BIOCHEMISTRY EXPERIMENT

ARTICLE

IDENTIFICATION OF AMINO ACID IN UNKNOWN SAMPLE THROUGH

ASCENDING TECHNIQUE OF PAPER CHROMATOGRAPHY

By
Ni Kadek Wahyuni Antari
1213031002
A

CHEMISTRY EDUCATION DEPARTMENT

FACULTY OF MATHEMATICS AND NATURAL SCIENCES
UNIVERSITAS PENDIDIKAN GANESHA
2015

Rubrik Penilaian Artikel Praktikum

Biokimia S1

Nama mahasiswa

: Ni Kadek Wahyuni Antari

NIM

: 1213031002

Tanggal

: 23 Maret 2015

Mata Acara praktikum

: Kromatografi Kertas

No.

Aspek yang dinilai

Bobot

Skor

(%)

(0-100)

Format

Abstract

10

3.

Introduction

20

4.

Materials and Methods

10

5.

Result and discussion

30

6.

Conclusion

10

7.

Acknowledgment

8.

References

9.

Clear

Total score

100

Singaraja, 23 Maret 2015

Penilai,

Dr. I Nyoman Tika, M.Si

Bobo x skor

IDENTIFICATION OF AMINO ACID IN UNKNOWN SAMPLE THROUGH

ASCENDING TECHNIQUE OF PAPER CHROMATOGRAPHY
By
Ni Kadek Wahyuni Antari
Chemistry Education Department, FMIPA, UNDIKSHA
Udayana Street Singaraja, Bali
Email: wahyuni.antari@yahoo.co.id
Abstract
Amino acids contained in a sample can be analyzed by using the paper chromatography technique. Paper
chromatography is one method of physical separation, in which the elements will be separated are distributed
between two phases, namely stationary and mobile phases. In this experiment, were used standard amino acid,
especially: phenylalanine, glycine, tryptophan, and L-cysteine. Technique that used in this experiment was
ascending technique. The objectives of this experiment were to determine the (1) distribution coefficient (Rf) from
various amino acid standard and unknown sample through paper chromatography with ascending technique and (2)
amino acid content in unknown sample through comparing Rf with those in amino acid standard. The Rf and content
of unknown sample were measured descriptively through physical and chemical change during the treatment and
comparing to those of standard amino acid. From this experiment, by using mixture of n-butanol, distilled water,
and glacial acetic acid as eluent, was obtained the Rf value of phenylalanine, glycine, tryptophan, and L-cysteine
as standard amino acid is 0.60, 0.19, 0.57, 0.20 respectively. The Rf values of unknown sample A are 0.17 and
0.23, while the Rf values of unknown sample B are 0.17, 0.56, and 0.58. Therefore, it can be identified that in
unknown sample A contains glycine and L-cysteine, while in unknown sample B contains glycine, tryptophan, and
phenylalanine.
Keywords: Amino Acid Content, Paper Chromatography, Ascending Technique

INTRODUCTION
Amino acids are organic molecules
with low molecular mass (100-200 Da)
containing at least one carboxyl group (COOH) and an amino group (-NH2). Amino
acids have an important role for human life,
ranging from ensuring that cell division occurs
perfectly, metabolic functions, maintaining
memory, growth, healing up as the building
blocks of proteins are linear chains of amino
acids (Tika, 2010). In general, the amino acid is
obtained as a result of hydrolysis of proteins,
using either enzymes or acid. In this way is
obtained a mixture of a variety of amino acids
and to determine the type and quantity of each
amino acid should be a separation between the
amino acids. There are several methods to
analysis the type quantity of each amino, e.g.
gravimetric, microbiology, chromatography,
and electrophoresis methods. Furthermore,
each type of amino acids has different
properties. Beside differ in isoelectrical point,
they also differ in distribution coefficient (Rf).
To separate the constituent components of the
sample, chromatography paper can be done.
Chromatography comes from the word
"chroma" and "graphein". In Greek, the word
means both "color" and "write (Tika, 2010).
The process of chromatography was discovered
in 1903 by Mikhail Tswett, who separated

solubilized plant pigments using solid

adsorbents (Voet, Voet, & Pratt, 2013).
Chromatography separation method is based on
the differential distribution of sample
components between two phases, namely the
stationary phase and the mobile phase.
Stationary phase can be either solid or liquid
that is bound to a solid surface (paper or an
adsorbent), while the mobile phase can be a
liquid called the eluent or solvent, or an inert
carrier gas. Movement of this mobile phase
resulted in differential migration of components
in the sample (Tika, 2010).
Paper chromatography is one type of
chromatographic mehod which has stationary
phase and mobile phase that does not mix with
each other. On paper chromatography (mixture
of substances) dropped by a capillary tube at the
paper, and then mixed solvent (eluent) migrate
through the spots (spot) with an upward
direction (ascending) or down (descending), as
the following figure:

Figure 1. Paper Chromatography

Tendencies that may occur in the

chromatographic process are as follows: (1)
Tendency of molecules component is dissolved
into liquid solvent; (2) Tendency of molecules
component attached to a smooth solid surface
(adsorbed); (3) Tendency of the molecules of
the components to react chemically (Tika,
2010). In this case, the separation of
components must be dissolved in the mobile
phase and it has the ability to interact with the
stationary phase by dissolving in it, adsorbed,
or interact chemically (ion exchange).
Separation is occurs based on differences in
migration of the substances components in a
sample. (Poedjadi, 1994).
In the chromatographic process, the
various components were separated by
differential affinity of these components to the
stationary phase (solid or liquid) carried by the
mobile phase (gas or liquid). (Soebagio, et al;
2003). In this systems typically use a saturated
O
C
C

O
R

OH
+

OH

H2N

C
COOH

(aq)

(aq)

+ NH3(aq) + CO2(g) + RCHO(aq)

OH
(aq)

Ninhydrin Reducted

O
Ninhydrin

solution of a nonpolar solvent (eg n-butanol)

and polar solvents (eg water). This solvent
mixture migrate throughout the paper, polar
components adsorbed on the supporting media
(cellulose) produced thousands of droplets are
adsorbed on the support. The droplets are
adsorbed stationary phase is by passed by the
mobile phase in the form of non-polar solvent
so that the partition or separation of the sample
mixture occurs.
In paper chromatography, the result
would be quite difficult observation. It is
because considering that dissolved amino acids
did not show a specific color. In anticipation of
this, after elution process, the position of the
solvent front is marked with a pencil, and
chromatography paper was dried and sprayed
with ninhydrin solution. Ninhydrin is used
because ninhydrin reacts with amino acids
produce colored compounds, mainly purple
color (Tika, 2010).

H
H

OH
+
OH
(aq)

N
H

H (aq)

O
C
N

HO

C
O

(aq)

(aq)

Complex Colored Compound

Figure 2. Ninhydrin reacts with amino acids produce purple compounds

By using paper chromatography, the
components of the sample can be determined by
comparing the Rf value of sample toward the Rf
value of standard amino acids. Each component
has a specific Rf value. The amount of Rf is
expressed degree of retention of a component in
the stationary phase. Therefore, Rf is also called
the factors reference. Rf can be calculated as the
distance taken by the components of mixture
from baseline divided by the distance taken by
eluent from baseline (Tika, 2010).
The Rf value of a compound in a paper
chromatography system depends on many

variables,
including
solvent
system,
temperature, length of run, and type of paper.
Because it is influenced by many variables, the
Rf of a compound that has been known to be
used as a standard or benchmark shopped
determine Rf other compounds. For qualitative
analysis, certain compounds are already known
to be used in conjunction with the compound to
be identified. Two different compounds in a
given solvent system can have the same Rf
value. Therefore, the analysis results obtained
by paper chromatography techniques must be
justified by other methods.

MATERIALS AND METHODS

This experiment was conducted at
Organic Chemistry Laboratory of Chemistry
Education Department, Faculty of Mathematics
and Natural Sciences, Ganesha Education
University on 9th of March 2015
Materials:
In conducting this experiment, it was
used n-buthanol solution, distilled water, acetic
acid
glacial,
phenylalanine,
glycine,
tryptophan, L-cysteine, unknown sample A and
B. All of them were needed at sufficiently
amount.
Equipment:
This experiment was used severaal
equipment namely, capillary pipe (3 unit),
chromatography chamber ( 1 unit), beaker
glass 100 mL (1 unit), beaker glass 250 mL (3
unit), separation funnel 500 mL ( 1 unit), ring,
stative and clamp (1 set), stirrer rod ( 1 unit),
ruler 30 cm (1 unit), spatula (1 unit), drop
pippete (2 unit), heater (1 unit), scissor (1 unit),
and tweezer (1 unit).
Procedures:
This experiment was carried out
accordance with the procedures adopted from
book
Penuntun
Praktikum
Biokima
developed by I Wayan Redhana and Siti
Maryam.
Making elution solution
A total 100 mL of n-butanol was added by
100 ml of distilled water and 24 mL of glacial
acetic acid. All of the were placed in a
separatory funnel and shaken. The two layers
were formed and then separated.

Preparation of chromatography paper

Chromatography paper was prepared with
appropriated size in chromatogaphy chamber
that is 15 cm x 16.5 cm. In the about 1.5 cm
from the bottom edge of the paper was marked
with a pencil.
Chromatography using eluent mixture of nbutanol, distilled water, and glacial acetic acid
Filter paper with a size of 15 cm x 16.5
cm spotted with a solution of phenylalanine,
glycine, tryptophan, L-cysteine, unknown
sample A and B colorless solution by using
capillary pipe. Spot distance between each
other was 1.5 cm. Things to note, that each
droplet must be dried first with aerated spotted
before the next drop. Spot should not exceed
0.4 cm. Paper was kept clean and untouched as
possible by finger. Furthermore, the paper was
hung in chromatographic chamber for several
hours in order to make elution run. After elution
solution runs 10 cm from the sample
boundary,
elution was
stopped and
chromatrography paper was removed from
chromatography chamber. Solution boundary
was marked with a pencil and paper filter was
dried at a temperature of 100-105C. The dried
paper was sprayed with ninhydrin solution.
Distance between eluent distance and distance
of color formed were measured. Then, the value
of Rf can be determined.
RESULT AND DISCUSSION
Result
From the experiment, it was obtained data as
follows:

Table 1. The result of paper chromatography by uisng eluent of mixture of n-butanol, distilled water,
and glacial acetic acid
Distance taken by
Distance taken by
Sample
component of
Rf
eluent (cm)
mixture (cm)
Phenylalanine
5.2
8.6
0.60
Glycine
1.6
8.6
0.19
Tryptophan
4.9
8.6
0.57
L-cysteine
1.7
8.6
0.20
1.5
8.6
0.17
Unknown sample A
2.0
8.6
0.23
1.5
8.6
0.17
Unknown sample C
4.8
8.6
0.56
5.0
8.6
0.58
Discussion
In this experiment, carried out paper
chromatography to determine the amino acids

contained in the mixture of amino acids by

using the ascending technique. In this case the

constituent components were separated by an

unknown differential distribution of the
unknown component between two phases,
namely stationary and mobile phases. The
amino acids contained in the mixture of amino
acids was determined by comparing the Rf
value of each detected amino acid in samples
with Rf value of predetermined standards
amino acid. Standard solution of amino acids
used were phenylalanine, glycine, tryptophan,
and L-cysteine. While the unknown sample
used namely unknown samples A and B.
Experiment was conducted by using a
mixture of n-butanol, glacial acetic acid, and
distilled water as the eluent. In this case, eluent
acts as a mobile phase was non-polar solvent
namely n-butanol, while the stationary phase
was polar solvent namely water. The presence
of acetic acid in the eluent is intended to
distribute the two solvents that do not mix with
each other, whereas the n-butanol and water can
be distributed equally in acetic acid so that the
specific volume ratio can be obtained. Mixtures
that contain n-butanol, acetic acid, and water
used for saturating chromatography paper, the
mixture migrate throughout the paper, whereas
the polar component which water will be
adsorbed on the supporting media (paper).
Water molecules (as the polar phase) will be
distributed on the surface of the paper. Whereas
the interaction is a very important thing in paper
chromatography.

differences in the distribution of amino acids

that make up the mixture of mobile phase and
with water (stationary phase).
Chromatography paper was taken by
using tweezers. This was done because if it was
taken by using hand there will be contact by
hand that will disrupt the process of
chromatography. In which, every moment our
body take out of sweat which has main
component namely organic substances such as
urea and oil. Therefore, if the hand is used to
pick up the paper these organic compounds will
also migrate as a non-polar phase (motion) that
will affect the results of observations on the
chromatographic process.
The solution to be tested was taken
with a capillary pipe and then dropped on the
chromatography paper. It was intended that the
amino acids in the paper does not exceed 0.4 cm
in diameter. If it exceeds then the results will
not perfect due to the permeation of the mobile
phase and the stationary phase becomes firm
and detected color diffuse. Each of drops was
dried first and then proceed with another drop
of solution to be tested.

Figure 4. Chromatography paper that already

spotted by colorless standard amino
acid, unknown sample A and B

Figure 3. The mixture of n-butanol, glacial

acetic acid, and distilled water
produce two layers
Water molecules (as the stationary
phase) which is adsorbed on the surface of the
paper, creating thousands of tiny droplets, this
allows water to act as the stationary phase.
Droplets of stationary phase which is adsorbed
is skipped by mobile phase namely non-polar
components in the eluent (n-butanol).
Therefore, this results partition or separation of
mixtures can be occured. It is occured since

The chromatography paper was hung in

chromatography chamber and dip the bottom
edge of the chromatography paper in eluent
(drop of sample and standard amino acids were
not submerged in eluent).

Figure 5. Chromatography paper was hung in

chromatography chamber

Elution process was stopped after

elution moving at a distance of 10 cm. After
it was dried, the paper was sprayed with
ninhydrin solution, but the stain of amino acids
cannot be directly detected its color, neither
standard amino acids nor amino acid samples.
This is because on the surface of
chromatogram, there was still adsorbed water
molecules so that do not react perfectly with
O

with amino acids distributed in the polar phase

(water) and mobile phase (n-butanol). Thus, it
necessary the heating in an oven at 105oC, the
temperature at which water molecules will
evaporate. After warm up, then purple stains
will be detected that indicate amino acids
distributed. The reaction of the formation of
blue wish purple pigment is as follow:

OH
+

OH

H2N

C
COOH

(aq)

(aq)

(aq)

Ninhydrin Reducted

+ NH3(aq) + CO2(g) + RCHO(aq)

OH

O
Ninhydrin

H
H

OH
+
OH
(aq)

N
H

H (aq)

O
C
N

HO

C
O

(aq)

(aq)

Complex Colored Compound

Figure 6. Ninhydrin reacts with amino acids produce purple colored compound

Figure 7. The Chromatography result after

sprayed nynhidrin solution of
amino acid standard and unknown
sample A and B solutions which
has purple color.
After
arising
of
stains
on
chromatography paper, then the value of its Rf
can be calculated. Whereas Rf is the ratio of the
distance taken by the components of mixture
from baseline divided by the distance taken by
eluent from baseline. In order to determine the
amino acids contained in the sample needs to be
compared that Rf value of standard solution
with sample solution. Whereas amino acids
with the same Rf value or close to one of the

standard amino acids were identified as the

same.
From the table result, it can be seen in
a paper chromatography using the eluent
mixture of n-butanol, acetic acid and water, the
Rf value of amino acid standard of
phenylalanine, glycine. Tryptophan, and Lcysteine are 0.60, 0.19, 0.57, 0.20 respectively.
The Rf values of unknown sample A are 0.17
and 0.23, while the Rf values of unknown
sample B are 0.17, 0.56, and 0.58. Thus by
comparing the Rf value of unknown sample A
and B toward standard amino acid, further can
be identified that the Rf values of unknown
sample A are close to glycine and L-cysteine,
so in sample A contained glycine and Lcysteine amino acids. While, the Rf values of
unknown sample B are close to glycine,
tryptophan, and phenylalanine, so in sample B
contained
glycine,
tryptophan,
and
phenylalanine.
CONCLUSION
Based on the explanation above, it can
be concluded that: (1) In paper chromatography
by using eluent a mixture of n-butanol, distilled
water, and glacial acetic acid was obtained the
Rf value of amino acid standard of
phenylalanine, glycine, Tryptophan, and L-

cysteine are 0.60, 0.19, 0.57, 0.20 respectively.

While the Rf values of unknown sample A are
0.17 and 0.23 and the Rf values of unknown
sample B are 0.17, 0.56, and 0.58; (2) Thus by
comparing the Rf value of unknown sample A
and B toward standard amino acid, further can
be identified that in sample A contained Lcysteine and glycine amino acids. While, in
sample B contained glycine, tryptophan, and
phenylalanine amino acids.
ACKNOWLEDGEMENT
In writing this article, the author has of
a lot of support, guidance and encouragement
from many quarters. For this reason, the author
respectfully thanks for Dr. I Nyoman Tika,
M.Si as lecturer on the practicum, Ms. Dewi as
lecturer assistant, Mr. Dewa Subamia as the
laboratory assistant and also all member of
RKBI12 of chemistry class for always as good
partners.

REFERENCES
Poedjadi, Anna dan Titin Supriyanti. 1994.
Dasar-Dasar
Biokimia.
Jakarta:
Universitas Indonesia
Redhana, I Wayan., Siti, Maryam. 2003.
Penuntun Praktikum Biokimia. Singaraja :
IKIP NEGERI SINGARAJA
Soebagio, Endang Budiasih, Sodiq Ibnu,
Hayuni Retno Widarti, Munzil. 2003.
Common Textbook (Edisi Revisi)
Kimia Analitik II. Malang: Universitas
Negeri Malang
Tika, I Nyoman. 2010. Buku Penuntun
Praktikum
Biokimia.
Singaraja:
Universitas Pendidikan Ganesha
Voet, D., Voet, J. G., & Pratt, C. W. (2013).
Fundamentals of biochemistry; life
at the molecular level. Danvers:
John Wiley & Sons, Inc.