1. Using chromatography to identify amino acids
27 April 2017
Using chromatography to identify amino acids
Learning Outcome
explain the principle of chromatography
• identify amino acids in a mixture
• interpret chromatograms and calculate Rf values from chromatograms.
Key words:
SOLVENT – a liquid in which chemicals dissolve to make a solution (e.g. water, ethanol)
SOLUTE – a substance that is dissolved in a solvent.
SOLUTION – a solute dissolved in a solvent
AQUEOUS SOLUTIONS – solutions where a solute is dissolved in water
NON-AQUEOUS SOLUTIONS – solutions where the solute is dissolved in a solvent that isn’t water (e.g ethanol)

2. Chromatography
A technique used to separate and identify chemicals in a mixture.
Relies on the movement of a gas or liquid through a medium.
The liquid or gas that moves is the mobile phase.
The medium that does not move is the stationary phase

3. Paper Chromatography
The stationary phase is water trapped between the fibres in the paper.
The mobile phase is a solvent
The choice of solvent depends on what is being separated.

4. Thin Layer Chromatography
The stationary phase is a thin coating of absorbent solid on a sheet of plastic or glass.
The mobile phase is a solvent.
TLC is quicker than paper chromatography and can be used for more substances.
Dyes in food products and clothing, drugs, medicines etc.

5. Paper Chromatography Paper chromatography is a technique used for the
separation & identification
of relatively small chemical
substances by a moving
solvent on sheets or strips
of filter paper
Filter paper
cylinder
clip
Solvent
Direction of
solvent
movement
Substances to be identified are “spotted” near one end of the filter paper
Concentrated
spot of
chemicals to
be separated
and identified
As the solvent moves up the paper, different molecules move at different rates with the smallest molecules moving the fastest
The technique is used for small molecules such as amino acids, small peptides and sugars

6. Developing the Chromatogram
The chromatogram is the paper or thin layer plate.
The solvent front is marked with a pencil line.
Coloured spots are marked in case the colour fades.
Invisible spots can be viewed using a UV lamp and then marked.
Some chemicals need to be developed – sprayed with a chemical that causes a colour change.

7. SIDE VIEW
Stationary phase
When doing the thin layer chromatography with painkillers, the stationary phase was solid coating on the card.
Test sample
When doing the paper chromatography with food colours, the stationary phase was the water in the paper.
FRONT VIEW

8. SIDE VIEW
Sample moves from stationary phase to mobile phase.
Mobile phase (e.g water, ethanol)
Solvent front – how far the solvent has moved
FRONT VIEW

9. SIDE VIEW
This substance has moved further because it is more attracted to the mobile phase
So, chromatography separates substances in a mixture because they are more or less attracted to the mobile phase.
FRONT VIEW

10. Identify Amino Acids In A Mixture
Mark the solvent
front & allow
paper to dry
Solvent
front
Spray the dry
paper with locating
agent (ninhydrin)
to make the spots
visible
Dry the paper
with gentle heat
to develop
the amino acid
spots
Purple spots
develop located at
different distances
from the origin line
Amino acid
spots
origin
solvent

11. Reference materials
Testing a sample alongside a set of reference samples can be used to identify the chemicals present.
The spots in the sample can be compared with the spots from the known reference samples.

12. What does this show about Unknown?
Contains 2 substances (2 spots)
They are E120 and E124 because the spots match the reference samples
Reference samples

13. Retardation Factors (Rf)
A chemical can also be identified by its retardation factor (Rf)
The formula is:
Rf = distance moved by sample
distance moved by solvent
The answer is never greater than 1.

14. Calculate Rf for the three spots.
Substance
Distance moved by sample (cm)
Distance moved by solvent front (cm) Rf
E120
7.2 10
0.72
E133
4.1
0.41
E124
9.5
0.95

15. The Rf Value Solvent front The Rf value is always a value lessX1 X5 X4 X3 X2 Y
The Rf value is
always a value less
than one as the
solvent front always
moves further than
the solute molecules
origin

16. Rf values
Any substance has a particular Rf value when used with a particular mobile and stationary phase.
This can be used to identify substances if you know the Rf values of the reference samples

17. Rf
E.g.
A sample moves 3cm, whilst the solvent front moves 12cm. Which chemical is present?
Chemical 1 Rf = 0.44
Chemical 2 Rf = 0.12
Chemical 3 Rf = 0.25
Chemical 4 Rf = 0.60

18. contain four different amino acids with arginine & leucine
Identifying
Unknown
Amino Acids
Solvent
front
The mixture of
unknown amino
acids is seen to
contain four different amino acids
Of these four
amino acids,
two can be positively
identified
Mixture of
unknown amino
acids
The mixture contains
four amino acids; two
unknown together
with arginine & leucine
origin
arg
leu
ala
met

19. Qualitative Analysis
Paper and TLC are both examples of qualitative analysis.
They don’t show how much chemical is present – instead they just show what is or isn’t present.
TLC gives better results and can be used for a wider range of chemicals.
TLC is quicker, more sensitive and produces a clearer separation.

20. Quantitative Analysis
Paper and TLC can give a rough idea of how much based on how intense the coloured spot is.
Sometimes the spots can be removed and processed to find an amount – giving quantitative data.

21. Paper dried and rotated
TWO-WAY PAPER CHROMATOGRAPHY
Solvent
front
Solvent
front
Paper dried and rotated
clockwise through
90o
Mixture of
amino acids on
origin line
Second solvent
Two-way paper chromatography is not on the AQA specification but you may wish to mention it briefly.
First solvent
Two-way chromatography provides better separation of
substances that behave in a similar fashion in the first solvent.
A second run in a different solvent resolves two very close spots
more clearly