068 47.6440 45.5121
Specific Gravity = ((S+B)- B)/((W+B)-B)
For Mustard oil = (45.6133-22.1459)/(47.4963-22.1459)= 0.9257
For Coconut oil = (44.5089-22.3072)/(46.6123-22.3072)=0.9134
For Blended oil = (45.5121-21.8068)/(47.6440-21.8068)=0.9174
Acid Value: Sample End point Sample wt
1. Monounsaturated (Mustard) 0.5 2.2573
2. Saturated (Coconut) 0.3 2.0570
3. Polyunsaturated (Blended) 0.2 2.0055
Table: 7
Acid Value = (56.1??0.1014??x)/(sample wt)
For monounsaturated fats = 1.2633
For saturated fats = 0.8296
For polyunsaturated fats = 0.5672

Free Fatty Acid:
FFA = (Acid value)/1.99
For monounsaturated fats = 1.2633/1.99=0.6348
For saturated fats = 0.8296/1.99=0.4168
For polyunsaturated = 0.5672/1.99=0.2850
Refractive Index:
Sno Sample RI value (nD)
1. Monounsaturated fats (Mustard) 1.4724
2. Saturated fats (Coconut)
3. Polyunsaturated fats (blended) 1.4737
Table: 8
CBH = 100 ' (Moisture content + Ash content +Fats + Protein) %
For monounsaturated fats ( Black mustard seeds) = 50.3676%
For saturated fats ( Coconut) = 37.7715%

Energy = 4 ( CBH + P) + (9*fat) kcal/100gm
For monounsaturated fats = 5.4428 kcal/gm
For saturated fats = 6.5830 kcal/gm
Peroxide Value:
Sno Sample Sample wt (S) End point
1. Monounsaturated fats 5.0328 1
2. Saturated fats 5.0252 0.2
3. Polyunsaturated fats 5.0242 0.8
Table: 9
Blank value = 0.2
Peroxide value = ((S-B) * Normality * 1000)/(sample wt)
For monounsaturated fats = ((1-0.2)*0.0475*1000)/5.0328=9.6268
For saturated fats= ((0.3-0.2)*0.0475*1000)/5.0252=0.9452
For polyunsaturated fats= ((0.8-0.2)*0.0475*1000)/5.0242=5.6725
Protein value : Sample Initial point Final point
1. Monounsaturated fats 10 9.98
2. Saturated fats 9 8.97
3. Polyunsaturated fats 8 7.98
Table: 10
Protein value of all the sample oils tends to be approximately 0.
Saponifiable value: Sample Sample Wt. (gms) (B-S)
1. Monounsaturated fats 2.0011 25-12.7=12.3
2. Saturated fats 2.0900 25-5.6=19.4
3. Polyunsaturated fats 2.0441 25-11.2=13.8
Table: 11
Blank value is 25.
Saponifiable Value= (56.1(B-S)*Normality(0.5))/(sample wt)

For monounsaturated fats S.V.= 172
For saturated fats S.V.= 260
For polyunsaturated S.V.= 190

Unsaponifiable matter: Samples Sample wt.(gms) (B-S)
1. Monounsaturated fats 2.0011 0.0240
2. Saturated fats 2.0900 0.0313
3. Polyunsaturated fats 2.0441 0.2452
Table: 12
Unsaponifiable matter=(100(B-S))/(sample wt.)

For monounsaturated fats U.V.= 1.20%
For saturated fats U.V.= 1.5%
For polyunsaturated U.V.= 1.2%
Iodine value: Samples Sample wt.(gms) (B-S)
1. Monounsaturated fats 0.4130 68.3
2. Saturated fats 0.4547 5.7
3. Polyunsaturated fats 0.4411 61.3
Table: 13
Iodine Value= (12.69 (B-S)*N)/(sample wt)

For monounsaturated fats U.V.= 100
For saturated fats U.V.= 8
For polyunsaturated U.V.= 90

Kries Test:

Figure: 2
Result indicates that:
Saturated fats are fine as no color change.
Monounsaturated fats are fine as no color change.
Polyunsaturated fats are pinkish therefore it is about to rancid.

Smoke/flash/fire point: Sample Smoke point (degree Celsius) Flash point(degree Celsius) Fire point(degree Celsius)
1. Monounsaturated fats 254 310 360
2. Saturated fats 177 225 280
3. Polyunsaturated fats 266 327 395
Table: 14

Rancimat Test:
For coconut oil:

Figure: 3

For mustard oil:

Figure: 5
For blended:


RFRAC , gave an immense support and opportunity to pursue my training in food technology. This government institute also gave me a number of resources to learn the pathological techniques which help me a lot in my future studies.
In laboratory, performing variety of tests for studying oils and their properties. And majorly I have actually learned and witnessed how government institutes actually work, how labs are maintained and operated, how members are allotted their entries and most importantly how samples are stored and tested along with their record maintainence.
As a form of essential laboratory knowledge, techniques such as furnish chamber, using refractometer, fat machine etc were learnt during the course of training. The practical knowledge gained throughout the training would always remain an important step and a major stepping stone in my learning curve of scientific research.


Alberts, Bruce; Johnson, Alexander; Lewis, Julian; Raff, Martin; Roberts, Keith; Walter, Peter. Molecular Biology of the Cell. New York: Garland Science, 2002, pp. 62, 118-119

Bell, N. et al. (2002). "Methyl iodide: Atmospheric budget and use as a tracer of marine convection in global models". Journal of GeophysicalResearch107: 4340. Bibcode:2002JGRD..107.4340B. doi:10.1029/2001JD001151.
Chemistry And Technology Of Oils And Fats Chemistry And Technology Of Oils And Fats By Dr. M.M. Chakrabarty
Harper, Douglas. "oil". Online Etymology Dictionary
Housecroft, C. E.; Sharpe, A. G. (2008). Inorganic Chemistry (3rd ed.). Prentice Hall. p. 541. ISBN 978-0131755536.
Kvenvolden, Keith A. (2006). "Organic geochemistry ' A retrospective of its first 70 years". Organic Geochemistry 37: 1.doi:10.1016/j.orggeochem.2005.09.001
Lyday, Phyllis A. "Iodine and Iodine Compounds" in Ullmann's Encyclopedia of Industrial Chemistry, 2005, Wiley-VCH, Weinheim, ISBN 978-3-527-30673-2 doi:10.1002/14356007.a14_381 Vol. A14 pp. 382'390.
"The Linear B word e-ra-wo". Palaeolexicon. Word study tool of ancient languages. "e-ra3-wo". Raymoure, K.A. "e-ra-wo". Minoan Linear A & Mycenaean Linear B. Deaditerranean.
Wells, A.F. (1984) Structural Inorganic Chemistry, Oxford: Clarendon Press.ISBN 0-19-855370-6.

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