Unit 13 P2 M1 Applied Science
Amino acid separation:
Aim: To separate the amino acids and identif them in a thin lafer using the chromatographf.
Equipment:
- 2% soluton oi individual amino acids
- Solvent mixture oi normal butanol, acetc acid and water in the rato 12:3:: bf volume
- Ninhfdrin reagent
- Capillarf tubes
- TLC plate
- TLC chamber
- Reagent spraf botle
- Conical fasss
- Beasers
Method:
1. Place the solvent mixture inside the TLC chamber and close immediately close it once the
solution has been placed into it.
2. Leave chambers for approximately 30 minutes to make sure that the atmosphere in the
chamber becomes saturated with the solvent.
3. Then cut the plate to ft the size and use a pencil draw a straight line across the plate of
about 2 cm from the bottom. Make sure that when you draw a straight line across the plate, the
line is very gentle and could barely be visible.
4. Use a capillary tube, once the drop of amino acid is spotted on surface of the line then let
the spot to dry.
5. Next, place the second amino acid onto the plate but make sure that there is enough space
between the spots.
6. Make sure to do repeats with the unknown acids.
7. Put the plate you’ve used into the TLC chamber and make sure it goes in as evenly as
possible and then place it against the side. Can be done by immersing the plate which will make
the line to go above the solvent mixture. Let the capillary to pull the solvent up the plate until it is
approximately 1 cm from the end.
8. Once that’s done remove the plate and draw a line across the top of the solvent. Then dry
the plate with a blow dryer. Then spray the plate with the ninhydrin reagent.
9. After which it is important to dry the plates in hot air oven at 105°C for about 5 min. In this
process the ninhydrin will react with the spots of amino acids which will make them purple and can
therefore be visible.
10. After that mark the centre of the spots and then measure the distance of the center of the
spots from the original and calculate the Rf values.
You can use the formula below to calculate the Rf values;
Amino acid separation:
Aim: To separate the amino acids and identif them in a thin lafer using the chromatographf.
Equipment:
- 2% soluton oi individual amino acids
- Solvent mixture oi normal butanol, acetc acid and water in the rato 12:3:: bf volume
- Ninhfdrin reagent
- Capillarf tubes
- TLC plate
- TLC chamber
- Reagent spraf botle
- Conical fasss
- Beasers
Method:
1. Place the solvent mixture inside the TLC chamber and close immediately close it once the
solution has been placed into it.
2. Leave chambers for approximately 30 minutes to make sure that the atmosphere in the
chamber becomes saturated with the solvent.
3. Then cut the plate to ft the size and use a pencil draw a straight line across the plate of
about 2 cm from the bottom. Make sure that when you draw a straight line across the plate, the
line is very gentle and could barely be visible.
4. Use a capillary tube, once the drop of amino acid is spotted on surface of the line then let
the spot to dry.
5. Next, place the second amino acid onto the plate but make sure that there is enough space
between the spots.
6. Make sure to do repeats with the unknown acids.
7. Put the plate you’ve used into the TLC chamber and make sure it goes in as evenly as
possible and then place it against the side. Can be done by immersing the plate which will make
the line to go above the solvent mixture. Let the capillary to pull the solvent up the plate until it is
approximately 1 cm from the end.
8. Once that’s done remove the plate and draw a line across the top of the solvent. Then dry
the plate with a blow dryer. Then spray the plate with the ninhydrin reagent.
9. After which it is important to dry the plates in hot air oven at 105°C for about 5 min. In this
process the ninhydrin will react with the spots of amino acids which will make them purple and can
therefore be visible.
10. After that mark the centre of the spots and then measure the distance of the center of the
spots from the original and calculate the Rf values.
You can use the formula below to calculate the Rf values;
