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Separation of Amino Acids by Thin Layer Chromatography Lab Questions 4. Why is it necessary to run TLC in a closed container and have the chamber saturated with solvent vapor? 5. Why must the spot be applied to the TLC plate above the level of development solvent? 6. What will be the result of adding too much sample to the TLC plate?

Separation of Amino Acids by Thin Layer Chromatography Lab Questions 4. Why is it necessary to run TLC in a closed container and have the chamber saturated with solvent vapor? 5. Why must the spot be applied to the TLC plate above the level of development solvent? 6. What will be the result of adding too much sample to the TLC plate?

Biochemistry
Biochemistry
9th Edition
ISBN:9781319114671
Author:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Publisher:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Chapter1: Biochemistry: An Evolving Science
Section: Chapter Questions
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Separation of Amino Acids by Thin Layer Chromatography Lab Questions

4. Why is it necessary to run TLC in a closed container and have the chamber saturated with solvent
vapor?

5. Why must the spot be applied to the TLC plate above the level of development solvent?

6. What will be the result of adding too much sample to the TLC plate?


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.
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].
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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