Science at Work in Healthcare
Post – 16 Science Education Pack
To familiarise students with the function of amino acids and how they can be identified using thin layer chromatography.
Students will be expected to:

Understand the difference between essential and non-essential amino acids.

Understand how disease can affect the level of different amino acids in the blood.

Follow a practical procedure to test a simulated blood sample and use the results to make a diagnosis.

Present their findings back to the group in a clear and logical manner.
By the end of this lesson:

All students must be able to understand the basic function and structure of amino acids in the human body. They should also be able to understand the basic principle behind thin layer chromatography.

Most students should be able to understand how the levels of different amino acids may fluctuate in the body due to the effects of disease. They should also be able to understand how the retention factor (R f
) value of a sample or component of a sample undergoing thin layer chromatography is characteristic of that sample or component of a sample.

Some students may be able to understand the detail of how TLC works and the relationship between deficiencies in particular amino acids and specific conditions. They may also be able to understand how diseases such as phenlyketonuria (PKU) have underlying genetic causes.
Approximately two hours for the sample testing, research and presentation of results.

Science at Work in Healthcare
Post – 16 Science Education Pack
Board
AQA
Biology
AQA
Human Biology
Edexcel
(concept approach)
Edexcel
(context approach)
OCR
Biology
OCR
Human Biology
3
6
1
3
6
F212
F213
F216
F221
F222
F223
F225
Unit
1
3
6
1
3
6
1
Spec.
3.1.2 (proteins and amino acids)
Investigative and practical skills
Investigative and practical skills
3.1.1 (proteins and amino acids)
Investigative and practical skills
Investigative and practical skills
Topic 2 Genes and health (proteins and amino acids)
Practical biology and research skills
Practical biology and research skills
Topic 2 Genes and health (proteins and amino acids)
Practical biology and research skills
Practical biology and research skills
2.1.1 (proteins and amino acids)
Practical skills in biology 1
Practical skills in biology 2
1.1.2 (proteins and amino acids)
2.2.2 (the role of amino acids and proteins in phenylketonuria)
Practical skills in human biology 1
5.1.1 (the role of amino acids and proteins in phenylketonuria)

Science at Work in Healthcare
Post – 16 Science Education Pack
Per student

Resource Sheet 7.1 – Thin Layer Chromatography

Resource Sheet 7.2 – Amino Acids
Per pair of students

2 TLC plates (5 cm x 4 cm and 5 cm x 3cm)

A 250 cm 3 beaker to act as a developing tank with a Petri dish or watch glass to act as a lid

A square piece of filter paper to sit inside the beaker

Pre-made TLC spotters or glass capillaries and a micro-burner so students can make their own

A pair of tweezers

Access to a hairdryer

A 15 cm rule

2 pairs of gloves

6 patient blood samples

Small beaker of eluting solvent

Access to ninhydrin visualising solution

Resource Sheet 7.3 – Analysing Patient Samples

Resource Sheet 7.4 – Making Your Recommendations
Students will also need access to an internet connected computer.

Science at Work in Healthcare
Post – 16 Science Education Pack
Intro activity:

Ask the students if they can explain what amino acids are and why they might be important to living organisms or a similar open question.

Follow up with discussion about where we get our amino acids from and what might happen if we do not get the correct ones in the right quantities. It is important that students realise that the body cannot store amino acids and understand the difference between essential and non-essential amino acids.

Explain that the presence of amino acids in the blood at elevated levels could be an indicator of disease. The particular disease will depend on the amino acid detected. This kind of analysis would be carried out using thin layer chromatography (TLC) by a biomedical scientist working in a clinical biochemistry lab .

Students can find out more about the work of biomedical scientists at www.nhscareers.nhs.uk
.
15 mins approx
Practical:

In this practical, students will take on the role of NHS biomedical scientists and work in pairs to analyse a simulated blood sample for elevated levels of the amino acid phenylalanine. This is an indicator of the condition phenylketonuria
(PKU), where the patient is unable to metabolise phenylalanine.

Three of the 6 samples they have been given will show no amino acids, two will contain phenylalanine and one will contain proline. After running the TLC, the students will visualise the plate using ninhydrin, where they will identify the 2 patients with phenylalanine at elevated levels in their blood and those with no amino acids in theirs. They will also see that one of the patients has an amino acid in their blood that isn’t phenylalanine. They can then run a second TLC, running the blood sample with the unknown amino acid against reference solutions of glycine, histidine and proline. After running and developing the plate, they should be able to identify that this patient has an elevated level of proline in their blood.

Information for producing the required samples is given in the Technician Notes at the end of this lesson plan.

Science at Work in Healthcare
Post – 16 Science Education Pack

Issue the students with the following resource sheets: o Resource Sheet 7.1 – Thin Layer Chromatography o Resource Sheet 7.2 – Amino Acids o Resource Sheet 7.3 – Analysing Patient Samples o Resource Sheet 7.4 – Making Your Recommendations

Once students have carried out the TLC procedure they will need to do some research into the effects and treatment of phenylketonuria and what condition might be indicated by high levels of proline in the blood. They can use Resource
Sheet 7.4 – Making your Recommendations to help them with this process.
Note: High levels of proline in the blood is a symptom of type I or type II hyperprolinemia, a condition which can be caused by liver disease and may lead to seizures and various psychiatric and neurological problems.

Students can then conduct their own research into what conditions may be represented by the particular amino acids they have detected and what the possible treatments might be.
60 mins approx
Plenary activity:

Students can present their findings back to the group. The group should be encouraged to question their findings.

Presentations should be no more than 5 minutes in length and should show clearly how the collected data supports any conclusion.

If time is short, rather than presenting their findings, students could compile a report. This report should have the same content as the presentation as outlined in Resource Sheet 7.4 – Making your Recommendations.

Science at Work in Healthcare
Post – 16 Science Education Pack

Students could research conditions which occur due to deficiency or excess of different amino acids and see if there is any commonality amongst the resulting conditions. They may find that a number of conditions resulting from amino acid deficiency include an impairment of neurological function in some way. This is due to the fact that glutamic acid, gamma aminobutyric acid
(GABA), aspartic acid and glycine all act as neurotransmitters and deficiency in these is detrimental to the passage of nerve impulses in the brain. Note that
GABA is not included on the list of amino acids on Resource Sheet 7.2 –
Amino Acids as it is not generally found in proteins or in mammalian tissue in general, other than in the nervous system.
It is the responsibility of the supervising teacher to carry out all risk assessments with regard to this activity and to make sure that any such risk assessment complies with the requirements of the particular institution in which it is being conducted.

Science at Work in Healthcare
Post – 16 Science Education Pack
Please read Resource Sheet 7.3 – Analysing Patient Samples.
Each pair of students will require the following:

2 TLC plates (5 cm x 4 cm and 5 cm x 3cm)

A 250 cm 3 beaker to act as a developing tank with a Petri dish or watch glass to act as a lid

A square piece of filter paper to sit inside the beaker

Pre-made TLC spotters or glass capillaries and a micro-burner so students can make their own

A pair of tweezers

Access to a hairdryer

A 15 cm rule

2 pairs of gloves

6 patient blood samples

Small beaker of eluting solvent

Access to ninhydrin visualising solution
Solutions of amino acids should be ~0.1M, dissolved in 50:50 methanol/water. The masses of amino acids needed to give 0.1M solutions in 25 cm 3 of solvent are given below:

Phenylalanine: 0.413g

Proline: 0.288g

Histidine: 0.388g

Glycine: 0.188g
These amino acids are available relatively cheaply, and only small amounts are needed.

Science at Work in Healthcare
Post – 16 Science Education Pack
6 patient samples labelled A-F should be prepared:

2 should contain phenylalanine in 1:1 methanol/water (you may choose to make them more dilute than the reference solutions).

1 should contain proline in 1:1 methanol/water.

The other 3 should be just 1:1 methanol/water with no amino acids.
10 g ninhydrin in 300 cm 3 ethanol in a 500 cm 3 glass beaker. Label: ‘Ninhydrin dip:
WEAR GLOVES – IT WILL STAIN YOUR SKIN!’
This is 1:1 methanol/water.
Any silica TLC plates, backed with aluminium or plastic will work fine. Packs of 10 or
25 can be purchased fairly economically and will last some time if cut to an appropriate size. Each student/pair/group will need 2 plates (suggested sizes 1: 5cm x 4cm, 2: 5cm x 3cm).
Each student will also need a developing tank (a 250 cm 3 glass beaker will be ideal) with a piece of square filter paper to sit inside the beaker.
These are easily made from glass capillaries (e.g. for melting points) by heating them in the middle using a microburner on a blue flame to soften them, before pulling on each end to draw out a fine spotting capillary. It might be wise for these to be made beforehand, but the students will probably enjoy doing this themselves if you’re happy for a few capillaries to be wasted as they get used to doing it.
If students are going to make their own spotters this should be done before the eluting solvent and visualising solution are made available.