AIM: To identify the components in a plant extract using thin layer chromatography.

APPARATUS/MATERIALS : Silica TLC plate (plastic backing), Capillary tube, Beakers,

Hexane, Acetone, Hibiscus (plant extract).

1. A baseline was drawn approximately 1cm from the bottom of the TLC plate.

2. The extract was obtained and dissolved in 60% hexane and 40% acetone,

3. A clean capillary tube was used to introduce dissolved extract into TLC plate.

4. The spotted TLC plate was placed into the beaker consisting of the suitable solvent –

60% hexane, 40% acetone.

5. The plate was removed when the solvent front was approximately 0.5cm from the top of

the plate.

6. All visible spots were circled with a dull pencil on the chromatogram.

7. Two spots were identified and used to calculate the RF value.

8. All observations and calculations made were recorded.

VARIABLES:

Manipulated – Solvents used

Responding – Order in which compounds are released from the mixture

Controlled – TLC plate used

OBSERVATION/RESULTS:

TLC PLATE SHOWING RESULTS OF THE SEPARATION OF COMPONENTS USING A SOLVENT MIXTURE OF HEXANE AND ACETONE

CALCULATIONS:

Retention Factor = Distance moved by solute / distance moved by solvent

Distance moved by solvent = 4.8cm

Therefore RF value = distance moved by solute / 4.8

For Spot A, distance moved = 2.2cm

Therefore RF = 2.2cm/ 4.8 cm

= 0.46

For Spot B, distance moved = 2.5cm

Therefore RF = 2.5cm/ 4.8 cm

= 0.52

For Spot C, distance moved = 2.7cm

Therefore RF = 2.7cm/ 4.8 cm

= 0.56

For Spot D, distance moved = 4.4cm

Therefore RF = 4.4cm/4.8cm

= 0.92

DISCUSSION:

Chromatography is a sophisticated method of separating and identifying mixtures of two or more compounds. The separation is accomplished by the distribution of the mixture between two phases, one that is stationary and one that is moving or mobile. Chromatography works on the principle that different compounds will have different solubilities and adsorption to the two phases, which will allow for their separation. Thin Layer Chromatography (TLC) is a solid-liquid technique in which the two phases are a solid, stationary phase and a liquid, mobile phase. The solid phase is a plastic plate covered with an adsorbent, in this case, silica gel. Aluminum is another common solid phase used. Because silica is a polar molecule, the components of the solution used are separated based on their relative polarities. The more polar a molecule, the higher affinity it will have for the more polar silica plate and will therefore spend less time in the mobile phase. As a result, it will move up the plate more slowly. Conversely, a less polar molecule will spend more time in the mobile phase and will therefore move up the plate more quickly. The speed at which the molecules will move up the plate thus depends on the relative difference in polarity between the stationary and mobile phases, and will vary depending on the nature of the stationary and mobile phases used for separation.

The plate is relatively small of around 2 cm by 6 cm. The mixture to be analyzed is spotted at the bottom of the TLC Plate and allowed to dry. This area is labelled as the base line or the lie of origin. The plate is placed in a closed vessel such as a covered container containing the mobile phase so that the liquid level is below the spot. TLC Is very efficient and the solvent ascends the plate by capillary action. During TLC the liquid is filling spaces between solid particles. This technique is usually done in a closed vessel to ensure that the atmosphere is saturated with solvent vapor and that evaporation from the plate is minimized before the run is complete. The partition here is between the solute adsorbed onto the silica or alumina particles via polar and hydrogen bonded interactions and to a lesser extent acid base attractions due to the slight acidity of the silica or alumina plate. The plate is removed when the solvent front approaches the top of the plate and the position of the solvent front is recorded before it is dried. This allows the calculation of the retention factor. The difference each molecule travels along the adsorbent in relation to how far the mobile phase has traveled is called the Retention Factor (Rf) and can be used to identify molecules, as the value is molecule specific. The Rf for any given molecule will vary depending on the mobile and stationary phases used. TLC has many uses such as determining the number of components in a mixture, the identity of two substances and to monitor the progress of a reaction.

PRECAUTIONS:

1. Use a very dull pencil instead of a sharp pencil to make markings on the TLC plate as the layer of silica powder is very fragile and thus will break easily.

2. Ensure TLC plate does not settle on the side of the container as it will cause non-uniform movement of solvent.

SOURCES OF ERROR/LIMITATIONS:

1. The TLC plate may have been damaged thus not allowing the even separation and identification of the various components.

2. Contaminations may have occurred while using the capillary tube.3. Enough solution may not have been spotted to ensure that all the components would be

produced.