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"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
Q2
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
Q5
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 100
c 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
March 2015
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
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