SEPARATION OF COMPOUNDS FROM A MIXTURE USING TLC

 

SEPARATION OF COMPOUNDS FROM A MIXTURE USING TLC

Objective:

To separate and identify individual compounds from a given mixture using Thin Layer Chromatography (TLC) technique.

Principle:

Thin Layer Chromatography is based on the differential adsorption of compounds on a stationary phase and their differential solubility in a mobile phase.

·         The stationary phase is a thin layer of adsorbent (e.g., silica gel or alumina) on a glass or plastic plate.

·         The mobile phase is a solvent or solvent mixture that moves up the plate via capillary action.

·         Compounds move at different rates depending on their polarity and affinity to the stationary and mobile phases.

Materials Required:

·         TLC plate (silica gel-coated)

·         Developing chamber (glass jar with lid)

·         Capillary tubes

·         Sample mixture (Ink/plant extract/standard dye mix)

·         Solvent system (hexane:ethyl acetate in 7:3 ratio)

·         UV lamp or iodine chamber for visualization

·         Pencil, ruler, gloves

Procedure:

1.   Preparation of the TLC Plate: Using a pencil, draw a light line 1 cm from the bottom of the TLC plate. Mark spots for sample application.

2.   Sample Application: Using a capillary tube, apply a small spot of the sample solution on the marked line. Allow the spot to dry and apply multiple times for concentration if needed.

3.      Development of Chromatogram: Pour the mobile phase (solvent) into the developing chamber to about 0.5 cm depth. Carefully place the TLC plate into the chamber without touching the solvent line to the sample spots. Cover the chamber and allow the solvent to rise up to about 1 cm from the top of the plate.

4.   Drying and Visualization: Remove the plate and immediately mark the solvent front. Dry the plate in air or with a dryer. Observe the separated spots under UV light or place the plate in an iodine chamber for visualization.

5.   Rf Calculation: Measure the distance moved by each spot and by the solvent front. Calculate the Retention Factor (Rf) for each compound:

Rf=Distance travelled by compound/Distance travelled by solvent front

Observation Table:

Compound

Distance by Spot (cm)

Distance by Solvent Front (cm)

Rf Value

Spot 1

 

 

 

Spot 2

 

 

 

 

Result

Compounds were successfully separated based on differences in their polarity and interaction with the stationary and mobile phases. Different compounds had distinct Rf values.

Conclusion:

TLC is an effective and simple method to separate and identify compounds in a mixture based on their Rf values and interaction with solvents.

Precautions:

·         Handle TLC plates with clean gloves or forceps.

·         Apply small, concentrated spots to prevent tailing.

·         Ensure the solvent level is below the sample spots.

·         Keep the developing chamber covered during development.

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