ORGANIC cHEMISTRY - Iv

Experiment 2

Aim of the Experiment

To prepare TLC plates, and identify unknown compounds in the given mixture and also to calculate the R_f values of unknown compounds.

Principle

Thin layer chromatography (TLC) is a purification technique used to purify the components from their mixture. It has a stationary phase and a mobile phase. The mobile phase flows through the stationary phase and carries the components of the mixture with it. Different components travel at different rates depending on their level of interaction with the stationary phase. TLC is done using a thin, uniform layer of Silica gel or Alumina coated onto a piece of glass, metal or rigid plastic. The Silica gel is the stationary phase and mobile phase is a suitable liquid, solvent or mixture of solvents.

Materials required

Ethyl acetate, Petroleum ether, Mixture and their corresponding standard compounds, Silica Gel GF 254, Filter Paper, TLC glass plate, TLC chamber, Capillary, Iodine chamber, UV Chamber.

Procedure

Making TLC plate: The first requirement for doing a TLC analysis is a TLC plate, which we make ourselves. Slurry of Silica gel G in a suitable volatile solvent (ethyl acetate) is made and then stirred to make uniform suspension. One glass plate is coated by dipping in the slurry and carefully withdrawing it so as to leave a thin layer of Silica gel G on glass surface. Place the coated plates horizontally on the table and allow them to dry out. This will take about 5 minute. These plates are called Chromaplates.

Spotting the samples: Mark a reference line about 1 – 2 cm from the bottom edge of the plate with the pencil gently. The standards provided to you must be applied as tiny dots on the starting line and well spaced from each other. You need to put 3 spots on the starting line corresponding to 2 standards and one unknown mixture. Draw a small amount of sample solution in a capillary say 1 – 2 cm and lightly touch the capillary to Silica surface so that sample is soaked up by Silica and forms a tiny spot. Do not use the capillary for another sample to avoid contamination.

TLC solvent run: After spotting, TLC plate is transferred to a glass chamber containing the solvent to be used as mobile phase (Ethyl acetate & Petroleum ether). The solvent level in the chamber must be few mm below the starting line. Allow the solvent to rise along the Silica surface and as soon as the solvent front reaches the finish line, remove the plate from the solvent chamber and allow the solvent to dry out. Repeat this procedure using different combination of mobile phase until the mixture is separated properly on TLC plate.

Visualization of spots: Transfer the plate to Iodine / UV chamber and watch the spots appear on the plate. In the Iodine chamber the sample spots absorb iodine and develop a yellow-brown color, the density of which depends on solubility of I₂ in the given sample.

After withdrawing the plate from the iodine chamber, circle the spots with pencil and measure the distance travelled by each spot. Draw the chromatogram in your journal sheet and calculate the R_f value for each spot and use it to identify the constituent in mixture. The components can also be identified by comparison of the color of spots with those given by standards.

Calculation

Distance travelled by the solvent = _____________________________ (X) cm

Distance travelled by Compound 1 = _____________________________ (Y) cm

Distance travelled by Compound 2 = _____________________________ (Z) cm

R_f of compound 1 = = _____________________

R_f of compound 2 = = _____________________

Result

Reference

Video link for Thin Layer Chromatography technique

www.youtube.com/watch?v=qdmKGskCyh8&t=19s

Questions

1. Definition of Rf value?

2. What is stationary and mobile phase?

3. Principle of TLC?

4. Factors affecting separation efficiency?

5. How to use TLC technique for non UV active compounds?