- The chromatography paper used must be of suitable dimensions to fit the jar. Measure 4cm from the end of the paper and draw a line. When doing this, a pencil (not a pen) is used. Six spots are uniformly marked along the line. Each of these spots is an origin. The origins are then labeled: (X is the unknown mixture)
Origin1 X
Origin2 Asp (for aspartic acid)
Origin3 Leu (for leucine)
Origin4 Pro ( for proline)
Origin5 Asp (for Asparagine)
Origin6 Ph (For Phenylalanine)
The amino acid is the added in spots to their corresponding origins. This is done using a capillary tube. Try to keep the spots as small as possible. You may want to practice the spotting of a solution on paper. Practice with water and a paper towel (or a piece of filter paper). Do not waste the chromatographic paper for practicing. What you want is a small spot of solution on the paper--about 2mm (.2 cm in diameter). If you want more material put on the paper, you should wait until the first spot has dried before applying a second spot of the same material. All in all about six spots are sufficient. To speed up time a hair dryer can be used to dry the amino acids quicker. Do not use the same capillary for each--you will contaminate each spot with the previous substance. Use a different capillary for each solution (or use the capillary that was present in the solution).
- The Paper rolled up and stapled is lowered into the jar inside the fume cupboard itself . The penciled end is lowered first so that the paper is upright in the jar. The bottom end should be 5mm into the solvent, thus keeping the line of origins above the solvent and the paper must not be touching the sides of the jar as much as possible.
- The top of the jar must be replaced and the apparatus is left in the fume cupboard until the solvent has run up the chromatography paper. The amino acids will move up the paper by capillary action at different rates depending on their relative solubilities in the developing solvent.
- The strip is best left overnight and then removed. The first thing that should be done is to mark the highest point reached by he solvent in pencil. The strip is hung in the fume cupboard to dry. Heat guns may be used at this stage.
Developing the chromatogram (this was done for us)
Dilute Ninhydrin solution in acetone is used to develop the chromatogram. This procedure involves the staining of the amino acids.
Ninhydrin is poisonous: It must be used only in a fume cupboard and protective glasses must be worn. Is any of it gets on your skin it must be washed off immediately. Avoid inhaling the fumes.
A small amount of ninhydrin is poured into a glass crystallizing dish and the chromatogram is slowly drawn through the liquid. The whole of the area between the two pencil lines must be soaked. The amino acids react with the ninhydrin (in the developing solution) spots.
The strip is then allowed to dry in the fume cupboard. It is then heated in an oven or by heat guns until the stains appear. Heating is then continued until the stains are as dense as possible.
Diagram- (on separate sheet)
Results-
The chromatogram is included separately.
The measurement of the RF value is used to identify the amino acids.
Rf is found dividing the “distance moved by the spot” by the “distance moved by the solvent”… both of which are found by measuring on the chromatogram.
The measurement is meant to be taken from the centre of each spot but since our results were rather elongated we measured the distance from the top of the elongated spot.
These were the RF results we obtained
Aspartic acid – 0.53
Luecine - 0.60
Proline - 0.516
Asparagine - 0.33
Phenylaline - 0.58
Discussion-
The official figures for the RF values for the corresponding amino acids are:-
Aspartic acid – 0.24
Luecine - 0.73
Proline - 0.43
Asparagine - 0.
Phenylaline - 0.68
The figures obtained and the official figures are almost the same. Anomalies are however quite obvious here due to experimental error. The largest error probably came from the measurement of the stains as they were not spots but elongated stains. The amino acids were also dabbed on the origins with a large drop at times which probably added to the inaccuracy of the corresponding stains. Some stains were longer than usual, probably because the amino acids mixed. Other sources of error include the fact that the amino acid solutions may have been impure. Blow drying the amino acids may have interfered with the amino acids. The chromatography paper may have been a little
The mixture X is found to contain Phenylalinine, proline, and aspartic acid.
We know that X had Proline as there was a yellow section. Only Proline stains yellow.
There was a similar stain(in shape and height) in x to that of aspartic acid therefore we know that aspartic acid was in X.
There was also another stain in X of the same height and shape as Phenylaline so obviously this amino acid was also present in X.
