Lipids

SEMINAR ON ASSAY lipids
Presented By
Hadiuz Zaman
M.Pharm
Dept. of Pharmaceutical Analysis
KARNATAKA COLLEGE OF PHARMACY
Bangalore-560064

CLASSIFICATION:-
Lipids are broadly classified into 5 types which are the
following:-
I. Simple lipids
II. Complex lipids
III. Derived lipids
IV. Neutral lipids and
V. Miscellaneous lipids

SIMPLE LIPIDS:-
• Esters of fatty acids with glycerol.
• Mainly of two types:-
Fats and oils: These are esters of fatty acids and glycerol. Difference between fats
and oils is physical.
Waxes : Esters of fatty acids+alcohol other than glycerol.
Cetyl alcohol is most commonly used.
COMPLEX OR COMPOUND LIPIDS:- Esters of fatty acids+Alcohol+other groups
like phosphate,Nitrogenous base,carbohydrate ,Protein.etc.
Based on the group present they are further classified into:-
1.PHOSPHOLIPIDS: F.A+Alcohol+phosphoric acid as nitrogenous base.
Based on the type of alcohol present they are again divided into:
Glycerphospholipids:Contain Glycerol as alcohol. Eg:lecithin &cephalin
Sphingophospholipids : Contain sphingosine as alcohol.
Eg:sphingomyelin

ii.GLYCOLIPIDS:-
Fatty acids+alcohol+carbohydrate as nitrogenous base.
They contain sphingosine as alcohol and hence also known
as GLYCOSPHINGOUPIDS.
Eg: Cerebrosides and Gangliosides.
iii.LIPOPROTEINS:-
Macromolecular complexes of lipids with proteins.
Eg:LDL,VLDL,Chylomicrons,HDL,etc
iv. Other complex lipids:-
Sulfolipids,Aminolipids and other Lipopolysaccharides come
under this.

DERIVED LIPIDS:-
These are the derivatives of hydrolysis of simple and complex
lipids which possess the characteristics of lipids.
These include:
• Lipid soluble vitamins
• Steroid hormones
• Hydrocarbons
• Ketone bodies
• Mono and diacylglycerol ,etc
NEUTRAL LIPIDS:-
These are the lipids which are uncharged and are reffered ro as
neutral lipids.
These are mono,di and triacylglycerols, cholestrol and
esters.

MISCELLANEOUS LIPIDS:
A large number of compounds possess characteristics of lipids,such compounds come
this category
Example:carotenoids,squalene,hydrocarbons like pentacosone and terpenes etc.
FATTY ACIDS:
• Carboxylic aicds with hydrocarbon side chains.
• Occur in esterified form
• They occur in even and odd carbon forms
• Saturated and unsaturated.
• Essential and non essential fatty acids.
• Essential fatty acids are: Linoleic acid ,Linolenic acid ,Arachidonic acid.

SPHINGOMYELINS:
• They contain amino alcohol
• It attaches to an amide group by fatty acxid to form ceramide.
• Sphingomyelins are important constituent of brain and nervous tissue.
LIPOPROTEINS:
 Lipids+ proteins
 Five types
LDL
VLDL
HDL Chylomicrons
Free fatty acids.

DETERMINATION OF ADULTERATION IN
FATS AND OILS
What is Adulteration??
Adulteration usually refers to mixing other matter of an inferior and
sometimes harmful quality with food or drink . As a result of
adulteration, food or drink becomes impure and unfit for human
consumption.

Determination of adulterants in fat and oil;
Ghee:
One teaspoonful of melted sample is taken and added equal quantity of conc. HCl
to it. Added a pinch of sugar ,shaken for one minute and kept aside for 5 minutes.
Appearance of crimson color indicates the presence of ghee.
Castor oil:
A small quantity of sample is dissolved in petroleum ether. Acidified with HCl, Few
drops of ammonium molybdate is added and shaken well. Appearance of white
turbidity indicates the presence of castor oil.
Linseed oil:
A small amount of sample is treated with solution of bromine in CCl4. yellow
precipitate indicates the presence of linseed oil.

Coconut oil:
3 ml of the sample is taken in a test tube,10 drops of alcoholic potash is
added and heated, a little amount of FeSO₄ and FeCl₃ is added and shaken
thoroughly. 3 ml of HCl is added, a blue color indicates the presence of oil.
Mustard oil :
Little amount of sample is taken in a test tube and added 20 drops of nitric
acid. The test tube is heated for 3 minutes. A red coloration indicates the
mustard oil.

Mineral oil:
Take 2 ml of the oil sample and add an equal quantity of N/2
Alcoholic potash. Heat in boiling water bath (dip in boiling water)
for about 15 minutes and add 10 ml of water. Any turbidity shows
presence of mineral oil.

Oil soluble coal tar colour:
Take 2 gms of the sample in a test tube, add few ml of solvent ether and
shake. Decant ether layer into a test tube containing 2 ml of dilute
Hydrochloric acid (1 ml HCL + 1 ml of water). Shake it, the lower acid layer
will be coloured distinct pink to red indicating presence of oil soluble
colour .
Adulteration of argemone oil in edible oils :
To small amount of oil in a test-tube, add few drops of conc. HNO3 and
shake. Appearance of red colour in the acid layer indicates presence of
argemone oil.

Detection of Dyes in Oils & Fats:
(a) Take 2 gm. of the melted and filtered fat in a test tube. Add 5 mL of light
petroleum to avoid its solidification and one mL of hydrochloric acid to it.
Shake the contents thoroughly and allow it to stand for some time.
Appearance of pink colour in the lower layer will indicate the presence of
dyes.
(b) Mix 1-2 mL of the fat with same amount of mixture of conc. Sulphuric
acid & glacial acetic acid (in 1:4 ratio) and heat the mixture nearly to boiling.
Pink or reddish colour of the solution will indicate the presence of dyes.
Detection of Paraffin Wax & Hydrocarbons:
Heat small amount of unsaponifiable matter of oils with acetic anhydride.
Droplets of oil floating on the surface of acetic anhydride indicates the
presence of wax or hydrocarbons.

Objectives of Refining
● In refining, physical and chemical processes are combined to remove undesirable natural as well as
environmental-related components from the crude oil.
● These components comprise for example phosphatides, free fatty acids, pigments (such as chlorophyll),
odors and flavors (including aliphatic aldehyde and ketone), waxes as well as heavy metals, pesticides etc.
● Removal of undesired products from crude oils
►free fatty acids (FFA)
► phospholipids (gums)
► oxidised products
► metal ions
► color pigments
► other impurities
● Preservation of valuable vitamins. (vitamin E ortocopherol-natural anti-oxidants)
● Minimize oil losses
● Protection of the oil against degradation

Methods o Refining
CHEMICAL REFINING:
The Chemical Refining process is used for oils and fats with low FFA and contains three basic steps.
•Neutralizing
•Bleaching
•Deodorizing
Residual soap and gums removal in neutralizing is accomplished by either water washing or using
silica adsorbent in bleaching.
PHYSICAL REFINING:
The Physical Refining process is used for oils and fats with high FFA and contains three basic steps.
•Acid Conditioning or Enhanced Degumming
•Bleaching
•Stripping and Deodorizing
The degumming process used depends on the oil or fat being refined.

Depending on the requirements, the following basic processes are implemented,
 degumming for removal of phosphatides,
 neutralization for removal of free fatty acids,
 bleaching for removal of color.
 deodorization to distill odors and flavors as well as free fatty acids and
 winterization for separation of waxes.

Chemical Refining
Neutralizing:
Objective:Removal of free fatty acids
Batch Neutralization:
Refining of vegetable oils is essential to ensure removal of gums. waxes,
phosphatides and free fatty acid from the oil; to impart uniform colour by removal of
colouring pigments and to get rid of unpleasant smell from the oil by removal of
odiferous matter.
Refining is carried out either on batch operation or as continuous operation. With
certain oils even physical refining can be carried out instead of chemical. For
processing less than thirty tones of oil per 24 hours, and when oil has F.F A. content of
1 % or less normally batch process is recommended. Batch process involves low
capital investment, simplicity of operation and low maintenance, making refining
economically a viable proposition even at capacity as low as 10 tonnes per 24 hours.

Bleaching:
Objective: bleaching for removal of color,
• Silica adsorption reduces water consumption, effluent treatment and bleaching
earth consumption
• Pre-bleaching oil minimizes bleaching earth consumption
Deodorizing:
Deodorizing oil removes the final traces of volatile components,
primarily those that contribute to flavor and odor. The goal of
deodorization is to produce a nearly flavorless, odorless oil with a light color.
Deodorization consists of steam sparging the oil under high vacuum (< 10 mm Hg) at
high temperatures (> 200°C). After deodorization and cooling of the oil, a chelating
agent, such as citric acid, may be added to deactivate trace metals. Antioxidants may
also be added to enhance stability.

PHYSICAL REFINING
Acid Conditioning or Enhanced Degumming
Objective : degumming for removal of phosphatides.
The aim of degumming operation:-
 The emulsifying action of phospholipids increases oil losses during alkali refining.
 Gums lead brown discoloration of oil after heating during deodorization.
 Salts may be formed with cooper, magnesium,calcium and iron, accelerating oxidative
of oil.
Different degumming processes are carried out to remove phosphatides. For efficient and economic
application of this procedure appropriate machines and equipments are used.
1. Water degumming
2. Acid degumming
3. Enzymatic degumming
4. Membrane degumming

Water Degumming Process Steps:
• Heat oil to 60 -70 °C
• Water addition and mixing
• Hydration mixing 30 minutes
• Centrifugal separation of hydrated gums
• Vacuum drying of degummed oil
• Gums - dried for edible lecithin or recombined in
meal.

Acid degumming
There are two type of Acid degumming
1- Dry acid degununitig
2- Wet acid degumming
Acid Degumming Process Steps
• Heat oil to 60 -70 °C
• Acid addition and mixing
• Hydration mixing 30 minutes
• Centrifugal separation of hydrated gums
• Vacuum drying of degummed oil
• Gums -recombined in meal

Enzymatic degumming:
Enzymatic degumming was first introduced by the German Lurgi Company as the Enzy Max
process. The Enzy Max process can be divided into four different steps:
i.The adjustment of the optimal conditions for the enzyme reaction i.e. optimal pH with a citrate
buffer and the optimal temperature;
ii. The addition of the enzyme solution;
iii. The enzyme reaction;
iv. The separation of lysophosphatide from the oil at about 75 °C.
Enzymes for enzymatic degumming;
Lecitase1OL (pancreatic phospholipase A2)
Lecitase Novo (microbial lipase)
Lecitase Ultra (microbial lipase)

Crude Oil Refined Oil
Triglycerides % 95-97 >99
Phosphatides % 1.5-2.5 0.00-0.045
Unsaponifiable matter% 1.6 0.3
Plant sterols% 1.6 0.13
Tocopherols% 0.15-0.21 0.11-0.18
Hydrocarbons (squalene)% 0.014 0.01
Free fatty acids % 0.3-0.7 <005
Trace metals: Iron ppm 1-3 0.1-0.3
Copper ppm 0.03-0.05 0.02-0.06
Average Compositions for Crude and Refined
Soybean Oil:

Determination of Total Lipid Concentration
Introduction
It is important to be able to accurately determine the total fat content of foods for a number of
reasons:
• Economic (not to give away expensive ingredients)
• Legal (to conform to standards of identity and nutritional labeling laws)
• Health (development of low fat foods)
• Quality (food properties depend on the total lipid content)
• Processing (processing conditions depend on the total lipid content)
The principle physicochemical characteristics of lipids used to distinguish them from the other
components in foods are their solubility in organic solvents, immiscibility with water, physical
characteristics and spectroscopic properties.
The analytical techniques based on these principles can be conveniently categorized into three
different types:
(i) solvent extraction;
(ii) non-solvent extraction and
(iii) instrumental methods.

Solvent Extraction
Sample Preparation
The preparation of a sample for solvent extraction usually involves following steps:
Drying sample: It is often necessary to dry samples prior to solvent extraction, because many organic
solvents cannot easily penetrate into foods containing water, and therefore extraction would be inefficient.
Particle size reduction: Dried samples are usually finely ground prior to solvent extraction to produce a
more homogeneous sample and to increase the surface area of lipid exposed to the solvent. Grinding is often
carried out at low temperatures to reduce the tendency for lipid oxidation to occur.
Acid hydrolysis: Some foods contain lipids that are complexed with proteins (lipoproteins) or
polysaccharides (glycolipids). To determine the concentration of these components it is necessary to break the
bonds which hold the lipid and non-lipid components together prior to solvent extraction. Acid hydrolysis is
commonly used to release bound lipids into easily extractable forms, e.g. a sample is digested by heating it for
1 hour in the presence of 3N HCl acid.

Solvent Selection:
The ideal solvent for lipid extraction would completely extract all the lipid components from a
food, while leaving all the other components behind. In practice, the efficiency of solvent
depends on the polarity of the lipids present compared to the polarity of the solvent. Polar lipids
(such as glycolipids or phospholipids) are more soluble in polar solvents (such as alcohols), than in
non-polar solvents (such as hexane). On the other hand, non-polar lipids (such as triacylglycerols)
are more soluble in non-polar solvents than in polar ones. The fact that different lipids have
different polarities means that it is impossible to select a single organic solvent to extract them all.
Thus the total lipid content determined by solvent extraction depends on the nature of the organic
solvent used to carry out the extraction: the total lipid content determined using one solvent may
be different from that determined using another solvent. In addition to the above considerations,
solvent should also be inexpensive, have a relatively low boiling point (so that it can easily be
removed by evaporation), be non-toxic and be nonflammable (for safety reasons). It is difficult to
find a single solvent which meets all of these requirements. Ethyl ether and petroleum ether are
most commonly used solvents, but pentane and hexane are also used for some foods.

Batch Extraction
Batch extraction is a very simple, yet widely used method of extraction.
The sample is mixed with one or more solvent which along with endogenous water forms multiple
layer of varying concentration. As the lipids are more soluble in non-polar solvents than in water, lipid
portion goes to the layer with more solvents and non-lipid component remains in the layer with more water.
The lipid part is then separated using a separating funnel. The separation is based on partition principle
hence multiple extraction of the aqueous phase is necessary to obtain most of the lipid. The weight of lipid
not extracted is given by the equation below:
Where Wm= weight of lipid remaining,
Vw+= the volume of aqueous layer,
Vo = volume of solvent in each extraction step,
D = the distribution ratio of lipid in solvent,
N = the number of extraction steps. The selection of solvent is then done using the
distribution ratio of lipids in known solvents.

Continuous Extraction
Continuous solvent extraction recycles the solvent used so that small amount of solvent can
accomplish the equivalent extraction of several steps. This process is preferred for solid samples
where the distribution ratio is low. These samples need multi step extraction as very little lipid is
extracted to the solvent in each step.
Soxhlet extractor is widely used extractor for lipid.
ADV:
Continuous extraction is faster and uses less solvents than batch extraction.
It is the most widely used extraction system.
The equipment used is not very expensive and can be used for extraction of other
materials.
DISAD:
These processes are slow and disposal of solvent is an everyday problem.

Supercritical Fluid Extraction:
Substance in temperature above its critical temperature and pressure is
called supercritical fluid. Solvent property of supercritical fluid was first
demonstrated in 1879 (Hannay & Hogarth). They have huge prospects in
extraction because they combine the solubility power of liquid with penetration
power of gas. Moreover, their solubility can be found by changing the pressure
and temperature. Carbon dioxide and water are the most promising fluid for
supercritical extraction due to their non-toxicity and environmentally nature.

Nonsolvent Liquid Extraction Methods:
A number of liquid extraction methods do not rely on organic solvents, but use other
to separate the lipids from the rest of the food. The Babcock, Gerber and Detergent methods are
examples of nonsolvent liquid extraction methods for determining the lipid content of milk and
other dairy products.
Babcock Method
 A specified amount of milk is accurately pipetted into a specially designed flask (the Babcock
bottle). Sulfuric acid is mixed with the milk, which digests the protein, generates heat, and breaks
down the fat globule membrane that surrounds the droplets, thereby releasing the fat.
 The sample is then centrifuged while it is hot (55-60oC) which causes the liquid fat to rise into
the neck of the Babcock bottle. The neck is graduated to give the amount of milk fat present in
wt%.
 The Babcock method takes about 45 minutes to carry out, and is precise to within 0.1%. It does
not determine phospholipids in milk, because they are located in the aqueous phase or at the
boundary between the lipid and aqueous phases.

Gerber Method
 This method is similar to the Babcock method except that a mixture of sulfuric acid and
isoamyl alcohol, and a slightly different shaped bottle, are used. It is faster and simpler to
carry out than the Babcock method.
 The fat can be separated from fat containing milk through the addition of sulphuric acid.
separation is made by using amyl alcohol and centrifugation. The fat content is read directly
on a special calibrated butyrometer
 This method is used mainly in Europe, whilst the Babcock method is used mainly in the USA.
As with the Babcock method, it does not determine phospholipids.

Detergent Method
 This method was developed to overcome the inconvenience and safety concerns associated
the use of highly corrosive acids.
 A sample is mixed with a combination of surfactants in a Babcock bottle.
 The surfactants displace the fat globule membrane which surrounds the emulsion droplets in
milk and causes them to separate.
 The sample is centrifuged which allows the fat to move into the graduated neck of the bottle,
where its concentration can then be determined.

Instrumental methods
 The are a wide variety of different instrumental methods available for determining the total lipid
content of food materials.
These can be divided into three different categories according to their physicochemical
principles:
(i) measurement of bulk physical properties,
(ii) measurement of adsorption of radiation, and
(iii) measurement of scattering of radiation.

Measurement of bulk physical properties
 Density: The density of liquid oil is less than that of most other food components, and so
is a decrease in density of a food as its fat content increases. Thus the lipid content of foods can
be determined by measuring their density.
 Electrical conductivity: The electrical conductivity of lipids is much smaller than that of
substances, and so the conductivity of a food decreases as the lipid concentration increases.
Measurements of the overall electrical conductivity of foods can therefore be used to determine
fat contents.
 Ultrasonic velocity: The speed at which an ultrasonic wave travels through a material depends
on the concentration of fat in a food. Thus the lipid content can be determined by measuring its
ultrasonic velocity. This technique is capable of rapid, nondestructive on-line measurements of
lipid content.

Measurement of adsorption of radiation:
 UV-visible: The concentration of certain lipids can be determined by measuring the absorbance of
ultraviolet-visible radiation. The lipid must usually be extracted and diluted in a suitable solvent prior to
analysis, thus the technique can be quite time-consuming and labor intensive.
 Infrared: This method is based on the absorbance of IR energy at a wavelength of 5.73 mm due to
molecular vibrations or rotations associated with fat molecules: the greater the absorbance the more
present. IR is particularly useful for rapid and on-line analysis of lipid content once a suitable
curve has been developed.
 Nuclear Magnetic Resonance: NMR spectroscopy is routinely used to determine the total lipid
concentration of foods. The lipid content is determined by measuring the area under a peak in an
chemical shift spectra that corresponds to the lipid fraction. Lipid contents can often be determined in
few seconds without the need for any sample preparation using commercially available instruments.
 X-ray absorption: Lean meat absorbs X-rays more strongly than fat, thus the X-ray absorbance
decreases as the lipid concentration increases. Commercial instruments have been developed which
utilize this phenomenon to determine the lipid content of meat and meat products.

Measurement of scattering of radiation
 Light scattering: The concentration of oil droplets in dilute food emulsions can be determined
using light scattering techniques because the turbidity of an emulsion is directly proportional to
the concentration of oil droplets present.
 Ultrasonic scattering: The concentration of oil droplets in concentrated food emulsions can be
determined using ultrasonic scattering techniques because the ultrasonic velocity and absorption
of ultrasound by an emulsion is related to the concentration of oil droplets present.

Determination of Lipid Composition
Separation and Analysis by Chromatography
Lipid fractions by TLC
 TLC is used mainly to separate and determine the concentration of different types of lipid
groups in foods, e.g. triacylglycerols, diacylglycerols, monoacylglycerols, cholesterol, cholesterol
oxides and phospholipids.
 A TLC plate is coated with a suitable absorbing material and placed into an appropriate solvent.
A small amount of the lipid sample to be analyzed is spotted onto the TLC plate. With time the
solvent moves up the plate due to capillary forces and separates different lipid fractions on the
basis of their affinity for the absorbing material.
 At the end of the separation the plate is sprayed with a dye so as to make the spots visible.
 By comparing the distance that the spots move with standards of known composition it is
possible to identify the lipids present. This procedure is inexpensive and allows rapid analysis of
lipids in fatty foods.

HPLC:
 High Performance Liquid Chromatography is now a preferred method for lipid analysis. This is
because it is more versatile than TLC and operates at room temperature, thus can be used to
analyses labile groups that cannot be done using GC.
GC:
 Gas Chromatography is the preferred method for analysis of trans fatty acid. It can also be used
triglycerides and fatty acids; however, methylation is necessary. Fatty acids are non-volatile, hence
before carrying out GC, the lipids are saponified and methylated to give Fatty Acid Methyl
Esters(FAME) which are volatile and can be used for GC.

Determination of Iodine Value:
Definition: The iodine value is a measure of the degree of unsaturation in an
oil. It is constant for a particular oil or fat. Iodine value is a useful parameter in
studying oxidative rancidity of oils since higher the unsaturation the greater the
possibility of the oils to go rancid.
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.

Materials
Hanus Iodine Solution
Weigh 13.6g of iodine and dissolve in 825mL glacial acetic acid by heating, and cool.
25mL of this solution against 0.1N sodium thiosulphate.
Measure another portion of 200mL of glacial acetic acid and add 3mL of bromine to it.
To 5mL of this solution add 10mL of 15% potassium iodide solution and titrate against 0.1N
sodium thiosulphate.
Calculate the value of bromine solution, to double halogen content of the remaining
of the above iodine solution as follows:
X = B/C
where X = mL of bromine solution required to double the halogen content,
B = 800 x thiosulphate equivalent of 1mL of iodine solution and
C = thiosulphate equivalent of one mL of bromine solution.
●15% Potassium Iodide Solution
● 0.1% Sodium Thiosulphate
●1% Starch

Procedure
1.Weigh 0.5 or 0.25g of oil into an iodine flask and dissolve in 10mL of chloroform.
2.Add 25mL of Hanus iodine solution using a pipette, draining it in a definite time.
Mix well and allow to stand in dark for exactly 30min with occasional shaking.
3.Add 10mL of 15% KI, shake thoroughly and add 100mL of freshly boiled and
cooled water, washing down any free iodine on the stopper.
4.Titrate against 0.1N sodium thiosulphate until yellow solution turns almost
colorless.
5.Add a few drops of starch as indicator and titrate until the blue color completely
disappears.
6.Towards the end of titration, stopper the flask and shake vigorously so that any
iodine remaining in solution in CHCl3 is taken up by potassium iodide solution.
7.Run a blank without the sample.

Calculation
The quantity of thiosulphate required for blank minus the quantity required for sample gives
thiosulphite equivalent of iodide adsorbed by the fat or oil taken for analysis.
Iodine number = (B – S) x N x 12.69
Weight of sample (g)
where,
B = ml thiosulphate for blank
S = ml thiosulphate for sample
N = normality of thiosulphate solution

Determination of Saponification Value:
Definition: The saponification value is the number of mg of potassium hydroxide required to
saponify 1 gram of oil/fat.
Principle: The oil sample is saponified by refluxing with a known excess of alcoholic potassium
hydroxide solution. The alkali required for saponification is determined by titration of the excess
potassium hydroxide with standard hydrochloric acid.
Apparatus:
a. 250 ml capacity conical flask with ground glass joints.
b. 1 m long air condenser, or reflux condenser (65 cm minimum in length) to fit the flask.
c. Hot water bath or electric hot plate fitted with thermostat.
Reagents:
(i) Alcoholic potassium hydroxide solution - Reflux 1.2 litre alcohol 30 minutes with 10 gm KOH
and 6 gm granulated Aluminium or Al foil. Distill and collect 1 litre after discarding first 50 ml. Dissolve
40 g of potassium hydroxide in this 1 litre alcohol keeping temperature below 15 0 C while dissolving
alkali. Allow to stand overnight, decant the clear liquid and keep in a bottle closed tightly with a cork
rubber stopper.

ii) Phenolphthalein indicator solution - Dissolve 1.0 g of phenolphthalein in 100 ml rectified
spirit.
iii) Standard hydrochloric acid: approximately 0.5N
Procedure:
 Melt the sample if it is not already liquid and filter through a filter paper to remove any
impurities and the last traces of moisture. Make sure that the sample is completely dry. Mix the
sample thoroughly and weigh about 1.5 to 2.0 g of dry sample into a 250 ml Erlenmeyer flask.
 Pipette 25 ml of the alcoholic potassium hydroxide solution into the flask. Conduct a blank
determination along with the sample. Connect the sample flasks and the blank flask with air
condensers, keep on the water bath, boil gently but steadily until saponification is complete, as
indicated by absence of any oily matter and appearance of clear solution. Clarity may be
achieved within one hour of boiling.
 After the flask and condenser have cooled somewhat wash down the inside of the condenser
with about 10 ml of hot ethyl alcohol neutral to phenolphthalein.

 Titrate the excess potassium hydroxide with 0.5N hydrochloric acid, using about 1.0 ml
phenolphthalein indicator.
Calculation:
Saponification Acid Value = 56.1 (B-S)N / W
Where, B = Volume in ml of standard hydrochloric acid required for the blank.
S = Volume in ml of standard hydrochloric acid required for the sample
N = Normality of the standard hydrochloric acid and
W = Weight in gm of the oil/fat taken for the test.
56.1= Molar mass of KOH

REFFERENCES:
 https://books.google.co.in/books?id=Nanp7jmUQZoC&lpg=PP1&pg=PA293#v=onepage&q
&f=false

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