RP12 - Separation of species by thin layer chromatography

Question 1

what’s the method for TLC

Answer 1

1) wearing gloves , draw a pencil line 1cm above the bottom of a TLC plate and make 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 solvent to a chamber or large beaker with a lid so that it’s no more than 1cm3 in depth
4) place TLC into the chamber , making sure that the level of the solvent is below the pencil line , replace the lid to get a tight seal
5) when the level of the solvent reaches about 1cm from the top of the late , remove the plate and mark the solvent level with a pencil . Allow the plate to dry in a fume cupboard
6) place the plate under UV 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 do we wear plastic gloves

Answer 2

to prevent contamination from the hands to the plate

Question 3

why do we use a pencil line

Answer 3

will not dissolve in the solvent

Question 4

why do we use tiny drops of solution

Answer 4

too big a drop will cause different spots to merge

Question 5

what happens if the solvent is too deep

Answer 5

it will dissolve the sample spots from the plate

Question 6

why do we add a kid

Answer 6

to prevent evaporation of toxic solvent

Question 7

why do we dry in a fume cupboard

Answer 7

as the solvent is toxic

Question 8

what do we use if the spots are colourless and not visible

Answer 8

UV lamp

Question 9

EQ : Outline the steps needed to locate the positions of the amino acids on the TLC plate and to determine their Rf values (4)

Answer 9

• spray with developing agent or use UV
• measure distances from initial pencil line to the spots
• measure distance from initial pencil line to solvent front line
• Rf value = distance spots travelled/ distance solvent front travelled