EXPERIMENT 5 : ANALYSIS OF LIPID USING THIN LAYER

CHROMATOGRAPHY

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1.0 Abstract
Lipids are usually defined as those components that are soluble in organic solvents but
are insoluble in water. The lipid fraction of a fatty food therefore contains a complex mixture of
different types of molecule. Even so, Triacylglycerols are the major component of most foods,
typically making up more than 95 to 99% of the total lipids present. In this experiment, it
demonstrates how to separate and identify lipids from sample and the amount of lipid from palm
oil and vegetable oil was determined. 1L of the sample on the TLC plate was spotted on to the
TLC plate was placed in the container containing the solvent system in a leaning position to the
wall of the container. The solvent was allowed to rise until it reaches the solvent front. Different
compounds in the sample mixture travel at different rates due to the differences in their attraction
to the stationary phase. By changing the solvent, the separation of components (measured by the
Rf value) can be adjusted. The Rf of olive oil which is 0.69 was higher than the Rf of palm oil;
0.31. Compound with the larger Rf value is less polar because it does not stick to the stationary
phase as long as the polar compound, which would have a lower Rf value. Lastly, The Rf value
can be improved by reducing the eluent polarity so that the spot will be lower down on the plate
and therefore the value will become smaller. Factor that affects the Rf value of TLC is the
polarity of the solvent. If the solvent is too polar all of the compounds can run right up the plate
resulting in poor separation. Other important factors that can affect Rf values is the absorbent
uniformity on the thin layer plate, same concentration, room temperature during the mobile
phase and development distance of the solvent during the mobile phase.

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2.0 Introduction
Lipids are organic compounds that contain the same elements as carbohydrates: carbon,
hydrogen, and oxygen. However, the hydrogen-to-oxygen ratio is always greater than 2:1
(Moulton, 2004). All lipids are hydrophobic, which means they are not soluble in water since
lipids are nonpolar molecules and water molecules are polar but soluble in nonpolar organic
solvent like chloroforms. This group of molecules includes fats and oils, waxes, phospholipids,
steroids (like cholesterol), and some other related compounds. Nearly all of the energy needed by
the human body is provided by the oxidation of carbohydrates and lipids. Whereas carbohydrates
provide a readily available source of energy, lipids function primarily as an energy reserve. The
amount of lipids stored as an energy reserve far exceeds the energy stored as glycogen since the
human body is simply not capable of storing as much glycogen compared to lipids. Lipids yield
9 kcal of energy per gram while carbohydrates and proteins yield only 4 kcal of energy per gram
(Anonymous, n.d).
Chromatographic separations take advantage of the fact that different substances are
partitioned differently between two phases, a mobile phase and a stationary phase. A thin layer
chromatography (TLC) plate is a sheet of glass, metal, or plastic which is coated with a thin layer
of a solid adsorbent usually silica or alumina (Carey, 2013). A small amount of the mixture to be
analyzed is spotted near the bottom of this plate. The TLC plate is then placed in a shallow pool
of a solvent in a developing chamber so that only the very bottom of the plate is in the liquid.
This liquid, or the eluent, is the mobile phase, and it slowly rises up the TLC plate by capillary
action. As the solvent moves past the spot that was applied, equilibrium is established for each
component of the mixture between the molecules of that component which are adsorbed on the
solid and the molecules which are in solution. In principle, the components will differ in
solubility and in the strength of their adsorption to the adsorbent and some components will be
carried farther up the plate than others.

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When the solvent has reached the top of the plate, the plate is removed from the
developing chamber, dried, and the separated components of the mixture are visualized. If the
compounds are colored, visualization is straightforward. Usually the compounds are not colored,
so a UV lamp is used to visualize the plates.

Figure 1: The Thin Layer Chromatography

TLC System consists of:

a) TLC plates preferably ready made with stationary phase: These are stable and chemically inert
plates on to whose surface a thin layer of stationary phase is applied. The stationary phase on the
plates is of uniform thickness and consists of fine particle size.

b) TLC chamber: This is used for the development of TLC plate. The chamber maintains
uniform environment inside for proper development of spots. It also prevents the evaporation of
solvents and keeps the process dust free.

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c) Mobile phase: This comprises of a solvent or solvent mixture recommended for the purpose.
The mobile phase used should be particulate free and of highest purity for proper development of
TLC spots. The solvents recommended are chemically inert with the sample, stationary phase.

d) A filter paper moistened in the mobile phase, to be placed inside the chamber. This helps
uniform rise in mobile phase over the length stationary phase.

The retention factor, or Rf, is defined as the distance traveled by the compound divided by the
distance traveled by the solvent.

For example, if a compound travels 2.1 cm and the solvent front travels 2.8 cm, the Rf is 0.75:

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The Rf for a compound is a constant from one experiment to the next only if the chromatography
conditions below are also constant:

solvent system
adsorbent
thickness of the adsorbent
amount of material spotted
temperature

Since these factors are difficult to keep constant from experiment to experiment, relative
Rf values are generally considered. "Relative Rf" means that the values are reported relative to a
standard, or it means that you compare the Rf values of compounds run on the same plate at the
same time.
The larger an Rf of a compound, the larger the distance it travels on the TLC plate. When
comparing two different compounds run under identical chromatography conditions, the
compound with the larger Rf is less polar because it interacts less strongly with the polar
adsorbent on the TLC plate. Conversely, if you know the structures of the compounds in a
mixture, you can predict that a compound of low polarity will have a larger R f value than a polar
compound run on the same plate.
The Rf can provide corroborative evidence as to the identity of a compound. If the identity of
a compound is suspected but not yet proven, an authentic sample of the compound, or standard,
is spotted and run on a TLC plate side by side (or on top of each other) with the compound in
question. If two substances have the same Rf value, they are likely (but not necessarily) the same
compound. If they have different Rf values, they are definitely different compounds.

EXPERIMENT 5 : ANALYSIS OF LIPID USING THIN LAYER

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2.1 Objective
1) To demonstrate how to separate and identify lipids from sample
2) To compare the amount of lipids (triglycerides and fatty acid) from palm oil and
vegetable oil

3.0 Material
1) Petroleum ether
2) Chloroform
3) Diethyl ether
4) TLC plate
5) Acetic acid
6) Eppendorf tube
7) Lipid standards (triglycerides and fatty acid)
8) Capillary tubes

EXPERIMENT 5 : ANALYSIS OF LIPID USING THIN LAYER

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4.0 Procedure

Cooking oil sample (palm oil or vegetable oil) was mixed in 1ml of petroleum etherchloroform (1:1; v/v)
The petroleum-chloroform lipid was transferred into an Eppendorf Tube

The thin layer chromatography plate was prepared using silica gel by making lines to
indicates the spots of samples to made and solvent

The solvent system of petroleum ether was prepared: acetic acid (80:30:100, v/v) and
poured the solvent system in the TLC container with a heavy lid. The depth of the solvent
should not exceed the line where the sample were spotted

The TLC plate was placed flat on the table and spot about 10L of the sample using
capillary tubes or pipette tips. Dried the spot immediately with a hair dryer to avoid
spreading and forming large diluted spots.
The TLC place was placed in the container containing the solvent system in a leaning
position to the wall of the container
The solvent was allowed to rise until it reached the solvent front

The plate was removed and allowed the solvent to evaporated at room temperature

The plate was placed at 110oC for 25 minutes and spots was detected by spraying with
50% iodine. The lipid the sample was identified

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5.0 Result

a) Palm Oil
Table 1: The retention value of palm oil
Group

Distance Travelled
for Sample
Distance Travelled
for Standard
Distance of Solvent

2.5

5.5

5.7

6.0

18.0

17.2

0.14

0.31

0.32

0.35

Retention value of
sample
Retention Value of
standard

b) Olive Oil
Table 2: The retention value of corn oil
Group

Distance Travelled
for Sample
Distance Travelled
for Standard
Distance of Solvent

3.8

12

10.1

13.5

18.9

17.5

0.20

0.69

0.53

0.77

Retention value of
sample
Retention Value of
standard

EXPERIMENT 5 : ANALYSIS OF LIPID USING THIN LAYER

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6.0 Discussion

Thin layer chromatography (TLC) is a method for identifying substances and testing the
purity of compounds. TLC is a useful technique because it is relatively quick and requires small
quantities of material. Separations in TLC involve distributing a mixture of two or more
substances between a stationary phase and a mobile phase. The stationary phase is a thin layer of
adsorbent (usually silica gel or alumina) coated on a plate. The mobile phase is a developing
liquid which travels up the stationary phase, carrying the samples with it. Components of the
samples will separate according to how strongly they adsorb on the stationary phase versus how
readily they dissolve in the mobile phase.
Different compounds in the sample mixture travel at different rates due to the differences
in their attraction to the stationary phase, and because of differences in solubility in the solvent.
By changing the solvent, or perhaps using a mixture, the separation of components (measured by
the Rf value) can be adjusted.

Separation of compounds is based on the competition of the solute and the mobile phase
for binding places on the stationary phase. For instance, if normal phase silica gel is used as the
stationary phase it can be considered polar. Given two compounds which differ in polarity, the
more polar compound has a stronger interaction with the silica and is therefore more capable to
dispel the mobile phase from the binding places. Consequently, the less polar compound moves
higher up the plate (resulting in a higher Rf value). If the mobile phase is changed to a more
polar solvent or mixture of solvents, it is more capable of dispelling solutes from the silica
binding places and all compounds on the TLC plate will move higher up the plate. It is
commonly said that "strong" solvents (elutants) push the analyzed compounds up the plate, while
"weak" elutants barely move them. The order of strength/weakness depends on the coating
(stationary phase) of the TLC plate.

EXPERIMENT 5 : ANALYSIS OF LIPID USING THIN LAYER

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The chromatographic tanks are developed in a 150-mL beaker or jar containing the eluent.
The tank need to be closed to keep the atmosphere in the beaker saturated with solvent vapor.
When the TLC plates are removed from the eluent, the solvent is allowed to evaporate. Oilseed is
colorless. The oilseed separating spots can be visualized by spraying the plate with a reagent that
will react with one or more of the components of the sample which is iodine solution.
There are three type of solvent mixture (eluent) with their respective ratio are used in this
experiment. These three solvents are not always necessary. Sometimes one solvent will be
appropriate. However, more than one solvent allow us to adjust the eluent polarity easily when a
developed plate does not give an appropriate result. A suitable eluent is one that gives a
retardation factor (Rf, also called retention factor) close to 0.5 upon developing the plate. Thus,
the most suitable solvent mixture (eluent) with respective ratio to extract the oilseed compound is
petroleum ether: diethyl ether: acetic acid (80:30:100, v/v).

During the chromatography, the eluent distributes the compounds present in the sample
over the silica. The compound sample which spotted onto the stationary phase on the plate has
two choices either adsorbs to the solid phase or being eluted by the mobile phase. Overall, the
eluent is competed with the sample for a space on the stationary phase) coated on the plate. The
more polar compound will win this competition and adhere to the stationary phase, while the rest
of the mixture will move with the mobile phase (upwards). The Rf value is the ratio of the
distance travelled by the center of the spot to the distance simultaneously travelled by the mobile
phase.

In this experiment, two samples were used; Palm oil and Olive oil. Each sample was
prepared by two groups. Firstly, measured 1 cm from the bottom of the thin layer
chromatography plate by using a pencil, draw a line across the plate at the 1 cm mark. This is the
origin where the sample will be spotted. Take care not to press so hard with the pencil that you
disturb the adsorbent. Then, spotted about 1L of the sample on the TLC plate

EXPERIMENT 5 : ANALYSIS OF LIPID USING THIN LAYER

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. The TLC plate was placed in the container containing the solvent system in a leaning
position to the wall of the container. The solvent was allowed to rise until it reaches the solvent
front. As the solvent slowly travels up the plate, the different components of the dye mixture
travel at different rates and the mixture is separated into different coloured spots. After a
separation is complete, individual compounds appear as spots separated vertically. Each spot has
a retention factor (Rf) which is equal to the distance migrated over the total distance covered by
the solvent. The Rf formula is,

Rf =
The Rf value can be used to identify compounds due to their uniqueness to each
compound. By definition the Rf values are always less than unity. They are usually given to two
decimal places. From the comparison of the standard that was used, the value of the standard
should be the same as the standard used by other group but there are certain group having
different standard value. This is because the plate might be overloaded, in other words the
solution used to spot the plate is too concentrated or there are simply too many components in
the mixture to be separated by thin layer chromatography. Moreover from the comparison of
palm oil from group 1 with the standard, the value of retention factor is too far to the retention
factor of standard. This is because either the adsorbent has flaked off the sides of the plate or the
sides of the plate are touching the sides of the container (or the paper used to saturate the
container) as the plate develops. Crooked plates make it harder to measure R f values accurately.
The value of reference standard for this experiment was 0.32 from group 1 and 0.35 from group
2 which as to the sample is palm oil. This showed that the palm oil does fall under required
standard which the value of retention factor is 0.14 from group 1 and 0.31 from group 2. Same
goes to the value of olive oil which is the value of the retention factor sample does fall under
required standard which the value from group 1 is 0.20 and from group 2 is 0.69 with the
standard value is from group 1 is 0.53 and group 2 is 0.77.

EXPERIMENT 5 : ANALYSIS OF LIPID USING THIN LAYER

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When comparing between Palm oil and Olive oil under the same conditions, the Rf of
olive oil which is 0.69 was higher than the Rf of palm oil; 0.31. The compound with the larger Rf
value is less polar because it does not stick to the stationary phase as long as the polar
compound, which would have a lower Rf value. The compound with the larger Rf is less polar
because it interacts less strongly with the polar adsorbent on the TLC plate. This shows that the
polarity of olive oil is higher than palm oil. Rf values and reproducibility can be affected by a
number of different factors such as layer thickness, moisture on the TLC plate, vessel saturation,
temperature, depth of mobile phase, nature of the TLC plate, sample size, and solvent
parameters. These effects normally cause an increase in Rf values. However, in the case of layer
thickness, the Rf value would decrease because the mobile phase moves slower up the plate.

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7.0 Conclusion
Chromatography works on the principle that different compounds will have different
solubility and adsorption to the two phases between which they are to be partitioned. As the
conclusion, the objectives of this experiment; to demonstrate how to separate and identify lipids
from sample and to compare the amount of lipids (triglycerides and fatty acid) from palm oil and
vegetable oil were achieved because thin layer chromatography is a useful technique when trying
to identify compounds and see how they separate. It is also a useful tool to see how polar or
nonpolar a compound is. Thin Layer Chromatography is an analytical tool widely used because
of its simplicity, relative low cost, high sensitivity, and speed of separation.
How to improve the Rf value? We can reduce the eluent polarity so that the spot will be
lower down on the plate and the Rf value therefore will become smaller as well. We can either
choose a different eluent or adjust the solvent ratio by increasing the percentage of the nonpolar
solvent relative to the polar solvent in the eluent. There are many advantages of calculating the
Rf value for each spot on the plate. Rf value can be used to identify a particular chemical
substance. It is like a 'fingerprint' to identify an unknown substance based on known Rf value of
a list of substance.

Besides, the factor that affects the Rf value of TLC is the polarity of the solvent. If the
solvent is too polar all of the compounds can run right up the plate resulting in poor separation.
The Rf value is also used to established the identity of the spots of TLC plate. In fact the Rf
value is ratio of the distance the solvent travels to the distance compound travels. Here are some
other important factors that can affect Rf values; the absorbent uniformity on the thin layer plate,
same concentration (spotting is too weak or strong), room temperature during the mobile phase
and development distance of the solvent during the mobile phase

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8.0 References
1) Anonymous. (n.d). Thin Layer Chromatography. [Online]. [Accessed 31th October
2014]. Available from : http://orgchem.colorado.edu/
2) Carey, J.L. (2013). Retention Factor. [Online]. [Accessed 31th October 2014]. Available
from : http://chemwiki.ucdavis.edu/
3) Moulton, R. (2004). Thin Layer Chromatography. [Online]. [Accessed 31th October
2014]. Available from : http://www.chemguide.co.uk/
4) RSC. 2010. Palm oil. [Online]. [Accessed 31th October 2014] Available from :
http://www.rsc.org/
5) Theoretical Basis of Thin Layer Chromatography (TLC). (2009). [pdf]. Available from :
file:///C:/Users/Dyan/Downloads/9783642107276-c1.pdf [Accessed 31th October 2014]

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9.0 Appendices
9.1 Calculation to find Retention Factor

Rf =
Distance of solvent =18cm
1. Sample Palm Oil =2.5cm

Rf =
= 0.14
2. Standard = 5.7cm

Rf =
= 0.32
3. Sample Olive Oil =3.8cm

Rf =
= 0.21
4. Standard = 10.1cm

Rf =
= 0.56

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9.2 Picture of Result Thin Layer Chromatography for each sample

Figure1: Result Thin Layer Chromatography of Palm oil

Figure1: Result Thin Layer Chromatography of Olive Oil