EXPERIMENT 8: Thin Layer Chromatography (TLC)

OBJECTIVE
To detect lipid and plant pigments using TLC

INTRODUCTION

Chromatography is used to separate mixtures of substances into their components. All

forms of chromatography work on the same principle. They all have a stationary phase (a solid,
or a liquid supported on a solid) and a mobile phase (a liquid or a gas). The mobile phase flows
through the stationary phase and carries the components of the mixture with it. Different
components travel at different rates. Thin layer chromatography is done exactly as it says using
a thin, uniform layer of silica gel or alumina coated onto a piece of glass, metal or rigid plastic.
The silica gel (or the alumina) is the stationary phase. The stationary phase for thin layer
chromatography also often contains a substance which fluoresces in UV. The mobile phase is a
suitable liquid solvent or mixture of solvents.

TLC is a simple, quick, and inexpensive procedure that gives the chemist a quick answer
as to how many components are in a mixture. TLC is also used to support the identity of a
compound in a mixture when the Rf of a compound is compared with the Rf of a known
compound (preferably both run on the same TLC plate). 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, an 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. 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 straight
forward. Usually the compounds are not colored, so a UV lamp is used to visualize the plates

METHODOLOGY

(A) Preparation of TLC plate

1. The TLC plates was handled

2. the TLC plate was cut into strips of 2 cm 10 cm in size.
3. a line was drawn with pencil approximately 1.5 cm from the short edge of the TLC plate
and another line was marked approximately 1.0 cm from the edge of the other side

1.5cmm 1 cm
(B) Lipid detection

1. 26mL acetone , 4 mL toluene, 4 mL water, and 1 mL ammonium hydroxide was mixed

into a beaker.
2. 10 mL of this solution was pured into the TLC chamber.
3. A micropipette tip was dipped into triglyceride solution and gently touch the end of it
onto the 1.5 cm line on TLC plate to make a spot as small as possible. the spot was dried
with hair dryer. step 3 was repeated on the same spot until the spot is clearly visible
4. The plate was developed in the closed TLC chamber until the solvent front reaches to
within 1 cm from the top of the TLC plate.
5. bromophenol blue solution was sprayed on the developed plate.
6. the spot was observed and its Rf value was calculated

Distance of compound from starting point

Retention factor =
Distance of solvent from starting point

(C) Plant pigment detection

The pigments in vegetables, flowers and leaves can be separated and identified by using thin-
layer chromatography. Green pigments, known as chlorophylls, serve as the main photoreceptor
molecules of plants. Carotenoids, yellow pigments, aid the plant in the photosynthesis process.
In addition, xanthophyll is contained in the chloroplasts which can be isolated and identified
using chromatographic techniques.

1. The sample was placed in a mortar.

2. In the fume hood, 5 ml of acetone was added approximately and the sample was grinded
with the pestle until a concentrated extract is obtained.
3. A micropipette tip was dipped into the solution and the end was touched gently of it onto
the 1.5 cm line on TLC plate to make spot as small as possible, about 1 mm in diameter.
 4. The spot was dried with hair dryer. Step 3 is repeated on the same spot if necessary.
5. The plate was developed in the TLC chamber with mobile phase (70:30 hexane-acetone
solvent) until the solvent front reaches to within 1 cm from the top of the TLC plate.
6. The individual spots was circled lightly with a pencil .
7. The spot was observed and its Rf value was calculated. Identify each spot was identify by
referred on table below.

Pigment Color Rf value

carotene yellow-orange 0.93
pheophytin a grey 0.55
pheophytin b light grey (may not be visible) 0.47-0.54
chlorophyll a blue-green 0.46
chlorophyll b green 0.42
xanthophylls yellow 0.41
xanthophylls yellow 0.31
xanthophylls yellow 0.17

RESULT
 6 cm

Figure 1 : Lipid detection

Distance of compound from starting point = 6cm

Distance of solvent from starting point = 9cm
Retention factor = 0.67cm

Figure 2 : Plant Pigment Detection

Table 1 : Result for hibiscus leave

Color of component Distance of component from Rf value Pigment

origin (cm)
yellow 1.7 0.20 Xanthophylls
yellow 2.9 0.35 Xanthophylls
yellow 3.0 0.36 xanthophylls
green 3.3 0.40 Xanthophylls
yellow 3.7 0.45 chlorophyll b
Green 4.3 0.52 pheophytin b
gray 5.7 0.67 Pheophytin a

DISCUSSION

Methods thin layer chromatography is based on the principle of separation. The

separation depends on the relative affinity of compounds towards stationary and mobile phase.
The compounds under the influence of mobile phase (driven by capillary action) travel over the
surface of stationary phase. During this movement the compounds with higher affinity to
stationary phase travel slowly while the others travel faster. Thus separation of components in the
mixture is achieved. Once separation occurs individual components are visualized as spots at
respective level of travel on the plate. Their nature or characters are identified by means of
suitable detection techniques.

This experiment was conducted to detect lipid and plant pigment by using thin layer
chromatography method. This experiment also to rind retention factor each pigment that separe
by using TLC method. There are two parts in this experiment which part 1 is lipid detection and
part 2 is plant detection. For part 1, solution from mixed acetone, toluene, water, and ammonium
hydroxide was used as a solvent and triglyceride solution was used as compound. Then, for part
2, extracted from pomegranate flower and hibiscus leave was used to detect compound.

Lipids are a diverse group of compounds that serve many key biological functions.
Lipids, together with proteins and carbohydrates, constitute the principal structural components of
all tissues. They account for the major (~50%) compositional and structural element of biological
membranes. A subgroup of lipids, triglycerides, is a major form of energy storage in animals and
plants. Lipids also play a signicant role as pathway intermediates in cell signaling
cascades Most commonly, lipids are categorized as a group of naturally occurring organic compounds
that are related by their solubility in non polar organic solvents (e.g. ether, chloroform, acetone and
benzene) and general insolubility in water. Analytical characterization of lipids typically requires
that samples undergo laborious, multistep preparative processes prior to analysis. Lipids can be
isolated byliquidliquid extraction and separated into classes, often derivatized, and
then analyzed. Conventional methods of analysis include measuring iodine value, elemental
phosphorus, acid value (saponication equivalent),peroxide value and radiochemical techniques.
More recently, classical methods have been replaced by thin-layer chromatography (TLC), gas
chromatography (GC),and high performance liquid chromatography (HPLC) as well as
mass spectrometry (MS)

From the result, for lipid detection, distance of compound from starting point is 6cm and
distance of solvent from starting point is 9cm so, by using formula (distance of component from
starting point / distance of solvent from starting point), its retention time is 0.67 cm. the basic
process involves separation of the sample on a thin layer of aluminium coated. Lipid are
separated based on differences in their molecular functional group, polarity, degree of saturation
and chain length. The obtained results include the amount of individual lipid classes and the total
amount of phospholipids to analyze both neutral and polar lipid. Lipid can be detect in this part
because lipids are categorized as a group of naturally occurring organic compounds that are related by
their solubility in non polar organic solvents (e.g. ether, chloroform, acetone and benzene) and general
insolubility in water.

Part 2 of this experiment is to detect plant pigment by using TLC method. There are 2 sample
was used for this part which is yield from pomegranate flower and yield from hibiscus leaf. From
the result in table 1, sample from pomegranate no spot of compound detected. This maybe due to
solvent cannot separate compound into the solution. Other than that maybe compounds in
pomegranate need more time to separate.

Based on the result at table 1, we can see that there are five spot means that have 5
component that separate in this experiment. For yellow spot at distance 1.7cm its Rf value is 0.20
and by refer to theory, pigment at this Rf value is Xanthophylls. In this experiment, there are 4
pigment that can extract from hibiscus leaf which is Xanthophylls, chlorophyll b, pheophytin a
and pheophytin b. Leaf extract containing a mixture of many compounds is spotted onto a TLC
plate and an organic solvent is allowed to move up the plate, potentially carrying with it the
various compounds in the leaf extract. The different components of a leaf extract are separated
based on their affinities for the stationary phase (the silica on the TLC plate) and for the mobile
phase (the solvent that is moving up the plate). Compounds with more affinity for the silica (i.e.
hydrophilic compounds) will not move very far, while compounds with a high affinity for the
organic solvent (i.e. hydrophobilic compounds) will move much farther.

This experiment is achieved with objective which is to detect lipid and plant pigments
using TLC. But in this experiment, the pomegranate flowers cannot extract the pigment, this is
maybe due to the spot at base to little and the pigment cannot separate. By refer to the table in
manual, the color of result for hibiscus leaf different with the Rf value. As see in the table, at
distance 4.3cm, it Rf value is 0.52 so by refer to manual, it color should be grey but in the TLC
result that has been done, the color appears is green. Same with distance of component at 4.3cm
with Rf value 0.52, it should be light-gray but in result, it shows that green color is appeared.
This result may be due some error occurred during an experiment.

CONCLUSION

Objective of this experiment is to detect lipid and plant pigment, for lipid part, it is
successful to detect component. From the result, for lipid detection, distance of compound from
starting point is 6cm and its retention time is 0.67 cm. Lipid has been separated based on
differences in their molecular functional group, polarity, degree of saturation and chain length.
The obtained results include the amount of individual lipid classes and the total amount of
phospholipids to analyze both neutral and polar lipid. Lipid can be detect in this part because
lipids are categorized as a group of naturally occurring organic compounds that are related by their
solubility in non polar organic solvents (e.g. ether, chloroform, acetone and benzene) and general
insolubility in water. For part 2, which is detection of plant pigments, pomegranate was failed to
detect and only hibiscus leaf can be detect their pigments. there are 4 pigment that can extract
from hibiscus leaf which is Xanthophylls, chlorophyll b, pheophytin a and pheophytin b. Leaf
extract containing a mixture of many compounds is spotted onto a TLC plate and an organic
solvent is allowed to move up the plate, potentially carrying with it the various compounds in the
leaf extract. The different components of a leaf extract are separated based on their affinities for
the stationary phase (the silica on the TLC plate) and for the mobile phase (the solvent that is
moving up the plate). Compounds with more affinity for the silica (i.e. hydrophilic compounds)
will not move very far, while compounds with a high affinity for the organic solvent (i.e.
hydrophobilic compounds) will move much farther.

There are some recommendations to ensure that experiment more successful in future
which is, for pomegranate should dip into chloroform solution to extract its compounds. The
chromatography paper should not dip into solution too long because it will make the color spot
disappear and cannot be detect.

REFERENCES

1. Thin layer chromatography. Retrieve November 5, 2015, from

http://www.wellesley.edu/chemistry/chem211lab/orgo_Lab_Manua/Appendix/Techniques/T
LC/thin_layer_chrom.html

2. Column Chromatography. Rietrieve November 5, 2015, from

http://www.chemguide.co.uk/analysis/chromatography/column.htm
3. Bayquen, A., Cruz, C., de Guia, R., Campa,F., Pea, G., Sarile, A., & Torres, P.(2009).
Laboratory Manual in Organic Chemistry. Quezon City: C&E Publishing Inc

4. Palleros, D. (2000).Experimental Organic Chemistry. California: John Wiley & Sons Inc