QUALITATIVE ANALYSIS OF AMINO ACID IN UNKNOWN SAMPLE THROUGH

PAPER CHROMATOGRAPHY TECHNIQUES WITH ELUENT OF

N-BUTANOL AND PHENOL

Gusti Ngurah Putu Eka Putra (1013031020)

rahekagusti@yahoo.com

Chemistry Education Department

Faculty of Math and Natural Sciences,Ganesha University of Education

ABSTRACT

This experiment aims of this experiment are 1) to compare the distribution coefficient (Rf)
of various amino acids, especially glycine, leucine, methionine and tyrosine, 2) to determine the
content of amino acids by paper chromatography by the ascending technique. In this experiment
used are paper chromatography, which has a stationary phase and a liquid phase that are not
intermingled. By using paper chromatography of amino acids then can be determined, the amino
acid content that is contained in the sample that is by comparing the Rf value of sample
components with standard Rf, so that the Rf value equal or close is identified as the same amino
acid. Eluent used was n-Butanol mixed with distilled water and glacial acetic acid and phenol
eluent. The result shows that amino acids that contained in the unknown sample II is glycine.
Key Words: Paper Chromatography, Identification, Amino Acids.

INTRODUCTION
Chromatography comes from the Greek
word is "Choma" which means the color and
"graphien" which means to write, so that
chromatography can be interpreted as the
writing of the color. With the development of
science, the use of chromatography is not
only limited to the color components, but
also can be separated compounds are
colorless. Chromatography is a separation
method based on differences in the
distribution of sample components between
two phases.
Chromatography always involves two
phases, namely the stationary phase
(stationary phase) and the mobile phase
(mobile phase). The stationary phase can be
either solid or liquid that is bound to a solid
surface (paper or adsorbent), whereas for the
liquid phase can be called eluent/solvents, or
inert carrier gas. Movement of this mobile
phase resulted in differential migration of
components in the sample (Tika, 2010). The
tendency in the chromatographic process is
as follows (Soebagio, et al: 2003).
1. The tendency of molecules to
dissolve in the liquid component.
2. Tendency for the component
molecules attached to a smooth
solid surface (adsorption)
3. The tendency of the molecules of
the components to react chemically
(ion exchange)
Separated components must dissolve in
the mobile phase must have the ability to
interact with the stationary phase by
dissolving in it, adsorbed or react chemically
(ion exchange). Separation occurs based on
differences in migration of the substances
that make up a sample. The results of
separation can be used for identification
(qualitative analysis), the assay (quantitative
analysis) and purification of a compound
(preparative work).
This separation technique has several
advantages over other separation methods,
such as: can be used to sample constituents to
the very small (semi-micro and micro), is
quite selective, especially for multi
components organic compounds, and the
separation process can be done in a relatively
short and simple.
One type of chromatography is a
paper chromatography. It is one that has the
type of chromatography stationary phase and
a liquid phase that is not mixed with each
other. These systems are typically used on a
saturated solution of a polar solvent (e.g. n-
butanol) and polar solvents (e.g. water). This
solvent mixture migrate throughout the
paper, polar components adsorbed on the
support medium (cellulose) produces
thousands of tiny droplets are adsorbed on
the support. The droplets are adsorbed
stationary phase is bypassed by a mobile
phase of non-polar solvent mixture so that the
partition or separation occurs. On paper
chromatography, samples were dropped on
the paper with a capillary tube, then mixed
solvent (eluent) migrate through the spot
(spot) with an upward direction (ascending)
or down (descending). The duration of the
migration process depends on the stability of
the paper, the selected solvent, and
temperature (Ledever, 1961).
In this experiment, would do a
qualitative analysis of a solution containing
various amino acids. Paper chromatography
can be either one or two dimensions. If not
too many different components, then the two-
dimensional manner is often required. For
that, we need two kinds of eluent solution,
the one required for the direction and the
second one to the other direction
perpendicular to the direction of the first
elution, after a dry chromatographic paper
(Tika, 2010).
By using paper chromatography, the
components of the sample can be determined
by comparing the samples with the standard
price of Rf. Each component has a specific
Rf prices. Rf scale suggests a degree of
retention of components in the stationary
phase. Therefore, the Rf is also called a
factor or factors Referrals reference. Price Rf
can be calculated as the distance traveled by
the components of the base line distance
divided by the eluent (mobile phase) from
baseline (Tika, 2010).
Rf value of a compound in the paper
chromatography system depends on many
variables, including solvent system,
temperature, duration of elution, and paper
type. Because it is influenced by many
variables, the Rf of a compound that has been
known to be a standard or benchmark to
determine Rf shopped other compounds. The
qualitative analysis of specific compounds
that are known to be used in conjunction with
the compound to be identified. Two different
compounds in a particular solvent system can
have the same Rf value. Therefore, the
analysis results obtained by paper
chromatographic techniques must be justified
by other methods.
EQUIPMENTS & MATERIAL
The equipment that are used in this
experiment are capiler pipe, chromatography
chamber, beaker glass 250 ml, beaker glass
100 ml, glass rod, spatula, ruler, scissors,
pinset, oven, drop pipette, and separated
funnel. Materials that is used in this
experiment are n-butanol solution, glacial
acetic acid, aquades, glycine solution, leusine
solution, metionine solution, tyrosine
solution, tryptophan solution, ninhydrin
solution, phenol, chromatography paper, and
unknown sample.
METHODS
This test uses qualitative methods to
determine the ratio of the distribution
coefficient (Rf) of various amino acids,
especially glycine, leucine, methionine,
tyrosine, and tryptophan as well as determine
the amino acid content of the unknown
samples II.
- Preparation of Elution Solution
Elution solution used is a mixture of
n-butanol, water, and glacial acetic acod
made by mixing a solution of 100 ml of n-
butanol is added with 100 ml of distilled
water and 24 mL of glacial acetic acid. The
third solution is placed in a separating funnel
and then shaken and separated. The second
elution solution was phenol mixed with water
in the ratio between phenol and water volume
was 100 mL: 39 mL.
- Preparation of Paper Chromatography
Paper chromatography prepared with
size 18 x 6 cm chromatography container
adapted to as many as 4 sheets. At the bottom
is marked with a pencil approximately 1.5 cm
from the bottom edge of the paper. The place
when put the samples is marked and given
sample spacing of 1.5 cm.
- Process Chromatography Using the
mixture of eluent n-Butanol, distilled
water, and Glacial Acetic Acid
In a prepared paper chromatography
then spotted with a capillary tube a standard
solution of amino acids and adjacent samples
with a distance of 1.5 cm from each other.
The solution was placed 2 cm from the tip of
the edge of the paper. Each droplet dried
first, efforts should be made so that enough
amino acids placed in the paper and the stain
should not exceed 0.4 cm. The paper used is
clean and not touched with a finger during
the chromatographic process.
Chromatographic chamber have been
saturated with vapor eluent before the paper
chromatography conducted. Paper
chromatography which already contains the
sample solution and the standard solution
enter into a container that already contains
the eluent chromatography, stains or spots
not participate submerged in eluent.
Comparison eluent mixture of n-butanol:
glacial acetic acid: water is 100 mL: 24 mL:
100 mL. Standard amino acid elution and
sample eluent was conducted about a
distance of 10 cm. Elution was stopped and
the distance is already taken eluent marked
using a pencil. Further chromatography paper
was dried in oven at 100-105oC, and then
sprayed with a solution of ninhydrin paper
that had been prepared beforehand. It put in
oven again at a temperature 100-105oC for 5
minutes. Amino acid stains will look on
paper chromatography, then measured and
determined Rf value of the amino acid as
well as the sample solution.
- Process Chromatography by Using
Eluent Phenol
Chromatography using eluent phenol,
the procedure is the same as the eluent
chromatography using a mixture of n-
butanol, distilled water, and glacial acetic
acid eluents only used in space
chromatographic are distinct eluent is used
phenol.
RESULTS AND DISCUSSIONS
On this experiment, two eluents were
used namely: phenol and n-butanol. The
eluent of n-butanol was made by mixing the
water, glacial acetic acid and n-butanol. Two
layers were formed during the mixing
process. The 1st layer was turbid meanwhile
the 2nd layer was colorless. It shown as the
figure bellow:
Figure 1. Two layers that formed from
mixture of n-butanol, distilled water and
acetic acid glacial.
 The addition of acetic acid to the and n-butanol) with an upward direction
mixture of n-butanol, water and glacial acetic (ascending) so that the separation of a
acid is to distribute to a solvent that does not mixture of samples occurs.
mix with each other. Water and n-butanol
would be equally distributed in acetic acid
solution so that at a certain volume ratio,
obtained a mixture containing n-butanol -
acetic acid - water in one phase. This mixture
migrated to the rest of the paper so that the
polar components of the water will be
adsorbed on the support media (paper),
wherein the composition of the cellulose
paper was then performed with the goal of
saturation is also conditioned to the
chromatographic run faster because when the
air is already saturated in chromatography as (a)
well as paper and then when the elution of
the sample begins to focus the work of n-
butanol is only elute the sample components.
In this case, water is a polar solvent acts as a
stationary phase, while n-butanol is non-polar
solvent acts as the mobile phase.
Chromatograms or paper
chromatography is then inserted into the first
two chromatograms chamber containing
phenol and a second eluent containing a
mixture of n-butanol, water and glacial acetic
acid. When the space has been saturated
paper chromatography and the elution can be
started. Elution was stopped after the eluent (b)
moves to a distance of 10 cm. That Figure 2. The Eluent of n-butanol (a)
occurred during the elution process is as The Eluent of phenol (b)
follows. Eluent (phenol or a mixture of n-
butanol, acetic acid and distilled water) will After the eluent moves with the
migrate to the rest of the paper. Polar distance about 10 cm, then the elution
component of this mixture (water) will be process is stopped and removed from the
adsorbed on the support media of paper, chromatography paper chromatography
wherein the composition of paper is chamber.
cellulose. Then there was an interaction
between the cellulose fibers with water vapor Furthermore, paper chromatography
causing a complexity. With the adsorption of was dried then sprayed by using solution of
water molecules on the surface of this paper ninhydrin. After drying back, the color can be
will produce thousands of tiny spots that detected, which is a bluish purple color
adsorbed on the support media. Small spots which indicates the presences of amino acids
were then passed by the mobile phase, the are distributed. In this case the reaction
non-polar component of the eluent (phenol occurs between amino acids and ninhydrin
with the following equation:
 O O
C R C
OH H
+ H2N C COOH + NH3 + CO2 + RCHO
C OH C OH
H
O O
Ninhidrin
Ninhydrin Ninhidrin tereduksi
Reducted Ninhydrin
O O O O
C H C C C
OH H
+ N + N
C OH H H HO C C C
O O O O
Blue complex compound biru
Kompleks berwarna

Figure 3. Reaction Between Amino Acid And Ninhydrin (Solomon, 2000)

After appear color then Rf can be
calculated as the distance traveled of amino
acid is proportional to the distance of eluent.
To determine what type of amino acid
contained in the unknown sample can be
done by comparing the Rf value of the
sample solution that is unknown to the Rf
standard amino acids leucine, cysteine, and
glycine. Unknown sample can be predicted
if the value is equal or close to Rf of the
standard.
Rf is the distance traveled by each
compound from baseline relative to the
distance the solvent/eluent, with the
following formula.

Table 1. Data of Rf value from amino acid and Unknown sample by using eluent n - butanol

Type of Jarak Tempuh Rf value

amino Component Phenol
acid (cm) (cm)
Leucine 9.8 10.2

Cystein 2.1 10.3

Glycine 5.2 10.3

Unknown
3.7 10.2
sample II

On the paper chromatography using about 10 cm then the chromatography paper

phenol as the eluent was done the same chromatography carried out from the drying
thing. After elution reached a distance of chamber and dried. After spraying by
ninhydrin, the color came ups then distance were measured. The data clearly state as
of each eluent and amino acids contained table below:

Table 2. Data of Rf value from amino acid and Unknown sample by using eluent phenol

Type of Jarak Tempuh

amino Component Phenol Rf value
acid (cm) (cm)

Leucine 10.1 10.1

Cystein 10 10.1

Glycine 9.7 9.8

Unknown
9.7 10
sample II

Considering to the table 1, it can be ACKNOWLEDGMENT

seen that the unknown sample II in phenol
eluent the Rf value close to glycine and
based on table 2, the unknown sample II in
the n-butanol eluent have Rf values close to
glycine. So the unknown sample II is
glycine.
CONCLUSION
Based on the above discussion, can
be obtained several conclusions, among
others:
1. Distribution coefficient (Rf) in the eluent
phenol for the amino acid leucine,
cysteine, and glycine, respectively are
1.0, 0.99, and 0.989, while in the eluent
n-butanol respectively are 0.96, 0.2, and
0.5.
2. Unknown samples II contains amino
acids of glycine with (Rf) values on
phenol eluent is 0.97 while in the eluent
n-butanol is 0.37.
The reporter wish to thank Dr. I
Nyoman Tika, M.Si as the lecture who gave
the guidance in several cases during the
experiment conducted, Mr. Lasya as the
laboran who kept care of us beyond the
experiment and the patience on us. The
classmate of RKBI 2010 who kept motivates
each other during the experiment. Thanks to
all of them, in particular for their attention to
detail.
REFERENCES
Solomons, Graham and Craig Fryhle.
(2000). Organic Chemistry Seventh
Edition. United State: John Wiley and
Sons, Inc.
Soebagio, dkk. (2003). Kimia Analitik II.
Malang : IMSTEP
Tika, I Nyoman. 2010. Penuntun Praktikum
Biokimia. Singaraja: Universitas
Pendidikan Ganesha