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Unit 19 - Practical Chemical Analysis Assignment 4 & 5 (Full)
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Unit 19 – ChemistryAssignment 4 & 5
Over a period of two weeks (09/03/2020 – 16/03/2020) Level 3 BTEC Applied Science (Forensic Science) Group
B students carried out two separate chemistry practicals regarding chromatography. The first practical I carried
out alongside Bethan Williams was the Chromatographic Separation of Amino Acids on 9th March 2020.
Chromatographic Separation of Amino Acids.
Aim
The aim of this experiment is to use paper chromatography for the separation and identification of individual
amino acids.
Introduction
Paper chromatography can be described as a useful technique for separating amino acids. The issue with the
determination and identification of amino acids within mixtures are the stimulant for the development of
modern paper chromatography. Since its discovery and application to amino acid studies, paper
chromatography has actually revolutionised the investigation of not only protein structure but also
biochemistry overall. Amino acid analysis is invaluable in the study of metabolism, the examination of enzyme
reactions as well as the determination of characteristic abnormal patterns located in urine and blood regarding
certain diseases.
Safety & Hazards
To ensure an individual is safe throughout this practical there are control measures which have been sternly
put in place and must be followed, these control measures are as follows;
A laboratory coat and safety goggles are mandatory to be worn by an individual during this practical,
to ensure no hazardous substances encounter the skin. A laboratory coat and safety goggles also are
enough to take account of the most hazards as well as risks deemed significant.
It’s important that immediately after use waste is placed in labelled containers, where waste can then
be correctly disposed of by a scientific technician.
A good laboratory practice is remined of for a safe working environment to be maintained.
Harmful/Irritant(s) -
Ninhydrin.
Ammonia.
Ethanol.
DL-Lysine .
DL-Leucine.
DL-Aspartic Acid.
Method
1. Firstly, the mobile phase needs to be made. This is created in the ratio of;
Butan-2-ol : Acetic Acid : Deionised Water
60% : 15% : 25%
However, only 20cm 3 of solution is required so the ratios needed to be in correspondence with the
solution measurements, by carrying out the following calculations this was able to be established;
Butan-2-ol : 60% x 2 = 120 ÷ 10 = 12cm
Acetic Acid : 15% x 2 = 30 ÷ 10 = 3cm
Deionised water: 25% x 2 = 50 ÷ 10 = 5cm
Then using a pipette, the above amounts of the three solutions individually need to be pipetted into a
measuring cylinder and accurately measured out (ensuring that the meniscus is on the line) before being
placed into a 1-litre glass beaker. Once all three of the solutions have been placed into the 1-litre glass beaker
a glass lid needs to be placed over the beaker. The reason why this is done first, and the lid placed over the
beaker is so that the solvent can equilibrate. Equilibrium is described as the state of balance or a stable
situation whereby opposing forces cancel each other out and no changes are taking place.
2. Whilst the solvents are equilibrating the paper slide needs to be prepared. Gloves must be worn when
handling the paper as fingerprints can be an issue on the paper during the later stages, as the amino
acids from the skin can contaminate the paper and affect the amino acids which were using, thus,
affecting our results. The paper used must be 10cm in width and 30cm in length. The paper needs to
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,Unit 19 – Chemistry
Assignment 4 & 5
be laid down before a line is drawn using a pencil and ruler, 1cm from the base of the paper going
landscape across the paper.
3. This sheet of paper needs to fit into the 1-litre glass beaker, in a cylinder shape without the paper
coming into contact with the beaker’s sides. Therefore, a clean 1-litre glass beaker needs to be used
to measure the paper to a suitable size, the paper will be placed in a cylinder shape
into the beaker and held in place by two paperclips. Below is an image giving a more
illustrative idea of this:
4. Once a suitable cylinder size has been determined, the paper needs to be taken out
from the glass beaker and where the paperclips were sitting marked with pencil (this
is to be aware of how big the cylinder shape needs to be to fit in the beaker.) By
using the line drawn across the sheet, 8 evenly spaced pencil dots need to be
marked going across the line.
5. Next, one drop of each of the 8 solutions (7 amino acids & 1 mixture (unknown))
using a toothpick need to be placed onto the 8 pencil dots.
One drop of Alanine needs to be placed onto the first pencil dot.
One drop of Cystine needs to be placed onto the second pencil dot.
One drop of Glycine needs to be placed onto the third pencil dot.
One drop of Leucine needs to be placed onto the fourth pencil dot.
One drop of Histidine needs to be placed onto the fifth pencil dot.
One drop of Tryptophan needs to be placed onto the sixth pencil dot.
One drop of Valine needs to be placed onto the seventh pencil dot.
One drop of the mixture (unknown what were trying to work out)) needs to be placed onto
the eighth pencil dot.
These drops need to be left to air dry for about 30 seconds before another drop of each solution needs to be
placed onto each pencil dot and left to air dry for another 30 seconds. Below are images showing these
solutions:
6. Using the previous two paperclips put the paper into the cylinder shape, before removing the lid off
the 1-litre glass beaker and placing the paper into the beaker, ensuring that the paper doesn’t touch
the sides of the beaker, before placing the lid back over the beaker.
7. The chromatogram needs to be left to run for around 45minutes, when the solvent has travelled up to
1cm from the top of the paper, the paper will need to be removed from the tank before the solvent
front marked using a pencil and ruler.
8. Next, the plate needs to be dried in an oven until its free from solvent and ammonia.
9. Once removed from the oven, ninhydrin needs to be sprayed onto the sheet of paper under the fume
cupboard.
10. Then, the chromatogram needs to be placed into the oven at 105C for around two minutes. Ideally,
the amino acids should form purple spots. Each of these spots need to be marked with a pencil.
11. Finally, each Rf values of the amino acids need to be measured before being compared to the
unknown to establish what the unknown is made up of.
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, Unit 19 – Chemistry
Assignment 4 & 5
Results
Rf Values = (distance spot travelled ÷ distance solvent travelled.)
Alanine = 5.7 ÷ 7.6 = 0.75 Rf
Cystine = 0.4 ÷ 7.6 = 0.053 Rf
Glycine = 2.9 ÷ 7.6 = 0.38 Rf
Histidine =1.8 ÷ 7.6 = 0.24 Rf
Leucine = 5.2 ÷ 7.6 = 0.68 Rf
Tryptophan = 5 ÷ 7.6 = 0.087 Rf
Valine = 4.9 ÷ 7.6 = 0.645 Rf
Mix (unknown) = 5 ÷ 7.6 = 0.087 Rf 2.9 ÷ 7.6 = 0.38 Rf
Conclusion
To conclude, from carrying out this experiment we established that the chromatogram didn’t produce purple
spots for the amino acids at the final stage, this was an issue as it meant we were unable to compare seven of
the different amino acids with the mixture, so, we were unable to establish what the unknown mixture was.
Therefore, Dr Emma Morgan provided us with a chromatogram sheet whereby we were able to compare the
known solutions with the unknown, from doing the comparison of the Rf values and colours this way we
established that the unknown mixture from measuring Rf values were Glycine and Tryptophan, by establishing
that the mixture had the same Rf value as Tryptophan and Glycine along with observing the colours we were
able to determine what the unknown mixture contained. Essentially, from carrying out this practical it allowed
us to come to grasp with how paper chromatography works. An advantage of this experiment is that it is a
relatively simple and cheap experiment to conduct particularly when the method is followed, it further allowed
us to establish the different Rf values of different amino acids and how these travel up the paper during the
chromatogram run. However, one of the negatives we established through conducting this practical was that
the solvent wasn’t rising equally up the paper, the fact that the paperclips used to hold the paper in a cylinder
shape within the 1-litre beaker was causing the solvent to rise up the paper quicker (only in the areas where
the paperclips were and not where they weren’t) the reason why the paperclips caused the solvent to rise up
the paper more quickly is due to the fact the paperclips were pushing the paper together, whereas, the areas
where the paperclips weren’t placed on the paper meant that the solvent had to rise up the paper through
capillary action, thus, taking longer. Another error is that the measurements for the Rf values might be slightly
inaccurate through human error, as it’s possible that Lauren or Beth might’ve measured the values incorrectly.
Through comparing this method to other chromatographic methods, such as; gas chromatography it isn’t as
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