PAPER CHROMATOGRAPHY

A. OBJECTIVE
After doing this lab-work, student is expected to determine the value of Rf (Retordation
Factor) of a compound.
B. LITERARY REVIEW
Paper chromatography, in analytical chemistry is technique for separating dissolved
chemical substances by taking advantage of their different rates of migration across sheets of
paper. It is an inexpensive but powerful analytical tool that requires very small quantities of
material. (encyclopedia Britannica online, 2015 )
Paper chromatography techniques was introduced by Consden, Gordon and Martin
(1994), which uses filter paper as stationary phase support. Paper is pure cellulose which has
affinity to water or other polar solvents. When water is adsorbed on paper, it will form a thin
layer which can be considered analogous to the column. Paper sheet acts as a buffer and water
acting as the stationary phase are absorbed in the pore structure of the paper. (Yazid,2003)
As the paragraph above, stationary phase in this chromatography is water bounded on
paper cellulose while the mobile phase is non polar organic solvent. According to both, paper
chromatography can be grouped in partition chromatography. (Soebagio et al, 2003)
In paper chromatography, mobile phase is permeated into paper because of capillary
effect. The seepage of mobile phase on paper can be carried out by ascending technique or
descending technique. Toward ascending technique, the seepage of mobile phase move upward
while on descending technique, mobile phase move downward. On descending technique, the
seepage of mobile phase beside is helped by capillary effect, it is also helped by gravitation
effect so that the seepage can move faster (Soebagio et al, 2003).
For ascending technique, paper is hung on the top of the beaker containing water until
the paper is immersed partially so that the solvent can move upward to paper by capillary effect.
For descending technique, the paper tightly fitted in a solvent tray at the top of the cabinets and
the solvent moves downward by capillary effect and assisted by gravitation effect. In the form
down, the paper tightly fitted in a solvent tray at the top of the cabinets and the solvent moves
downward by capillary power is assisted by gravity. After the face solvent finished moving in
almost all paper, then tape is taken, dried and examined. In a successful case, solutes from the

original mixture will move along the paper at different speeds, to form a series of separate stains.
If a colored compound, certainly taints can be seen (Day & Underwood, 1990).
Components of compounds that will be analyzed,can be separated and differentiate using
the value of Rf (Retordaction Factor) which states the ratio of the travel distance of a component
with travel distance eluent (mobile phase) at the same time. The easy distributed part in the
water will be quickly adsorbed by paper and the travel is shorter. While the part that is not
distributed in the water, but in the eluent, will flow continuously to the top and the travel is
further. It means that the Rf is higher than the shorter travel. The stains of component in the
analyzed compound will be lined up in a straight line or tape. Eluent was allowed to rise to near
the upper edge of the paper, then paper is marked line (Regina Tutik et al, 2003 ).
Paper chromatography is very useful for the separation of organic substances, inorganic,
and biochemistry in very small amounts. paper chromatography proved invaluable in
biochemistry which is often found small samples and complex. a mixture of simple amino acids
can be separated with eluent water-saturated phenol as a developer (Soebagio et al, 2003)

C. Chemicals and equipments

Equipments:
1. Filter paper (Whatman paper)
2. Expander Container
3. Hanger Stick
Chemicals:
1. Ink which is dissolved in aquadest
2. Aquadest

D. Procedures
Prepare Filter paper with size (3 x 12) cm

Flat line 1 cm from the bottom

Make freckles

Red ink and blue ink with the distance 2 cm

Eluen (distilled water)

Expander Container

Hanger stick

Dry

Measure the distance travel of stain that move up

E. Scheme of Equipment

F. Observation Data
Filter paper

Ink Color

Component Color

Distance (cm)

Red

Orange
Pink
Yellow

6,6 cm
0,9 cm
1 cm

Blue

Purple
Light Blue
Dark Blue

8 cm
0,8 cm
1,1 cm

(Whatman Paper )
1

G. Calculation
Rf =

travel ditance of component

travel distane of eluent

Ink Color: Red

# Orange component Rf =

# Pink component Rf =

# Yellow component Rf =
Ink Color: Blue
# Purple component Rf =

6,6 cm
10 cm

0,9 cm
10 cm

= 0,66

= 0,09

1 cm
10 cm

= 0,1

8 cm
10 cm

= 0,8

# Light Blue component Rf =

0,8 cm
10 cm

= 0,08

# Dark Blue component Rf =

1,1 cm
10 cm

= 0,11

H. Discussion
In this experiment titled Paper Chromatography, the objective is determining the value
of Rf (Retordation Factor) of a compound. Sample compound used in this experiment is ink
of red marker and blue marker.
Paper chromatography is partition chromatography in which the mobile phase is
supported by the water molecules from the cellulose paper. Paper used in this experiment is
whatman filter paper, because it has big pores so that the stain can seep quickly and regularly.
The separation method in Chromatography paper is divided into 3 steps: freckling, expansion
and identification. Where the stationary phase is water bound to the paper (cellulose) and the
mobile phase is a expander solution consist of aquadest. The purpose of this chromatography
is to separate basic dye ink, because the ink color known to consist of several constituent
components of color.
1. Freckling Step
In this step, size of whatman paper used to be freckled is (3x12) cm and horizontal lines
made is within 1 cm of the lower end of the paper. Start line on paper using a pencil

because pencil is made of graphite which is insoluble in the eluent while if the ink pen
will dissolve so that it can interfere with the appearance of stains. After start line has been
made, red marker and blue marker is freckled in the line with the distance of each color is
2 cm and let the ink dry for a moment. Finish line is also made before expansion step, it
is within 10 cm from the start line.
2. Expansion Step
In this step, ends of paper near start line that has been freckled with the sample (ink), is
immersed in the solvent (eluent) in expander container with the paper is hung on the stick
so that the start line is not immersed in the solvent and paper is not touched container
wall. Because, if the paper touches container wall, it will affect the propagation of stains.
When eluent seeped through the freckles (sample), the components of the sample will be
carried away by the seepage of the sample. This is because the capillary effect of the
paper. The solubility difference of sample components in eluent will cause moving speed
of components in the paper are different too. The difference of moving speed of these
components is generally called deferential migration. The separation of these components
are occurred because of deferential migration. The separation will appear as colored
stains on the paper with varying distance from the start line. These stains is then called
chromatogram. In this experiment, stains from blue ink is moving faster than stains in red
ink. The seepage of eluent is stopped when the eluent has reached finish line. Paper is
then taken out from the container to be dried and identified.
3. Identification Step
In this identification step or can be called as appearance of stains, the distance travel from
start line of stains that have been seen are measured to determine the value of Rf
(Retordation factor ). Retordation factor is a retention degree of component in stationary
phase. The color stains seen of red ink (red marker) are orange, pink, and yellow with the
distance travel of each color from start line respectively are 6,6 cm;0,9cm;1cm. While the
color stains seen of blue ink (blue marker) are purple, light blue, and dark blue with the
distance travel of each color from start line respectively are 8cm;0,8cm;1,1cm. Thus,
from those datas, the value of Rf can be determined using formula:
Rf =

travel ditance of component

travel distane of eluent

In this case, the travel distance of eluent (solvent) is 10 cm. Every components has its
own Rf. So, from datas obtained in this experiment, the Rf value of components color in
red marker( orange, pink, yellow ) respectively are 0,66 cm;0,09cm;0,1 cm and Rf value
of components color in blue marker (purple, light blue, dark blue) respectively are
0,8cm;0,08cm;0,11cm.

This chromatography experiment is using ascending technique because the eluent is

moving upwards.

I. Conclusion
Based on his experiment titled Chromatograpy Paper, it can be conclude that:
1. Paper chromatography, in analytical chemistry is technique for separating dissolved
chemical substances by taking advantage of their different rates of migration across
sheets of paper.
2. The Rf value of each component are:
Filter paper

Ink Color

Component Color

Rf Value

(Whatman Paper )
Red

Orange
Pink
Yellow

0,66
0,09
0,1

Blue

Purple
Light Blue
Dark Blue

0,8
0,08
0,11

J. Assesment
1. The value of Rf of each color components:
Ink Color: Red
# Orange component Rf =

# Pink component Rf =

# Yellow component Rf =
Ink Color: Blue
# Purple component Rf =

6,6 cm
10 cm

0,9 cm
10 cm

= 0,66

= 0,09

1 cm
10 cm

= 0,1

8 cm
10 cm

= 0,8

# Light Blue component Rf =

0,8 cm
10 cm

= 0,08

# Dark Blue component Rf =

1,1 cm
10 cm

= 0,11

3. The conclusion that can be suggested in this experiment is:

Paper chromatography, in analytical chemistry is technique for separating dissolved
chemical substances by taking advantage of their different rates of migration across
sheets of paper. Components that are easily distributed in water will be quickly
distributed by the paper, or in other words it has shorter way. While a distributed
component in the eluent, will continue to flow upwards and journey further, thus
having a greater Rf. In this experiment, blue ink has greater Rf than red ink because
the travel distance of components of blue mark is further than the travel distance of
components of red mark.

K. REFFERENCES

Day & Underwood. 1990. Analisa Kimia Kuantitatif. Edisi Keempat. Jakarta: Erlangga
Paper Chromatography. 2015. Encyclopdia Britannica Online. Retrieved 07 November, 2015,
from http://www.britannica.com/science/paper-chromatography
Soebagio, et al. 2003. Kimia Analitik II. Malang: UM press
Tutik, Regina et al. 2010. Petunjuk Praktikum Analisis II. Yogyakarta: FMIPA
Yazid, Estien. 2005. Kimia Fisik untuk Paramedis. Yogyakarta: ANDI