INDEX

Determination of peroxide value in oil

EXPERIMENT NO 1 and fat by iodometric titration.

Determination of moisture and volatile

EXPERIMENT NO 2 matter by air oven method

Determination of iodine value in lipid

EXPERIMENT NO 3

Determination of saponification value

EXPERIMENT NO 4

Measurement of free fatty acid or acid

EXPERIMENT NO 5 value in lipid

Chlorophyll pigment in crude vegetable

oil
EXPERIMENT NO 6

Determination of slip melting point

EXPERIMENT NO 7

Insoluble Impurities In Oil And Fats

EXPERIMENT NO 8

Determination of specific gravity of oil.

EXPERIMENT NO 9

1
 EXPERIMENT NO 01
OBJECT
Determination of peroxide value in oil and fat by iodometric titration.

THEORY
IODOMETRY
The term “iodometry” describes the type of titration that uses a standardised sodium thiosulfate
solution as the titrant, one of the few stable reducing agents where oxidisation of air is
concerned. Iodometry is used to determine the concentration of oxidising agents through an
indirect process involving iodine as the intermediary. In the presence of iodine, the thiosulphate
ions oxidise quantitatively to the tetrathionate ions.

To determine the concentration of the oxidising agents, an unknown excess of potassium iodide
solution is added to the weakly acid solution. The iodine, which is stoichiometrically released
after reduction of the analyte, is then titrated with a standard sodium thiosulphate solution
(Na2S2O3)

INDICATOR IN IODINE/THIOSULPHATE TITRATION

Titration involving iodine commonly uses a starch suspension as indicator. This suspension is a
watery solution of starch with a few drops of bactericide added to prevent decomposition, as this
would stop the starch behaving as an indicator.

Once the bond between the iodine (I2) and the helical chain of beta-amylose is formed it turns an
intense blue.

IMPORTANT CONSIDERATIONS
Iodometric titration needs to be done in a weak acid environment which is why we need to
remember that:

1. The iodine solution used needs to be at pH < 8.5 because at a base pH iodine
disproportionates (a particular kind of oxidoreduction reaction where one substance
partly oxidises and partly reduces);
2. Sodium thiosulphate needs a neutral or weak acid environment to oxidise with
tetrathionate (in an alkaline solution we would get sulphate oxidation);
3. In a strong acid environment thiosulphate decomposes to S2;
4. In acid environments the iodide is oxidised to iodine as in the reaction below:

O2 + 4I- + 4H+ ↔2I2 + 2H2O

2
OXIDATIVE PROCESS
 Initiation
in the presence of a catalyst, alkyl
radicals (L·) form and these tend to
accumulate.
 Propagation
the alkyl radicals which have
formed react with atmospheric
oxygen to form a peroxyl radical
(LOO· ). This radical can react with
another available hydrogen atom
(LH) to form another free radical
(L·) and a hydroperoxide (LOOH).
 Termination
hydroperoxides, which are highly
unstable chemicals, decompose to Reaction of radicals responsible for formation of
produce additional free radicals and /or hydroperoxides in edible fats and oils. LH is a
monosaturated or polyunsaturated acid
secondary oxidation products which
accumulate and so increase the rancidity of oil.

WHAT IS OIL OXIDATION?

Oil oxidation is an undesirable series of chemical reactions involving oxygen that degrades the
quality of an oil. Oxidation eventually produces rancidity in oil, with accompanying off flavours
and smells. All oil is in a state of oxidation - you cannot stop it completely - but there are ways to
reduce it. Attempts should therefore be made to reduce oxidation at each stage of oil
manufacture.

Oxidation is not one single reaction, but a complex series of reactions. When oil oxidizes it
produces a series of breakdown products in stages,

 starting with primary oxidation products (peroxides, dienes, free fatty acids),
 then secondary products (carbonyls, aldehydes, trienes)
 and finally tertiary products.

Oxidation progresses at different rates depending on factors such as

 temperature,
 light,
 availability of oxygen,
 and the presence of moisture and metals (such as iron).

3
 The type of oil also influences the rate of oxidation. Marine oils (including fish, mussel) are
highly susceptible to oxidation due to the large number of polyunsaturated fatty acids (PUFA)
they contain. These unsaturated fatty acids have reactive double bonds between their carbon
atoms, whereas saturated fats have no double bonds so they oxidise more slowly.

HOW DO WE MEASURE OXIDATION?

Measuring oxidation involves testing for the primary and secondary breakdown products. The
most common test is peroxide value (PV). However, very rancid oils can have a reduced PV
therefore the anisidine value (AV) and a Totox value are used to show the whole oxidation story.
Other measurements of oxidation are the acid value (free fatty acid FFA), thiobarbituric acid
value (TBA) and iodine value (IV). Head space volatiles (smell) can also be tested using
artificial nose technologies.

PEROXIDE VALUE (PV)

 BACKGROUND

Primary oxidation processes in oil mainly form hydroperoxides, which are measured by the PV.
In general, the lower the PV, the better the quality of the oil. However PV decreases as
secondary oxidation products appear. Most customers will require a PV of less than 10 in marine
oils, but PV may need to be as low as 2, depending on the market.

The PV test is a good way to measure the amount of primary oxidation products in fresh oils.
Oils with significant levels of peroxides may still be odourless if secondary oxidation has not
begun. If oxidation is more advanced, the PV may be relatively low but the oil will be obviously
rancid.

 DEFINITION
The peroxide value (PV) is the concentration of hydroperoxide, the primary oxidation products
present in the sample.

Peroxides are the primary oxidized products produced, which on further oxidation would
degrade to aldehydes, ketones, esters, etc., which are the secondary oxidized products.

 PRINCIPLE
The principle involves peroxides liberating iodine from potassium iodide, i.e.

ROOH+KI→ROH+KOH+I2

The amount of ROOH is then determined by measuring the amount of iodine formed, which is
done by titration with sodium thiosulfate and using a starch indicator:

4
 I2+starch+2Na2S2O3(blue)→2NaI+starch+Na2S4O6(colorless)

The amount of peroxides is calculated back by the amount of sodium thiosulfate (Na2S4O6)
consumed. It is expressed as peroxide value (PV) in units of milli-equivalents (meq) peroxide per
1 kg of fat extracted from the food. A general rule is that PV should not be above 10–20 meq/kg
fat to avoid rancidity flavor

IMPORTANCE OF DETERMINING PEROXIDE VALUE

The peroxide value (PV) is a very important characteristic of lipid quality. The assessment of
hydroperoxides provides an estimate of the overall oxidation status for lipids and lipid-
containing foods especially in the primary phase of oxidation, generally known as the induction
period.

From this value, the propagation step of the free radical chain mechanism and the accumulation
of hydroperoxides can be followed. However, it is not possible to use the peroxide value alone to
judge the quality of edible oils, because hydroperoxides decompose during storage. This
decomposition can take place faster than the formation of new hydroperoxides, depending on
certain storage conditions such as temperature, light or metal traces. Although the oil has already
been damaged by oxidation, and higher levels of degradation products have already formed, the
speed of hydroperoxide decomposition can result in falsely low levels of these compounds. In
this instance, the peroxide value tells us nothing about the real quality of the product. In order to
avoid misinterpreting peroxide values, it is necessary to know the history of the sample.
However, the peroxide value is a suitable parameter for measuring the deterioration of quality
over time. After the induction period, during which the peroxide value increases slowly, a steep
increase indicates that the oil has gone bad.

In general, the aim of oil production should be to produce oils with peroxide values as low as
possible, without the formation of secondary reaction products. A higher peroxide value at the
beginning of the storage period has a negative effect on the storage stability of the oil. For
refined oils, producers should aim for a peroxide value below 1, better 0.5 meq O2/kg oil, while
the peroxide value for virgin oils can be higher, up to 3 meq O2/kg oil.

MATERIALS
 Oil or lipid extract sample
 3:2 (v/v) acetic acid/ chloroform solution
 Saturated potassium iodide solution
 Sodium thiosulfate 0.1 N solution
 1%(w/v) starch indicator solution
 250mL glass-stopperedErlenmeyer flasks
 10 or 25 mL graduated glass burette `

5
 PROCEDURE
Accurately weigh 5.00 ± 0.005 g oilinto a 250ml glass stoppered Erlenmeyerflask Or use table
according to expected PV. Each sample should be assayed atleast in duplicate.

Add 30ml of 3:2 acetic acid/chloroform solution and swirl flask until oil is dissolved.

Add 0.5ml saturated potassium iodide solution to flask.

Allow solution to stand exactly 1 min with occasional shaking and then add 30 ml distilled water.

Using a 10 or 25 ml biurette ,gradually add standardized 0.1 N sodium thiosulfate to flask to titrate
solution until the yellow color has almost disappeared. Shake constantly and vigorously.

Add 0.5ml of 1% starch indicator solution and continue titration, shaking flask vigorously near the end point to
liberate all iodine from the chloroform layer. Add sodium thiosulfate solution dropwise until the violet color
disappears. Record the total added sodium thiosulfate volume.

Conduct a blank determination of the reagents by following steps 2 to 7 (i-e without the addition of oil
or lipid extract sample) The blank titration must not exceed o.i ml sodium thiosulfate

OBSERVATION AND CALCULATION

SODIUM PV VALUE
STANDARD
SAMPLE WEIGHT (g) THIOSULFATE (meq active
DEVIATION
(ml) O2/kg)
Blank
Sample 1
Sample 2
Sample 3

PV= [(S-B)xNx1000]/W
Where S is the volume (ml) of sodium thiosulfate required to titrate the sample

B is the volume (ml) of sodium

thiosulfate required for the blank

6
N is the calculated normality of the standardized sodium thiosulfate solution

W is the weight of the sample (g)

From this equation, the PV is expressed as meq active oxygen/kg sample and is equal to mmol
active oxygen/ 2kg sample.

DISCUSSION
1. Why iodine is used in hydroperoxide measurements?
The iodine can be detected by its colour. The detection of the iodine can be enhanced by the
addition of starch solution.

Iodine dissolves in the iodide-containing solution to give triiodide ions, which have a dark brown
color. The triiodide ion solution is then titrated against standard thiosulfate solution to give
iodide again using starch indicator:

2. How does starch indicate iodine?

When starch is mixed with iodine in water, an intensely colored starch/iodine complex is formed.
Many of the details of the reaction are still unknown. But it seems that the iodine (in the form of
I5- ions) gets stuck in the coils of beta amylose molecules (beta amylose is a soluble starch). The
starch forces the iodine atoms into a linear arrangement in the central groove of the amylose coil.
There is some transfer of charge between the starch and the iodine. That changes the way
electrons are confined, and so, changes spacing of the energy levels. The iodine/starch complex
has energy level spacings that are just so for absorbing visible light- giving the complex its
intense blue color.

The complex is very useful for indicating redox titrations that involve iodine because the color
change is very sharp. It can also be used as a general redox indicator: when there is excess
oxidizing agent, the complex is blue; when there is excess reducing agent, the I5- breaks up into
iodine and iodide and the color disappears.

3. Why do we not use starch in the beginning of iodometric titration?

The starch indicator is added to the solution near the end of the titration, at the point where dilute
iodine imparts a pale yellow color to the solution.

There are two reasons why the indicator is not added at the beginning of the titration when the
iodine concentration is high.

 First, a diffuse endpoint would result from the slow dissociation of the starch-iodine
complex if a large amount of iodine were absorbed in the starch.

7
  Second, iodometric titrations are carried out in strongly acid media, a situation that
promotes the reaction between oxidizing agents and iodide. Unfortunately starch has a
tendency to hydrolyze (decompose) in acidic media, destroying its indicator qualities.

4. What are the advantages of using starch as an indicator in iodometric

titration?
1. Starch give deep blue color which is helpful for detection of endpoint.
2. Starch made complex with iodide when heated with water decomposition occure ,beta-
amylose formed which combined with iodine to form starch/iodine complex of highly
intense color.
3. Freshly prepared starch is used because it is biodegraded.
4. Starch added at the end of reaction because in the beginning pale yellow color of iodine
present but when starch added it turn into dark blue color show the end point of reaction.

5. Why is a conical flask place in dark in iodometric titration?

Iodine is weakly soluble in water and easily lost out of solution because it is volatile. Keeping
the solution in the dark slows down the loss of iodine from the water.

6. What is the purpose of mixture of acetic acid and chloroform?

The role of chloroform and acetic acid in peroxide value is to dissolve fat or oil.

The following reaction takes place on adding acetic acid

2ROOH+2H++2I−⟶2ROH+I2+H2O

The acidic conditions (excess acetic acid) prevents formation of hypoiodite (analogous to
hypochlorite), which would interfere with the reaction
Acetic acid also has the fortunate property of being miscible with many polar and non-polar
solvents. Because this is a method for the analysis of oil samples, it is necessary to chose a non-
polar solvent capable of dissolving the sample that is also miscible with the acetic acid.
Chloroform is one solvent that fits that description nicely.

8
 EXPERIMENT NO 02
OBJECT
Determination of moisture and volatile matter by air oven method

MATERIALS
 Air oven
 Moisture dish
 dessicator

THEORY
HOT AIR OVEN
A hot air oven is a type of dry heat sterilization.

Dry heat sterilization is used on equipment that cannot be wet and on material that will not melt,
catch fire, or change form when exposed to high temperatures. Moist heat sterilization uses water
to boil items or steam them to sterilize and doesn't take as long as dry heat sterilization.
Examples of items that aren't sterilized in a hot air oven are surgical dressings, rubber items, or
plastic material.

Items that are sterilized in a hot air oven include:

 Glassware (like petri dishes, flasks, pipettes, and test tubes)

 Powders (like starch, zinc oxide, and sulfadiazine)
 Materials that contain oils
 Metal equipment (like scalpels, scissors, and blades)

Hot air ovens use extremely high temperatures over several hours to destroy microorganisms and
bacterial spores. The ovens use conduction to sterilize items by heating the outside surfaces of
the item, which then absorbs the heat and moves it towards the center of the item.

The commonly-used temperatures and time that hot air ovens need to sterilize materials is

 170 degrees Celsius for 30 minutes,

 160 degrees Celsius for 60 minutes,
 and 150 degrees Celsius for 150 minutes.

Pure oils and fats contain practically no water, but processed

lipid-rich materials may contain substantial amounts of water (from about

9
15% in margarine and mayonnaise to 40% in salad dressings).

PROCEDURE
1. Weight accurately 5gm of test sample into a tare moisture dish that has been dried and
cooled previously in a dessicator.
2. Place in air oven and dry for 90 minutes at 130C.
3. Remove from the oven and cool to room temperature in dessicator and weight.
4. Perform three tests and calculate standard deviation among the results.

OBSERVATION AND CALCULATION

moisture and %moisture and
Sample weight Sample weight
volatile volatile
(gm) after drying (gm)
matter(gm) matter(%)
5.0883 5.085 0.0033 0.064

%𝒎𝒐𝒊𝒔𝒕𝒖𝒓𝒆 𝒂𝒏𝒅 𝒗𝒐𝒍𝒂𝒕𝒊𝒍𝒆 𝒎𝒂𝒕𝒕𝒆𝒓 =

𝑺𝒂𝒎𝒑𝒍𝒆 𝒘𝒆𝒊𝒈𝒉𝒕 𝒈𝒎 − 𝑺𝒂𝒎𝒑𝒍𝒆 𝒘𝒆𝒊𝒈𝒉𝒕 𝒂𝒇𝒕𝒆𝒓 𝒅𝒓𝒚𝒊𝒏𝒈 (𝒈𝒎)

∗ 𝟏𝟎𝟎
𝑺𝒂𝒎𝒑𝒍𝒆 𝒘𝒆𝒊𝒈𝒉𝒕 (𝒈𝒎)

𝟓. 𝟎𝟖𝟖𝟑 − 𝟓. 𝟎𝟖𝟓
∗ 𝟏𝟎𝟎
𝟓. 𝟎𝟖𝟖𝟑
%moisture and volatile matter= 0.064%

DISCUSSION
1) What is moisture content?
Moisture content is the amount of water present in a food sample. Water in food found in three
forms i.e. free, bound and capillary. The moisture content is determined by measuring the mass
of a food before and after the water is removed by drying.

2) Why sometimes weight of oil increases rather than decreasing after

drying?
The most common method applied for the determination of moisture content is oven drying
method. But in case of fats & oils determination of moisture content, this method is not suitable

10
without creating a vacuum. Because the oxygen present in the oven oxidize the oil sample and
instead of decreasing weight due to moisture loss, the weight will increase.

In an oven drying method, the sample is heated under specified conditions, and the loss of weight
is used to calculate the moisture content of the sample. The amount of moisture determined is
highly dependent on the type of oven used, conditions within the oven, and the time and
temperature of drying.

11
 EXPERIMENT NO 03
OBJECT
Determination of iodine value in lipid

THEORY
 Fats and oils are a mixture of triglycerids. Triglycerides are made up of three fatty acids
linked to glycerol by fatty acyl esters. Fatty acids are long chain hydrocarbons with
carboxyl groups (COOH groups). These fatty acids can be classified into saturated or
unsaturated based on the number of double bonds present in the fatty acid.
 Saturated fatty acids contain only single bond between the carbon atoms and are tend to
be solids at room temperature.
 Unsaturated fatty acids contain double bonds between the carbon atom in addition to the
single bonds present in the fatty acid chain. They are likely to exists as liquids at room
temperature. The double bonds present in the naturally occurring unsaturated fats are in
the Cis form. Trans fatty acids are associated with health problems and cardiovascular
diseases.
 Unsaturated fatty acids can be converted into saturated by the process of hydrogenation.
Depending upon the degree of unsaturation, the fatty acids can combine with oxygen or
halogens to form saturated fatty
acids.
 So it is important to know the
extend to which a fatty acid is
unsaturated. There are different
methods for checking the unsaturation level in fatty acids, one among them is by
determining the iodine value of fats. Iodine value or number is the number of grams of
iodine consumed by 100g of fat. A higher iodine value indicates a higher degree of
unsaturation.

IODINE VALUE
Iodine value is the measure of the unsaturation of fats and oilsand is expressed in terms of the
number of centigramsof iodine absorbed per gram of sample (% iodine absorbed).

PRINCIPLE
The oils contain both saturated and unsaturated fatty acids. Iodine gets incorporated into the fatty
acid chain wherever the double bond exist. Hence, the measure of the iodine absorbed by an oil,
gives the degree of unsaturation. Iodine value/number is defined as the „g‟ of iodine absorbed by
100g of the oil.

12
REACTION MECHANISM
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

Saturated fatty acids will not give the halogenation reaction. If the iodine number is between 0-
70, it will be a fat and if the value exceeds 70 it is an oil.

Starch is used as the indicator for this reaction so that the liberated iodine will react with starch
to give purple coloured product and thus the endpoint can be observed.

IODINE MONOCHLORIDE
Iodine monochloride is an interhalogen compound with the formula ICl. It is a red-brown
chemical compound that melts near room temperature. Because of the difference in the
electronegativity of iodine and chlorine, ICl is highly polar and behaves as a source of I+.

Iodine monochloride is produced simply by combining the halogens in a 1:1 molar ratio,
according to the equation

I2 + Cl2 → 2 ICl

The Wijs solution, iodine monochloride dissolved in acetic acid, is used to determine the iodine
value of a substance

13
  HAZARDS
 Iodine Monochloride is a CORROSIVE CHEMICAL and contact can severely irritate
and burn the skin and eyes with possible eye damage.
 Breathing Iodine Monochloride can irritate the nose and throat.
 Breathing Iodine Monochloride can irritate the lungs causing coughing and/or shortness
of breath.

POTENTIAL HEALTH EFFECTS OF WIJ’S SOLUTION

 Eye:

Vapors are irritating to the eye, liquid contact may result in clouding of the cornea, erosion, up to
total corneal opacification and loss of the eye. Corrosive vapors above 5ppm.

 Skin:

Skin contact may result in irritation, pain, burns, blisters, and brown or yellow stains Corrosive
irritant/sensitizer. Ingestion:

 Ingestion

results in irritation/corrosion of mucous membranes, mouth, stomach with edema (severe) of the
pharynx, larynx; abdominal spasms, nausea, vomiting, colitis, hypotension, circulatory collapse,
delerium, coma possible. One ounce (30 mL) can burn holes in throat; five ounces can cause
death.

 Inhalation:

Nose, throat and lung irritant above 10ppm. Intolerable at 50 ppm for most; severe respiratory
tract irritation, coughing, choking, throat swelling, fluid in the lungs, headache and dizziness.

 Chronic:

Sensitization, hypersensitivity, dental erosion, ulceration of jaw, nose, throat; throat and lung
disease (bronchitis, pneumonia, laryngitis), gastrointestinal disturbances, "iodism", tissue
necrosis,skin blackening, painful joint swelling,conjunctivitis

PREPARATION OF SOLUTIONS
WIJ’S SOLUTION
 16 gram ICL in 50 ml glacial acetic acid in iodine flask
 Leave for few minutes

14
POTASSIUM IODIDE SOLUTION (10 %)
 Dissolve 5gm KI in 50ml water
 Keep it in dark place

STARCH INDICATOR
 Take few amount of starch and dissolve it in water after that microwave for few seconds
to form the solution

SODIUM THIOSULFATE SOLUTION (0.1 N)

 Dissolve 6.25gm sodium thiosulfate in 250 ml water in a volumetric flask.

PROCEDURE

filter the sample through filter paper

add 20 ml cyclohexane
add 25 ml wijs solution

seal the flask with a glass stopper and place in dark place for 1 or 2 hours depending on
expected Iodine value

Add 20 ml of KI solution and 100 ml of Distilled Water. Add starch indicator. Mix and titrate
with 0.1 N sodium thiosulfate until the color disappear

For blank, follow the same procedure from step number 2

15
OBSERVATION AND CALCULATION
ERROR

IV =
𝒎𝒍 𝒐𝒇 𝑵𝒂₂𝑺₂𝑶₃𝒇𝒐𝒓 𝒃𝒍𝒂𝒏𝒌−𝒎𝒍 𝒐𝒇 𝑵𝒂₂𝑺₂𝑶₃𝒇𝒐𝒓 𝒔𝒂𝒎𝒑𝒍𝒆 × 𝑵𝒂₂𝑺₂𝑶₃𝒏𝒐𝒓𝒎𝒂𝒍𝒊𝒕𝒚 ×𝟏𝟐.𝟔𝟗
𝒔𝒂𝒎𝒑𝒍𝒆 𝒘𝒆𝒊𝒈𝒉𝒕

each milliliter of sodium thiosulfate solution is equivalent to 12.69 mg of available iodine.

DISCUSSION
1. What is Saturated Fat?
Saturated fat is a fat molecule that comprises of two kinds of smaller molecules known as
monoglyceride and fatty acids. These fatty acids in the saturated fat molecule have only single
bonds. Fatty acids are made of long chains of carbon (C) atoms, and some of these carbon atoms
are connected by single bonds (-C-C-) only. Fatty acids containing double bonds can react with
hydrogen to form saturated fats. Most animal fats are considered as saturated fat. When saturated
food is exposed to the atmosphere, they are not susceptible to further oxidation and rancidity.

2. What is Unsaturated Fat?

Unsaturated fat is a fat or fatty acid that has both single bonds and double bonds. In other words,
these fat molecules should have at least one double bond within the fatty acid chain. Unsaturated
fats can be again subdivided into two categories. They are monounsaturated and polyunsaturated
fats. Monounsaturated fat contains only one double bond whereas polyunsaturated fat contains
more than one double bond. When unsaturated food is exposed to the atmosphere, they are
susceptible to further oxidation and rancidity.

3. What is the importance of iodine value test?

The most important application of the iodine value is to determine the amount of unsaturation
contained in fatty acids. This unsaturation is in the form of double bonds which react with iodine
compounds. The higher the iodine value, the more unsaturated fatty acid bonds are present in a
fat.

The higher the degree of unsaturation the more liquid the product will be at room temperature.
Also the increased degree of unsaturation means the product will be less stable and may need
protection from light, oxygen etc.

16
 4. Difference between saturated fat and unsaturated fat.

SATURATED FAT UNSATURATED FAT

Definition
A saturated fat is a fat, or fatty acid that has An unsaturated fat is a fat, or fatty acid has
only single bonds. both single bonds and double bonds.
Categorization
Unsaturated fat is subdivided into two
Saturated fat is not further dived into
categories. They are monounsaturated and
subcategories.
polyunsaturated fats.
Physical Nature
Due to their chemical structure, unsaturated
Due to their chemical structure, saturated fats
fats have a liquid consistency at room
have a solid consistency at room temperature.
temperature.
Most Common Sources
Most animal derived fats are considered as Most plant-derived fats are considered as
saturated fat. saturated fat.
Analysis Methods
Unsaturated fatty acids are separated by gas
Saturated fatty acids are separated by gas chromatography of methyl esters or thin-layer
chromatography of methyl esters or thin-layer chromatography and gas chromatography, and
chromatography and gas chromatography, and mass spectroscopy can be used to analysis
mass spectroscopy can be used to analysis these molecules. In addition to that, iodine
these molecules. value can be used to determine the proportion
of unsaturated fat.
Susceptibility to Oxidation
When saturated food is exposed to the When unsaturated food is exposed to the
atmosphere, they are not susceptible to further atmosphere, they are susceptible to further
oxidation and rancidity. oxidation and rancidity.
Health Aspects
High amount of saturated fats consumption Unsaturated fats are associated with various
may be harmful to health. health benefits.
Most Common Examples
Palmitic acid, stearic acids, lauric acid and Palmitoleic acid, oleic acid, myristoleic acid,
myristic acid are common examples of linoleic acid, and arachidonic acid are common
saturated fat. examples of unsaturated fat.

17
 5. Why saturated fats have 0 iodine value?
Saturated oils, fats, and waxes take up no iodine; therefore their iodine value is zero; but
unsaturated oils, fats, and waxes take up iodine. (Unsaturated compounds contain molecules with
double or triple bonds, which are very reactive toward iodine.) The more iodine is attached, the
higher is the iodine value, and the more reactive, less stable, softer, and more susceptible to
oxidation and rancidification is the oil, fat, or wax.

6. The more the unsaturation in the triglycerides, the more the degree of
oxidation. Explain.
Unsaturated fats are more susceptible to oxidation than are saturated fats, meaning the more
polyunsaturated a fat is, the faster it will go rancid. This is due to the more unstable double
bonds, which allow more oxygen to react at those points. Oils generally dont suddenly go rancid;
they tend to slowly become more oxidized over time.

18
 EXPERIMENT NO 04
OBJECT
Determination of saponification value

THEORY
SAPONIFICATION
Saponification is the process by which the fatty acids in the glycerides of the oil are hydrolyzed
by an alkali.

SAPONIFICATION VALUE
Saponification value is the amount (mg) of alkali required to saponify a definite quantity (1g) of
an oil or fat. This value is useful for a comparative study of the fatty acid chain length in oils.

It is expressed as mg potassium hydroxide (KOH) per g sample

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 hydrolysed. It is also considered as a measure of
the average molecular weight (or chain length) of all the fatty acids present. The long chain fatty
acids found in fats have low saponification value because they have a relatively fewer number of
carboxylic functional groups per unit mass of the fat and therefore high molecular weight .

𝒎𝒍 𝒐𝒇 𝑯𝑪𝒍 𝒃𝒍𝒂𝒏𝒌−𝒎𝒍 𝑯𝑪𝒍 𝒇𝒐𝒓 𝒔𝒂𝒎𝒑𝒍𝒆 ×𝑯𝑪𝒍 𝑵𝒐𝒓𝒎𝒂𝒍𝒊𝒕𝒚 ×𝟓𝟔.𝟏

Saponification value = 𝒔𝒂𝒎𝒑𝒍𝒆 𝒘𝒆𝒊𝒈𝒉𝒕 (𝒈)

Where 56.1 is the molecular weight of KOH

 Principle
A known quantity of oil is refluxed with an excess amount of alcoholic KOH. After
saponification, the remaining KOH is estimated by titrating it against a standard acid.

The procedure involves the use of excess alcoholic KOH, which catalyzes the
saponification/release of the free fatty acids from the glycerol backbone. The unreacted KOH is
then back-titratedwith standardized HCl using phenolphthalein as the indicator. The amount and
normality of the HCl used for neutralizationcan then be used to calculate the saponification
value. The saponification value provides evidence as to the relative chain lengths of the fatty
acids in the system.

19
 MATERIALS
 Fat or oil sample to be analyzed
 Standardized HCl (0.5 or 0.1 N)
 Alcoholic potassium hydroxide (see recipe)
 Phenolphthalein solution
 250mL Erlenmeyer flasks or flat-bottomboiling flasks with 24/40 ground glass joints
 250mL round-bottom flasks boiling flasks with 24/40 ground glass joints
 Waterbath or heating mantles
 Air condensers (65cm long, for use with boiling waterbath)
 Water cooled condensers (65cm long for use with heating mantles)
 50 mL burette

PROCEDURE
Melt the sample if it is not already liquid and filter through paper to remove any impurities and the last traces of
moisture. The sample must be completely moisture-free.

Weigh 4-5g sample into the flask. Add 50mL of alcoholic KOH from burette by allowing it to drain for a definite
period of time.

Prepare a blank also by taking only 50mL of alcoholic KOH allowing it to drain at the same duration of time.

Connect air condenser to the flasks and boil them gently for about 1h.

After the flask and condenser get cooled, rinse down the inside of the condenser with a little distilled H 2O and then
remove the condenser.

Add about 1mL of indicator and titrate against 0.5N HCl until the pink color just disappears.

OBSERVATIONS AND CALCULATIONS

Error (experiment was not performed)

20
DISCUSSION
1) What is back-titration?
A back titration is a titration method where the concentration of an analyte is determined by
reacting it with a known amount of excess reagent. The remaining excess reagent is then titrated
with another, second reagent. The second titration's result shows how much of the excess reagent
was used in the first titration, thus allowing the original analyte's concentration to be calculated.

A back titration may also be called an indirect titration.

2) What reaction occur during saponification?

The hydrolysis of triglycerides basicly involved two stage which is

1. Formation of glycerol through the steam hydrolysis

2. Fatty acid is neutralized in KOH to
form the soap.

R COOH + KOH → R COOK + H2O

KOH + HCl → KCl + H2O

3) What are soaps?

Soaps are sodium or potassium salts of fatty acids. They are produced from the hydrolysis of
natural oils or fats in a chemical reaction called saponification. Soap is an excellent cleanser
which often occurs in form of solid bars or in liquid form.

4) If there are two oils one having higher SV value than the other
then,Why SV of two oils are different? The SV of two oils is different
because of the difference in chain length.
The shorter the chain of fatty acid, the higher is the saponification value, the long chain fatty
acids found in fats have a low saponification value because they have a relatively fewer number
of carboxylic functional groups per unit mass of the fat as compared to short chain fatty acids.

5) What does SV measure?

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 hydrolysed.

21
It is also considered as a measure of the average molecular weight (or chain length) of all the
fatty acids present. The long chain fatty acids found in fats have low saponification value
because they have a relatively fewer number of carboxylic functional groups per unit mass of the
fat and therefore high molecular weight .

22
 EXPERIMENT NO 05
OBJECT
Measurement of free fatty acid or acid value in lipid

THEORY
INTRODUCTION
Free Fatty Acids (FFA) Fats and oils are primarily composed of various combinations of fatty
acids bonded to a glycerine backbone. When fats and oils become rancid, individual fatty acids
are "freed" and make the material slightly acidic. The FFA test measures this acidity and then
expresses it on a fatty acid basis. The presence of high concentrations of free fatty acids in feed-
grade fat, particularly "whole" animal fats may mean the fat is rancid. However, some fat sources
such as acidulated soap stocks, can contain high amounts of free fatty acids without being rancid.

WHAT ARE FREE FATTY ACID?

Fatty acids are straight-chain carboxylic acids (either saturated or unsaturated). They are derived
from the hydrolysis of fats or can be synthesized from two carbon units:
individual fatty acids can be found as:
 free fatty acids (FFA)
 the non-esterified fatty acids
Free fatty acids (FFA) are produced by the hydrolysis of oils and fats.They are non-esterified
fatty acids.

WHAT IS ACID VALUE?

The acid value (AV) is a common parameter in the specification of fats and oils. It is defined as
the weight of base in mg needed to neutralize the organic acids present in 1g of fat and it is a
measure of the free fatty acids (FFA) present in the fat or oil.

DETERMINATION OF FREE FATTY ACID

A small quantity of free fatty acids is usually present in oils along with the triglycerides. The free
fatty acid content is known as acid number/acid value. It increases during storage
Determination of free fatty acid level in fats and oils have much importance. The level of FFA
depends on time, temperature and moisture content because the oils and fats are exposed to various
environments such as storage, processing, heating or frying. FFA are less stable than neutral oil, they
are more prone to oxidation and to turning rancid. Thus, FFA is a key feature linked with the quality
and commercial value of oils and fats.
There are various methods to determine the free fatty acid of fats and oil. The rapid method for
determination of FFS are as follows:

23
TITRATION
the titration method for determination of the acid number and free fatty acids (FFA) in fats and
oils is suitable for edible fats and oils such as butter, olive, palm, or sunflower oil.

PRINCIPLE
The free fatty acid in oil is estimated by titrating it against base(NaOH) in the presence of
phenolphthalein indicator. The acid number is defined as the mg KOH required to neutralize the
free fatty acids present in 1g of sample. However, the free fatty acid content is expressed as oleic
acid equivalents.

PROCEDURE
1. filter the sample through filter paper. The filter paper will remove any solid debris and
can be handle small amounts of water, however, if unsure, use anhydrous sodium
sulphate(Na2SO4) to remove the water. This can be done either by placing some Na2SO4
in the filter paper and slowly pouring the oil through(this would be the more efficient) or
by outing Na2SO4 in the oil for several hours(with occasional swirling) and then filtering
the oil. If sample is a fat(solid or semi solid), then one must melt it prior to filtering; be
careful to not heat>10 degree centigrade over its actual melting point. Once a clean and
dry sample is obtained, it needs to mixed sufficiently to give a representive sample.
2. Expected FFA level selected from the table and weigh according to the condition(to
0.001g) into a 250ml Erlenmeyer flask
3. Add volume of neutralized alcohol preheated 50 to 65 degree centigrade and add 1ml of
phenolphthalein.
4. Titrate immediately with NaOH sol. Swirl vigorously throughout the titration(use hot
plate stir)
5. Titrate to the permanent pink colour and record the volume of sodium hydroxide solution
used.
OBSERVATION
INITIAL READING FINAL READING DIFFRENCE
2ml 7.9ml 5.9ml
CALCULATION
FFA values are usually reported as oleic acid :however in oils such as coconut and palm kernel,
FFA is reported in terms of lauric acid or palmitic acid respectively.
𝑚𝑙 𝑜𝑓 𝑁𝑎𝑂𝐻 ×𝑁𝑎𝑂𝐻 𝑛𝑜𝑟𝑚𝑎𝑙𝑖𝑡𝑦 ×28.2
FFA as oleic acid= 𝑤𝑒𝑖𝑔 𝑕𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 (𝑔)

5.9×0.1×28.2
FFA as oleic acid= 20

FFA as oleic acid= 0.8319mg

24
FOR 1 G SAMPLE
FFA as oleic acid=0.8319/20
FFA as oleic acid=0.04159 mg
RESULT
FFA as oleic acid=0.04159 mg

DISCUSSION
1. What Is The Significance Of Acid Value Or Free Fatty Acid?
The acid value (AV) is a common parameter in the specification of fats and oils.. An increment
in the amount of FFA in a sample of oil or fat indicates hydrolysis of triglycerides (Structure on
the left). Such reaction occurs by the action of lipase enzyme and it is and indicator of inadequate
processing and storage conditions (i.e., high temperature and relative humidity, tissue damage).
The source of the enzyme can be the tissue from which the oil or fat was extracted or it can be a
contaminant from other cells including microorganisms. Besides FFA, hydrolysis of triglycerides
produces glycerol.
acidity represents a basic indicator of the genuineness of the product.. The test is particularly
important during the refining of oils and fats, for the assessment of the processing cycle and for
the definition of product categories.

2. Why We Use Alcohol?

We use alcohol as neutralizing the medium. If solvent is not neutral it will interfere in acidity
test.The free fatty acids that are present in hydrolyzed form from triglycerides. The neutral
solvents helps find the exact acidity from free fatty acids present in a fat sample.. if solvent is
not neutral it may consume more sodium hydroxide (if it acidic) and less if solution is
basic,leading to wrong estimation of fee fatty acid analysis

3. Draw Back Of Titration Method

The AOAC(association of agricultural chemist) method to determine AV in fats and oils is based
on a titration in ethanol using phenolphthalein as indicator. Disadvantages of this and similar
methods are
1. the use of organic solvents (volume and toxicity)
2. the need for heating the reaction media
3. incomplete solubility of the oil/fat
4. the need to pre-neutralize the solvents
5. use of large amounts of sample
6. the possibility of error to detect the color change of the indicator when analyzingcolored
samples.

25
There are some non-titration methods designed to overcome these disadvantages. However, in
spite its drawbacks, the titration method is still the most used due to the fact that it does not
require expensive equipment.

26
 EXPERIMENT NO 06
OBJECT
Chlorophyll pigment in crude vegetable oil

THEORY
WHAT IS VEGETABLE OIL?

Vegetable oil is oil that is extracted from various types of fruits, seeds, grains, and nuts (all
considered vegetables for this purpose). The most popular oils are made from canola, coconut,
corn, cottonseed, olive, palm, palm-kernel, peanut, safflower, soybean, and sunflower. Vegetable
oil is used to add flavor, assist with texture, and to cook food
WHAT IS CRUDE VEGATBLE OIL?
Every vegetable contains varying amounts of vegetable oil. This "crude vegetable oil" is the
unrefined and unprocessed oil produced from vegetables - and how it is found in the natural
vegetable oil state when it is first extracted from the vegetable, whether the vegetable oil comes
from corn, soybeans, oil palm, jatropha, cottonseed, etc. To make the crude vegetable oil ready
for use, it must undergo further processing and refining to take it from its crude form to
a "refined vegetable oil" state.
WHAT IS CHLOROPHYLL?
Chlorophyll,is the green pigment found in green parts of plants such as green leaves,stems,seeds.
It is an integral part of photosynthesis. It is an important agent in photosynthesis as it helps plants
to absorb energy from sunlight.Chlorophyll is a key part of the plant growth and survival
process.

CHLOROPHYLL CONTENT IN VEGETABLE OIL

Seeds are important to the plant as a major means of reproduction and to humans and animals as
a major source of food. Seeds are storage reserves of protein, starch and oil that are utilized by
the plant during development and maturation. In addition, chlorophyll that
is present in the seed is partially broken down during seed development.
Chlorophyll a is present in appreciable quantities in the testa of physiologically mature seeds that
is photosynthetically inactive.
Chlorophyll and other related pigments are present, in high concentrations, in the seeds of
oilseed rape at the end of seed development and before ripening. During ripening, the
chlorophyll pigments are steadily degraded, but some may remain at harvest. Most of the
residual chlorophyll is then extracted along with the oil during the commercial crushing process

27
DETERMINATION OF CHLOROPHYLL IN VEGETABLE OIL

In the literature, several works have been published about analytical methods able to identify and
quantify pigments in oil matrices. These methods can be divided into two main categories based
on the physical principle:
(1) chromatographic techniques (characterized by pretreatment of the samples, such as extraction
and/or saponification)
(2) spectroscopic techniques (without pretreatment of the samples).

SPECTROPHOTMETER
spectrophotometers are devices that can measure a light beam's intensity as a function of its color
(wavelength).

Spectrophotometers work on the underlying principle known as Beer-Lambert‟s law, also simply
referred to as Beer‟s law, which states that absorbance of a material is directly proportional to its
thickness or path length.

PRINCIPLE OF SPECTROSCOPY:
UV spectroscopy obeys the Beer-Lambert law, which states that:
“When a beam of monochromatic light is passed through a solution of an absorbing
substance, the rate of decrease of intensity of radiation with thickness of the absorbing
solution is proportional to the incident radiation as well as the concentration of the solution.”
The expression of Beer-Lambert law is-
A = log (I0/I) = Ecl
Where, A = absorbance
I0 = intensity of light incident upon sample cell
I = intensity of light leaving sample cell
C = molar concentration of solute
L = length of sample cell (cm.)
E = molar absorptivity
Spectrophotometery can be used to measure of the concentration of a compound in a solution by
using the compounds ability to absorb light of a given wavelength.

SPECTROPHOTOMETRIC DETERMINATION OF CHLOROPHYLL

DEFINITION
This method is used to determine mg/kg (ppm) of chlorophyll in cold pressed oil (e.g., virgin
olive oil) and chlorophyll-related pigments (predominantly pheophytin a) in crude heated oils
from spectrophotometric absorption measurements at 630, 670 and 710 nm
The content of chlorophyll pigments in vegetable oils is expressed as mg of pheophytin a in 1 kg
of oil.

28
PRINCIPLE
Chlorophyll pigments are determined by measuring the absorbance at 630,670 nm, and 710 nm
correcting the result for the background absorption, and calculating the content with use of the
absorptivity of pheophytin a, which is the main chlorophyll pigment in crude vegetable oils

PROCEDURE
1. Turn on the spectrophotometer
2. Place 50mm cuvette containing ch2cl2 and zero the instrument
3. Adjust the temperature of the oil to 25 degree centigrade. The oil test sample must be
clear and free of moisture,haze and particle. Fill sample with the oil test sample and place
in spectrophotometer.
4. Measure absorption of oil sample at 630, 670 and 710 separately.
5. All the absorbance value should preferably fall between 0.3 and 0.8 use the maximum
cuvette size to give the desire values.

OBSERVATION
S.NO WAVELENGTH ABSORBANCE

1 630 1.450

2 670 0.300

3 710 1.440

CALCULATION
CONCENTRATION OF CHLOROPHYLL PIGMENT
𝐴670−(𝐴630+𝐴710)/2
Mg/kg(ppm)= 𝐹×𝐿

Where,
C=chlorophyll
A=absorbance
L=cuvette length,cm
F=factor specific to each spectrophotometer

29
 0.300−(1.450+1.440)/2
Mg/kg(ppm)= 1×3.5

Mg/kg(ppm)= -0.31857 mg/kg

RESULT
Mg/kg(ppm) of pheophytin= -0.31857 mg/kg

DISCUSSION
1. Is Chlorophyll Content Is Desirable In Seed Oil?
Chlorophyll and other related pigments are present, in high concentrations, in the seeds of
oilseed rape at the end of seed development and before ripening. During ripening, the
chlorophyll pigments are steadily degraded, but some may remain at harvest. Most of the
residual chlorophyll is then extracted along with the oil during the commercial crushing process.
Chlorophyll pigments in oil lead to a number of problems.
 They can interfere with processing,
 they can result in rancidity
 they produce an undesirable colour.
The pigments, therefore, have to be removed during refining which increases costs significantly
and results in a loss of oil.
However unlike other oil chlorophyll is desirable in olive oil.

2. Significance Of Chlorophyll In Olive Oil

The unique colour of olive oil related to its pigment content varies from a light gold to a rich
green. Green olives produce a green oil because of the high chlorophyll content, while ripe olives
yield a yellow oil because of the carotenoids (yellow red). The exact combination and
proportions of pigments determine the final colour of the oil. The role of chlorophylls as natural
pigments accounting for greenish colours and in photosynthesis is well known. There are also
some reports about the benefits of dietary chlorophylls for human health.
The structure of chlorophyll pigments, consisting of one tetrapyrrole macrocycle, coordinated to
a Mg++ ion to form a planar complex, is responsible for the absorption in the visible region of
the spectrum of olive oils. Here, both the bluish‐green chlorophyll‐a and the yellowish‐green
chlorophyll‐b can be found. Chlorophylls in olive oils are mostly converted to pheophytins, due
to the exchange of the central Mg++ ion with acid protons. Pheophytin‐a is predominant with
respect to pheophytin‐b. In the case of bad storage conditions, pheophytins are further degraded
to pyropheophytins

3. What Is The Difference Between Pheophytin And Chlorophyll?

chlorophyll is chlorophyll (green pigment) while pheophytin is (biochemistry) a chlorophyll
from which the central magnesium atom has been removed.

30
 4. Why Content Of Chlorophyll Pigments In Vegetable Oils Is Expressed
As Mg Of Pheophytin?
Pheophytin is a common degradation product of chlorophyll. The structure of chlorophyll
pigments, consisting of one tetrapyrrole macrocycle, coordinated to a Mg++ ion to form a planar
complex, is responsible for the absorption in the visible region of the spectrum of oils.
Chlorophylls in vegetable oils are mostly converted to pheophytins, due to the exchange of the
central Mg++ ion with acid protons. Measurement of pheophytin gives and indirect
concentration of chlorophyll

5. What Is The Scope Of This Method?

This method is applicable to cold pressed and expelled oils. This method is not applicable to
hydrogenated oils, deodorized oils and finished products because in these processed oils the
absorption maximum does not occur at 670 nm.

31
 EXPERIMENT NO 07
OBJECT
Determination of slip melting point

THEORY
WHAT IS SLIP MELTING POINT?
The slip point (open tube melting point) is an index of temperature at which fat softens or
becomes sufficiently fluid to slip or run.
The Slip melting point (SMP) or "slip point" is one conventional definition of the melting
point of a waxy solid.

METHODS FOR DETERMINATION OF SLIP POINT OF FATS AND

OILS
Fats are invariably the mixture of different triglycerides and have different melting points.
Therefore they do not melt at fixed temperature but over a range of temperature. The melting
point is the temperature at which solid and liquid phases are in equilibrium. While melting point
of fat is related to its physical and thermal properties. In addition, it provides the information
about the purity and identity of the compound.

A pure solid has a sharp melting point whereas impurity decreases it.There are varieties of
methods for the determination of fat. This includes
 capillary tube methods
 slipping point
 Wiley melting point
 Mettler dropping point
 photoelectric point

Each method gives different results depends on the condition under which tests are performed.
But according to AOAC, Wiley method and capillary tube method are the official methods of
analysis.

OPEN TUBE CAPILLARY-SLIP METHOD | FAT MELTING POINT

ANALYSIS
This mostly used method is known by different names. It is described as slip point method in
Great Britain, melting point rise in Germany and as a softening point method by AOCS. But
overall it is known as open tube capillary method.

PRINCIPLE

32
A capillary tube containing a column of the fat which has been crystallized under controlled
conditions is immersed to a specified depth in water, the temperature of which is increased at a
specified rate. The temperature at which the column is observed to start rising in the capillary
tube is recorded.

WORKING

SMP using open capillary tube method is determined by casting a 10 mm column of the solid in
a glass tube with an internal diameter of about 1 mm and a length of about 80 mm, and then
immersing it in a temperature-controlled water bath. The slip point is the temperature at which
the column of the solid begins to rise in the tube due to buoyancy, and because the outside
surface of the solid is molten.

This is a popular method for fats and waxes, because they tend to be mixtures of compounds
with a range of molecular masses, without well-defined melting points
ADVANTAGES OF OPEN TUBE CAPILLARY-SLIP METHOD
This method is used in international trade for palm oil and hard fats. It is also the good method
for coconut oil, hydrogenated fats and hard tallows.

DISADVANTAGES OF OPEN TUBE CAPILLARY-SLIP METHOD

One of the disadvantages is 16 hrs stabilization time, as a result, it requires long processing time.
This method is less satisfactory for lard compounds, animal fats, the mixture of hard and soft fats
and emulsions

PROCEDURE
1. Melt the test sample and filter through paper to remove any impurities and the last trace
of moisture. It is essential that the test portion be absolutely dry.
2. Dip at least three clean capillary tubes in a completely liquid test portion so that the test
portion 10mm high in the tubes. Chill the test portion at once by holding the ends of the
tubes that contain the test portion against a piece of ice until the fat solidifies.
3. Place the tubes in a beaker and hold in refrigerator at 4-10 degree centigrade.
4. Remove the tubes from refrigerator and attach with a rubber band to the thermometer so
that the lower ends of melting point tubes are even with the bottom of the mercury bulb
of the thermometer.
5. Suspend the thermometer in a 600ml beaker of clear distilled water. The bottom of the
thermometer is immersed in water to the immersion mark.
6. Adjust the starting bath temperature 8-10 degree centigrade below the slip point of the
test portion. Agitate the water bath with a small stream so that temperature increases at
rate of 1C/min
7. Continue heating until the fat column rises in each tube. observe the temperature at each
column rise. Calculate average temperature of all the tube and report it as slip point.

33
OBSERVATION
The slip melting point observe of ghee sample is=23 ◦c

RESULT
The slip melting point obtain of ghee sample is=23 ◦c

DISCUSSION
1. ERROR IN RESULT
The slip melting point obtain of ghee sample is=23 ◦c. This is not the expected result on
comparing it with the standard value
Error may occur due to following reason
 Inappropriate condition of tube in which the sample has been taken
 Handling error
2. WHAT IS THE DIFFRENCE BETWEEN SLIP MELTING POINT
AND MELTING POINT?
melting point is the temperature at which the solid melts to become a liquid. Conventionally
melting point refers to the sharp melting point, and this is exhibited by pure chemical compounds
that do not decompose. Slip melting point usually refers to a technique for measuring the point at
which a waxy solid "slips" in a tube -waxy solids such as hydrocarbons produced from
petroleum oil are mixture and melt over a range and this is near reproducible way of obtaining a
diagnostic single "melt" temperature for the mixture.
3. DOES THE SAMPLE SIZE AFFECTS THE MELTING POINT?

f the sample is too small- then the melting point observed will be less than the actual melting
point because it will melt very quickly. b) If the sample is too large - Then it will take more time
to melt and the reading would be wrong

34
 EXPERIMENT NO 08
OBJECT
Insoluble Impurities In Oil And Fats

THEORY
WHAT ARE INSOLUBLE IMPURITIES?
Insoluble impurities in fats and oil includes dirt, minerals, resins, oxidized fatty acids, alkaline
soaps of palmitic and stearic acids, and proteins that are suspended in the oil.

Impurities' are non-hazardous filterable materials not soluble in petroleum ether. However,
impurities can create physical problems as they settle to create tank sludge and ultimately clog
valves, lines and nozzles. Impurities could be meat and bone particles remaining in the tallow
after the rendering operation even though it is filtered or it could be foreign materials such as
sand or metal particles picked up after processing, during storage and/or transport.

METHODS FOR DETERMINATION OF INSOLUBLE IMPURITIES OF

FATS AND OIL
Insoluble impurities in fats and oil may be determine by various method principle for standard
method of determination of insoluble impurities are as under:

PRINCIPLE
The same sample that moisture was determined from is filtered through a fine filter paper using a
solvent. The weight of the material left on the filter paper is a measure of the insoluble
impurities. The impurities which is filtered out and expressed as percentage(%).

GOOCH CRUCIBLE
A Gooch crucible is a filtration device for laboratory use. It is also called a Gooch filter. It is
convenient for collecting a precipitate directly within the vessel in which it is to be dried,
possibly ashed, and finally weighed in gravimetric analysis.

The device was originally a standard platinum laboratory crucible with a perforated base into
which asbestos pulp was placed to form the filter mat. The crucible was then heated in an oven to
dry out until it attained constant weight. The use of these materials meant that after filtration, the
crucible and its contents could be subjected to high temperature to dry the filtrate and possibly
oxidize or ash it to minimum weight. However, because of the high cost of platinum, versions

35
made of porcelain were introduced. Some Gooch crucibles, such as the one in the drawing,
permit two layers of asbestos to be used, separated by a perforated porcelain plate.

Other inorganic fibers, notably glass, have been used in place of asbestos. Gooch crucibles made
of borosilicate glass with fritted glass bases are more common today.

PROCEDURE
1. Use the residue from the moisture and volatile matterdetermination
2. Add 50 mLof kerosene to residue and heat In a water bath to dissolve the f at
3. Filter through the prepared Gooch crucible with the aid of a vacuum. Wash with five 10-
mLportions of hot kerosene, allowing each portion to drain before adding the next.
4. Wash thoroughly with petroleum ether to remove all of the kerosene. Dry the crucible
and contents to aconstant weight at 101±1°C.cool to room temperature in a desiccator
and weigh

OBSERVATION
Mass of oil=10.0664gm

Mass of filter paper=1.0882 gm

Mass of filer paper+impurities=1.7058gm

CALCULATION
(Mass of filer paper +impurities )−Mass of filter paper
Insoluble impurities(%)= Mass of oil

1,7058 −1.0882
Insoluble impurities(%)= 10.0664

Insoluble impurities(%)= 0.06176%

RESULT
Insoluble impurities(%)= 0.06176%

DISCUSSION
1. What Is The Purpose Of Adding Kerosene?
Kerosene is an organic compound which is non-polar in nature. Being same nature as oil(i.e. they
both are non-polar in nature) they dissolve oil due to which it passes out from filtration more
easily. Also impurities are insoluble in impurities which again aids in filtration. Thus the
function of adding kerosene is to make filtration more efficient.

36
 2. What Is The Purpose Of Adding Petroleum Ether?
The purpose of using petroleum ether is washing out the remains of kerosene on filter paper.
After filtration some of the kerosene along with left on the filter paper. It is necessary that for
accurate reading or amount of impurities filter paper must contains only impurities otherwise
result may not be accurate.

3. What Is The Significance Of This Test?

For high quality of oil must not contain any kind of impurities. This test is one way of
determining the amount of insoluble impurities an oil sample may contain. It is helpful in
assessing the quality of and oil.

37
 EXPERIMENT NO. 9
OBJECT:
Determination of specific gravity of oil.

THEORY:
CHEMICAL COMPOSITION OF VEGETABLE OIL:
Vegetable oils are a group of fats that are derived from some seeds, nuts, cereal grains,
and fruits. The main components of edible fats and oils are triglycerides. The minor components
include mono- and diglycerides, free fatty acids, phosphatides, sterols, fat-soluble vitamins,
tocopherols, pigments, waxes, and fatty alcohols. Other than the free fatty acids, crude vegetable
oils contain approximately two percent of these minor components. Animal fats contain smaller
amounts.

A triglyceride consists of three fatty acids attached to one glycerol molecule. If all three fatty
acids are identical, it is a simple triglyceride. The more common forms, however, are the
"mixed" triglycerides in which two or three kinds of fatty acids are present in the molecule. The
fatty acids in a triglyceride define the properties and characteristics of the molecule.

SPECIFIC GRAVITY:
Specific gravity is a ratio, expressed decimally, of the weight of a substance to the weight of an
equal volume of a substance chosen as a standard (Water for liquids and solids; while hydrogen
for gases).
𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝒂 𝒔𝒖𝒃𝒔𝒕𝒂𝒏𝒄𝒆
Specific gravity = 𝒘𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝒆𝒒𝒖𝒂𝒍 𝒗𝒐𝒍𝒖𝒎𝒆 𝒐𝒇 𝒘𝒂𝒕𝒆𝒓

Substances that have a specific gravity less than 1 are lighter than water. Substances that have a
specific gravity greater than 1 are heavier than water. Temperature must also be taken into
account when determining specific gravity, since density changes in relation to temperature. the
standard temperature for specific gravities is 25°C.

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METHODS FOR DETERMINING SPECIFIC GRAVITY OF LIQUIDS:
Two methods are commonly used for determining the specific gravities of liquids. One method
uses the hydrometer, an instrument that gives a specific gravity reading directly. A second
method, called the bottle method, uses a specific-gravity bottle, i.e., a flask made to hold a
known volume of liquid at a specified temperature (usually 20°C). The bottle is weighed, filled
with the liquid whose specific gravity is to be found, and weighed again. The difference in
weights is divided by the weight of an equal volume of water to give the specific gravity of the
liquid.

SPECIFIC GRAVITY OF OIL:

This method determines the mass per unit volume (“liter weight”) of oils and fats in order to
convert volume to mass or mass to volume, and involves the measurement of the mass, at the
required temperature, of a volume of the oil or fat contained in a pycnometer that has been
calibrated at the same temperature. The mass per unit volume (“liter weight”) of an oil or fat is
defined as the ratio of the mass of the oil or fat to its volume at a given temperature and is
expressed in grams per milliliter (g/mL) or kilograms per liter (kg/L).

This method,is used in the international trade of oils and fats.

SPECIFIC GRAVITY DETERMINATION BY PYCNOMETER:

Construction and working of pycnometer:

The specific gravity was usually determined with

Pycnometers. As the density depends on
temperature, the specific gravity depends on the
temperatures to which the densities of the substance
and the reference relate.

A pycnometer is a device used for also measuring

the density of liquids.The pycnometer (from the
Greek puknos, meaning "density", also called
pyknometer or specific gravity bottle), is a flask with a close-fitting ground glass stopper with a
fine hole through it, so that a given volume can be accurately obtained. This fine hole releases a
spare liquid after closing a top-filled pycnometer and allows for obtaining a given volume of
measured and/or working liquid with a high accuracy. This enables the density of a fluid to be
measured accurately, by reference to an appropriate working fluid such as water or mercury,

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using an analytical balance. Pycnometers are generally available for laboratory use in volumes
ranging from 1 mL to 50 mL.

In using a pycnometer, The Pycnometer is weighed empty, then full of a liquid with a known
density (normally water) and full of the liquid whose specific gravity is to be determined. For the
later two weighings the Pycnometer must be thermos statted precisely to the reference
temperature. If both weighings are performed at 20°C Specific gravity is yielded. The actual
volume of the Pycnometer has no influence on the result and the result does not depend on the
calibration of the balance either.

PRINCIPLE:

Since specific volume is temperature-dependent, hence the pycnometer is immersed in a water

bath maintained at a standard temperature. specific gravity of a liquid determined is based on
Archimedes' principle, which states that a body immersed in a liquid displaces an amount of the
liquid equal to its own volume and suffers an apparent loss in weight equal to the weight of the
displaced liquid.

Important considerations while using pycnometer:

 The pycnometer is delicate and expensive; exercise care when handling it.

 The bulb and stopper of the pycnometer are both engraved with the same number. Be
sure that you do not inadvertently switch stoppers with someone else; check the numbers
occasionally.

 The pycnometer must be clean and DRY before the initial weighing.

 To fill the pycnometer with liquid, use a Pasteur pipet to fill the bulb to about halfway up
the neck (there is usually a white mark). Then slowly insert the capillary stopper.

 When full, there should be NO air bubbles in the bulb or capillary of the pycnometer, and
no air space at the top of the capillary.

 Before weighing the full pycnometer, the outside should be perfectly dry.

Advantages of pycnometer:

The major advantages of the pycnometric method for the determination of density are:

 high accuracy of measurement (to 105g/cm3)

 the possibility of using small quantities of material (0.5 to 100 cm3)
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  the small area of free surface of the liquid in thepycnometer (which virtually
eliminates evaporation of the liquid and absorption of moisture from the air)
 the separationof the operations of thermostatic control and subsequent weighing.

PROCEDURE:
1. Weigh the empty clean dry pycnometer with the thermometer in it.

2. Fill pycnometer with oil and replace the thermometer, so as not to include air bubbles in
it.

3. Place the filled pycnometer in the water bath or oven maintained at temperature 25℃
required for the determination until the sample reaches this temperature.

4. Remove pycnometer from water bath and wipe the surplus from top of the outlet.

5. Weight the full pycnometer.

6. Record the volume of pycnometer at the maintained temperature.

OBSERVATIONS:
 Mass of empty pycnometer= Mo = 13.1418g
 Mass of pycnometer + oil= M2 = 21.9701g
 Mass of oil = M2-Mo = 21.9701-13.1418= 8.8283g
 Volume of pycnometer at temperature T (ml)= VT = 10ml

CALCULATIONS:
𝑴𝒂𝒔𝒔 𝒐𝒇 𝒐𝒊𝒍
Specific gravity or density of oil =
𝑽𝒐𝒍𝒖𝒎𝒆 𝒐𝒇 𝒐𝒊𝒍 𝒂𝒕 𝒕𝒉𝒆 𝒈𝒊𝒗𝒆𝒏 𝒕𝒆𝒎𝒑𝒆𝒓𝒂𝒕𝒖𝒓𝒆

𝑀2−Mo
= 𝑉𝑡

8.823
= 10

Specific gravity of oil = 0.88283 gm/ml or 0.9 gm/ml

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RESULT:
Specific gravity of oil is found to be = 0.88283 gm/ml or 0.9 gm/ml.

DISCUSSION:
1. What Is The Influence Of Specific Gravity In Oil?
Specific gravity is the ratio of the density of the material to density of the equal volume of water.
The temperature at which the density has been measured must be known because density
changes as temperature changes. As temperature increases the density of fluids decreases.
Specific gravity is influenced by the chemical composition of the oil.

An increase in the amount of aromatic compounds in the oil results in an increase in the
specificgravity. Aromatics are very stable under heat and are chemically active to a moderate
degree. The aromatic compounds contain a higher proportion of carbon than the other
hydrocarbon types.An increase in the saturated compounds results in a decrease in the specific
gravity.

2. Why Specific Density Of Oil Is Less Than Water?

The specific density of oilis 0.919 and that of water is always 1 so it shows that oil is less dense
than water. This is so because the molecules that make up the oil are larger than those that that
make up water, so they cannot pack as tightly together as the water molecules can. They take up
more space per unit area and are less dense. While Water molecules are packed more closely
together than the long molecules that make up oil. The oxygen atoms in water are also smaller
and heavier than the carbon atoms in oil. This contributes to making water more dense than oil.

3. Why Oil And Water Molecules Do Not Mix Together?

Oil and water do not mix together because they are made of different kinds of chemical bonds.
This is because Molecules of water are made of oxygen and hydrogen that form polar bonds and
are strongly attracted to each other. While Oil is made of carbon and hydrogen atoms that form
nonpolar bonds and are hydrophobic or „water-fearing‟ so it is less dense than water and floats
on top.

4. Why Specific Density Of Fats Is Less Than Oils?

Fats are generally solids and oils are generally liquids at ordinary room temperature. Solid fats
contain more saturated fats and/or trans fats than oils. While oils contain more monounsaturated
and polyunsaturated fats and are unsaturated. So an increase in saturated compounds results in

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decrease in specific gravity. Fats are less dense than oil as they contain more saturated
compounds than oils.

5. Importance Of Specific Gravity/ Density In Oil Industry:

Standard specific gravity used by the oil industry, compares the density of oil to that of water
through a calculation designed to ensure consistency in measurement. Less dense oil or “light
oil” is preferable to more dense oil as it contains greater quantities of hydrocarbons that can be
converted to gasoline.Liquid density is an important characteristic used to provide information
concerning composition, concentration, mass flow in fuels, and caloric content.Knowing the
volume and the density helps determine the best way of transporting those fluids.

6. Why Is It Necessary To Remove The Air Bubble While Filling Up The

Sample In Pycnometer?
Air bubbles trapped in the liquid take up space, lowering the density of the liquid and inflating
the volume measurement slightly. Thus in the calculation density = mass/volume the
denominator is increased by the volume of the bubbles and when you increase the denominator
of a fraction the value of the fraction decreases. Thus the density you obtain from this calculation
is too small.

7. Difference Between Specific Gravity And Density:

DENSITY SPECIFIC GRAVITY

1. Density is also called as volumetric It is also known as relative density.
mass density of a substance.
2. Density is defined as mass per unit It is defined as ratio between density of a
volume of a substance substance to the density of a given reference
material ( preferably water)
𝑀𝑎𝑠𝑠 𝜌𝑠𝑢𝑏𝑠𝑡𝑎𝑛𝑐𝑒
3. Formula= 𝑉𝑜𝑙𝑢𝑚𝑒 Formula= 𝜌𝑟𝑒𝑓𝑒𝑟𝑒𝑛𝑐𝑒

4. S.I unit= 𝜌 No unit

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