CHAPTER I

THIN LAYER CHROMATOGRAPHY

1.1 INTROUCTION

TLC stands for Thin Layer Chromatography, and it is a scientific technique for identifying distinct
substances in a mixture. The stationary phase, which normally comprises a solid, and the mobile
phase, which can be either a liquid or a gas and can be a pure solvent or a combination of solvents,
are the two phases. The stationary phase is provided by TLC plates, which have a solid surface
such as glass, metal, or plastic with a thin adsorbent later on the surface. The solid, stationary phase
will be silica gel, and the 99:1 ethyl acetate will be used in the lab. Acetic acid will be used to
simulate the liquid, mobile phase. Because the compound is polar, it will attach to the stationary
phase if it generates dark spots on the TLC plate. They tend to create patches along the TLC plate's
starting, genesis line. The spots, on the other hand, will flow with the mobile phase since the
compound is less polar than the other. Nonpolar compounds will also generate spots that go a
greater distance than slightly polar substances. A more polar substance will bond to the silica gel
more firmly than a less polar compound. A retention factor (Rf) is determined for each location to
compare the distances between each substance. It is calculated by dividing the distance travelled
by the chemical by the distance travelled by the solvent. The polarity of the solvent can be
increased to increase the Rf value, while the polarity of the chemical can be decreased to decrease
the Rf value.

1.2 OBJECTIVE

1. To obtain the retention factor for each component.

 2. To determine the purity of mixture.

1.3 APPARATUS

1. TLC plate

2. Pyrene

3. Fluorene

4. Hexane

5. Developing chamber

6. UV lamp

7. Pencil and ruler

1.4 METHOD

1. Prepare a suitable size of TLC plate that can fit into chamber that fill with hexane as
solvent.

2. Lightly mark a line, termed the Origin Line, one centimeter above the bottom of the TLC
plate with a ruler and pencil. Then, make 4 mark with A, B and C.

3. Using capillary pipe, make spotting of solutions at the mark have made. Place the plate in
the ascending position inside the chamber after spotting. Ensure that the solvent component
does not meet the spots.
 Figure 1 TLC plate in solvent

4. Remove the TLC Plate from the developing chamber after few minutes when the solvent
reaches the top of the plate (approximately a centimeter from the top). Then, on the solvent
front, make a mark (the boundary between the dry and wet silica).

5. Then gently moving the TLC Plate under a UV light source. It should be able to notice
dark dots if look closely. To locate the locations on the plate with visible light, outline the
separated dots using a pencil.

Figure 2 TLC plate under UV lamp

 6. Remove the TLC plate from UV lamp and calculate the Rf for each component.

1.5 RESULT

Figure 3 shows the result of Thin Layer Chromatography

Rf of solvent A = dA/dS

=7/7.5 = 0.93

Rf of solvent B = dB/dS

=7.1/7.5 = 0.95

Rf of solvent C = dC/dS

= 7/7.5 = 0.93

1.6 DISCUSSION

Thin layer chromatography, or TLC, is a method for analyzing mixtures by separating the
compounds in the mixture. TLC can be used to help determine the number of components in a
mixture, the identity of compounds, and the purity of a compound. By observing the appearance
of a product or the disappearance of a reactant, it can also be used to monitor the progress of a
reaction. TLC is a laboratory technique in which the mixture is dissolved within a solvent and
placed near the bottom TLC plate. The TLC plate can be compromised of glass, metal or plates
which is covered in a thin layer of adsorbent serving as the stationary phase. TLC also includes a
mobile phase which a pure solvent or a mixture of solvents. The stationary phase used the
experiment is silica gel. It acts as a polar adsorbent of partition of chromatography of organic
compounds. Polar molecules with dipole-dipole bonding and hydrogen bonding can be adsorbed
by the stationary phase, silica gel. The more polar the compound the more strongly the compound
will bind to the stationary phase.

Experiment proved that a less polar solvent will bring higher Rf values than those of a more polar
solvent. This is because when the solvent is more polar, the compound tends to stick easier causing
it to move more slowly. Rf value of solvent A which is pyrene have low value which is 0.93 which
is pyrene is more polar than the fluorene. Rf value of solvent B which is fluorene shows high value
than pyrene and mix which is 0.95. This shown that fluorene has less polar than the other solvent.
Rf value of solvent C which is mixe pyrene-fluorene shows same value as pyrene. There’s might
be error while doing this experiment.

There is a few error occur in this experiment which is over-large spot. Spotting sizes of smple
should not be larger than 1-2 mm diameter. The component spots will never be larger than or
smaller than the sample origin spot. If have an-over large spot, this could overlapping of othe
components spot with similar Rf values on the TLC plate. If overlapping occurs, it would prove
difficult to resolve the different components. Next error is streaking. If the sample spot is too
concentrated, the substance will travel up the stationary phase as a streak rather than a single
separated spot. In other words, the solvent can not handle the concentrated sample and in result,
moves as much of the substance as it can up the stationary phase. The substance that it can not
move is left behind. This can be eliminated by diluting the sample solution. To ensure that you
have enough solution, use a short-wave UV light to see if the spot is visible. Lastly the error is
spotting. The sample should be above the solvent level. If the solvent level covers the sample, the
sample spot will be washed off into the solvent before it travels up the TLC plate
1.7 CONCLUSION

In conclusion, TLC proved to be successful method of determining the polarity of substances. The
Rf value of the solvent A which is pyrene is 0.93 (more polar). The Rf value of solvent B which is
fluorene is 0.95 ( less pyrene) . The Rf values were calculated by measuring the distance from the
baseline to the spot on the plate that was easily visible with the naked eye. The more polar
component will be held tightly by the silica gel and while other elutes up the TLC plates along
with hexane.