Year 12 – Diploma Biology
ANALYSIS OF AMINO ACIDS BY CHROMATOGRAPHY
This is a multi-faceted investigation, involving research and comprehension, an experimental investigation, and
then the interpretation of the results. The whole investigation, in all its parts, should be presented digitally,
through Google Drive. The 7 specific questions to be researched and/or investigated are boxed and are shown in
bold. The over-riding question for investigation is:
"Can the amino acids which are present in the proteins of raw fruit juice, be
identified?"
It should be possible to determine which amino acids are present in proteins which have first been hydrolysed,
using chromatography.
Q1
What do you understand by the term “hydrolysis”, when this word is applied to a protein or a
polypeptide chain?
Hydrolysis is adding water to break the chemical bond.
Q2
Why is it necessary to hydrolyse protein or polypeptides before doing this investigation?
By hydrolysing the protein or the polypeptides the different proteins will be separated depending on their
polarity and that can later be distinguished from one another.
This practical involves:
▩ A first chromatogram, with a mixture of known amino acids, from which can be established some Rf values.
This procedure is a ‘rehearsal’ and will allow you to perfect your technique.
▩ A second chromatogram, testing fresh fruit juices, to find out which amino acids are present in the juices.
You may be able to compare two different juices. We might be able to identify the amino acids lysine, aspartic
acid and leucine in juice.
Chromatography is a difficult technique to get right, but if it works, some excellent results can be obtained.
Take care!
THE CONCEPT
Simply put, the principle of chromatography involves the passage of a solvent along absorptive
(chromatography) paper, passing through a small spot where has been placed a concentrated mixture of the
chemicals which are being analysed. The solvent picks up the mixture and carries it along but the different
factors of the mixture are not carried along at the same 'speed' - some almost keep up with the solvent 'front'
while others lag behind. When the flow of the solvent is stopped, the factors will therefore be spread out
behind the line the solvent took along the paper. The 'speeds' of these factors, relative to each other in any
solvent, are known, so if the factors can be seen (ie like pigment colours of leaves), then it is possible to
establish what each of the factors is.
STEP 1
A small amount of solvent is put at the bottom of a chromatography jar. A strip of absorptive paper
(chromatography paper), with a concentrated spot of the mixture of the amino acids at the bottom (but above
the surface of the solvent), is suspended in the jar so that the end dips in the solvent. The solvent then moves
slowly up the paper, passing through the concentrated spot of amino acids, and carries the amino acids with it.
The amino acids travel at different speeds, and thus separate one from another. Look at the drawing and
understand how this works.
Q3
Why do the amino acids travel up the chromatography paper at different speeds and thus
separate one from another? (Be careful – the answer is not as simple as might be thought. Research!)
They travel at different speeds because each amino acids have different R groups that determine the polarity
of the amino acids and it is that polarity that determines who much the solvent can carry them up the
chromatography paper.
STEP 2
If you were separating coloured pigments (eg. chlorophyll, xanthophyll, carotene, etc.) from green plant
leaves, we would straightaway be able to see where the pigments are on the paper strip. In the case of amino
acids, which are colourless, we have to use a reagent to stain them in order to detect and identify them. The
reagent is called Ninhydrin and its action often gives a "Eureka!" moment!
STEP 3
An important principle of chromatography is that always (assuming that the investigation is conducted
properly!) the amino acids move at the same speed in relation to each other. Depending upon the solvent
used, they may move at different speeds or different distances on the chromatography paper, but their
relationships always remain the same for that solvent. If this relationship is known, it is therefore possible to
identify the different amino acids in a mixture. Of course the relationship is known. The amino acids are given
a value, known as the Rf value, for any given solvent, and it is this Rf value which is known. Known Rf values
can be compared to the ones which are obtained in the investigation, and thus it is possible to determine
which amino acids are present in the mixture. This is all fun to do, but difficult to get right in a small school
laboratory!
Q4
What does Rf stand for, in paper chromatography.
The Rf stands for retention factor which is calculated by the ratio the amino acids move compared to where
the solvent has travelled.
Q5
Compare paper chromatography with (paper) electrophoresis.
Paper chromatography separates the particles through capillary action whereas electrophoresis separates
depending on the molecular size and the ionic charges as well.
THE PRACTICAL INVESTIGATION
Collect the apparatus and solutions:
 Glass jar, with lid and a device (a hook or pin?) for suspending the chromatogram
 Chromatography paper – this should be cut to a length just a little longer than the height of the gas jar. (Be
careful not to touch the face of the paper with your fingers.)
 Scissors
 Fine capillary tube – for spotting on to the chromatography paper
 Crystallising dish (watch glass)
 Gloves




About half a test-tube of solvent (4 parts Butan-1-ol: 1 part glacial Ethanoic acid: 1 part water)
Small amount of a mixture of known amino acids, including lysine, aspartic acid and leucine
Small amount of fresh juice – lemon and/or orange
Ninhydrin (in the fume cupboard)
 You will also need access to the fume cupboard and to a hair drier.
►Set up the glass jar with solvent to a depth of about 3cm. Put the lid on the jar and leave aside in the place
where you will make the chromatogram. The solvent must saturate the environment inside the jar.
►Carefully cut a piece of chromatography paper to a width that will fit easily inside the jar, without touching
the sides, and a length that will enable the paper to hang from the top of the jar and be suspended in to the
solvent. Look at the drawing.
►Rule a pencil line across the paper, about 4cm from the bottom of the paper – this will mean that, when the
paper is suspended, the line will be about 1 cm above the solvent.
►Using the fine pipette, place a small drop of the amino acid mixture in the centre of the line. Let this dry
completely. (You can speed it up with a hair drier, but NOT a Bunsen flame.) Repeat at least 6 times – making
sure that the drop is dry each time. You should finally have a small spot of concentrated mixture.
►Very carefully lower the paper with the spot in to the gas jar, so that the bottom dips in to the solvent but
does not cover the spot. Be very careful not to move the jar and don’t swill the solvent around. Attach the
top of the paper to the top of the jar, gently put the lid on, and leave until the solvent front has almost reached
the top of the chromatography paper.
►Carefully remove the lid from the jar and slowly take out the chromatography paper. Rule a pencil line at the
point on the paper to where the solvent front has reached. Let the chromatogram dry completely.
►At this point, it is not possible to see the amino acids. They have to be stained and ‘fixed’ by means of a dilute
solution of ninhydrin. Take care! Use the fume cupboard and gloves. Place some of the Ninhydrin in the
crystallising dish. Hold the paper at both ends and pull the paper backwards and forwards through the
Ninhydrin. Place the paper in an oven at 100〫c and dry rapidly. Purple spots will appear, each spot
representing a different amino acid. Use a pencil to mark the spots before they disappear.
Interpreting the results
Each purple spot corresponds to one (or more) amino acids. To identify them we make use of the Rf values.
This is the ratio of the distance moved by the spot to the distance moved by the solvent:
Rf
=
Distance moved by the spot
Distance moved by the solvent
Draw a horizontal line through each spot and calculate its Rf value. By comparing your Rf values with those in
the table, try to identify the amino acids responsible for each spot in your chromatogram.
QUESTIONS AND PRESENTATION OF RESULTS (DCP & CE)
Q6
Draw the chromatogram, showing the spots, and label each spot with its Rf value and the name
of the amino acid. Show how you have worked out the Rf values. (You may add the
chromatograms, if you have them.) Scan or photograph these results.
Q7
Make a thorough evaluation of the investigation – what worked well and what worked poorly?
How could you improve your technique? (This is important because we will later do another
chromatography investigation, to identify photosynthetic pigments.)
In our experiment we had a bit of a problem with the length of the chromatography paper we had it too
Small and that meant we had the jar opened for more time letting the fumes escape so the concentration the
Chromatograph was not what it could have been. Another thing is that the drops of the juice were not put in
the same place and that meant the area of the circle grew and that when the solvent went up it didn’t take all
of the same concentration of the amino acids that made it later harder to identify the amino acids. What we
could of done to improve it is have the strip of chromatography paper measured so that we were sure it
wouldn’t touch the sides of the jar or the bottom to avoid it getting bent and in some way affect the results.
For the drops to be more exact apart from having the X marked on the chromatography paper we could of
done it slower so the drops end up in the same place as well as choosing the person who had the most
steady hand.
ASSESSMENT:
OBJECTIVE B: Application of Knowledge & Problem Solving
OBJECTIVE C: Research and Communication
OBJECTIVE D: Analysis of Data and Making Conclusions
OBJECTIVE E: Experimental Investigation
IB PRACTICAL ASSESSMENT CRITERIA:
Data collection and processing (DCP)
Conclusion and evaluation (CE)
(Manipulative skills)
John Osborne
January 2014
For the technician:
Three groups. Each group requires:
 Chromotography jar – gas jar, with lid, and a means of suspending the chromatography paper
are some Rf values
acids in thelonger
solvent
used
this investigation.
These
Chromotography
paperfor
– aamino
few centimetres
than
theinheight
of the chromatography jar, and
wide enough to be suspended iside the jar, without touching the sides.
 Scissors
Amino acid
Rf value
 Fine capillary tube for chromatography
0.38
 Crystallising dish alanine
 Gloves
arginine
0.20
 About half a test-tube of solvent (4 parts Butan-1-ol: 1 part glacial Ethanoic acid: 1 part water)
0.5acid and leucine
 Small amount of aasparagine
mixture of known amino acids, including lysine, aspartic
 Small amount of fresh
juiceacid
– lemon and/or orange
aspartic
0.24
 Ninhydrin (in the fume cupboard)
cysteine
0.4
 The students will also
need access to the fume cupboard and to a hair drier.
glutamine
0.13
glutamic acid
0.30
glycine
0.26
histidine
0.11
isoleucine
0.72
leucine
0.73
lysine
0.14
methionine
0.55
phenylalanine
0.68
proline
not a true amino acid - shows up as yellow
0.43
serine
0.27
threonine
0.35
tryptophan
0.66
tyrosine
0.45
valine
0.61