Title

Experiment 1 Determination of Acidity Number in Fatty Acid

Objective
To determine the acidity number in fatty acid which indicates the amount of carboxylic acid
groups by using different type of oil
Introduction
Fatty acids are a non polar molecules consist of a long chain of carbons with an oxygen and
a hydroxyl group at the end. Fatty acid are normally found especially in animal and vegetable
fats and oils and have a general formula of CnH2n+1COOH. Fatty acids are the building
blocks of fat sources in living organisms and fats or oil are made up of 3 fatty acids attached
to a glycerol backbone to make up a triglyceride. Fatty acids are necessary to create essential
building blocks such as triglycerides and appear as lipids breakdown products and are
therefore good indicators of degradation. Fatty acids can be divided into two which are
saturated and unsaturated fatty acids.A fatty acid is said to be saturated if a carbon is joined to
its adjacent carbons by a single bond. If one or more double bonds are present, the fatty acid
is said to be unsaturated. Unsaturated fatty acids are classified based on the position of the
first double bond from the end of the chain by counting starts at the end of the fatty acid
molecule without the oxygen. Some of the examples of saturated fatty acids includes palmitic
acids , cerotic acids and lauric acids. On the other hand, examples of unsaturated fatty acids
are sapienic acids , oleic acids and erucic acids.
Acid value of an fat or oil is actually a indication of the amount of acids present on that
particular fat or oil. Actually, it measures the amount of carboxylic acid groups in a chemical
compound such as a fatty acid. In simple words, it can be defined as mass of a metal
hydroxide requires to neutralize a unit quantity of chemical substances. The acid value of a
oil or fats can be find by using the titration method between a neutralized solvent mix with a
type of oil or fats and metal hydroxide commonly used sodium hydroxide. In this method to
find acid value, the fats or oil used have to be different. This causes the fats or oil which is a
triglycerides to undergo rancidification thus are converted into fatty acids and glycerol which
cause an increase in acid number. Factors that can cause rancidification of an oil or fats are
exposure to air, heat and light. In this presence of this factors, it aid in the process of oil or

fats to breakdown into fatty acids. The more saturated fat, the less susceptible it is to rancidity
and the greater the amount of unsaturated fat in an oil, the more likely it is to become
rancidity.
The free fatty acids (FFA) can be measured by using this titration method which can be very
useful to us and usually indicated in percents. This is the equation to measure the FFA of an
oil or fats in this experiment.
FFA % = (25.6 x N x V) / W
where N is the normality of metal hydroxide solution used usually sodium hydroxides
solution, V is the volume of metal hydroxide solution used in mili litre and W is the weight
of different type of oil or fats used in the experiments whereas the 25.6 is the molecular
weight of palmitic acid divided by 10 because of unit conversion process. Fats or oil with a
low FFA usually indicates high quality oil while a high FFA almost always indicates poor
quality oil.

Materials: Standard sodium hydroxide solution, 0.02N, phenolphthalein indicator solution,

Isopropanol.
Apparatus:
Burette,25mL, with graduation in 0.05mL divisions.
Erlenmeyer flask,250mL
Measuring cylinder,50 mL
Beaker,50 mL
Hot plate with temperature control
Electrical balance
Funnel
Thermometer
Retort stand
Dropper

Results
Sample oil A1:
Experiment set

Initial reading of NaOH (mL)

14.00

19.20

Final reading of NaOH (mL)

15.50

20.80

Titre value of NaOH (mL)

1.50

1.60

Average titre value of NaOH

(mL)

1.55

Sample oil A2:

Experiment set

Initial reading of NaOH (mL)

15.50

17.90

Final reading of NaOH (mL)

16.80

19.20

Titre value of NaOH (mL)

1.30

1.30

Average titre value of NaOH

(mL)

1.30

Calculations
FFA % as palmitic acid =

25.6 x N x V
W

where, N = normality of NaOH solution = 0.02, V = volume of average of NaOH solution

used in mL , W = weight sample

Sample oil A1 :
By taking average weight of sample oil A1 = 2.00g, and average titre value of NaOH in mL =
1.55

FFA % as palmitic acid =

25.6 x 0.02 x 1.55

2.0

= 0.397

Sample oil A2 :
By taking average weight of sample oil A2 = 2.00g, and average titre value of NaOH in mL =
1.30
FFA % as palmitic acid =

25.6 x 0.02 x 1.30

2.0

= 0.333
Sample oil A3 :
By taking average weight of sample oil A3 = 2.01g, and average titre value of NaOH in mL =
0.70
FFA % as palmitic acid =

25.6 x 0.02 x 0.70

2.01

= 0.178
Sample oil A4 :
By taking average weight of sample oil A4 = 2.01g, and average titre value of NaOH in mL =
0.90
FFA % as palmitic acid =

25.6 x 0.02 x 0.90

2.01

= 0.229
Sample oil A5 :
By taking average weight of sample oil A5 = 1.81g, and average titre value of NaOH in mL =
0.60
FFA % as palmitic acid =

25.6 x 0.02 x 0.60

1.81

= 0.170
Sample oil A6 :

By taking average weight of sample oil A6 = 1.89g, and average titre value of NaOH in mL =
0.80
FFA % as palmitic acid =

25.6 x 0.02 x 0.80

1.89

= 0.217

Discussion
Acidity Number is a measure of biodiesel rather than waste vegetable oil (WVO), is
the amount of KOH or NaOH in mg needed to react with the organic acids present in one
gram of fat and to measure the free fatty acids (FFA) present in the fat or oil. It indicates
hydrolysis of triglycerides when the amount of FFA in a sample of oil or fat increase. Such
reaction occurs by the action of lipase enzyme. It is an indicator of inadequate processing and
storage conditions such as high temperature and relative humidity or 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. As oilfats rancidify, triglycerides are converted into fatty acids and glycerol, causing an increase in
acid number. Kardash, E. and Turyan, Y. I. (2005). Factors which lead to a high free fatty
acidity in an oil include fruit fly infestation, delays between harvesting and extraction, fungal
diseases in the fruit, prolonged contact between oil and vegetation water after extraction, and
careless extraction methods. Goulart (1980).
The breakdown of oil or biodiesel will produce Free Fatty Acids (FFA). FFA% is used
to describe the FFA content of oils, while Acidity Number (AN) is used to describe the FFA
content of finished biodiesel. The weight to weight ratio of FFA found in an oil sample
represent by FFA%. J. Van Gerpen, B. Shanks, and R. Pruszko (2004).
In this experiment, we use chemical titration method. This method uses
phenolphthalein as an indicator. We observe the colour of oil sample with phenolphthalein
added to turn pink to obtain the alkali (NaOH) needed to neutralise the oil sample which is
acidic. From the experiment, we found that Sample oil A1 gives the highest FFA% value
which is 0.397 while Sample oil A5 gives the lowest FFA% value which is 0.170. The
sequence of the FFA% value of the sample oil in increasing order is A5, A3, A6, A4, A2 and
A1. As we know that the higher the FFA% value, the higher the acid number of the oil, which
indicated that the quality of the oil is lower. Therefore through this experiment, we can
concluded that Sample oil A5 is the best oil to consume among these six oil sample while
Sample oil A1 is the most unhealthy oil among all.
Other than chemical titration method, there are a few of alternative ways to determine
the acidity number of oil. The first alternative method is thermometric titration method. This
method uses a method where free fatty acids are titrated with a standard solution of KOH in
isopropanol. The first excess of hydroxyl ion after neutralization of fatty acids in a measured
sample of lipids catalyzes a strong exothermic reaction between components of the solvent
mixture (acetone/chloroform, 25/2, v/v) dissolving the fat or oil. A thermistor is used to sense
the change in solution temperature which signals the sharp and unequivocal titration
endpoint. Smith TK, J Am Oil Chem Soc (2003).
Another way to determine acidity number is by measuring the metal fatty acid
complexes. 50 ml heptane solution of fatty acids varying from 0.1 to 200 nmoles, 100 ml
chloroform/heptane mixture (4/1, v/v), and 10 ml of reagent solution containing 100 nmoles
Co nitrate, and 1 to 7 x 105 cpm 60Co are added to a small test tube. The tube is stoppered,

vortexed for 30 s, and centrifuged for 20 min at low speed. A 100 ml aliquot of the upper
phase is collected for radioactivity counting. Counts are corrected for background and the
amount of fatty acids is calculated from the counts obtained from standard fatty acid
solutions. Ho RJ et al., Anal Biochem (1969).
The another method is enzymatic method. This method introduce acyl-CoA
synthetase that enable quantify fatty acids in biological fluid in coupling the formation of
activated fatty acids with three enzymatic reactions which lead to a fluorimetric detection of
NADH consumption. The method was described for the determination of plasma free fatty
acids but may be adapted to small lipid extracts dissolved in Triton X-100 solution and it is
very simple and inexpensive and well suited for automation. The presence of up to 10 nmoles
fatty acids in the sample tube is accurately determined with minimum interference from other
compounds. Jebens E et al. Scand J Clin Lab Invest (1992).
The another method is using a fatty acid binding protein. This method is useful for the
measurement of very low concentrations of fatty acids such as those found for the free fatty
acid pool in biological fluids. Using a fluorescent probe ADIFAB, composed of a fatty acid
binding protein derivatized with the fluorescent molecule acrylodan can directly measure
FFA in aqueous solutions. When excited at 385 nm, the ratio of light emission at 505/432 nm
can be converted to accurate FFA concentrations. This measurement can be used with serum
and also to monitor FFA production by cells or enzyme activities. Richieri GV et al.
Biochemistry (1993).
There are several precautions need to be take care of before or during the experiment. First
and foremost, we should wear glove before handling alkali solution (NaOH) even though the
concentration is relatively low. Besides, the filling of isopropanol should be done inside a
fume hood to prevent us from inhale in the isopropanol as it is bad for our health. Moreover,
when measuring the temperature of the mixture of isopropanol and oil, we must make sure
the bulb of the thermometer must not touching the bottom of Erlenmeyer flask as this will
cause the thermometer to record down the temperature of the flask instead of the mixture.
Lastly, we should titrate the standard alkali slowly into the flask to prevent excess usage of
standard alkali which will cause inaccuracy.

Conclusion:
Different fatty acids have different acidity number by using titration method using
phenolphthalein as an indicator. The higher the FFA% value in the oil, the higher the acid
number of the oil, thus the lower the quality of the oil. From the experiment, sample oil A5
has the lowest FFA% value, thus it is the highest quality oil among the six sample while
sample oil A1 has the highest FFA% value, which is the lowest quality oil among all.

References:
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