NAME: Merve MURAT 22.03.

2017
ID: 2004141
SECTION / GROUP: 3 / 1
SUBMITTED TO Evrim ELN

PAPER CHROMATOGRAPHY
CALCULATIONS

-1
0
-1
0
0
0
0
1

-1
1

0 0 0 2

-1 1

Table 1: pKa and pI values of 6 amino acids and their ionic forms in different solvent systems

1.3 cm 11 cm 0.118 1.1 cm 10 cm 0.110

1.8 cm 11 cm 0.164 2.1 cm 10 cm 0.210

6.6 cm 11 cm 0.600 6.0 cm 10 cm 0.600

2.6 cm 11 cm 0.236 0.9 cm 10 cm 0.090

3.2 cm 11 cm 0.291 1.0 cm 10 cm 0.100

4.9 cm 11 cm 0.445 3.2 cm 10 cm 0.320

3.1 cm 11 cm 0.282 0.9 cm 10 cm 0.090

4.9 cm 11 cm 0.445 3.1 cm 10 cm 0.310

6.6 cm 11 cm 0.600 6.4 cm 10 cm 0.640

Table 2: Rf value of aas for both solvent systems
RESULTS

Pic 1. Distances travelled by solvent front and amino acids in Tank 1 (Group 1)
Pic 2. Distances travelled by solvent front and amino acids in Tank 2 (Group 2)
DISCUSSION
In this experiment, six amino acids (Aspartic acid, Glutamic acid, Leucine, Lysine, Histidine
and Proline) and an unknown amino acids mixture are used for separation by paper
chromatography.
There are some important and error points of this experiment. When loading, 10 L of the
amino acid samples must be dropped step by step and by drying after each droplet. If not,
sample spreads too much and then it can mix with other samples on the paper. In addition,
taking proper amount of sample by using micropipette is important because it may be ended
up with mixing of sample. For instance, the sample taken excessively by micropipette can
spread extremely on the paper and can mix adjacent amino acid. After loading, the paper must
be linearly placed into tank that includes mobile phase. If not, solvent front cannot move in a
linear way and Rf calculations cannot be done properly. Also, the cover of tank must be
removed at the same time when the paper is placed into the tank. If not, vapor equilibrium is
broken and separation cannot take place properly. To visualize the separation, the paper must
be treated with ninhydrin. It reacts with free alpha-amino group of amino acid and gives
purple color (Block, 1958). In addition to this, ninhydrin is oxidizing agent and very reactive,
so it must be handled carefully. When working with it, gloves must be worn because hands
also contain amino acid and they can be easily painted. The last important point is to mark
solvent front before it dries. Solvent front must be marked before paper dries to calculate R f
value. In addition, it is important to mark the center of amino acid dots on the paper in order
to calculate correct Rf value.
There are some important factors which have an effect upon Rf value: Temperature (e.g.
affects the solubility of samples in mobile phase or equilibrated gas in the tank), The
composition of mobile and stationary phase (e.g. if sample is polar, it will move easily in
polar phase), distance that solvent front must travel on paper (e.g. if this distance is too short,
separation cannot take place properly), humidity (it can be change the level of paper moisture
content). (Clarke & Hawkins, 1963; Ritchie, 1963)
As mentioned above, ninhydrin gives a reaction (ninhydrin reaction) with free alpha-amino
group of amino acid; then it can be seen as purple (diketohydrindylidenediketohydrindamine
(DYDA)) on the paper. When looking at the results, it can be easily seen that the color of
Proline is yellow after ninhydrin treatment. The reason is that Proline does not have free
alpha-amino group, so it cannot dye Proline although ninhydrin can react with Proline.
(HAMILTON & ORTIZ, 1950)

Pic 3. Ninhydrin reaction

http://vlab.amrita.edu/?sub=3&brch=63&sim=1094&cnt=1

On the other hand, Histidine has imidazole, amino group (positively charged) and carboxylate
group (negatively charged) in the form of zwitter ion. Deprotonated amino group of Histidine
that can be observed with its protonated form in equilibrium and the carbonyl group of
ninhydrin condensate; then, lone-pair of electrons of amino nitrogen attack to the carbonyl
carbon of ninhydrin. After decarboxylation, Schiffs base which is unstable and yellow forms.
Then, it hydrolyses and 2-amino indanedione (D 1) and an aldehyde form. D1 interact with
different ninhydrin molecule in order to give DYDA. Hydrindantin which is side product of
reaction can be observed at low pH and low temperature. It reduces the yield of DYDA if it
forms. By the help of hydrolysis, ammonia that can interact with hydrindantin to form DYDA
forms from 2-iminoindanedione (F) (in pathway C). Because of these different patways,
Histidine may be observed as blue. (Kabir-Ud-Din, Rafiquee, Akram, & Khan, 1999)
Pic 4. Reaction of Histidine with ninhydrin.
http://onlinelibrary.wiley.com.sci-hub.cc/doi/10.1002/(SICI)1097-4601(1999)31:2%3C103::AID-
KIN4%3E3.0.CO;2-4/abstract

The polarity of two tanks is close to each other, but Tank 2 is a little bit more polar because its
solvent moves slower. The solvent of Tank 2 form H-bond with cellulose and fall behind in
the solvent of Tank 1. On the other hand, the solvent of Tank 1 has less affinity for cellulose
due to less polarity. In addition, pHs are too different: the pH of Tank 1 is 9 and the pH of
Tank 2 is 3. Thus, the migration rate and distance travelled by amino acids are different.
According to results, Aspartic acid (polar) travels a little bit longer way in Tank 1. Actually,
this is not expected because it should travel longer distance in Tank 2. The solvent of Tank 1
is less polar (moves faster) and Aspartic acid is polar and negatively charged at pH 9; thus,
Aspartic acid and mobile phase interaction is less in Tank 1. In addition to interaction,
Aspartic acid should move slower due to its charge. On the other hand, the solvent of Tank 2
is more polar and Aspartic acid is polar and found as in the form of zwitter ion (no net charge)
at pH 3. Thus, aspartic acid spends most of its time in mobile phase in Tank 2 and should
travel longer distance. This may be caused by some experimental error: wrong amino acid
may be loaded to paper or contamination caused by other amino acids. The results of
Glutamic acid is expected due to the same reasons with Aspartic acid. When looking at the
results of both Asp and Glutamic acid, Glu has one more -CH 2 group. This difference make
Glu more non-polar although both of them are polar. Thus, Glu travels longer distance than
Asp. Lysine is polar and positively charged in both tank. It traveled longer distance in Tank 1.
This case is also expected because it is in the form of zwitter ion in Tank 1. Histidine is also
polar, positively charged in Tank 2 and negatively charged in Tank 1. The result of His is
expected because it moved faster in Tank 1 due to its aromatic side chain. When looking at the
results of both Lys and His, His is faster than Lys because of aromatic side chain of His. This
side chain provides less affinity to His for stationary phase. Leucine is non-polar and has no
net charge in both tank. Tank 1 is also more non-polar than Tank 2. Because of this, it is
expected that it should travel longer distance in Tank 1, but it moves the same rate in both
tank. This may be caused by some experimental error: contamination caused by other amino
acids. Proline is polar and is in the form of zwitter ion form in both tank. Its result is expected
because of its side chain that gives Pro non-polarity.
In Tank 1 (basic), Leu, Lys, His and Pro has no net charge. However Leu is non-polar and
moves the fastest in basic solvent. Pro comes after Leu because of its uncharged R-group, and
His (due to its aromatic side chain) and Lys come. The charged ones are Asp and Glu. Glu is
faster than Asp because Glu is more non-polar than Asp. In Tank 2 (acidic), Asp, Glu, Leu and
Pro has no net charge. However Leu is non-polar and moves the fastest in acidic solvent. Pro
comes after Leu because of its uncharged R-group, and Glu (due to its non-polarity) and Asp
comes. The charged ones are Lys and His. His is faster than Asp because Glu is more non-
polar (aromatic side chain).
According to unknown results, Unknown 1 is Leu Unknown 2 is Pro and Unknown 3 is His
(with respect to its RF value).
ASSINGMENT
One of the usage area of paper chromatography is forensic chemistry because the nature color,
the behavior in UV light, purity and radioactivity of substances under investigation can be
easily found. For instance, in a suicide case, it may be important to specify whether chemical
used in suicide and obtained from the stomach of dead person comes from a certain bottle.
This can be decided by the help of chromatogram. Also, in a murder case, the ink on a paper
which is evidence for case can be separated and analyzed in a short time. In addition to ink,
for instance, a gum can be an evidence for some cases. By the help of chromatographic
separation for gum, its component and even its relative concentrations can be determined.
After separation of gum, its data are compared with the data of known sugars and their
appearance in toffees and sweets on chromatogram. So, the determination of criminal become
easier with paper chromatography. (Curry, 1953)
REFERENCES
Block, R. J. (1958). Manual of Paper Chromatography and Paper Electrophoresis. Elsevier
Science. Retrieved from https://books.google.com.tr/books?
id=aC4XBQAAQBAJ&pg=PA4&dq=paper+chromatography&hl=tr&sa=X&ved=0ahU
KEwjJipLosvfSAhWmDpoKHSkCBzQQ6AEIIDAB#v=onepage&q=paper
chromatography&f=false
Clarke, E. G. C., & Hawkins, A. E. (1963). A NOTE ON THE FACTORS AFFECTING RF
VALUES ON CITRATE BUFFERED PAPER CHROMATOGRAMS. Journal of
Pharmacy and Pharmacology, 15(1), 390393. https://doi.org/10.1111/j.2042-
7158.1963.tb12803.x
Curry, A. S. (1953). Journal of Criminal Law and Criminology The Application of Paper
Chromatography to Forensic Chemistry. Retrieved from
http://scholarlycommons.law.northwestern.edu/jclc
HAMILTON, P. B., & ORTIZ, P. J. (1950). Proline and hydroxyproline: purification, reaction
with ninhydrin, and some properties of their N-nitroso derivatives. The Journal of
Biological Chemistry, 184(2), 60715. Retrieved from
http://www.ncbi.nlm.nih.gov/pubmed/15428444
Kabir-Ud-Din, Rafiquee, M. Z. A., Akram, M., & Khan, Z. (1999). Kinetics of interaction of
Histidine and Histidine Methyl Ester with Ninhydrin in micellar media. International
Journal of Chemical Kinetics, 31(2), 103111. https://doi.org/10.1002/(SICI)1097-
4601(1999)31:2<103::AID-KIN4>3.0.CO;2-4
Ritchie, A. S. (1963). Some factors influencing the constancy of RF values in paper
chromatography under australian field conditions. Journal of Chromatography A, 10,
281283. https://doi.org/10.1016/S0021-9673(01)92310-6