Kishan Ajai Subramanian/Experiment 1

Introduction
Proteins are a diverse group of large, complex molecules that perform
astonishing variety of functions in a living cell 4. In order to understand the chemical and
structural complexity of proteins, it is crucial to determine the monomeric units (amino
acids) that make up protein molecules 5. An insight into the structural assembly of this
macromolecule will pave way for understanding cellular processes that have many
pharmacological applications including customized drug therapy 5. The purpose of the
following experiment is to identify an unknown dipeptide through widely known
dansylation sequencing techniques1. Initially, known standard amino acids along with
the unknown dipeptide were prepped by mixing it with dansyl chloride. A thin layer
chromatography was used to isolate the fragments of the peptide and identify them 1.
In this experiment, the N-terminal amino acid was identified by reacting the
amino groups of the unknown dipeptide with 1-dimethylaminonaphthalene-5-sulphonyl
chloride (dansyl chloride) through a substitution reaction in which the dansyl chloride
attached to the amino group1. This was followed by acid hydrolysis which released the
dansyl amino acid from the unreacted amino acid 1. By taking advantage of the
fluorescence property of dansyl chloride, peptides were tagged and analyzed using thin
layer chromatography (TLC) technique in which a glass plate was uniformly coated with
a thin layer of silica gel or cellulose was used to isolate the amino acids 5. When
developing the TLC plate, as the liquid rises up by capillary action, the non-polar
peptides move with the non-polar solute component of the liquid, forming the mobile
phase. Likewise, water in the developing liquid constitutes as the stationary phase 1.
Since the solutes have different solubility in two phases, they will migrate up the plate
at a unique rate which depends on their relative partition coefficients 3. By comparing
the TLC contours of known standards with the unknown peptide, dansyl amino acid was
identified by viewing under UV light. Rf indicates the relative mobility of solute where
lower value means the solute is more soluble 7. Furthermore, to determine the Cterminus amino acid, cellulose TLC was used in which the stationary phase, the
cellulose matrix is very polar1. While developing, the solvent separates into two
components where water binds to the cellulose, forming the stationary phase, and the
mobile non-polar carries the amino acids along the plate 1. The relative solubility of
amino acids are highly influenced by the side chain since all amino acids have the same
amino- and carboxy- terminals2.

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While analyzing the TLC plates, standard dansyl chloride and the control are
expected to migrate the same length. Dansylated phenylalanine and alanine are
strongly non-polar and would migrate the farthest 6. Glycine, being non-polar but
charged, would migrate very little as it binds to the stationary phase while glutamic
acid is polar and should migrate relatively similar to glycine 6. By comparing the
migration of dipeptide on silica gel plate and cellulose plate, its N terminus amino acid
and C terminus amino acid were deduced 6.

RESULTS
(a)

(b)

Figure 1 (a) represents the Silica gel plate after development in a solution containing
chloroform/methanol/acetic acid at a ratio of 95:10:1 while (b) represents cellulose plate
after development in a liquiid mixture of butanol/acetone/acetic acid/water at a ratio of
10:10:5:2 respectively1. The circles on (a) represent marking of the migration distance
of each amino acid under UV long wage light. Letters B and G indicate Blue and Greenyellow fluorescence that was visualized under the UV light. Purple spots on (b) mark the
migration distance of the amino acids on cellulose plate. These purple spots were
marked under visible light. A diagram showing each lane and the distance migrated can
be found in the appendix.

Table 1: Summary of results from dansylation of unknown dipeptide # 13 and analysis

through TLC
Dipeptide #:

N-Terminal amino acid

Rf of
Rf of

C-Terminal amino acid

Rf of
Rf of
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Kishan Ajai Subramanian/Experiment 1

dipeptide
12
0.587
Dipeptide
Structure
and name

matching
dipeptide
standard
0.60
0.71
N-Alanylphenylalanine-C

matching
standard
0.72

Through a series of calculations, Pmixture of the solvent used for silica TLC was found to be
4.21 while the solvent used for cellulose TLC was found to be 5.24. Refer to appendix
for calculations

DISCUSSION
The objective of this experiment was to sequence a given unknown dipeptide (#13) by
determining its amino and carboxyl terminal amino acid. Reacting the dipeptide with
dansyl chloride at a high pH range of 9.5-10 is critical to avoid the unwanted effect of
the aqueous hydrolysis of dansyl chloride at the thiol group and in deprotonating the
amine group to increase its nuclepophilicity 4. Addition of Sodium bicarbonate plays an
important role in normalizing the reactive side groups 4. Strong Acid hydrolysis which is
used to break the peptide bond, also cleaves all other amide bonds as well 1. Thus, this
sequencing method cannot be used on longer polypeptides. As mentioned in the results
section, the calculated polarity index for the silica gel TLC solvent was calculated to be
4.21. Lower polarity index indicates less polar solvent 5. It is imperative that the solvent
stays relatively polar to ensure the amino acids migrate with the mobile phase. Upon
examining figure 1(a), the N-terminus amino acid was deduced to be Alanine. This is
because the Rf value of the unknown N terminus amino acid was very close to that of
Alanine.
Upon analyzing the cellulose TLC plate, the C-terminal amino acid was identified
to be Phenylalanine. Non-dansylated amino acid standards were spotted along with the
unknown because there was no longer a need to protect the N-terminal from cleaving 3.
Cellulose was chosen as the medium because it is more polar than silica and will
prevent polar unbound amino acids from migrating 1. The solvent used to develop
cellulose TLC had a polarity index of 5.24, which is much higher than the one used for
silica. This is because cellulose, being more polar than silica, requires a solvent with
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Kishan Ajai Subramanian/Experiment 1

stronger polarity to migrate the amino acids. A compound known as Ninhydrin was
sprayed on cellulose plate, allowing it to react with the amide groups of the amino
acids3. It forms aldehydes, carbon dioxide and ammonia as products in which ammonia
and ninhydrin condense to produce purple pigment called Ruhemanns purple 3. CTerminal amino acid was determined using the same deductive methods as the NTerminal amino acid. C-Terminal amino acid was found to be Phenylalanine with an R f
value of 0.71
On TLC plates, known standard amino acids migrated as per the prediction where
non polar amino acids migrated the farthest while polar and charged amino acids
migrated relatively short distances. The control and standard dansyl chloride migrated
the same distance reaffirming the validity of the experiment and indicating absence of
any contamination.
In order to improve results, the buffer used to normalize the peptide can be
changed to Phosphate based buffer could be used as it has a high pK value and would
maintain the reaction pH of about 9.5 at a lower salt concentration. This is significant
because higher salt concentrations could affect the dansylation reaction by reducing
the nucleophilicity of the amine group.
Overall, the experiment was successful in identifying the given unknown
dipeptide (#13)
References:
1. Habeeb, Zeeshan. Experiment 1 Determination of amino terminal groups in
peptides and proteins CHM372 Lab manual 2015 pages 1 to 13.
2. Gray, W. R., End-group Analysis Using Dansyl Chloride. Methods Enzymol. 1972,
25, 121-138.

3. Wigfield, D., Buchanan, G., Croteau, S., On Ruhemanns Purple. Canadian Journal
of Chemistry 1980 58(3), 201.
4. Gros, C.; Labouesse, B. Study of the Dansylation Reaction of Amino Acids, Peptides and
Proteins. European Journal of Biochemistry 1969, 7, 463-470.

5. Bartzatt, R. Fluorescent labeling of drugs and simple organic compounds containing amine
functional groups, utilizing dansyl chloride in Na2CO3 buffer. J. Pharmacol. Toxicol. Methods 2001,
45, 247-253.

6. Sutton, M. R.; Bradshaw, R. A. Identification of dansyl dipeptides in the dansyl-Edman peptide

degradation. Anal. Biochem. 1978, 88, 344-346.

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Kishan Ajai Subramanian/Experiment 1

7.

Rf =

distance migrated by solute

distance migrated by solvent front

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