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 Penicillin G Unknown

Substance (cm) 11.8 11.5

Solvent (cm) 13.9 14.1

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

Penicillin G

Unknown

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;

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 R value of a solution is zero, the solute remains in the stationary phase and thus it is immobile. Whereas if the R 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 R value of the unknown sample was compared with the R 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. 159173. 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.