Introduction:

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.

A 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). 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. 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

Objectives:

To detect Pennicilin G using TLC plates.

To determine the unknown solution tested.

To observe the size and the intensity of the spots.

Procedure:

26 mL acetone, 4 mL toluene, 4 mL water, and 1 mL ammonium

hydroxide was mixed.

24 mL of the solution was poured into the TLC development bag.

It was develop until the solvent front reaches to within 1 cm from

the top of the TLC plate.

The plate was dried and observed under UV light at 254 nm.

The Rf was calculated.

Result:

Sample Substance (cm) Solvent (cm)

Penicillin G 11.8 13.9

Unknown 11.5 14.1

Table 1: Results of the distances travelled by the substances and the solvent

R f=ba= migration distance : substance

migrationdistance :solvent front

Penicillin G

R f=ba=11.8 cm

13.9cm=0.8489

Unknown

R f=ba=11.5 cm

14.1 cm=0.8156

Discussion:

In this experiment, the objectives are to detect the Pennicilin G and to make a

comparison of the results of the unknown solution with the results of Pennicilin G. Firstly in

this experiment, a mixture of acetone, toluene, water and ammonium hydroxide was

prepared. Then it was prepared in the TLC development bag. After the Pennicilin G and the

unknown sample was placed onto the silica plate using a pipette. Both samples were

developed until it reaches 1 cm from the top of the TLC plate. It was then dried and observed

under UV light at 254 nm. Under the UV light, the distance of the samples travelled onto was

measured. It was then the Rf value was calculated using the formula;

R f=ba= migration distance : substance

migrationdistance :solvent front

The Rf value is the retention factor which is the ratio of the distance travelled by the

substance to the distance travelled by the solvent. It is stated that if the Rƒ value of a solution

is zero, the solute remains in the stationary phase and thus it is immobile. Whereas if the R ƒ

value = 1 then the solute has no affinity for the stationary phase and travels with the solvent

front. The theory is in order to determine the unknown solution, the Rƒ value of the unknown

sample was compared with the Rƒ value of Pennicilin G. In Table 1, it is shown that the

solvent travel further than the substances in both of the Pennicilin G and the unknown

sample. The Pennicilin G travelled 11.8 cm while the solvent travelled 13.9 cm. For unknown

sample, it travelled 11.5 cm while the solvent travelled 14.1 cm. The reason behind this is in

silica gel, the dominant interactive forces between the adsorbent and the materials to be

separated are of the dipole-dipole type. Highly polar molecules interact fairly strongly with

the polar SiOH groups at the surface of these adsorbents, and will tend to stick or adsorb onto

the fine particles of the adsorbent while weakly polar molecules are held less tightly. Weakly

polar molecules generally tend to move through the adsorbent more rapidly than the polar

species.

From the migration distance of both substance and solvent, the Rf value was

calculated for both Pennicilin G and unknown sample. The Rf value for Pennicilin G is

0.8489 and the Rf value for unknown sample is 0.8156. When comparing two different

compounds run under identical chromatography conditions, the compound with the larger R f

is less polar because it interacts less strongly with the polar adsorbent on the TLC plate. The

result shown that even though the value Rf value of the unknown sample is slightly lower

than the Rf value of Pennicilin G, it does not mean that it is a different compound. This is

because the Rf value can be affected by many factors such as solvent system, adsorbent,

thickness of the adsorbent, amount of material spotted and temperature.

Conclusion:

From this experiment, it can be concluded that the experiment was successful and the

objectives were achieved. The unknown sample was determined to be Pennicilin G also. This

is because even though the Rf value is slightly lower, many factors can affect the results

which may cause the Rf value obtained was slightly low. In order to obtained better result, it

is advisable that the experiment should be conducted on the same TLC plate with standard

and unknown sample side by side, instead of doing it on a different plate.

References:

1. Harry W Lewis, Christopher J. Moody. Experimental organic chemistry: Principles

and Practice (Illustrated edition ed.). pp. 159–173. ISBN 978-0-632-02017-1.

2. Vogel's Textbook of Practical Organic Chemistry (5th Edition) (Hardcover) by A.I.

Vogel (Author), A.R. Tatchell (Author), B.S. Furnis (Author), A.J. Hannaford

(Author), P.W.G. Smith ISBN 0-582-46236-3.

3. Reich, E.; Schibli A. High-performance thin-layer chromatography for the analysis of

medicinal plants (illustrated edition). Thieme: New York, 2007. ISBN 3-13-141601-7

4. ^Fair, J. D.; Kormos, C. M. J. Chromatogr. A 2008, 1211(1-2), 49-54.