Separation of Amino Acids by Thin Layer Chromatography Lab Questions1. Describe in detail Thin layer chromatographic experiment. Example: the theory behind it, how youwould prepare the materials to spot on the plate with different mobile amino acids and unknown andhow 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 cmfrom the origin?3. In a TLC experiment using a 70:30 mixture of Petroleum ether and ethyl acetate, a student noted thedevelopment of spots in the origin, what can you suggest about this observation? Separation of Amino Acids by Thin Layer ChromatographyTheory ProcedureRequirements:Self Evaluation1. TLC plate.2. TLC chamber.3. Capillary tubes.4. Reagent spray bottle.5. Conical flasks.6. Beakers.Procedure:AnimationRf value can be calculated using the formula:RfSimulator AssignmentReference1. 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 cmfrom 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 thesolvent). 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 aspurple 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 Rfvalues.FeedbackNPTEL Videodistance moved by the substance from origindistance moved by solvent from originThe 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 ChromatographyTheoryProcedureSelf EvaluationAnimationNote: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-HRSimulator Assignment34All 20 of the common amino acids [standard amino acids] are a-amino acids. They have a carboxyl group and an amino group bonded to thesame carbon atom (the a- carbon). They differ from each other in their side chains, or R groups, which vary in structure, size, and electriccharge. The interaction of the amino acids with the stationary phase like silica varies depending on their 'R' groups. The amino acid thatinteracts strongly with silica will be carried by the solvent to a small distance, whereas the one with less interaction will be moved further. Byrunning controls [known compounds] alongside, it is possible to identify the components of the mixture.a -amino acidCOO™H₂N-C-HHGlycineCOO™1H₂N-C-HReferenceCH₂ОНTyrosineCOOIH₂N-C-HCH₂IFeedbackC CHNHTryptophanNPTEL VideoSince amino acids are colourless compounds, ninhydrin is used for detecting them. To identify this, after development, the TLC plate issprayed with ninhydrin reagent and dried in an oven, at 105°C for about 5 minutes. Ninhydrin reacts with a- amino acids that results inpurple 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 Rfvalue for each known compound should remain the same provided the development of plate is done with the same solvent, type of TLCplates, method of spotting and in exactly the same conditions].

Chromatography is a technique used to separate mixtures of substances into individual components. It…

Answered: Separation of Amino Acids by Thin Layer…

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

Problem 1P