Thin Layer Chromatography Of Amino Acids

Question 1

What is the use of chromatography?

Answer 1

To separate mixtures of substances into their components

Question 2

How is separation achieved?

Answer 2

Achieved by the distribution of the mixture between a stationary phase and a mobile phase

Question 3

What is the principle of chromatography?

Answer 3

Different substances are partitioned differently because they have different affinities for the two phases and therefore varying rates of migration

Question 4

What are the types of chromatography?

Answer 4

• Gas chromatography (GC)
• Column chromatography
• paper chromatography
• Thin layer chromatography
• High performance liquid chromatography(HLPC)

Question 5

What is the goal of thin layer chromatography?

Answer 5

To obtain well separated and well defined spots

Question 6

Explain the value of TLC

Answer 6

• determining of components in a mixture
• identifying substances in a mixture
• ascertaining the effectiveness of a purification
• monitoring the progress of a reaction

Question 7

How is TLC used in forensics?

Answer 7

Detection of document falsifications, poisoning investigations, dye analysis

Question 8

How is TLC used for clinical applications?

Answer 8

Lipids, metabolism studies, drug screening, doping control, presence of drug abuse

Question 9

How is TLC used for pharmaceutical purposes?

Answer 9

• presence or absence of substance in production

- purity of drug

Question 10

How does. food processing use TLC?

Answer 10

Food dye analysis, lipid analysis

-drug residues, additives, pesticides and insecticides

Question 11

What are the advantages of TLC?

Answer 11

• simple
• faster than paper chromotagraphy
• greater resolvingpower because there is less diffusion if spots
• wide choice of Absorbants
• relatively low cost

Question 12

What are the precautions of TLC?

Answer 12

• Do not breath solvent fumes (developing chambers are kept in fume hood)
• Wear Hans gloves when handling plates and chemical spray
• avoid touching surface of absorbent on plates
• Wear protective clothing (lab coat, safety goggles and proper shoes)

Question 13

Explain how it makes the mobile phase

Answer 13

• In TLC the sample is continuously fractioned as it migrates through the absorbent layer
• Competition for active absorbent sites between materials to be separated and the developing solvent will produce continuous fractionation
• A portion of the material being separated will be found in the mobile phase and a portion will be absorbed to the solid absorbent particles
• As the TLC continues, the various components of the mixture migrate different distances. Their migration will depend on their relative affinities for the absorbent as compared with the migrating solvent
• In general, the more polar compounds are held back by the absorbent while less polar material advance further

Question 14

How to determine affinity of a molecule?

Answer 14

The more polar a molecule, the higher it’s affinity for the more polar silica plate, and will therefore spend less time in the mobile phase
-As a result, it will move up the plate more slowly

A less polar molecule will spend more time in the mobile phase and
-will therefore move up the plate more quickly

The speed at which the molecules will move up the plate will vary depending on the nature of the stationary and mobile phases

Question 15

Contrast glycine and glutamic acid

Answer 15

Glycine= neutral non-polar (will move with the mobile phase)

Glutamic acid= polar(will remain with the stationary phase)

Question 16

What does the rate of migration depend on?

Answer 16

• size (in turn depends on AA side chain)
• polarity
• Absorbent medium
  
  • Silica gel = polar
• Solvent system
• Spot concentration and size
• temperature

Question 17

Visualization of chromatogram developed with:

Answer 17

• UV light
• Chemical sprays
  • All naturally occurring amino acids will react with Ninhydrin to give a purple colour, except proline which gives a yellow colour

Question 18

What is the retention factor?

Answer 18

The retention factor (Rf), is a quantitative indication of how far a particular compound travels in a particular solvent

• It gives a good indication of whether an unknown compound and a known compound are similar or not
• Distance traveled by each compound from the origin relative to the solvent front= Rf(retention factor )

Question 19

What is the purpose of Rf?

Answer 19

• Rf can be valuable in identifying a compound
• Rf is calculated from spots on the same plate
• compounds with larger Rf are less polar because they interact less strongly with the polar absorbent on the TLC plate