Biochemistry 1b

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Biological chemistry 1B

Experiment 1: Thin layer chromatography [TLC]

Aim
The aim of this experiment is to apply the technique of TLC to first separate out a mixture of coloured dyes and then to do the same for selected range of amino acids.

Procedure
2 MMs of each different colour were collected into separate beakers. Approximately 5 drops of water is added to each of the beakers containing the MMs and the tablets were stirred until almost all of the surface colourings were dissolved.

A line was drawn [pencil] onto a TLC plate 1cm from the bottom, and 6 dots were drawn separated from each other. Then a spotter was used to apply the dissolved colourings onto each of the dots drawn. This was done for all the beakers. The plate was then placed into a beaker fill to the height of ~0.5cm with a 12:1:1 mix of ethanol:water:ammonia solution.

Next the above is repeated on another plate, but instead of MM colouring, a selection of amino acids was applied onto the spots drawn. Also the solution that the plate was placed into is 70:30 ethanol:water instead. Once the solvent front had reached approx. 1cm from the top of the 2 plates, they were taken out to be dried. In addition the second plate was applied with a Ninhydrin spray, before being placed in a heating oven for 15min.

Results
Part 1
| MM colour |
measured distances of [cm] | blue | red | green | yellow| Orange| brown| tartrazine| component 1| 5.3| 0| 4.7| 1.1| 2.8| 2| 4.7|
component 2| | 4.6| 5.1| 4.6| 4.4| 3.2| |
component 3| | | | | | 4.8| |
component 4| | | | | | 5.1| |
|
Fig 1
| MM colour |
Rf value of | blue | red | green | yellow| Orange| brown| tartrazine| component 1| 0.77| 0.00| 0.68| 0.16| 0.41| 0.29| 0.68| component 2| | 0.67| 0.74| 0.67| 0.64| 0.46| |

component 3| | | | | | 0.70| |
component 4| | | | | | 0.74| |...
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