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 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?
Q2 Why is it necessary to hydrolyse protein or polypeptides before doing this
investigation?

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!)
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.
Q5 Compare paper chromatography with (paper) electrophoresis.

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.)

John Osborne

For the technician:
Each group requires:

 Chromotography jar – gas jar, with lid, and a means of suspending the chromatography
paper
 Chromotography paper – a few centimetres longer than the height of the chromatography
jar, and wide enough to be suspended iside the jar, without touching the sides.
 Scissors
 Fine capillary tube for chromatography
 Crystallising dish
 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
These are some Rf values for amino acids in the solvent used in this
investigation.

Amino acid Rf value
alanine 0.38
arginine 0.20
asparagine 0.5
aspartic acid 0.24
cysteine 0.4
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
0.43
not a true amino acid - shows up as yellow
serine 0.27
threonine 0.35
tryptophan 0.66
tyrosine 0.45
valine 0.61