Experiment number:5
Title: I. Paper Chromatography – Amino Acids Identification
Objectives:
1. To learn how to use the paper chromatography technique.
2. To identify and calculate the Rf of each amino acid.
3. To compare the Rf values and determine the two unknown amino acid.
Materials and Methods:
- Refer to UESB 2142 laboratory manual, page 12 and 13.
Results:
Table 1: The colour, distance migrated and the Rf of the amino acids samples
Amino acid Paper chromatography 1 Paper chromatography 2 Rf values
Colour Distance Rf Colour Distance Rf (average)
of migrated values of migrated values
amino by amino by
acid amino acid amino
acid acid
(cm) (cm)
Glutamic Purple 3.40 0.2982 Purple 3.85 0.3156 0.3069
acid
Lysine Purple 1.80 0.1580 Purple 2.10 0.1721 0.1651
Methionine Purple 6.10 0.5351 Purple 6.00 0.4918 0.5135
Threonine Purple 3.60 0.3158 Purple 3.70 0.3033 0.3096
Unknown 1 Purple 3.70 0.3246 Purple 3.60 0.2951 0.3099
Unknown 2 Purple 6.20 0.5439 Purple 6.20 0.5082 0.5261
Solvent front for paper chromatography 1 was recorded as 11.4cm.
Solvent front for paper chromatography 2 was recorded as 12.2cm.
Calculation:
The relationship of the distance moved by an amino acid to the distance moved by the
solvent front is calculated as below:
Distance moved by amino acid (cm)
Rf =
Distance from amino acid origin to solvent front (cm)
Paper chromatography 1:
For Glutamic acid: For Lysine:
Rf = 3.4 cm / 11.4 cm Rf = 1.8 cm / 11.4 cm
= 0.2982 = 0.1580
For Methionine: For Threonine:
Rf = 6.1 cm / 11.4 cm Rf = 3.6 cm / 11.4 cm
= 0.5351 = 0.3158
For Unknown 1: For Unknown 2:
Rf = 3.7 cm / 11.4 cm Rf = 6.2 cm / 11.4 cm
= 0.3246 = 0.5439
Paper chromatography 2:
For Glutamic acid: For Lysine:
Rf = 3.85 cm / 12.2 cm Rf = 2.1 cm / 12.2 cm
= 0.3156 = 0.1721
For Methionine: For Threonine:
Rf = 6.0 cm / 12.2 cm Rf = 3.7 cm / 12.2 cm
= 0.4918 = 0.3033
For Unknown 1: For Unknown 2:
Rf = 3.6 cm / 12.2 cm Rf = 6.2 cm / 12.2 cm
= 0.2951 = 0.5082
Observation:
Length of
Solvent front is
4.7 cm
Yellowish and
Purple Color reddish
(Movement of (Contamination)
amino acids)
Length of
Solvent front is
5.3 cm
L-M : L-Methionine G : L-Glutamic acid
L-P : L-Phenylalanine U1 : Unknown 1
U2 : Unknown 2 L : L-Lysine
Observation:
There are present of purple color, and yellowish or reddish colors on the
chromatography paper.
There were presence of purple, yellowish and reddish colours on the chromatography
paper.
Discussions:
In order to identify the different types of amino acids in this practical, a series of
procedures was followed. A chromatography paper was obtained by wearing gloves and a
line was drawn 2 cm from the bottom of the paper. Six marks were made at 1.5 cm
intervals along the line. Each amino acid sample was taken and lightly dotted on each
pencil mark by using a micropipette. The paper was dried with hair-dryer. These
processes should then be repeated at least 8 times, applying the same amount of amino
acid to the same pencil mark, to build up a concentration. This step was necessary for the
chromatogram results to be clear and distinct. Next, the BAW solvent was poured into the
chromatography tank and the chromatography paper was stand in the tank where its
bottom edge was in the solvent but the remaining paper did not touch the tank (including
the sample spots). The paper was left in the tank for about 2.5 to 3 hours for the solvent
movement. The damp chromatography paper was removed before the solvent front
reached the top of the paper. Position of the solvent front was marked with a pencil and
the paper was dried in the fume cupboard. Then, the paper was sprayed with ninhydrin.
The ninhydrin should have the effect of bringing the amino acid dots to visibility. The
paper was dried again and the position of each spot that developed was marked. Finally,
the colour present for the amino acids could be observed, and the Rf values for each
amino acid could be calculated. The amino acid composition of the unknown samples,
unknown 1 and unknown 2 could then be determined.
As the amino acids have no colour, all the procedures were carried out “blind” in
this experiment and the results would only be seen after spraying the revealing agent
(ninhydrin) onto the chromatogram. Ninhydrin is also known as Triketohydrindane
hydrate, a chemical used to detect ammonia or primary amines. When the ninhydrin
reacts with the free amines, a deep blue or purple colour is evolved. Hence, it is
commonly used to detect fingerprints, monitor deprotection in solid phase peptide
synthesis and used in amino acid analysis of proteins. Simultaneously, the solvent
solution used is a mixture of Butan-1-ol : Acetic acid : Water ,in the ratio of 60 : 15 : 25.
To obtain better result, the solvent used must be freshly prepared. Our experimental
results for the different amino acids might not be that accurate since the separation time is
short. Hence, the solvent front was not run to the top of the paper and the amino acids
sample might not be finished separating as we stopped and took out the paper from the
tank. In actual, the solvent front was supposed to be run in the undisturbed tank for about
2.5 to 3 hours. However, as a result of time consuming (as our practical class had only
two hours time), the solvent front was run for only 2 hours. Hence, the results obtained
might not be so accurate. The Rf value for each of the amino acids obtained from the
experiment was compared with the expected result (standard Rf values). The comparison
was recorded the table below:
Amino acid sample Rf values obtained Expected Rf values
Glutamic acid 0.3069 0.3000
Lysine 0.1651 0.1400
Methionine 0.5135 0.5500
Threonine 0.3096 0.3500
From the table above, it could be seen that the results obtained from this
experiment were different from the expected results (standard Rf values) . However, by
observing and comparing the sequences of these sets of data carefully, there were
similarities. Both sets of data shown that Methionine has the highest Rf value, followed
by Threonine and Glutamic acid. Lysine has the lowest Rf value.
From the observations, the presence of purple, reddish and yellowish colours on
the chromatography paper could be observed. The purple colour indicated the presence of
the amino acids sample while the presence of reddish and yellowish colours might due to
contamination.The contamination might due to our fingerprints as we might accidentally
take out a chromatography paper without wearing gloves at the beginning of the
experiment or during the drying process after spraying the ninhydrin solution onto the
paper as we hold the paper on hand. After recording the distance migrated by the solvent
front and the amino acids samples, the Rf values of each sample were calculated. By
comparing the Rf values, Methionine had the highest Rf (0.5135) .Conversely, Lysine had
the lowest Rf value (0.1651). This meant that Lysine was very attracted to the
chromatography paper but not too much to the BAW solvent so it did not move much as
the solvent moved up the paper and gave a low Rf value. In contrast, Methionine was
very attracted to the BAW solvent so it moved along with the solvent front and gave a
higer Rf value compared to that of Lysine. Next, the two unknown amino acids were
being compared by using the Rf values obtained. It was predicted that the Unknown 1
(0.3099) was Threonine as it had a very close Rf value with Threonine, which was
0.3096. The Unknown 2 was predicted as to be Methionine (0.5261) as it had a very close
Rf value with that of Methionine, which was 0.5135.
There were some precaution steps that should be taken into consideration when
handling this experiment in order to improve the quality of observations and obtain
accurate results. First, gloves should be worn when handling the chromatography paper
to avoid the depositing of amino acids from our fingers. Apart from this, nitrile gloves
should be worn rather than latex gloves as latex is soluble in many of the reagents but
nitrile is resistant to chemicals. Futhermore, the spray of ninhydrin should be done in the
fume cupboard to avoid inhalation and injury as the ninhydrin is carcinogenic. Besides
that, lead pencil should be used to make marks on the chromatography paper instead of
using the ink ball-pen. This is to get a better and more accurate result as the solvent front
might solubilize with the ball-pen ink as moving up to the top of the paper.
Conclusions:
1. The Rf value obtained for Glutamic acid was 0.3069, Lysine was 0.1651,
Methionine was 0.5135, Threonine was 0.3096, unknown 1 was 0.3099 and
unknown 2 was 0.5261.
2. By comparing the obtained Rf values, the unknown 1 was predicted to be
Threonine and the unknown 2 was predicted to be Methionine.
3. The presence of purple colour on the chromatography paper indicated the
separation of the amino acids but the presence of the reddish and the yellowish
colours might be due to the contamination of our fingerprints.
4. The obtained Rf values were not so accurate as the time for the movement of
solvent front was only 2 hours due to time consuming of our practical class.