Results of centrifuged juice + alcohol:
Pair no. Mass of 1st tube Mass of 2nd tube Places in centrifuge
1 10.29 g 10.29g 12,6
2 7.32 g 7.32 g 1,7
3 7.69 g 7.68 g 8,2
Making the chromatogram
Before you begin spotting the chromatogram, you should:
- Wash your hands (sweat contains amino acids)
- Hold paper at edges
- Place paper on two clean sheets of file paper
- Only use pencil for markings on the paper
Use a micropipette to spot the various substances on the chromatogram. They should be in the following order:
Juice, Juice + Alcohol, Amino acid mix, Asp, Leu, Lys, Pro.
We can now spot these substances onto the chromatogram. Dip the micropipette into a substance and ‘touch’ the paper 5 times on the same spot, waiting a few seconds after each ‘touch.’ However when spotting the juice + alcohol, the paper must be ‘touched’ 7 times instead of 5 (Make sure to use the same micropipette for each component). After spotting all the substances, put the chromatogram onto a frame along with all the other chromatograms and place the frame into the fume cupboard and into the solution for the chromatography to take place.
After chromatography had taken place:
- Carefully remove chromatograms from the frame.
- Spray with ninhydrin and then supply with heat for the reaction between the amino acids and the ninhydrin to take place, causing the colorless amino acids to show up in a certain color.
- Put an ‘x’ in the darkest region of color for each substance and outline the entire regions of color too.
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Measure the distance between the origin and the solvent front, and the distances between the origin and the top, bottom and darkest region of each amino acid. With these measurements, calculate the Rf Values.
My Rf Values:
Rf = Distance moved by component from origin
Distance moved by solvent front from origin
The order of calculations go up the chromatogram, h
Therefore the 1st amino acid (aa) will be the one closest to the origin for that particular substance.
Points on the region of color (the amino acid) are indicated as shown below:
Juice
1st aa:
x = 10/154 = 0.065
y = 20/154 = 0.13
z = 0/154 = 0
2nd aa:
x = 33.5/154 = 0.22
y = 46/154 = 0.30
z = 28/154 = 0.18
Juice + Alcohol
1st aa:
x = 9/154 = 0.058
y = 15/154 = 0.097
z = 1/154 = 0.0065
2nd aa:
x = 34/154 = 0.22
y = 43.5/154 = 0.28
z = 21/154 = 0.14
Amino Acid Mix
1st aa:
x = 33/154 = 0.21
y = 44.5/154 = 0.29
z = 15.5/154 = 0.10
2nd aa:
x = 54.5/154 = 0.39
y = 73/154 = 0.47
z = 48.5/154 = 0.31
3rd aa:
x = 80/154 = 0.52
y = 85.5/154 = 0.56
z = 74/154 = 0.48
4th aa:
x = 104/154 = 0.68
y = 117.5/154 = 0.76
z = 90/154 = 0.58
Asp.
x = 35/154 = 0.23
y = 45.5/154 = 0.30
z = 17/154 = 0.11
Leu.
x = 77/154 = 0.50
y = 114/154 = 0.74
z = 60.5/154 = 0.39
Lys.
x = 52.5/154 = 0.34
y = 72/154 = 0.47
z = 45/154 = 0.29
Pro.
x = 75/154 = 0.49
y = 90/154 = 0.58
z = 64.5/154 = 0.42
Percentage Variation (PV)
Juice
1st aa:
20-10/10*100 = 100%
0-10/10*100 = -100%
PV = 0.065 +- 100%
2nd aa:
46-33.5/33.5*100 = 37.3%
28-33.5/33.5*100 = -16.4%
PV = 0.22 + 37.3% or 0.22 – 16.4%
Juice + Alcohol
1st aa:
15-9/9*100 = 66.7%
1-9/9*100 = -88.9%
PV = 0.058 + 66.7% or 0.058 – 88.9%
2nd aa:
43.5-34/34*100 = 27.9%
21-34/34*100 = -38.2%
PV = 0.22 + 27.9% or 0.22 - 38.2%
Amino Acid Mix
1st aa:
44.5-33/33*100 = 34.8%
15.5-33/33*100 = -53%
PV = 0.21+ 34.8% or 0.21 - 53%
2nd aa:
73-59.5/59.5*100 = 22.7%
48.5-59.5/59.5 = -18.5%
PV = 0.39 + 22.7% or 0.39 – 18.5%
3rd aa:
85.5-80/80*100 = 6.9%
74-80/80*100 = -7.5%
PV = 0.52 + 6.9% or 0.52 - 7.5%
4th aa:
117.5-104/104*100 = 12.9%
90-104/104*100 = -13.5%
PV = 0.68 + 12.9% or 0.68 - 13.5%
Asp
45.5-35/35*100 = 30%
17-35/35*100 = -54.4%
PV = 0.23 + 30% or 0.23 - 51.4%
Leu
114-77/77*100 = 48.1%
60.5-77/77*100 = -21.4%
PV = 0.34 + 37.1% or 0.34 - 14.3%
Lys
72-52.5/52.5*100 = 37.1%
45-52.5/52.5*100 = -14.3%
PV = 0.34 + 37.1% or 0.34 - 14.3%
Pro
90-75/75*100 = 20%
64.5-75/75*100 = -14%
PV = 0.49 + 20% or 0.49 - 14%
Diagram of chromatogram in frame:
Evaluation
The amino acid found in the lemon juice was Aspartic acid, this supports my hypothesis. We can prove this because the ‘x’ on the aspartic acid and the ‘x’ on the 2nd amino acid in the juice are aligned, and so have ‘traveled’ approximately the same distance from the origin, hence telling us that they must be the same amino acids. There is one other amino acid in the juice that we cannot identify using my chromatogram as it was not present in the amino acid mix. The ‘spot’ did not align with any other spot on the chromatogram, and so showing that it is a different amino acid to the ones present. Not all the spots were the same size; some were a lot bigger. This could have been caused by too much of that substance being spotted onto the chromatogram, and so causing the amino acid to spread too much. After chromatography had taken place, the chromatogram was sprayed with ninhydrin and then heated. When a certain temperature is reached, a sudden reaction occurs between the ninhydrin and amino acids, causing each amino acid to give out a particular color. This reaction is helpful in making the amino acids visible to the naked eye. Because of the large spread of some amino acids, we end up with a large spread of color. This makes it difficult to determine the exact darkest region of that color, and so may cause the results to be slightly inaccurate. An example of this is with Leucine. I may have added too much Leu onto the chromatogram, causing it to spread quite largely, resulting in a large purple spot. I had marked what I thought was the darkest region of the spot with an ‘x.’ However that mark was aligned with the mark for Pro and so indicating that they are the same amino acid. This is impossible, but may have been caused by either:
Misinterpreting the darkest region of the spot (marking the wrong place)
Accidentally using the same micropipette for two substances
It would make more sense if the mark on the spot for Leu was closer to the top of the spot, where it would be aligned to one of the spots in the amino acid mix. The 1st amino acid spot for the juice had a smaller spot within it. The smaller spot even showed up as a different color to that of the larger spot, so it must be just another amino acid, not present in the amino acid mix.
I could have improved this experiment by making sure that I do not add too much of a substance onto the chromatogram, by getting a second opinion on whether I had marked the darkest region of a spot or not and/or by using a wider variety of amino acids to identify the other amino acids in the juice which do not align with any others.
