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INTRODUCTION

Chromatography is a process which involves a sample extract that being dissolved either
in a liquid, a gas or a supercritical fluid which often called as mobile phase. Then, the mobile
phase is force to contact with the stationary phase and the selection of the mobile phase and
stationary phase are chosen based on the solubility in each phase (J. G. Sherma., 2005). A
component which is quite soluble in the stationary phase will take longer to travel through it than
a component which is not very soluble in the stationary phase but very soluble in mobile phase.
One of the chromatography type that commonly used in bioprocessing is thin-layer
chromatography so called as TLC. Thin-layer chromatography (TLC) normally was choose
technique in synthetic chemistry for identifying compounds, purity and following the reaction
progress. The difference of TLC is it only require small amount of compound compare to other
column chromatography and it is also faster among others. Basically, based on research by James
F. Lawrence, 1992, it consists of thin layer plastic or metal film which made up of silica gel or
similar material as the stationary phase. It is a crucial to avoid touching the TLC plate on the side
with the white surface and in order to obtain an imaginary start line, make two notches on each
side of the TLC plate or draw a line by using pencil.
TLC is normally done on a small glass or plastic plate coated with a thin layer of a solid,
the most common are silica (SiO2) or alumina (Al2O3). This is the stationary phase. The mobile
phase is an organic solvent or solvent mixture. The solvent climbs up the plate by capillary
action, carrying the sample mixture along with it. Each compound in the mixture moves at a
different rate, depending on its solubility in the mobile phase and the strength of its absorption to
the stationary phase. When the solvent gets near the top of the plate, it is allowed to evaporate,

leaving behind the components of the mixture at various distance from the point of origin. The
ratio of the distance a compound moves to the distance the solvent moves is the R f value
(retention factor).

Figure 1.0: The calculation for retention factor (Rf).

According to F. Vitello, 1978, the principal is begin by producing the chromatogram by


placing the sample on the line drawn on TLC plate. When the sample dry, the plate is placed in a
shallow layer of solvent and it is really crucial to cover the beaker in order to ensure the
atmosphere to be saturated with solvent vapor. After a certain time period, 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 colored spots. In order to identify the spot on the thin layer
plate, normally fluorescence is needed to make the spot visible especially for colorless sample.
Besides that, in some cases, it is possible to make the spots visibly just by reacting them with
some chemicals that produce color such as spray with ninhydrin. There are two factors that affect
how fast the compound travel on the plate which are the solubility of the compound in the
solvent and amount of compound that stick to the stationary phase. The compound that have
hydrogen bond will stick on the plate tightly compare to other.

OBJECTIVES

The objectives of this experiment is to detect the lipid and plant pigments by using thin
layer chromatography (TLC).

METHODOLOGY

Firstly, this experiment was start with the preparation of TLC plate where the TLC was
cut into strips of 3 cm x 10 cm in size. A gloves must be wore when handling the TLC plates to
avoid the silica dust enter the pores of fingers. Then, a line was drawn approximately 1.0 cm
from the short edge of the TLC plate and 0.5 cm for the other side of the plate by using pencil.
Besides, this experiment consist of two part which the first part is lipid detection and the second
part is the plant pigment detection. For the first part, the mixed solvent was prepared (26 mL of
acetone, 4 mL of toluene, water and 1 mL of ammonium hydroxide) in the fume hood. Then,
about 10 mL of this mixed solvent was poured into the schott bottle. Next, the micropipette tip
was dipped into the olive oil sample and gently touch onto the 1.0 cm line on TLC plate and dry
the spot by using hair dryer. The plate was placed on the schott bottle and waited until the solvent
front reaches to within 0.5 cm from the top of the TLC plate. After that, the plate wad dried and
spray with the bromophenol blue solution on the developed plate. Lastly, the spot was observed
and the Rf value was calculated.
Next, for the second part, there are two samples which are frangipani flower and spinach
leaves. The sample were placed in a mortar and about 5 ml of acetone was added approximately
in the fume hood. The sample was carefully grinded until it become concentrated extract. Then,
the micropipette tip was dipped into the olive oil sample and gently touch onto the 1.0 cm line on
TLC plate and dry the spot by using hair dryer. The plate was placed on the schott bottle with the
mobile phase (70:30 hexane-acetone solvent) and waited until the solvent front reaches to within
0.5 cm from the top of the TLC plate. After that, the TLC plate was dried and the spot appeared
was observed and measure the spot with the ruler. Lastly, the Rf value was calculated.

RESULTS

Figure 1.0: The result forming after the plate was spray with bromophenol blue.

Figure 1.1: The result formed after the plate was dried.
Table 1.0: The distance measured for every migration of compound appeared.

Samples
Frangipani flower

Migration of compound
No migration appear

Rf value
0.00

Colors
Colorless

Pigment
None

Point 1: 0.3 cm

0.06

Green

None

Point 2: 0.8 cm

0.17

Yellow

Xanthophylls

Point 3: 1.3 cm

0.27

Yellow

Xanthophylls

Point 4: 1.4 cm

0.29

Green

Chlorophyll b

Point 5: 1.8 cm

0.38

Green

Chlorophyll b

solution

Spinach solution

DISCUSSION

Thin-layer chromatography (TLC) is an extremely valuable analytical technique in the


organic lab. It provides a rapid separation of compounds and thereby gives an indication of the
number and nature of the components of a mixture. Besides, this TLC can also be used to
identify certain compounds by comparison with the known samples, to check the purity of a
compound or to monitor the progress of a reaction, an extraction or a purification procedure.
Basically, TLC is normally done on a small glass or plastic plate coated with a thin layer of a
solid which is silica (SiO2). Hence, this is the stationary phase for this chromatography. Besides,
the mobile phase is an organic solvent or solvent mixture. There are two part for this experiment
which the first part is for lipid detection where the sample use was olive oil while for the second
part is the plant pigment detection which the samples used are frangipani flower and spinach.
When the samples dipped on the TLC plate and were dipped into the solvent, the solvent
climbs up the plate by capillary action, carrying the sample mixture along with it. Each
compound in the mixture moves at a different rate, depending on its solubility in the mobile
phase and the strength of its absorption to the stationary phase. For the lipid detection, when the
solvent gets near the top of the plate, it is allowed to evaporate, leaving behind the components
of the mixture at various distances from the point of origin. Then, after the solvent reached the
top, the TLC plate was spray by using bromophenol blue solution and detected by UV light. But,
unfortunately, for olive oil sample, there was no spot appeared and no migration occurred, as
shown in Figure 1.0. This might due to the actual Rf value of the olive oil is high. Due to the
theory, if the actual Rf value of oil is higher, the compound with the higher R f value is less polar
(D. F. Galanos., et al., 2009). Thus, it does not stick to the stationary phase and the compound
with higher Rf is less polar because it interacts less strongly with the polar absorbent on the TLC

plate. For instance, if normal phase silica is used as the stationary phase it can be considered as
polar. Therefore, the olive oil sample does not have any migration in the TLC plate.
Next, for the plant pigment detection, there is no migration occurred for sample
frangipani flower while there is a five spot appeared for spinach sample and there is a migration
occurred. Then, the spot is measure by using the ruler. The result obtained shows that spinach
compound at point 5 has the highest Rf value. This is because of the further the compound travel
along the TLC plate, the higher the Rf value and the lower the polarity of the compound. Besides,
a polar solvent will compete for silica absorption sites, disallowing the compounds to do so. In
the other hand, the type of pigment that might present in the spinach is, for point 2, the R f value
obtained is 0.17 which is xanthophylls pigment. For point 3, the R f value obtained is 0.27 which
is close to 0.31 Rf value for xanthophylls pigment. Lastly for point 4 and 5, the R f value obtained
is 0.29 and 0.38 respectively which is close to 0.42 Rf value for chlorophyll b pigment.
These result obeyed the principle of chromatography that works with different
compounds which will have different solubilities and adsorption to the two phases between
which they are to be partitioned. Moreover, TLC plate show a great separation of the pigments
because it provide a great affinity as the surface of TLC consists of silica that allows for a greater
separation of the pigments (D. F. Galanos., et al., 2009).

CONCLUSION

The objectives of this experiment which to detect the lipid and plant pigments by using
thin layer chromatography (TLC) was achieved. Unfortunately, for olive oil sample, there was no
lipid detection as there is no spot appeared and no migration occurred. This is because of the
olive oil does not stick to the stationary phase and the compound is less polar. Besides, there is
no plant pigment detection for sample frangipani flower while there is a migration and spot
appeared on the TLC plate for spinach sample. The spinach compound at point 5 has the highest
Rf value because the further the compound travel along the TLC plate, the higher the R f value
and the lower the polarity of the compound. Plus, a great separation of the pigments are achieved
which are xanthophylls pigment and chlorophyll b pigment in the spinach sample.

REFERENCES

D. F. Galanos., et al., 2009. Detection of Adulteration of Olive Oil by Argentation Thin Layer
Chromatography. [Online]. [Accessed 19-12-2016]. Available from world wide web:
http://link.springer.com/article/10.1007/BF02540162

F. Vitello, J. P. Zanetta., 1978. Thin-layer Chromatography of Phospholipids. Journal of


Analytical Biochemistry. Vol 24

J. Sherma., 2005. Thin-layer Chromatography Principles and Techniques. Journal of Food


Analysis. Vol. 2

James F. L., 1992. Residue Analysis of Organophosphorus Pesticides. Journal of


Chromatography A.

Nitin V., TLC Applications. [Online]. [Accessed 19-12-2016]. Available from world wide web:
http://www.pharmastuff4u.com/2012/12/applications-of-thin-layer-chromatographyTLC.html

TUTORIAL/QUESTIONS

1. APPLICATION OF TLC

TLC or Thin Layer Chromatography have several applications that are applicable in
various kind of fields that are available nowadays. First and the most used of TLC applied in the
industries is to purifying a sample. The purifying process is done between the purified sample
and the standard sample, where when any impurities were detected, extra spots can be seen on
the TLC, thus can be detected easily. Second, related to the first application, TLC is used for
identifying compound in a solution or mixture. TLC can be employed in several process, such as
in purification, isolation and identification of natural products. Third application is, used for
examination of reactions. This examination is done to identify whether the reaction in a process
is complete or not, and also applicable in identifying other separation processes, distillation and
molecular distillation. TLC also applicable for biochemical analysis. This process extremely
useful especially when dealing with the separation or isolation of biochemical metabolites or
constituent form the blood plasma, serum or solution. Another application of TLC is in the
Pharmaceutical industry. TLC is widely used for the detection of impurity in those
pharmacopoeial chemicals. TLC also used in the chemical industry, to separate and isolate the
compound that are closely related to one another, based on the cations and anions in organic
chemistry. Thin Layer Chromatography also applied in the food and cosmetic industry in
determining the colours, preservatives, sweetening agents, and other types of cosmetic products.

2.

Safety precaution in using Organic Solvent

Organic solvent is a compound that contain carbon-based molecules that are capable of
dissolving other substances. Solvents are among the most commonly used chemicals in the
industry and all solvent should be considered as hazardous. It is important to takes precaution
when handling these chemicals. It is important to always making sure that the lid of the solvent
container is always closed tightly when not in used and during storage. This is to prevent any
spillage and contact with the skin. When storing the solvent, only an allowable volume of
chemicals needed for the process is used without any excess and waste of the solvent. Third,
when handling the solvent, it is important to working in the upward position to avoid inhaling the
vapor from the organic solvent and prevent any accident from happening. Fourth and foremost,
suitable and fully protective clothing and equipment need to be wear and used when handling the
organic solvent. By using the suitable equipment, the error when working with the solvent can be
minimized and reduced the probability for error in the results. The protective gears will help the
wearer to be safe from any direct contact with the solvent, thus reducing the percentage for
accident from happening.

APPENDIX

Calculation for retention factor (Rf):

Rf

b
Migration dis tan ce : subs tan ce

a Migration dis tan ce : solvent front

For Frangipani sample:

Rf =

b
a

0
0

=0

For Spinach sample (POINT 1):


b

Rf = a

0.3
4.75

= 0.06

For Spinach sample (POINT 2):


b

Rf = a

0.8
4.75

0.17
For Spinach sample (POINT 3):
b

Rf = a

1.3
4.75

= 0.27

0.29
For Spinach sample (POINT 5):
b

Rf = a

1.8
4.75

= 0.38

For Spinach sample (POINT 4):


b

Rf = a

1.4
4.75