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Thin-Layer Chromatography
of Amino Acids
HASPI Medical Biology Lab 15b
Background
Macromolecules
There are four major types of biological macromolecules that make up the human body: nucleic acids
(DNA & RNA), carbohydrates, proteins, and fats. The following diagram summarizes the polymers
and monomers of the four major macromolecules.

http://teachers.henrico.k12.va.us/henrico/smith_d/4macromo.jpg
Amino Acids
There are 20 essential amino acids used to form a protein. Each amino acid has a slightly different
chemical structure. Proteins are responsible for many important functions in the body and the table
below summarizes only a few of these functions.
Function Protein Class Example
Defense Antigens Mark foreign proteins, like those on bacteria, for the
immune system to destroy
Transport Membrane Carry glucose into cells to be converted into energy
Increase chemical
reactions
Enzymes Break down carbohydrates, proteins, and fats in the body
during digestion
Support Fibers Form cartilage, hair, and nails
Regulation Binding Store calcium in the bones
Movement Muscle Contract muscle fibers
Storage Hormones Control the amount of glucose in the blood
The shape and function of a protein depends on the number and order of the amino acids in the
protein, just as the combination of letters in the alphabet can create a different word, sentence, or
paragraph.

Name: ____________________________

Period: ___________ Date: ___________
Name: ________________________________________ Date: __________ Period: _____________

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Thin-Layer Chromatography
Chromatography is a process used to separate a mixture of different
substances back into their individual forms. For example, if you mixed
red, yellow, green, and blue food coloring together chromatography could
be used to separate them back into their individual colors. There are
many different types of chromatography depending on the substances that
need to be separated. The most commonly used methods include paper
chromatography, gas chromatography, liquid chromatography, and thin-
layer chromatography.

Thin Layer Chromatography (TLC) is used to separate solids from a liquid.
The most common use is to separate amino acids from a liquid and each
other. A spot of the sample is placed on a sheet of glass treated with an
absorbent substance. The glass is then placed in a solvent that will travel
up the absorbent surface and cause the solid to move out of the liquid with
it. Different solids will move different distances on the sheet, but the
distance will remain constant no matter how many times chromatography
is done. This distance is calculated into an amount called the Rf value,
which can be used to determine the identity of the substance.


Rf values
The distance a substance travels related to the distance the solvent travels is called the Rf value.
The Rf value can be calculated by measuring the distance of the substance from its starting point in
millimeters, as well as the distance the solvent traveled from its starting point in millimeters, then
dividing the substance distance by the solvent distance. The equation is:





It does not matter if the solvent moves 10 mm or 100 mm, the Rf value of a substance will remain the
same. For example the average Rf value for a dye called methylene blue is 0.50. SO, that means if
the substance moved 5 mm, the solvent moved 10 mm. It also means if the substance moved 50
mm, the solvent moved 100 mm.

Phenylketonuria
Phenylalanine is an amino acid. A genetic disorder in
which the body is not able to break down or use
phenylalanine is called phenylketonuria or PKU. If an
individual with PKU consumes too much phenylalanine
he or she can suffer from brain damage that may cause
mental retardation, seizures, and growth delays.

Phenylalanine is found in beef, milk, eggs, and artificial
sweeteners, so individuals with PKU have to be very
careful about what they eat. Any product containing
phenylalanine must have a warning to help those with
PKU avoid those products.
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Example:
Name: ________________________________________ Date: __________ Period: _____________

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Materials
Chromatography plate Capillary tubes (3) Amino acid solution A
Chromatography solvent Hinge-topped vial Amino acid solution B
Ruler UV black light Phenylalanine solution
Graduated cylinder Paper towels Calculator
Procedure
Purpose: The goal of this lab will be to use thin-layer chromatography to determine whether a
patient who has been diagnosed with PKU may have accidentally consumed phenylalanine.

Scenario:
A 15-year-old male patient that has been diagnosed with PKU was recently
brought into the ER at HASPI Hope Hospital suffering from screaming fits and
seizures. His PKU was not recognized until he was 2 years old and much of the
damage to his brain up to that point is irreversible. Since his diagnosis, the
parents have been extremely careful with his diet to make sure he does not
consume any phenylalanine. In order to determine whether his current seizures
are the result of him accidentally consuming phenylalanine, or due to something
else, testing needs to be performed.

He was eating some pork sausage and drinking a mixed juice about an hour
before his seizures started. You have been asked to determine if either of these
items he consumed may have had unlabeled phenylalanine in them.

Thin-layer chromatography will be used to compare a prepared phenylalanine solution to a solution of
the mixed juice (Amino Acid Solution A) and a solution of the pork sausage grease (Amino Acid
Solution B).

1. Obtain a TLC plate. Avoid touching the coated surface, since fingerprints
can leave significant quantities of protein and therefore ruin them.
2. Draw a light pencil line about 1 cm from the bottom of the plate.
3. Evenly space 3 marks across the pencil line. These will be the places where
you spot your amino acid samples on the plate. Label each mark at the
bottom of the plateP for the phenylalanine solution, A for amino acid solution
A , and B for amino acid solution Bso that you can identify them later.
4. Take your plate to the phenylalanine solution. Place the tip of the capillary
tube into the phenylalanine solution and place the tip on the first mark labeled
P. The spot should be small: more is not better! This step is very sensitive
and large spots lead to incorrect results.
5. Allow the sheet to dry for 15-20 seconds and spot-mark P with the
phenylalanine solution capillary tube again.
6. Spot a small sample of amino acid solution onto each mark with a
capillary tube. Spots should be small: more is not better here!
The method is very sensitive. Large spots lead to imprecise results.
7. Measure out 2 mL of chromatography solvent and CAREFULLY pour it into the
bottom of the hinge-topped vial. Try to not allow the solvent to run down the sides of the vial.
8. Place the end of the TLC plate with the P, A, and B spots into the hinge-topped vial. MAKE
SURE THAT THE LINE AND SPOTS DO NOT TOUCH THE SOLVENT!
9. Close the top on the vial to prevent the solvent from evaporating.
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Figure A
P A B
TLC Plate
Name: ________________________________________ Date: __________ Period: _____________

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10. Watch the chromatography plate closely as the solvent moves up the strip. If the solvent
reaches the very top before it is stopped, the experiment is invalid because an Rf value cannot
be determined.
11. You will not see the amino acids moving on the plate since they are colorless. We will use a
UV black light to see them.
12. Allow the solvent to move until it gets approximately 1 cm from the top of the plate. Remove
the plate and place it flat on the paper towel to dry. If there is not enough time to allow the
solvent to move that far, remove the plate regardless of the distance traveled since Rf values
can still be calculated.
13. Make a mark in pencil on the strip where the solvent was stopped.
14. Pour any leftover solvent in the vial back into the chromatography solvent bottle.
Analysis
Analyzing the TLC Plate
1. Place the TLC plate under a black light source. The black light
source should make any amino acids visible. Using the pencil,
make a mark on the plate where the amino acids present in any
of the samples stopped moving.
2. Measure the distance in millimeters from the start (pencil mark
at the bottom of the strip) to the end of the solvent movement.
Record that distance below.
3. Measure the distance in millimeters from the starting point to
where each of the amino acids stopped. Record the distances in
Data Table 1.
4. Calculate the Rf values for each amino acid. Refer to the
background information for details and examples.
5. Compare your calculated Rf values to Data Table 2 to determine the possible amino acids
found in each solution. Complete the Analysis Section.
Solvent Distance: _____________ mm
Data Table 1
Phenylalanine
Solution
Amino Acid Solution A
(Mixed Juice)
Amino Acid Solution B
(Sausage)
Amino
Acid
Distance
Moved
Rf
Value
Possible
Amino Acid
Distance
Moved
Rf
Value
Possible Amino
Acid
Distance
Moved
Rf
Value

Phenylalanine








Data Table 2 Average Rf Values of Amino Acids
Amino
Acid
Rf
Range
Amino
Acid
Rf
Range
Amino
Acid
Rf
Range
Alanine 0.30-0.46 Histidine 0.12-0.32 Proline 0.24-0.48
Asparagine 0.21-0.24 Isoleucine 0.53-0.73 Serine 0.26-0.30
Aspartic Acid 0.24-0.29 Glycine 0.25-0.26 Threonine 0.30-0.36
Arginine 0.16-0.32 Lysine 0.12-0.24 Tryptophan 0.57-0.61
Cysteine 0.37-0.41 Leucine 0.61-0.71 Tyrosine 0.44-0.55
Glutamine 0.25-0.29 Methionine 0.51-0.60 Valine 0.44-0.66
Glutamic Acid 0.31-0.35
http://www.reachdevices.com/TLC_aminoacids.html
http://www.wpi.edu/Academics/Depts/
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Solvent Distance
Start
Name: ________________________________________ Date: __________ Period: _____________

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Analysis Questions - on a separate sheet of paper complete the following
1. From reading your TLC plate, did either of the amino acid solutions contain phenylalanine?
Which one?
2. What would you advise the patient and physician in terms of the possibility of the symptoms
being caused by phenylalanine?
3. Why was it necessary to test the phenylalanine solution at the same time as amino acid
solutions A and B?
4. Did the phenylalanine solution Rf value that was calculated match the Rf value in Data
Table 2? If not, what may have caused the difference?
5. Why does touching the TLC plate with your hands potentially contaminate the results?
6. You dropped and mixed up your samples. You know that one container has valine,
alanine, and glycine. The other container contains leucine, lysine, and methionine. How
could you use TLC to determine which container has which amino acids?
7. You run a TLC plate with amino acid samples and obtain three Rf values: 0.70, 0.36, and
0.14. What are the possible amino acids in the sample?
8. CONCLUSION: In 1-2 paragraphs summarize the procedure and results of this lab.
Review Questions - on a separate sheet
of paper complete the following
1. What are the four major types of
macromolecules?
2. List the monomer for each of the four
macromolecules.
3. How many essential amino acids make up
proteins?
4. How is the structure of a protein similar to
the structure of a paragraph?
5. What are the main functions of proteins in
the body?
6. What is chromatography?
7. What is thin-layer chromatography?
8. How is the Rf value calculated?
9. What is the cause of phenylketonuria
(PKU)? What are the symptoms of PKU?
10. In what food sources is phenylalanine
found? Why is a warning label on food
products with phenylalanine important?
11. What is the Rf value of tryptophan?
12. Look at Figure B. If an individual was not
receiving enough glycine what organs
could be affected?
13. What if they were not receiving enough
methionine?
http://athletes-diets.com/home/wp-
content/uploads/2011/04/Amino-Acids-in-the-body.jpg
Figure B
Name: ________________________________________ Date: __________ Period: _____________

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