Experiment Report of BioChemistry 1

I.

TITLE OF EXPERIMENT

: Quantitative Test Of Lipid

II.

DATE OF EXPERIMENT

: November 28th 2014

III.

END OF EXPERIMENT

: November 28th 2014

IV.

PURPOSE OF EXPERIMENT

: Determine the peroxide number and

free fatty acid

V.

BASIC THEORY

A. LIPIDS

Lipids differ from the other

classes

of

naturally

occurring

biomolecules (carbohydrates, proteins,

and nucleic acids) in that they are
more soluble in non-to-weakly polar
solvents

(diethyl

ether,

hexane,

dichloromethane) than they are in water. They include a variety of structural types,
a collection of which is introduced in this chapter. In spite of the number of
different structural types, lipids share a common biosynthetic origin in that they are
ultimately derived from glucose. During one stage of carbohydrate metabolism,
called glycolysis, glucose is converted to lactic acid. Pyruvic acid is an
intermediate.

In most biochemical reactions the pH of the medium is close to 7. At this pH,

carboxylic acids are nearly completely converted to their conjugate bases. Thus, it
is common practice in biological chemistry to specify the derived carboxylate
anion rather than the carboxylic acid itself. For example, we say that glycolysis
leads to lactate by way of pyruvate. Pyruvate is used by living systems in a number
of different ways. One pathway, the one leading to lactate and beyond, is
concerned with energy storage and production. This is not the only pathway
available to pyruvate, however. A significant fraction of it is converted to acetate

Experiment Report of BioChemistry 1

for use as a starting material in the biosynthesis of more complex substances,
especially lipids. By far the major source of lipids is biosynthesis via acetate and
this chapter is organized around that theme. Well begin by looking at the reaction
in which acetate (two carbons) is formed from pyruvate (three carbons).
In most biochemical reactions the pH of the medium is close to 7. At this pH,
carboxylic acids are nearly completely converted to their conjugate bases. Thus, it
is common practice in biological chemistry to specify the derived carboxylate
anion rather than the carboxylic acid itself. For example, we say that glycolysis
leads to lactate by way of pyruvate.
Pyruvate is used by living systems in a number of different ways. One
pathway, the one leading to lactate and beyond, is concerned with energy storage
and production. This is not the only pathway available to pyruvate, however. A
significant fraction of it is converted to acetate for use as a starting material in the
biosynthesis of more complex substances, especially lipids. By far the major source
of lipids is biosynthesis via acetate and this chapter is organized around that theme.
Well begin by looking at the reaction in which acetate (two carbons) is formed
from pyruvate (three carbons).

B. FATS, OILS, AND FATTY ACIDS

Fats are one type of lipid. They have a number of functions in living systems,
including that of energy storage. Although carbohydrates serve as a source of
readily available energy, an equal weight of fat delivers over twice the amount of
energy. It is more efficient for an organism to store energy in the form of fat
because it requires less mass than storing the same amount of energy in
carbohydrates or proteins. How living systems convert acetate to fats is an
exceedingly complex story, one that is well understood in broad outline and
becoming increasingly clear in detail as well. We will examine several aspects of
this topic in the next few sections, focusing mostly on its structural and chemical
features.
Fats and oils are naturally occurring mixtures of triacylglycerols, also called
triglycerides. They differ in that fats are solids at room temperature and oils are
liquids. We generally ignore this distinction and refer to both groups as fats.

Experiment Report of BioChemistry 1

Triacylglycerols are built on a glycerol framework.

All three acyl groups in a triacylglycerol may be the same, all three may be
different, or one may be different from the other two. Figure 26.2 shows the
structures of two typical triacylglycerols, 2-oleyl-1,3- distearylglycerol (Figure
26.2a) and tristearin (Figure 26.2b). Both occur naturallyin cocoa butter, for
example. All three acyl groups in tristearin are stearyl (octadecanoyl) groups. In 2oleyl-1,3-distearylglycerol, two of the acyl groups are stearyl, but the one in the
middle is oleyl (cis-9-octadecenoyl). As the figure shows, tristearin can be
prepared by catalytic hydrogenation of the carboncarbon double bond of 2-oleyl1,3- distearylglycerol. Hydrogenation raises the melting point from 43C in 2oleyl-1,3- distearylglycerol to 72C in tristearin and is a standard technique in the
food industry for converting liquid vegetable oils to solid shortenings. The
space-filling models of the two show the flatter structure of tristearin, which allows
it to pack better in a crystal lattice than the more irregular shape of 2-oleyl-1,3distearylglycerol permits. This irregular shape is a direct result of the cis double
bond in the side chain. Hydrolysis of fats yields glycerol and long-chain fatty
acids. Thus, tristearin gives glycerol and three molecules of stearic acid on
hydrolysis. Table 26.1 lists a few representative fatty acids. As these examples
indicate, most naturally occurring fatty acids possess an even number of carbon
atoms and an unbranched carbon chain.
The structure of Fatty Acids :

Experiment Report of BioChemistry 1

A few fatty acids with trans double bonds (trans fatty acids) occur naturally,
but the major source of trans fats comes from the processing of natural fats and
oils. In the course of hydrogenating some of the double bonds in a triacylglycerol,
stereoisomerization can occur, converting cis double bonds to trans. Furthermore,
the same catalysts that promote hydrogenation promote the reverse process
dehydrogenationby which new double bonds, usually trans, are introduced in the
acyl group.
Fatty acids occur naturally in forms other than as glyceryl triesters, and
well see numerous examples as we go through the chapter. One recently
discovered fatty acid derivative is anandamide.

Anandamide is an ethanolamine (H2NCH2CH2OH) amide of arachidonic

acid (see Table 26.1). It was isolated from pigs brain in 1992 and identified as the
substance that normally binds to the cannabinoid receptor. The active component

Experiment Report of BioChemistry 1

of marijuana, 9-tetrahydrocannabinol (THC), must exert its effect by binding to a
receptor, and scientists had long wondered what compound in the body was the
natural substrate for this binding site. Anandamide is that compound, and it is now
probably more appropriate to speak of cannabinoids binding to the anandamide
receptor instead of vice versa. Anandamide seems to be involved in moderating
pain. Once the identity of the endogenous cannabinoid was known, scientists
looked specifically for it and found it in some surprising placeschocolate, for
example. Fatty acids are biosynthesized by way of acetyl coenzyme A. The
following section outlines the mechanism of fatty acid biosynthesis.
The formula to determination perecentage of Free Fatty Acids (FFA) :
(

C. PEROXIDE NUMBER
Peroxide number is index of fat or oil amount that have been oxidize. Peroxide
number is very important to identfy oil oxidation action.Oil that contain
unsaturated fatty acid can oxidized by oxygen that produce peroxide compound.
The way that often to determine the peroxide number is iodometry.
Determination of peroxide less well with regular iodometric way though
peroxide reacted with alkaline iodine. This is because other types peroxide reacts
only partially. In addition, errors may occur caused by the reaction between alkali
iodide with oxygen from the air.
Oxidation processes stimulated by the metal if it goes with intensive will lead
to rancidity and discoloration (darkening). This situation is obviously very
detrimental because the quality of palm oil is lowered. When the fat is heated, at a
certain temperature arises thin bluish smoke. This point is called the point of
smoke (smoke point). When heating is achieved forwarded flash point, namely the
oil starts to burn (visible flame). If the oil is burned regularly called fire point. The
temperature of the smoke point will vary and are influenced by the amount of free
fatty acids. If a lot of free fatty acids, the third temperature will go down. Similarly,
if a low molecular weight, temperature is lower third. All three of these properties
is important in determining the quality of fat used as cooking oil.
Determination peroxide number is based on measured of amount of iod that
free from potassium iodide by oxidize reaction by peroxide in room temperature in

Experiment Report of BioChemistry 1

acid acetic medium/ chloroform. Free fatty acids is determined as a containing of
fatty acid that presence much in certain oil. To reference oil is palm oil (the much
acid fat is palmiat), coconut oil (laurat acid), corn oil (linoleat acid), milk (oleat
acid).
The structure of palmiat acid is :

The formula to determination peroxide number :

( )

D. IODOMETRY TITRATION
Iodometry is titirmetric analayzed indirect to a compound that have oxidator
characteristics like Iron and zinc. In this experiment is peroxide oil ROOH that
contain peroxide ion O22- . This compounds will oxidized iodide that adding
formed iodine. Iodine that formed is determinate with used standard sodium
thiosulphate solution :
The reaction : 2e + I2 2IS2O32- S4O62- + 2e
I2 + 2S2O32- 2I- + S4O62Indicator that used in this method is strach (amylum) and the end point of titration
is signed bluish purple color.

E. HYDROLYSIS
In the hydrolysis reaction, fats and oils are converted into free fatty acids and
glycerol. Hydrolysis reactions resulting in damage to fats and oils. This happens
because there are a number of water in the fat and oil.

Experiment Report of BioChemistry 1

VI.

TOOLS AND MATERIALS

Tools :
1.

Beaker glass

(1 item)

2.

Drop pipette

(10 items)

3.

Burette

(1 item)

4.

Stative and clamp

(1 & 1 item)

5.

Erlenmeyer

(6 item)

6.

Erlenmeyer flask

(1 item)

7.

Measured glass 10 ml

(1 item)

8.

Measured glass 30 ml

(1 item)

9.

Balanced

(1 item)

10. Tissue

(Conditionally)

Materials :
1. Oil
2. Acetic acid-chloroform solution
3. Saturated of KI solution
4. Na2S2O3 0.05 N solution
5. Amylum solution 1 %
6. NaOH solution 0,1 N
7. Indicator pp
8. Alcohol 96 % solution

Experiment Report of BioChemistry 1

VII.

PROCEDURE

1. Determination of Peroxide Number

Oil / Lipid
- Weighed in 3 grams
- Entered in erlenmeyer Flask
- Added 30 mL of acetic acid-chloroform solution
- Shake until perfectly dissolved

- Added 0.5 mL of KI solution

- Let it until 20 minutes (sometimes shaked)
- Added 30 mL of distilled water
- Titrated with Na2S2O3 solution until yellow color
almost disappear
- Added 0.5 mL of amylum solution
- Titrated again with Na2S2O3 0.1 N until saturated
Volume of Na2S2O3

2.

Blanco Solution
Distilled water
- Weighed in 5 grams
- Entered in erlenmeyer Flask
- Added 30 mL of acetic acid-chloroform solution
- Shaked until perfectly dissolved

- Added 0.5 mL of KI solution

- Let it until 20 minutes (sometimes shake)
- Added 30 mL of distilled water
- Added 0.5 mL of amylum solution
- Titrated again with Na2S2O3 0.1 N until saturated
Volume of Na2S2O3

Experiment Report of BioChemistry 1

3. Determination of FFA (Free Fatty Acid)
Oil/ Lipid
- Balance 6 grams
- Entered in erlenmeyer flask
- Added 10 mL of alcohol 96%
- Added 5 drops of PP indicator
- Titrated with NaOH 0.1 N
Volume of NaOH

4. Blanco Solution
Distilled water
- Weighed 6 grams
- Entered into erlenmeyer flask
- Added 10 mL of alcohol 96%
- Added 5 drops of PP indicator

- Titrated with NaOH 0.1 N

Volume of NaOH

Experiment Report of BioChemistry 1

IX.

DATA ANALYSIS

1. Determination of peroxide value

This experiment purpose is to determine the peroxide value in
oil. Peroxide value is affected by the formation of peroxide
compounds resulting from the reaction of the oil with O2 in the air so
that the oil undergoes oxidation. Peroxide value is determined based
on the amount of iodine liberated from KI through oxidation by
peroxide in fats / oils at room temperature in acetic acid-chloroform
medium.
Blanco Solution
Steps are weighing 5 grams of distilled water is clear and colorless
in erlenmayer. Then add 30 mL of acetic acid-chloroform make the
solution cloudy and there are two phases, phase upon clear and
colorless while the bottom of the froth phase. In the upper phase was
distilled water mixed with acetic acid, while the lower phase was
chloroform can not be mixed with distilled water. The addition of
acetic acid-chloroform was used as a solvent, a solution of acetic
acid is a polar solvent while the chloroform solution of a non-polar
solvent. Then added 0.5 mL of saturated KI solution is clear and
colorless, permanent solution there are two phases in which the upper
phase is colorless and the bottom of the froth phase. Then allowed to
stand for 20 minutes while occasionally shaken. Furthermore, plus 30
mL of distilled water so that the solution becomes cloudy. Afterwards
plus 0.5 mL of 1% starch solution but no change in the mix.
Subsequently titrated with 0.1 N Na2S2O3 solution colorless until the
solution in the phase above erlenmayer be clear, this color change
indicates that the presence of I2. From these experiments obtained
for the blank solution titration volume of 0.2 mL.

Experiment Report of BioChemistry 1

Samples used cooking oil (minyak jelantah)
The first step taken was to prepare 3 erlenmayer and weigh 5
grams of cooking oil brownish yellow in 3 erlenmayer. Then added to
30 mL of acetic acid-chloroform (colorless) with a ratio of 3: 2. The
function of the addition of acetic acid-chloroform was as solvent.
because oil is a group that includes the class of lipids, which is an
organic compound found in nature and is not soluble in water, but
soluble in non-polar organic solvents for example, chloroform
(CHCl3), benzene and other hydrocarbons, fats and oils can be
dissolved in a solvent because oil has the same polarity as the
solvent.
Acetic acid is a polar solvent that will dissolve in the oil
component is polar, while the chloroform is a nonpolar solvent that
will dissolve the components in a nonpolar oil. In addition, a solution
of acetic acid-chloroform also serves to provide acidic conditions on
the mixture. Once added to a solution of acetic acid-chloroform, then
erlenmayer shaken to form two phases. Under the yellow phase (+)
which is a fatty acid that is soluble in chloroform, while the upper
phase yellow (-) is glycerol soluble in acetic acid. Chloroform was in
the bottom layer because it has a specific gravity greater than acetic
acid.
After the mixture was added 0.5 mL of a solution of KI (colorless),
produces yellow (+) color. Then allowed to stand for 20 minutes while
occasionally shaken. The function of the addition of KI is to liberate
iodine which marked the formation of a yellow color in the sample
and to determine the peroxide value, as KI will be oxidized by
peroxide into I2. After 20 minutes, the mixture was added 30 mL of
distilled water (colorless), is intended that the solution can be
mixed evenly and produce a mixture of the two phases upper layer is
yellow turbid and lower layer is brownish orange. The reaction was as
follows:
CH3(CH2)14COOH + 2 KI + H2O I2 + 2 KOH + CH3(CH2)14COH

Experiment Report of BioChemistry 1

Before doing titrated with Na2S2O3 solution was added a solution

of 1% starch in advance. The addition of starch serves as an
indicator of the presence of I2 formed 2 layers, the upper layer is light
yellow and the lower layer is yellow (+). After that titrated back with
Na2S2O3 solution until the top phase clearer than ever before. Reactions
involved as follows:
O22-

2e-

2O2-

Ion peroxidw

O2- bond in group OH-

2I-

I2

+ 2e-

Reaction I2 dengan sodium thiosulphate as follows :

2e + I2 2IS2O32- S4O62- + 2e
I2 + 2S2O32- 2I- + S4O62Starch solution serves as an indicator of the presence of I2
oxidized by peroxide compounds. From these experiments obtained
Na2S2O3 volume required for titration of 10.5 mL; 10.8 mL; and
11.0mL. It can be obtained from the calculated volume peroxide.
The purpose of this peroxide Numbers determination is to
determine the level of damage to the fat or oil, where the damage is
caused by a reaction that produces peroxide oxide, fatty acids,
aldehydes, and ketones. Peroxide value is determined based on the
amount of I2 are exempt from KI through oxidation by peroxide
compounds contained in oil at room temperature in acetic acidchloroform medium. Peroxide value indicates the amount of peroxide
contained in the oil / fat. The level of peroxide can determine the quality
of the oil. That is, high semakain peroxide value, then the oil / fat is
getting ugly. Because we use oil sample is likely to contain high
antioxidant that makes the sample can not be titrated so that we have not
been able to perform a quantitative analysis of the determination of
peroxide value.
Based on the calculation of the average peroxide for used cooking
oil that we use is 210,92 meq / g. The resulting peroxide differ much

Experiment Report of BioChemistry 1

from the theory that 6 meq. This indicates that the oil is not good to use
because it can be harmful to health. The high rate of peroxide caused by
the oil used has been warming many times so that the oil has been
oxidised.

2. Determination of Free Fatty Acids FFA

This experiment is to determine the free fatty acid (FFA) in the
sample. Sample used was a brownish yellow cooking oil because it is
often used for cooking. In the oil contained glycerol and fatty acids,
palm oil fatty acids are palmitic acid.
Blanco Solution
The first step is making blanco solution with weighing 6 grams of
distilled water (colorless) and colorless into erlenmayer flask then
added 10 mL of 96% alcohol solution is clear and colorless, the result
remains clear colorless solution. Alcohol solution serves as an organic
solvent. Then add 5 drops of indicator PP clear and colorless so formed
two layers, the upper layer is brownish yellow and the lower layer is
yellow color. Furthermore, the solution was titrated with 0.1 N NaOH
solution titrated. The purpose of this is to determine the volume of
NaOH used for titration analyte, the titration was stopped after the
color changes to red color in the upper layer and lower layer in yellow
color which indicates that the endpoint has been reached, the color is
due to the indicator changes color at the equivalence point. The
volume of NaOH required is 0,4ml. With the reaction :

H2O +

H3O+ +

H3O+ + NaOH Na+ + 2H2O

Experiment Report of BioChemistry 1

Free Fatty Acids

Making blanco solution is used to compare with the sample
solution. After that determine the free fatty acids in the oil sample,
which performed the same steps as in the manufacture of blank solution
but replace distilled water with oil. 6 grams of oil which has been
weighed in erlenmayer then added alcohol 96% as much as 10 mL, the
results formed two layers in which the upper layer is brownish yellow
and the lower phase is yellow. Alcohol is a polar solvent that is used
to dissolve the oil components in the polar ie glycerol that is left is a
nonpolar component is a fatty acid.
Then the sample is added as much as 5 drops PP indicator and
titrated with 0.1 N NaOH solution formed 2 . Titration was stopped
after the color changes to red brick which indicates that the endpoint
has been reached. Titration was repeated 3 times, volume of NaOH
required to titrate a row was 1,5 mL; 1,5 mL; and 1,6 mL. Titration of
volume data, can be calculated levels of free fatty acids in the oil
sample. Limit fatty acids are good for oil is 2%. In this experiment the
levels of free fatty acids in the oil sample is 0.48%. This shows that the
oil is still in good condition. With the Reaction :

+ NaOH

X.

CONCLUSION
From the experiments it can be concluded as follows:
1. Numbers peroxide for used cooking oil sample was 210,92 meq. It shows
that oil is in bad condition because has get much oxidation.

Experiment Report of BioChemistry 1

2. The content of free fatty acids in the sample was 0.48% used cooking oil.

Experiment Report of BioChemistry 1

REFFERENCES

Agustini, Rudiana dkk. 1993. Petunjuk Praktikum Biokimia. Surabaya: Unipress;

IKIP
Carey, A Francis. Organic Chemistry 4th Ed. Ebook: University of Virginia
Lehninger, Albert L. 1990. Dasar-dasar Biokimia Jilid 1. Terjemahan.
Jakarta;:Erlangga
Nelson, D.L. Lehninger. 1990. Principle of Biochemistry 4th. New York:
W.H.Freeman
Tim Penyusun. 2012. Petunjuk Praktikum Biokimia. Surabaya: Unesa Press

Experiment Report of BioChemistry 1

ANSWER OF QUESTION
1. Write down all reaction that include in fatty acid test in this
experiment !
Answer:
2e + I2 2IS2O32- S4O62- + 2e
I2 + 2S2O32- 2I- + S4O62-

2. Mention the essential fatty acid that presence in body, Why arakidonat
acid is not essential fat ?
Answer:
For humans, the essential fatty acids to include the plural unsaturated fatty
acids (polyunsaturated fatty acids, PUFAs) cis types, in particular from the
group of Omega-3 fatty acids, such as -linolenic acid (ALA),
eicosapentaenoic acid (EPA), and acid docosahexaenoic (DHA) and
Omega-6 fatty acids, such as linoleic acid. The human body is unable to
produce denaturase enzyme but is able to elongate and remodel PUFA.
3. Whats the differences of saturated fatty acid and unsaturated in
oxidation process ?
Answer:
Saturated fatty acids have only single bonds between carbon atoms
constituent, while unsaturated fatty acids having at least one double bond
between carbon atoms constituent. Warming (oxidation) will cause the
saturated fatty acids transformed into saturated fatty acids.

4.

Whats the differences between oil and fat seen by the molecule
structure ?

Experiment Report of BioChemistry 1

Answer:
In the structure of the oil has a double bond structure in the carbon chain
C, in the presence of the oil heating process can transform into fat that
structure does not have a double bond in the carbon chain C.
As an example of a hydrogenation reaction:

Experiment Report of BioChemistry 1

CALCULATED ATTACHMENT
1. Determination of peroxide number
Given :
N Na2S2O3

= 0,1 N

V1 Na2S2O3

= 10,5 mL

V2 Na2S2O3

= 10,8 mL

V3 Na2S2O3

= 11,0 mL

m1

= 5,0142 g

m2

= 5,0125 g

m3

= 5,0028 g

V blanko

= 0,2 mL

Asked : peroxide number ?

Answered :
(

)
( )

V1 Na2S2O3 = 10,5 mL
(

V2 Na2S2O3 = 10,8 mL

V3 Na2S2O3 = 11,0 mL

Peroxide number average

(

Experiment Report of BioChemistry 1

2. Determination of Free Fatty Acid (FFA)

Given :
N NaOH

= 0.1 N

V1 NaOH

= 1,5 mL

V2 NaOH

= 1,5 mL

V3 NaOH

= 1,6 mL

m1

= 6,0129 g

m2

= 6,0285 g

m3

= 6,0124 g

V blanco

= 0,4 mL

BM fatty acid

= 256 g/mL

Asked : % FFA?
Answered

)
( )

V1 NaOH = 1,5 mL
(

V3 NaOH = 1,6 mL
(

V2 NaOH = 1,5 mL
(

Average of %FFA
(

Experiment Report of BioChemistry 1

PICTURE ATTACHEMENT

No

Picture

Explanation

Determination of Peroxide Number

1
5 grams sample in
erlenmayer (V1), the color is
brownish yellow

5 grams sample in
erlenmayer (V2) the color is
brownish yellow

5 grams sample in
erlenmayer (V3) the color is
brownish yellow

Sample oil is brownish

yellow

Experiment Report of BioChemistry 1

Oil + acetic acid +

chlorofoam formed 2 layers :
- Upper layer is yellow(+)
- Lower layer is yellow (-)

Oil + acetic acid +

chlorofoam + KI become
yellow (+)
And let for 20 minutes

Oil + acetic acid +

chlorofoam + KI + distillate
water formed 2 layers :
- Upper layer is yellow turbid
- Lower layer is brownish
orange

After first titration + amylum

formed 2 layers :
- Upper layer is light yellow
- Lower layer is yellow (+)

Experiment Report of BioChemistry 1

After titrated with Na2S2O3

formed 2 layers :
- Upper layer is colorless
- Lower layer is yellow

Blanco Solution
2

5 grams blanco solution id

colorless

Blanco solution after titration

is colorless

Blanco Solution
3
6 grams Blanco solution is
colorless

Experiment Report of BioChemistry 1

Blanco solution after titrated

with NaOH become pink
color

Determination of FFA (Free Fatty Acid)

4
6 grams Sample solution
(V1), the color is brownish
yellow

6 grams Sample solution

(V2), the color is brownish
yellow

6 grams Sample solution

(V3), the color is brownish
yellow

Experiment Report of BioChemistry 1

Sample solution after added

alcohol and PP indicator is
brownish yellow

All sample solution after

titrated by NaOH become
redish yellow