Amino Acids, Proteins and DNA: Proteins - Thin-Layer Chromatography

Question 1

How can amino acids be separated and identified?

Answer 1

• By using thin-layer chromatography (TLC)

Question 2

What is chromatography?

Answer 2

• Analytical method used to separate substances in a mixture

Question 3

What are the two phases of chromatography?

Answer 3

• Mobile phase

- Stationary phase

Question 4

What is a mobile phase?

Answer 4

• Phase where molecules can move

- Always a liquid or gas

Question 5

What is a stationary phase?

Answer 5

• Phase where molecules cannot move

- Must be a solid, or a liquid on a solid support

Question 6

What factors affect the distance each substance moves?

Answer 6

• Solubility in the mobile phase

- Retention by the stationary phase

Question 7

What is retention?

Answer 7

• How strongly adsorbed (attracted) a substance is to the stationary phase

Question 8

What is the mobile phase in TLC?

Answer 8

• Liquid solvent

- E.g ethanol

Question 9

What is the stationary phase in TLC?

Answer 9

• Thin layer of silica (silicon dioxide) or alumina oxide fixed to a glass or metal plate

Question 10

Outline the method for TLC

Answer 10

• Draw a line in pencil near the bottom of the TLC plate (the baseline)
• Put a small drop of each mixture to be separated on the line
• Allow spots on plate to dry
• Place plate in beaker with small volume of solvent (mobile phase)
• Solvent level must be below baseline
• Cover top of beaker with watch glass
• Solvent will start to move up plate. As it moves, the solvent will carry substances in mixture with it
• Leave beaker until solvent has moved almost to top of plate
• Remove plate from beaker and draw pencil line to mark how far solvent travelled before it evaporates
• Place plate in fume cupboard and leave to dry
• Result is a chromatogram

Question 11

Amino acids are colourless, so how can amino acids be located on a chromatogram?

Answer 11

• By using ultraviolet (UV) light

- By using locating agents like ninhydrin

Question 12

How does using UV light to identify amino acids work?

Answer 12

• When UV light is shone on TLC plate, dark patches where the amino acids are can be seen

Question 13

How does using locating agents to identify amino acids work?

Answer 13

• Spray locating agent like ninhydrin onto chromatogram

- Ninhydrin turns chemical spots purple

Question 14

How can you identify the types of amino acids present on a chromatogram?

Answer 14

• Calculate Rf value

Question 15

How do you calculate an Rf value?

Answer 15

• Rf Value = distance moved by compound(dot) / distance moved by solvent