Chromatography Experiment


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Chromatography Experiment

Implementing

The plan that I was going to follow was altered in many ways. These
alterations were needed to improve the experiment and therefore
improve the results. Therefore the real method that was carried out is
explained below.

First of all a piece of Chromatography paper was taken and a line was
drawn 2 cm above the bottom of the page. This was the origin line. On
this line 10 crosses were drawn which would be the starting point of
the samples.

This paper was measured so that it would fit into the Chromatography
tank. After this the Chromatography paper was laid down and 6 drops of
the appropriate solution i.e. Aspartic acid was added using a
capillary tube. This was done 6 times so that the spot could be as
concentrated as it could without it being too big.

After this the solvent that would move up the Chromatography paper was
added. This had to be done carefully because the solvent could not go
over the origin line. Once this was done the Chromatography paper was
curved up into a cylindrical shape and stapled. The side with all the
information was left on the outside so that it could be noticed when
the solvent has reached the further most limits.

The solvent had to be added right at the end as it causes ventilation
problems therefore the lid was put on the Chromatography jar as soon
as the solvent was added. This was then left for a 3 hours (approx).
The longer the Chromatogram was left the better as the spots would
have moved up further therefore giving us better results.

After the Chromatogram was dried and sprayed with Ninhydren the spots
were examined. The students didn't spray the Chromatogram because it
was a real toxic solvent therefore the spraying was done by the lab
technicians.



Results and observations
========================

The results of this Chromatography experiment is to find out the
distance travelled by the spots and by the origin line.

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This will help
us to compare these results and then make accurate observations of
what has been produced. The measuring of the spots will be done in mm,
which is a very accurate measurement.

Sample

Distance travelled (mm)

Average (mm)

Test 1

Test 2

Phenylalanine

86

91

88.5

Aspartic acid

10

10

10

Fresh orange juice

10

10

10

Orange juice Brand A

11

89

10

86

10.5

87.5

Orange juice Brand B

10

9

9.5

The origin line moved up 134-mm. This is now known as the solvent
front as the solvent has moved the origin line.

The following observations have been made from my Chromatogram. This
should prove that some of the spots were found to be similar and
therefore resulting in them being present in the solution.

Aspartic acid has one spot, which is at the bottom of the
Chromatogram. Its colour is light purple and shows similarity to the
colour of Phenylalanine. This was found in all the samples as you can
see from the Chromatogram. It seems as if orange juice Brand A
contains both the amino acids. This shows that orange juice Brand A
contains Aspartamine. The fresh orange juice and the orange juice of
brand B were very similar. They have exactly the same path as both
contain Aspartic acid.



Precautions
===========

The following precautions were taken so that the experiment could be
completed as accurately as possible. These precautions are:

Ø To obtain a clear and accurate Chromatogram the students were
advised to use gloves. This was because the students fingerprints
excreted amino acids which would make the experiment and the results
inaccurate.

Ø Strict safety measures were taken in the adding of the solvent. This
was to ensure that the solvent didn't go above the origin line. The
reason for this is that if the solvent and the samples mixed this
would cause unreliable and faulty results.

Ø For the chromatogram to be accurate the lid was placed over the
Chromatography tank as soon as the solvent was added. This was because
as the solvent gas rises it takes some of the sample with it therefore
not putting the lid on would produce a loss in concentration of the
spots.

Ø The adding of the spots was carefully monitored, as small,
concentrated spots were what we were after. Large spots would cause a
defect in the Chromatogram by joining to neighbouring spots causing
imprecision.

Ø Since the students could not rely on just one result repeats were
done. This was to give the student a more accurate average, which is
more precise than just one result.

Ø Since the solvent was very toxic, and the Ninhydren is harmful their
handling by students was inappropriate. This was the reason that the
lab technicians carried this part of the experiment out for the
students. This was carried out in a well-ventilated area and most
probably in a fumes cupboard.



Analysing evidence
==================

The purpose of Chromatography is separating and identifying different
amino acids and therefore showing the different density of the
different samples. Chromatography is particularly approved for its
accuracy in distinguishing between each compound, which it does by
separating the chemicals according to their Relative Molecular Mass,
solubility in solvent and attraction to paper.

[IMAGE]

Before explaining the results that were obtained, there is one point
that has not been pointed out. This is that all the samples were first
hydrolysed which then meant that the tracing of any amino acid could
be made. This was done so that the comparison of already hydrolysed
Aspartic acid and Phenylalanine could be made with the different
orange juices. If both of these amino acids were present then the
conclusion could be brought about that that sample contained the
sweetener Aspartamine. If only one of the two amino acids was present
then Aspartamine was not in that sample.

My Chromatogram clearly indicates that orange juice A consists of both
amino acids Aspartic acid and Phenylalanine. Therefore it has to be
said that orange juice A does contain Aspartamine. Since the fresh
orange juice and orange juice A are very similar they have the same
properties, which are that they do not contain any of the two amino
acids. This statement an be backed up by the working out of the
Relevant Front values which is more commonly known as the Rf values.
These values are made that the smallest value is the heaviest. This is
because the smallest value means that the sample has moved the least
therefore proven that this sample is heavy.

The use of this type of calculation is useful to make comparisons
between the values worked out from the amino acids and the values from
the orange juice A. If the two values are similar then we can safely
predict that both the amino acids are in the orange juice A solution.
This value is worked out by the following formulae:

Distance travelled by spot = Rf value

Distance travelled by the solvent from

Now I will be able to work out the Rf values of the two amino acids
and thereafter the Aspartamine in the orange juice A sample.

Solution/amino acid



Rf Values
=========

Phenylalanine

88.5/134 = 0.66

Aspartic acid

10/134 = 0.075

Fresh orange juice

10/134 = 0.075

Orange juice brand A

Spot 1

10.5/134 = 0.078

Spot 2

87.5/134 = 0.65

Orange juice brand B

9.5/134 = 0.071

These results show the density of the different samples. The results
that I have obtained show me that Phenylalanine is the lightest sample
and the Aspartic acid is the heaviest. Looking at my Chromatogram this
is also visible, as the Aspartic acid has moved the least therefore
being the heaviest. Some figures of actual Rf values have been given
to the students and from comparison to the results obtained here they
are accurate. The result obtained here for Phenylalanine is 0.66 and
the result given to the students is 0.68. This comparison is very
close and would be very accurate keeping in mind the facilities and
apparatus used. The Aspartic acid on the other hand has a different
story altogether. Its result here is 0.075 whereas the result given is
0.24. this fluctuation can be because of many reasons which may
include the following. Chromatographic retention times are sensitively
and unpredictably dependent on the conditions; if a student runs
chromatography on a particular sample three consecutive times (using
the "same conditions") the student will achieve different Relative
front values (Rf) for the same substance each time. That's not to say
that the values will be wildly different. In sensitive techniques such
as gas chromatography you get almost the same values for the same
substance on consecutive runs.

Looking at the values of orange juice Brand A, these are very similar
to both Phenylalanine and to the Aspartic acid. The value of
Phenylalanine in the results is 0.066 and the result for orange juice
Brand A is 0.65. This is a difference of just 0.01. The result of
Aspartic acid is 0.075 whereas the result of the orange juice Brand A
is 0.078. This is a difference of 0.003, which in regular terms is
minuet. This certainly shows that Phenylalanine and Aspartic acid must
be present in orange juice Brand A.

It is also visible that Fresh orange juice and orange juice Brand B
are very similar. They both contain Aspartic acid and have Rf values
of 0.075 and 0.071 respectively. This shows that the orange juice
Brand B is very close to being fresh. This proves that this brand is
the more original.





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