Using chromatography to
identify amino acids
12 March
2016
Learning Outcome
• explain the principle of chromatography
• identify amino acids in a mixture
• interpret chromatograms and calculate Rf values from chromatograms.
https://youtu.be/J8r8hN05xXk
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)
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
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.
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.
Paper Chromatography
clip
Filter paper
cylinder
Solvent
Direction of
solvent
movement
Concentrated
spot of
chemicals to
be separated
and identified
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
Substances to be
identified are “spotted”
near one end of the filter
paper
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
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.
SIDE VIEW
Stationary phase
When doing the
thin layer
chromatography
with painkillers,
the stationary
phase was solid
coating on the
card.
Test sample
FRONT VIEW
When doing the
paper
chromatography
with food colours,
the stationary
phase was the
water in the paper.
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
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
Identify Amino Acids In A Mixture
Mark the solvent
front & allow
paper to dry
Spray the dry
paper with locating
agent (ninhydrin)
to make the spots
visible
Solvent
front
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
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.
Reference samples
What does this show
about Unknown?
-Contains 2 substances (2
spots)
-They are E120 and E124
because the spots match
the reference samples
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.
Calculate Rf for the
three spots.
Substance
E120
E133
E124
Distance moved by
sample (cm)
7.2
4.1
9.5
Distance moved
by solvent
front (cm)
10
10
10
Rf
0.72
0.41
0.95
The Rf Value
Rf 
distance from origin to solute (spot)
distance from origin to solvent front
X
Rf 
Y
The Rf value is
always a value less
than one as the
solvent front always
moves further than
the solute molecules
Solvent
front
X3
Y
X1
X4
X2
X
5
origin
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
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
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
The mixture contains
four amino acids; two
unknown together
with arginine & leucine
Mixture of
unknown amino
acids
origin
met arg
leu
ala
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.
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.
TWO-WAY PAPER CHROMATOGRAPHY
Solvent
front
Paper dried and
rotated
clockwise
through
90o
Solvent
front
Mixture of
amino acids on
origin line
Second solvent
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