Download MS Word - Wonderstruck

KS5 Biology
Lesson Plan 7 – Thin Layer Chromatography
Science at Work in Healthcare
Post – 16 Science Education Pack
Resource Sheet 7.3 – Analysing Patient Samples
You are part of a team of NHS biomedical scientists and you have been asked to analyse
blood samples from six patients who have been diagnosed with suspected phenylketonuria
(PKU).
To find out more about the work of biomedical scientists, visit www.nhscareers.nhs.uk.
PKU is a condition characterized by a deficiency in the enzyme phenylalanine hydroxylase
(PAH) which means that the sufferer cannot metabolise the amino acid phenylalanine to the
amino acid tyrosine. PKU is an autosomal recessive genetic disorder.
PKU can cause problems with brain development, leading to progressive mental retardation
and seizures. However, once detected it can be controlled by maintaining a diet low in
phenylalanine and high in tyrosine. Early detection is crucial as there is no cure and any
damage done is permanent and irreversible. This is why new born babies are given a heel
prick test to check their blood for elevated levels of phenylalanine.
The blood samples you will be given have already been treated to remove blood cells etc so
that they will now only contain any amino acids that may be present in excess.
Your task
Your task is to use thin layer chromatography (TLC) to conduct an analysis of these samples
to check for the presence of phenylalanine and any other irregularities.
KS5 Biology
Lesson Plan 7 – Thin Layer Chromatography
Science at Work in Healthcare
Post – 16 Science Education Pack
The procedure
Set up your TLC plate as shown below:
Set up a TLC tank using a beaker, Petri dish and filter paper as shown in Resource Sheet
7.1 – Thin Layer Chromatography.
Add the eluting solvent (1:1 methanol/water) to a depth of 0.5 cm in the bottom. Then place
the TLC plate carefully into the tank using tweezers. If the TLC plate falls over, or slips into
the solvent, then you will have to start again, so take great care.
Watch as the solvent moves up the plate. Once the solvent front is about 0.5 cm from the
top of the plate, remove it using tweezers and dry it using a hairdryer.
Using tweezers, dip the whole TLC plate into ninhydrin solution, remove it again and place it
on some tissue paper to soak up any excess ninhydrin solution. Make sure that you wear
gloves when using the ninhydrin as it is a stain and it will stain your hands just as
well as it stains a TLC plate.
KS5 Biology
Lesson Plan 7 – Thin Layer Chromatography
Science at Work in Healthcare
Post – 16 Science Education Pack
Holding the plate with tweezers, use a hairdryer to warm it and visualise the spots. Now
calculate the retention factor (Rf) values of each component. You should be able to see
clearly which patients have excessive phenylalanine in their blood by comparing the Rf
values with that of your phenylalanine reference.
Record which patients have excess phenylalanine in their blood, and those that don’t appear
to have any excess amino acids in their blood.
Did you notice anything about any of the other patients? It is possible that some of them
could have excess levels of other amino acids in their blood. Did you see any evidence for
this? If so, how can you investigate this?
Your second task: if you have a sample containing an unknown amino acid
If you have found that
one of the patients has
an unknown amino acid
in their blood, you will
need to compare the
sample with some other
reference solutions to try
to identify the unknown
amino acid.
The three reference
solutions you have,
contain the amino acids
glycine, proline and histidine. Set your plate up as shown above and run it in the tank you
prepared earlier. You can then follow the same visualisation process described above to
identify which amino acid is present in this patient’s sample.
Once you have completed your analysis you will need to present your findings back to the
rest of the group. Resource Sheet 7.3 – Making Your Recommendations will give you some
guidelines on how to do this.