JOURNAL OF BIOCHEMISTRY EXPERIMENT

(LIPIDE QUANTITATIVE TEST )

Created By :

Airiza Dian Luthfiana

PKU 2018
18030194032

CHEMISTRY DEPARTMENT
FACULTY OF MATHEMATIC AND SCIENCE
STATE UNIVERSITY OF SURABAYA
A. TITLE OF EXPERIMENT :
Lipide Quantitative Test
B. DATE AND TIME OF EXPERIMENT
Wednesday, October 27th 2021 at 13.00 p.m.
C. EXPERIMENT FINISHED
Wednesday, October 27th 2021 at 15.30 p.m.
D. PURPOSE OF EXPERIMENT
To Determine The Number Of Peroxides And Free Fatty Acids
E. BASIC THEORIES
1. Lipids
Lipids are considered one of the most elemental nutrients for humans.
Lipid metabolism generates many bioactive lipid molecules, which are
fundamental mediators of multiple signaling pathways and they are also
indispensable compounds of cell membranes. Any kind of changes in lipid
metabolism can result in modification of membrane composition and
subsequently in changes in its permeability. It may also lead to disruption of
signaling networks and could be associated with some pathological states,
such as cancer, cardiovascular, neurodegenerative, and metabolic diseases,
and similarly with inflammatory complications. (Orsavova, Misurcova,
Ambrozova, Vicha, & Mlcek, 2015).
In general, the classification of lipids is based on the basic framework
into complex lipids and simple lipids. The first group can be hydrolyzed,
while the second group cannot be hydrolyzed. Simple lipids include fatty acid
esters with various alcohols. Examples of simple lipids include:
1. Fats are esters of fatty acids with glycerol.
2. Oil is fat in a liquid state
3. Wax is an ester of fatty acids with alcohol monohydrate with a high
molecular weight.
In contrast to simple lipids, complex lipids are fatty acid esters containing
groups other than alcohols and fatty acids, such as phospholipids and
glycolipids. Phospholipids are lipids that contain a phosphoric acid residue,
in addition to fatty acids and alcohol, while glycolipids are lipids that contain
fatty acids, sphingosine, and carbohydrates (Mamuaja, 2017).
Lipids consist of fatty acids (FAs) classified mostly according to the
presence or absence of double bonds as saturated (SFAs—without double
bonds), monounsaturated (MUFAs—with one double bond) and
polyunsaturated fatty acids (PUFAs—with two or up to six double bonds);
further, as cis or trans based on the configuration of the double bonds and as
n-3 or n-6 PUFAs depending on the position of the first double bond from the
fatty acid methyl-end. The human body cannot synthesize PUFAs with the
first double bond on C3 and C6 from the methyl-end because of the absence
of appropriate enzymes. Thus, these fatty acids are essential (EFAs) and they
have to be obtained from a diet, particularly by the consumption of fish and
fish oils (Orsavova, Misurcova, Ambrozova, Vicha, & Mlcek, 2015)
Unsaturated fatty acids can exist in a cis- or trans-configuration. The
former configuration is found in most naturally occurring unsaturated fatty
acids, the latter configuration is the result of technology processing, such as
hydrogenation. Cis-unsaturated fatty acids are potent inducers of adiposomes
known as lipid droplets, which have important roles in cell signaling,
regulation of lipid metabolism and control of the synthesis and secretion of
inflammatory mediators. Lipid droplets are sites for eicosanoid generation in
cells during a process of inflammation and cancer (Orsavova, Misurcova,
Ambrozova, Vicha, & Mlcek, 2015).
1.1 Free Fatty Acid
Generally FFA are the hydrolysis product generating as a result of the
oil and fat oxidation during long time storage or processing at elevated
temperature and heating or frying. Initially, AOCS 2 , AOAC3 and
European Commission Regulation (EEC)4 have recommended standard
methods for the determination of FFA. These methods are almost similar
and based on the titration of oil (3.5-56.4 g). Oils or fats are dissolved in
 hot neutralized 95% ethanol (50-100 ml) or ethanol/diethyl ether (50-150
55 ml), against a strong base using phenolphthalein as an indicator.
Although, these titrimetric methods are very simple but extremely
laborious and need large amount of expensive chemicals associated with
environmental issues. Furthermore, the analysis of dark crude oils in the
presence of colorimetric indicator is very problematic to find out the
accurate end point during titration (Khaskheli, Mahesar, & Sherazl,
2014).
Hydrolysis of the ester linkage of triacylglycerols (TAG) to produce
FFAs, diglycerides (DAG), monoglycerides (MAG), and glycerol leads to
various undesirable changes, including production of off-flavours,
decrease of smoke point and acceleration of further hydrolysis.
Hydrolysis also leads to a drop of surface tension of the oil thereby,
increasing oxygen accessibility during frying and thus promoting
oxidative degradation of oil. The acid– base titration of FFA, using
phenolphthalein for endpoint determination has been the most commonly
used in routine assessment of frying oil quality. Although the method was
significantly improved following the introduction of technologies such as
potentiometric endpoint determination, it still suffers from several
drawbacks, including requirements for large amounts of organic solvents
and a large sample size (He & Bazena, 2018).
2. Peroxide
Peroxide is formed in the oxidation initiation step, at this stage
hydrogen is taken from the compound olefins generate free radicals. The
presence of light and metals play a role in the process uptake of the hydrogen.
The free radicals formed react with oxygen to form peroxy radicals, can then
take hydrogen from other unsaturated molecules to produce new peroxides
and free radicals. Peroxide can speed up the process of developing a rancid
odor and unwanted odors in food. If the amount of peroxide is more than 100
meq peroxide/kg of oil will be very toxic and have an unpleasant odor.
 Ascension the peroxide number is an indicator that the oil will smell rancid
(Husnah & Nurlelah, 2020).
The peroxide number is an index of the amount of fat or oil that has
undergone oxidation. The peroxide value is very important for the
identification of the oxidation state of the oil. The oil that containing
unsaturated fatty acids can be oxidized by oxygen to produce a peroxide
compounds. The most commonly used method for determining the peroxide
value is by iodometric titration method. The spoilage of the oil will affect the
quality and nutritional value of the food which is fried. Heating cooking oil to
a very high temperature will cause partially oxidized oil. Oil damaged by the
oxidation process will produce food unattractive color and unpleasant taste,
as well as damage to some vitamins and fatty acids essential in the oil. The
oxidation process occurs when the oil is in contact with some oxygen. The
oxidation reaction will also cause a rancid odor to the oil and fat (Ketaren, S.
1986). In addition to causing a rancid odor, free radicals can also be formed
due to oxidation which has the effect of damaging cells and body tissues.
This is due to radical free is very reactive (Husnah & Nurlelah, 2020).
3. Titration
The definition of titration is valid unchanged in its core: We need a
stoichiometric reaction, a precisely dosable, stable reagent and a detection of
the end of the reaction end or a curve showing the course of the reaction. In
acid-base or neutralization titration, acids are titrated with a base (or vice
versa). The detection of the equivalence point can take place by colour
indicators or potentiometrically with a glass electrode. The reaction is the
same for all acid/base titrations, water results from a proton and a hydroxide
ion.
H+ + OH-  H2O
The manual titration can be carried out with simple glass burettes or
with piston burettes. It still has its legitimacy when it comes to carrying out
very few individual content determinations with minimal effort. Manual
 titration is still included in many older standards as prescribed methods.
However, the automated methods have prevailed today. They can be
implemented analogously to the "old" methods, optimize and accelerate the
processes. In manual titration, an indicator is usually used that changes its
colour at the EQ (Reining, 2016).
In a redox titration, oxidizing components are titrated with a reducing
agent, or vice versa. The oxidation states of the reactants and thus the redox
potential of the sample change. The detection of the EQ can be carried out by
colour change (of colour indicators or the sample solution),
potentiometrically with a redox electrode (usually a Pt electrode) or
biamperometrically with a double platinum electrode (Reining, 2016).
Permanganometri is a titration method that uses the principle of
reduction and oxidation reactions. This method is a method that is often used
because permanganometri has advantages, among others, Permanganometri is
a strong oxidizing agent, does not require indicators, is easy to obtain and
affordable. The drawback of this method is that this solution is not stable in
storage, so it must be standardized frequently (Sugiarso, 2016).
F. Tools And Material
1. Tools
- Beaker glass 3 pieces
- Burette 1 unit
- Erlenmeyer 6 pieces
- Volume pipette 1 piece
- Pro pipette 1 piece
- Measuring cup 3 pieces
- Drop pipette 6 pieces
- Analitycal balance 1 unit
- Clamping stand 1 set
2. Materials
- Oil/ fat 5 ml and 6 grams
- 0.1 N NaOH solution sufficiently
- 1% PP indicator sufficiently
- Ethanol 96% 20 ml
- Aquades 51 ml
- H2SO4 solution 4N 15 ml
- KMnO4 solution 0.1N sufficiently
- Standard solution of 0.1N oxalate sufficiently
G. PROCEDURE
1. Determination of Peroxide Number
a. Sample

5 mL oil sample (new and used)

1. Put each oil into the erlenmeyer

2. Added 45 mL of aquades
3. Added 15 mL H2SO4 4 N
4. Titrate with 0.1 N KMnO4 solution

Pink solution (stable color)

5. Titrate 3 times
6. Record the volume of 0.1 N KMNO4 solution used
7. Calculated peroxide number

Pink solution (stable color)

Reaction:
2KMnO4 (aq) + 5H2O (aq) + 3H2SO4 (aq) → 2MnSO4 (aq) + K2SO4 (aq) +
8H2O (l)
Half cell reaction:
Reduction : MnO4 - (aq) + 8H+ (aq) + 5e-→ Mn2+ (aq) + 4H2O (l) (×2)
Oxidation : H2O2 (aq) → O2 (g) + 2H+ (aq) + 2e- (×5)
_______________________________________________
2MnO4- (aq) + 16H+ (aq) + 10e-→ Mn2+ (aq) + 8H2O(l)
5H2O2 (aq) → 5O2 (g) + 10H+ (aq) + 10e-
_______________________________________________
2MnO4- (aq) + 6H+ (aq) + 5H2O2 (aq) → 2Mn2+ (aq) +
8H2O (l) + 5O2 (g)
2. Determination of Free Fatty Acids (FFA)
a. Blank solution

6 grams of aquades

1. Put in erlenmeyer
2. Added 10 mL of 96% alcohol
3. Added 5 drops of PP indicator
4. Titrate with standardized 0.1 N NaOH

Pink solution (stable color for 30 seconds)

5. Note the volume of 0.1 N NaOH used

Blank volume

b. Sample solution

6 grams of oil sample (new and used)

1. Put in erlenmeyer
2. Added 10 mL of 96% alcohol
3. Added 5 drops of PP indicator
4. Titrate with standardized 0.1 N NaOH

Pink solution (stable color for 30 seconds)

5. Titrate 3 times
6. Note the volume of 0.1 N NaOH used

Blank volume

7. Titrate 3 times
8. Note the volume of 0.1 N NaOH used

Free fatty acid content (% FFA)

 Reaction:
CH3(CH2)14COOH (aq) + NaOH (aq) → CH3(CH2)14COONa (aq) + H2O (l)

H. REFERENCES

He, J., & Bazena, N. (2018). Analysis of Fatty Acid Profiles of Free Fatty Acids
Generated In Deep-Frying Proces. Journal of Food Science and
Technology .

Husnah, & Nurlelah. (2020). Analisa Bilangan Peroksida Terhadap Kualitas

Minyak Goreng Sebelum dan Sesudah Dipakai Berulang. Palembang:
Universitas PGRI Palembang.

Khaskheli, A. R., Mahesar, S. A., & Sherazl, S. T. (2014). Analytical

Approaches For Free Fatty Acids Assesment in Oil and Fats. Analytical
Methods .

Mamuaja, C. F. (2017). LIPIDA. Manado: Unsrat Press.

Orsavova, J., Misurcova, L., Ambrozova, J. V., Vicha, R., & Mlcek, J. (2015).
Fatty Acids Composition of Vegetable Oils and Its Contribution to Dietary
Energy Intake and Dependence of Cardiovascular Mortality on Dietary
Intake of Fatty Acids. J. Mol. Sci .

Reining, R. (2016). Titration Handbook. Germany.

Sugiarso, F. A. (2016). Perbandingan Metode Analisis Permanganometri dan

Serimetri dalam Penentuan Kadar Besi(II). JURNAL SAINS DAN SENI ITS
, 2337-3520.