1. Learning aim C: Undertake chromatographic techniques to identify components in mixtures

2. What you’ll be able to do:
Understand how and why chromatography is used as a separation technique.
2. Be able to calculate Rf values.
3. Recall the different phases involved in chromatography.

3. Chromatography
Chromatography is used to SEPARATE & IDENTIFY different components in a mixture.

4. When is it used? Forensic analysis Breathalyser Food testing
- Pharmaceutical industry

5. Calculating the Rf Valuesb
A = Distance travelled by component
B = Distance travelled by solvent

6. PRACTICAL What you have to do….
Correctly use chromatographic techniques to separate a mixture of amino acids producing clear chromatograms – This means REPEATS.
Use chromatography to separate different coloured pigments in plants.
EVALUATE the chromatographic techniques used in relation of outcomes and suggest improvements.

7. Paper and Thin layer chromatography (TLC)
PAPER CHROMATOGRAPHY
Separates components in a mixture that are coloured
i.e. in plant pigments
TLC
Separates components in a mixture and can be used to identify what components make up a mixture.
i.e. Mixtures of amino acids

8. The different phases of chromatography
STATIONARY PHASE – A solid surface on which a component attaches to and a mobile phase runs through.
MOBILE PHASE – The phase which moves up the paper/silica plate carring the components/compounds in a mixture up the plate and separates them.

9. 4. Be able to explain the two factors which affect how a components moves through the mobile phase.

10. Factors affecting the mobility of amino acids
There are 2 main things which affect the rate at which a component will separate In chromatography:
1 – POLARITY OF THE AMINO ACID LIKE FOR LIKE
A POLAR amino acid will interact more strongly with the polar stationary phase (silica). If this is the case, the polar amino acid will not move up the plate that much
A NON-POLAR amino acid will not interact strongly with the polar stationary phase and will be pushed up the plate. This is because the polar silica plate will prefer to interact with the polar mobile phase which will DISPLACE a non-polar amino acid.

11. Factors affecting the mobility of amino acids
2 – SIZE OF THE AMINO ACID
The LARGER the amino acid, the SLOWER it moves up the plate.

12. Spotting the TLC/Paper chromatogram
Making the spotter
What safety issues were identified when making spotters?

13. Why do you think we made spotters from capillary tubes?
So that we could accurately spot each compound on to the TLC plate/Paper

14. How did we spot the plate/paper?
Labelled
Spread out
PENCIL – Why?

15. Handling the Silica plate
Not touching the silica plate with our hands
Not contaminate the plate with grease from our skin
Not to come into contact with silica (Safety)
How did we minimise contact?

16. Solvents
A mixture of Butan-1-ol, ammonium hydroxide and ethanol was used as a solvent for the amino acid chromatography
6:2:2 ratio
What are the hazards identified when using these solvents?

17. Placing the plate/paper into the beaker
What precautions did we take when placing the plate into the beaker and why?

18. Plant pigment chromatography
Carrying out paper and thin layer chromatography on plant pigments to see the different colours found.