Separation of Amino Acids by Thin Layer Chromatography Lab Questions 1. Describe in detail Thin layer chromatographic experiment. Example: the theory behind it, how you would prepare the materials to spot on the plate with different mobile amino acids and unknown and how TLC Plate is developed and the reasoning behind which solvent/ solvent mixture should be used, along how to correctly identify of the unknown. 2. Calculate the Rf value if a solute travelled 5 cm from the base spot and the solvent front is 10 cm from the origin? 3. In a TLC experiment using a 70:30 mixture of Petroleum ether and ethyl acetate, a student noted the development of spots in the origin, what can you suggest about this observation?
Separation of Amino Acids by Thin Layer Chromatography Lab Questions 1. Describe in detail Thin layer chromatographic experiment. Example: the theory behind it, how you would prepare the materials to spot on the plate with different mobile amino acids and unknown and how TLC Plate is developed and the reasoning behind which solvent/ solvent mixture should be used, along how to correctly identify of the unknown. 2. Calculate the Rf value if a solute travelled 5 cm from the base spot and the solvent front is 10 cm from the origin? 3. In a TLC experiment using a 70:30 mixture of Petroleum ether and ethyl acetate, a student noted the development of spots in the origin, what can you suggest about this observation?
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Chapter15: Instrumentation In Medical Imaging
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Separation of Amino Acids by Thin Layer Chromatography Lab Questions
1. Describe in detail Thin layer chromatographic experiment. Example: the theory behind it, how you
would prepare the materials to spot on the plate with different mobile amino acids and unknown and
how TLC Plate is developed and the reasoning behind which solvent/ solvent mixture should be used,
along how to correctly identify of the unknown.
would prepare the materials to spot on the plate with different mobile amino acids and unknown and
how TLC Plate is developed and the reasoning behind which solvent/ solvent mixture should be used,
along how to correctly identify of the unknown.
2. Calculate the Rf value if a solute travelled 5 cm from the base spot and the solvent front is 10 cm
from the origin?
3. In a TLC experiment using a 70:30 mixture of Petroleum ether and ethyl acetate, a student noted the
development of spots in the origin, what can you suggest about this observation?
![Separation of Amino Acids by Thin Layer Chromatography
Theory Procedure
Requirements:
Self Evaluation
1. TLC plate.
2. TLC chamber.
3. Capillary tubes.
4. Reagent spray bottle.
5. Conical flasks.
6. Beakers.
Procedure:
Animation
Rf value can be calculated using the formula:
Rf
Simulator Assignment
Reference
1. Pour the solvent mixture in to the TLC chamber and close the chamber.
2. The chamber should not be disturbed for about 30 minutes so that the atmosphere in the jar becomes saturated with the solvent.
3. Cut the plate to the correct size and using a pencil (never ever use a pen) gently draw a straight line across the plate approximately 2 cm
from the bottom.
=
4. Using a capillary tube, a minute drop of amino acid is spotted on the line.
5. Allow the spot to dry.
6. Spot the second amino acid on the plate [enough space should be provided between the spots].
7. Repeat the above step for spotting the unknown acid.
8. Place the plate in the TLC chamber as evenly as possible and lean it against the side(immerse the plate such that the line is above the
solvent). Allow capillary action to draw the solvent up the plate until it is approximately 1 cm from the end.
9. Remove the plate and immediately draw a pencil line across the solvent top.
10. Under a hood dry the plate with the aid of a blow dryer.
11. Spray the dry plate with ninhydrin reagent.
12. Dry the plates in hot air oven at 105°C for 5 min. [Ninhydrin will react with the faded spots of amino acids and make them visible as
purple coloured spots.]
13. After some time, mark the center of the spots, then measure the distance of the center of the spots from the origin and calculate the Rf
values.
Feedback
NPTEL Video
distance moved by the substance from origin
distance moved by solvent from origin
The Rf values with butanol-acetic acid- water solvent are as follows: alanine 0.24, glutamic acid 0.25, glycine 0.2, leucine 0.58, valine 0.4,
lysine 0.58, tyrosine 0.42.](/v2/_next/image?url=https%3A%2F%2Fcontent.bartleby.com%2Fqna-images%2Fquestion%2Fac0eaf72-2e70-49c0-b17b-430ecce861ed%2Fa84283b9-ce1d-49da-abb4-eaef2e33f609%2Fydn4dsh_processed.png&w=3840&q=75)
Transcribed Image Text:Separation of Amino Acids by Thin Layer Chromatography
Theory Procedure
Requirements:
Self Evaluation
1. TLC plate.
2. TLC chamber.
3. Capillary tubes.
4. Reagent spray bottle.
5. Conical flasks.
6. Beakers.
Procedure:
Animation
Rf value can be calculated using the formula:
Rf
Simulator Assignment
Reference
1. Pour the solvent mixture in to the TLC chamber and close the chamber.
2. The chamber should not be disturbed for about 30 minutes so that the atmosphere in the jar becomes saturated with the solvent.
3. Cut the plate to the correct size and using a pencil (never ever use a pen) gently draw a straight line across the plate approximately 2 cm
from the bottom.
=
4. Using a capillary tube, a minute drop of amino acid is spotted on the line.
5. Allow the spot to dry.
6. Spot the second amino acid on the plate [enough space should be provided between the spots].
7. Repeat the above step for spotting the unknown acid.
8. Place the plate in the TLC chamber as evenly as possible and lean it against the side(immerse the plate such that the line is above the
solvent). Allow capillary action to draw the solvent up the plate until it is approximately 1 cm from the end.
9. Remove the plate and immediately draw a pencil line across the solvent top.
10. Under a hood dry the plate with the aid of a blow dryer.
11. Spray the dry plate with ninhydrin reagent.
12. Dry the plates in hot air oven at 105°C for 5 min. [Ninhydrin will react with the faded spots of amino acids and make them visible as
purple coloured spots.]
13. After some time, mark the center of the spots, then measure the distance of the center of the spots from the origin and calculate the Rf
values.
Feedback
NPTEL Video
distance moved by the substance from origin
distance moved by solvent from origin
The Rf values with butanol-acetic acid- water solvent are as follows: alanine 0.24, glutamic acid 0.25, glycine 0.2, leucine 0.58, valine 0.4,
lysine 0.58, tyrosine 0.42.
![Separation of Amino Acids by Thin Layer Chromatography
Theory
Procedure
Self Evaluation
Animation
Note:
Chromatographic Separation of Amino acids:
The present experiment employs the technique of thin layer chromatography to separate the amino acids in a given mixture.
COO™
H₂N-C-H
R
Simulator Assignment
34
All 20 of the common amino acids [standard amino acids] are a-amino acids. They have a carboxyl group and an amino group bonded to the
same carbon atom (the a- carbon). They differ from each other in their side chains, or R groups, which vary in structure, size, and electric
charge. The interaction of the amino acids with the stationary phase like silica varies depending on their 'R' groups. The amino acid that
interacts strongly with silica will be carried by the solvent to a small distance, whereas the one with less interaction will be moved further. By
running controls [known compounds] alongside, it is possible to identify the components of the mixture.
a -amino acid
COO™
H₂N-C-H
H
Glycine
COO™
1
H₂N-C-H
Reference
CH₂
ОН
Tyrosine
COO
I
H₂N-C-H
CH₂
I
Feedback
C CH
NH
Tryptophan
NPTEL Video
Since amino acids are colourless compounds, ninhydrin is used for detecting them. To identify this, after development, the TLC plate is
sprayed with ninhydrin reagent and dried in an oven, at 105°C for about 5 minutes. Ninhydrin reacts with a- amino acids that results in
purple coloured spots [ due to the formation of the complex - Rheuman's purple] [http://vlab.amrita.edu/?
sub=3&brch=63&sim=156&cnt=1]. Rf values can be calculated and compared with the reference values to identify the amino acids. [The Rf
value for each known compound should remain the same provided the development of plate is done with the same solvent, type of TLC
plates, method of spotting and in exactly the same conditions].](/v2/_next/image?url=https%3A%2F%2Fcontent.bartleby.com%2Fqna-images%2Fquestion%2Fac0eaf72-2e70-49c0-b17b-430ecce861ed%2Fa84283b9-ce1d-49da-abb4-eaef2e33f609%2F8vlfm_processed.png&w=3840&q=75)
Transcribed Image Text:Separation of Amino Acids by Thin Layer Chromatography
Theory
Procedure
Self Evaluation
Animation
Note:
Chromatographic Separation of Amino acids:
The present experiment employs the technique of thin layer chromatography to separate the amino acids in a given mixture.
COO™
H₂N-C-H
R
Simulator Assignment
34
All 20 of the common amino acids [standard amino acids] are a-amino acids. They have a carboxyl group and an amino group bonded to the
same carbon atom (the a- carbon). They differ from each other in their side chains, or R groups, which vary in structure, size, and electric
charge. The interaction of the amino acids with the stationary phase like silica varies depending on their 'R' groups. The amino acid that
interacts strongly with silica will be carried by the solvent to a small distance, whereas the one with less interaction will be moved further. By
running controls [known compounds] alongside, it is possible to identify the components of the mixture.
a -amino acid
COO™
H₂N-C-H
H
Glycine
COO™
1
H₂N-C-H
Reference
CH₂
ОН
Tyrosine
COO
I
H₂N-C-H
CH₂
I
Feedback
C CH
NH
Tryptophan
NPTEL Video
Since amino acids are colourless compounds, ninhydrin is used for detecting them. To identify this, after development, the TLC plate is
sprayed with ninhydrin reagent and dried in an oven, at 105°C for about 5 minutes. Ninhydrin reacts with a- amino acids that results in
purple coloured spots [ due to the formation of the complex - Rheuman's purple] [http://vlab.amrita.edu/?
sub=3&brch=63&sim=156&cnt=1]. Rf values can be calculated and compared with the reference values to identify the amino acids. [The Rf
value for each known compound should remain the same provided the development of plate is done with the same solvent, type of TLC
plates, method of spotting and in exactly the same conditions].
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