Chromatograhy

Question 1

What is the stationary phase?

Answer 1

This is the chromatography paper or thin-layer chromatography (TLC)

Question 2

What is the mobile phase?

Answer 2

This is the solvent of the biological molecules

Question 3

How to calculate the Rf value?

Answer 3

Distance the dot has travelled from the start to finish divided by the distance the solvent has travelled from the start to finish

Question 4

Method for chromatography

Answer 4

1) In a beaker add 2 cm of water or ethanol and cover with a lid
2) Draw a straight 5cm above the bottom with a pencil
3) Put an X where the solute should go
4) Using a capillary tube add 1 drop of the solute on the X
5) Wait for the drop to dry then repeat 5 more times with each solvent
6) Place the chromatography paper in the beaker and cover with lid
7) Wait for 3 hours or till the solvent stops moving
8) Measure the length from the pencil line to the top of the solvent
9) Measure the length from the pencil line to the top of the dot on each of the biological molecules
10) Calculate the Rf value of each molecule

Question 5

What is chromatography used for?

Answer 5

To separate different components of a liquid

To monitor the progress of reactions

Question 6

What is the thin-layer chromatography (TLC) made of?

Answer 6

A sheet of plastic, coated with a thin layer of silica gel or aluminium hydroxide

Question 7

Why is pencil used on the chromatography paper?

Answer 7

So it doesn’t bleed and effect the outcome

Question 8

Why and how is iodine used?

Answer 8

Place the paper into a closed jar with iodine crystals and their gas will bind to the amino acids making them visible
It is used when the solute is colourless

Question 9

Why and how is ninhydrin used?

Answer 9

Spray onto the paper and it will make the amino acids brown or purple spots
It is used when the solute is colourless

Question 10

Why and how is UV light used?

Answer 10

Shine onto the paper and see where it does not flourese to see the molecules
It is used when the solute is colourless

Question 11

How does solubility of the solute affect the Rf value?

Answer 11

The more soluble a molecule the faster it will travel up the paper

Question 12

How does polarity of the solute affect the Rf value?

Answer 12

The more polar a substance the less it will travel because the form hydrogen bonds and other dipole interactions with the free -OH molecules in the paper.
A non-polar substance will move faster and further because it doesn’t make as strong bonds with the TLC paper