Post Laboratory Report on

Exercise 6
Dipeptide Sequence Determination

Aldrin John N. Alviar

Chem 160.1 8L
2nd Semester, A.Y. 2016-2017

Groupmates:
Bane, Danica
Rabino, Mikaela Bernadette
Santos, Maura Mercedes

Prof. Marvin Bilog

Table 6.1. Rf values of amino acid standards from paper chromatography.
Amino acid standards Distance travelled by Distance travelled by Retardation factor
the sample (cm) the solvent (cm) (Rf)
Glycine 1.2 0.16
Alanine 2.5 0.33
Valine 4.3 0.57
Leucine 5.2 7.5 0.69
Glutamic acid 2.0 0.27
Phenylalanine 6.9 0.92
Methionine 3.2 0.43

Table 6.2. Amino acid composition of the acid hydrolysed dipeptide sample.

Amino acid Distance Distance Retardation Identity of Possible

sample travelled by travelled by factor (Rf) the amino identity of the
the sample the solvent acid dipeptide
(cm) (cm) sequence
H1 A 5.4 0.72 Leucine Leucine-
B 4.5 7.5 0.60 Valine Valine
H2 C 2.6 0.35 Alanine Alanine-
D 2.0 0.27 Glutamic Glutamic acid

Sample Calculation:
()
Rfglycine = ()
1.2
= 7.5 = 0.16

Table 6.1 shows the distance travelled by the solvent and sample (i.e. amino acid
standards) and their corresponding retardation factor (Rf). On the other hand, table 6.2 also
shows the distance of samples and solvent and their corresponding Rf, also their possible identity
of the dipeptide sequence. According to the data, leucine-valine and alanine-glutamic acid are
the possible identity of the dipeptide due to the comparison of the Rf value of the amino acid
standards. Also, the two peptide in the dipeptide sequence were labelled from A to D to
differentiate and get the corresponding amino acid based on the standard. In addition to that, it
can also be concluded that there is a direct relationship between the distance travelled by the
sample and Rf value. As the distance travelled by the sample increases, the Rf value also
increases. This can be proven by glycine and phenylalanine having lowest and highest Rf value,
respectively. Glycine has the shortest distance travelled while phenylalanine has the longest
distance travelled in the chromatogram.
Figure 6.1. Result of the Paper Chromatography of the labeled Dipeptide sequence (H1, H2)
alongside with amino acid standards.

After paper chromatography, the samples were run in thin layer chromatography (TLC) to
determine the N-terminal of the dipeptide sequence.

Table 6.3 Rf values of DNP-amino acid standards from TLC.

Amino acid standards Distance travelled by Distance travelled by Retardation factor
the sample (cm) the solvent (cm) (Rf)
Methionine 6.4 0.68
Phenylalanine 8.1 0.86
Glycine 2.2 0.23
Valine 1.3 9.4 0.14
Leucine 5.5 0.59
Glutamic acid 0.6 0.06
Alanine 4.1 0.44
Table 6.4. Rf value of the N-terminal of the dipeptide sequence and its corresponding identity.

Amino acid Distance Distance Retardation Same Rf in Identity of the

sample travelled by travelled by factor (Rf) amino acid DNP-amino
the sample the solvent standard acid
(cm) (cm)
DP1 5.1 9.4 0.54 Leucine DNP-Leucine
DP2 3.9 0.41 Alanine DNP-Alanine

Table 6.3 shows the Rf values of amino acid standards from TLC. This data also shows
the direct relationship of distance travelled by the sample and the Rf. On the other hand, table 6.4
shows the Rf values of the two dipeptide sequence (DP1 and DP2) and its corresponding DNP-
amino acid identity. The amino acid sample (H1 and H2) were added with 1-fluoro-2,4-
dinitrobenzene (FDNB) resulting to DP1 and DP2. After TLC, the resulted band only consists the
N-terminal of the amino dipeptide sequence. The distance travelled by N-terminal of the two
samples were also measured and compared to the amino acid standards to determine the identity
of the N-terminal. The Rf value of the N-terminal of DP1 has 0.54 which is the closest to the Rf
value of leucine while DP2 has 0.41 which is closest to alanine. Therefore, it can be concluded
that the N-terminal of DP1 (H1+FDNB) and DP2 (H2+FDNB) is leucine and alanine, making it to
be DNP-Leucine and DNP-Alanine, respectively.

Figure 6.2. Result of TLC of the DP1 (H1+FDNB) and DP2 (H2 +FDNB) alongside amino acid
standards.
 +

H1 (Leucine-Valine) FDNB DNP-Leucine-Valine

Figure 6.3. Reaction of the dipeptide sequence (H1) with FDNB.

Paper chromatography is used as a qualitative analytical chemistry technique for

identifying and separating colored mixtures like pigments and amino acids. (Coppens, 2016).
Ninhydrin is one of the common reagent to detect -amino acids, free amino acid, and
carboxylic acid groups on proteins and peptides. Despite its side effects (i.e. impotence), it is
mostly used in tests wherein detection of amino acids are involved. The reaction of ninhydrin with
amino acids will form purple color due to the degradation of amino acids into aldehydes, ammonia,
and CO2 with series of reactions (see figure 6.4). Eventually, according to Senese (2010), these
series of reactions will produce hydrindantin, hen the partially reduced ninhydrin will condense
with ammonia that leads to the production of the purple color in the amino acids.

Figure 6.4. Reaction mechanism of Ninhydrin with amino acid until purple colored product.

In running paper chromatography, it is advisable to run standards alongside of the sample

to ensure that the tests were run under the same condition. Using of literature Rf value standards
can yield error in the totality of the experiment. This is due to the presence of contamination,
human error (i.e wrong execution of the method), and the quality of the materials being used. In
addition, the immediate visual comparison between the bands in the chromatogram is much
easier to distinguish.
 In paper chromatography, comparing the Rf of the samples to the amino standards the
dipeptide sequence of H1 is Leucine and Valine while H2 consists Glutamic acid and Alanine.
Aside from this, we can also concluded that the components in dipeptide sequence or higher
number of peptides separate in paper chromatography and each amino acid has its own distinct
Rf value and can be determined when compared to the standards.
Next in determining the components of the dipeptide sequence, determination of the N-
terminal of the dipeptide sequence was also done through TLC. Thin-layer chromatography (TLC)
is known as a chromatography technique used in the separation of the non-volatile mixtures. The
principle behind this is the use of proper stationary and mobile phase making the different analytes
ascent in the TLC plate at different rates, resulting to the separation (Lewis & Moody, 1989).
The use of TLC also involved the use of correct visualizing agent depending on the
purpose. According to Boulander (2012), the examples of visualizing agents use in TLC and its
corresponding target group are bromcresol green: carboxylic acids; fluorescamine: primary amine
detection; iodine vapour: hydrocarbons; ninhydrin: amino acid; rhodamine 6G: lipids. It varies
depending on the type of molecules being analysed in the chromatography.
In the second extraction, ether phase was recovered instead of the aqueous phase since
the addition of hydrocholoric acid cleaves the N-terminal to the rest of the dipeptide with cation.
Then, addition of ether was done in the mixture to remove the non-polar N-terminal DNP-amino
acid due to the presence of large hydrophobic group in the DNP (i.e. benzene). The other amino
acids in the sequence now contains cation and is available in the aqueous phase of the mixture.
In thin layer chromatography, the ether phase was run and separate having different Rf
values. The Rf values of DP1 and DP2 were compared to the amino acid standard. DP1 has the
closest value to leucine while DP2 to alanine. It can be concluded the N-terminal amino acid of
H1 and H2 (result of paper chromatography) are leucine and alanine, respectively.
Apart from chromatography, there are also other techniques in determining the protein
sequence of dipeptide or polypeptide. It can be chemical techniques or enzymatic processes each
serving different cleavage styles and specific recognition of group. Example in chemical
techniques are Cyanogen bromide, Sangers reagent which uses fluorodinitrobenzene and
Edman Degradation which utilizes phenyl isothiocyanate. On the other hand, the use of enzymes
digest peptide with a higher degree of specificity (see table 6.5).
Table 6.5. Different enzymes and its specificity of cleavage procedures for sequence analysis.
Enzyme Cleavage site Recognition site
Trypsin Carboxyl side Basic amino acids (arg,lys)
Chymotrypsin Carboxyl side Aromatic amino acids and
leucine
Thermolysin Amino side Aromatic amino acids and
bulky non-polar side chains
amino acids
Pepsin Amino side Aromatic amino acids, acidic
amino acids, isoleucine
Aminopeptidase Carboxyl side N-terminal
Carboxypeptidase Amino side C-terminal

By the use of these enzymes, cleaving the peptides at specific sites to determine the
specific arrangement of amino acids can be done (Touchstone, 1992).
References:
Badani, H., Freeman, T. 2011. PepDraw. Wimley Laboratory. Tulane University. New
Orleans, Louisiana. United States.
Boulander, W. (2012). Thin Layer Chromatography. School of Chemical Sciences, Roger
Adams Lab Penthouse. 600 S. Mathews Avenue, Urbana, IL 61801. Retrieved on
http://scs.illinois.edu/hpl/tlc.php.
Coppens, T. June 15, 2016. What is Paper Chromatography and How Does it Work?
Owlcation. Retrieved from https://owlcation.com/stem/What-is-Paper-Chromatography-and-
How-does-it-Work

Harry W. Lewis and Christopher J. Moody (1989). Experimental Organic Chemistry:

Principles and Practice (Illustrated ed.). WileyBlackwell. pp. 159173.
Polypeptides and Proteins. [PDF file]. Retrieved from
http://www.hull.ac.uk/php/chsanb/PP/POLYPEPTIDES%20AND%20PROTEINS.pdf
Senese, F.(2010). What is a simple test for the presence of amino acids?. Retrieved April
11, 2015. Retrieved from http://antoine.frostburg.edu/chem/senese/101/organic/faq/amino-
acid-test.shtml.
Touchstone , J.C. 1992. Practice of Thin Layer Chromatography. 3rd Edition. NY.