RQP 12: Separation of a species by thin-layer chromatography

Question 1

Describe the detailed method of thin-layer chromatography

Answer 1

1. Wearing gloves, draw a pencil line 1cm above the bottom of a TLC plate and mark spots for each sample, equally spaced along the line.
2. Use a capillary tube to add a tiny drop of each solution to a different spot and allow the plate to air dry.
3. Add the solvent to a chamber or large beaker with a lid that is no more than 1cm in depth.
4. place the TLC plate into the chamber, making sure that the level of the solvent is below the penicl line. Replace the lid to get a tight seal.
5. When the level of the solvent reaches about 1cm from the top of the plate, remove the plate and mark the solvent level with a pencil. Allow the plate to dry in the fume cupboard.
6. Place the plate under a U.V lamp in order to see the spots. Draw around them lightly in pencil.
7. Calculate the Rf values of the observed spots.

Question 2

Why should you wear gloves when doing thin layer chromatography

Answer 2

To prevent contamination from the hands to the plate

Question 3

Why do you draw the starting line for thin layer chromatography in pencil

Answer 3

So it doesnt dissolve into the solvent

Question 4

Why is only a small drop of each substance added to the TLC plate

Answer 4

Too big a drop will cause different spots to merge

Question 5

Why must the level of the solvent be below the pencil line

Answer 5

To prevent the sample spots on the tLC plate from dissolving in the solvent

Question 6

Why must a lid be used when undertaking thin layer chromatography

Answer 6

To prevent evaporation of the toxic solvent

Question 7

When are the most accurate TLC values obtained

Answer 7

When the solvent has risen to near the topmof the plate- however thew Rf value can be calculated if the solvent front does not reach the top of the plate.

Question 8

Why do you dry the TLC plate in a fume cupboard

Answer 8

Because the solvent is toxic

Question 9

What is the equation for Rf value

Answer 9

Distance travelled by spot(travelled by each amino acid)/distance travelled by solvent

Question 10

What does separation by thin-layer chroatography depend on

Answer 10

The balance between solubility in the moving phase and retention in the stationary phase

Question 11

What does a solid stationary phase separate by

Answer 11

Adsorption

Question 12

What does a liquid stationary phase separate by

Answer 12

Relative solubility

Question 13

If the stationary phase was polar and the moving phase was non-polar, how would the movement of polar and non-polar compound compare

Answer 13

• Non-polar compounds would pass through the plate more quickly than polar compounds.
• This is because they would have a greater solubility in the moving phase.