Exercise No.

2: Paper
Chromatography
Introduction
One of the most common procedures used for the separation of mixtures is chromatography. The
term chromatography is derived from a Greek word, “chroma” meaning color and “graphein" meaning to
write. The chromatography technique uses paper strips or sheets as the adsorbent being the stationary phase
through which a solution is made to pass is called paper chromatography.
Paper Chromatography was first proposed in Russia around 1903 by M.S. Twett after successfully
separating a mixture of plant pigments. It is considered the simplest and most widely used of the
chromatographic techniques because of its applicability to identification, isolation, and quantitative
determination of organic and inorganic compounds.
All forms of chromatography work on the same principle. They have a stationary phase (a solid or
a liquid supported on a solid) and a mobile phase (a liquid or a gas). The stationary phase in paper
chromatography is a very uniform adsorbent paper, while the mobile phase is a suitable liquid solvent or
mixture of solvents. The mobile phase flows through the stationary phase and carries the components of
the mixture with it.
The distance of each compound moves, divided by the distance traveled by the solvent (distance
from the spot or origin to the solvent front), is called the Rf value for that compound. The Rf can be varied
and, in some cases, even reversed by using different developing solvents and various adsorbents. Thus, the
Rf value is a characteristic for each compound with a specific adsorbent and solvent system.
Rf = distance traveled by compound
distance traveled by the solvent
In paper chromatography, the principle involved can be partition chromatography or adsorption
chromatography. The substances are partitioned or distributed between liquid phases or adsorbed between
solid and liquid phases. When the mobile phase moves, the separation of the mixture takes place. Based on
the differences of the affinity of compounds in the mixture, compounds separate themselves towards
stationary and mobile phase solvents under the capillary action of pores in the paper.
There are many applications of paper chromatography. Some of the uses of paper chromatography
that is used in different fields are:
1. To detect the contaminants in drinks and foods.
2. To study the process of fermentation and ripening.
3. To determine dopes and drugs in humans and animals.
4. To examine the reaction mixtures in biochemical laboratories.
5. To inspect cosmetics.
6. To check the purity of pharmaceuticals.
7. To detect the adulterants.
There are types of paper chromatography:
1. Ascending Paper Chromatography
It is a technique wherein the solvent moves in an upward direction.
2. Descending Paper Chromatography
The movement of the flow of the solvent due to the gravitational pull and capillary action
is downwards.
3. Ascending-descending Paper Chromatography
In this technique, the movement of the solvent occurs in two directions after a particular
point. Initially, the solvent's movement is upwards on the paper, folded over a rod, and
after crossing the rod, it continues with its travel in the downward direction.
4. Radial or Circular Paper Chromatography
In this version of paper chromatography, the sample is deposited at the circular filter paper
center. When the spot is dried, the filter paper is tied horizontally on a petri dish that
contains the solvent.
5. Two-Dimensional Paper Chromatography
In this technique, the substances with the same R f values can be resolved with this type of
paper chromatography.
Learning Outcomes
1. To be able to understand the importance of paper chromatography.
2. To demonstrate the separation of components of two mixtures using paper chromatography.
Materials
 Mortar and pestle
 Pencil
 Scissors
 Filter paper/Oslo paper circles
 Ruler
 Empty ink chamber of a ballpen
 Watch glass
 Graduated cylinder
 Petri dish, glass (Do not use plastic)
 Glass bottle, 200 – 250 ml Figure 1 Encircled figure is an ink
chamber of a ball point pen

Developing the Leaf-extract Chromatogram

 Extract of any colored leaves; 3 ml
 Acetone; 10 ml (or use noncolored acetone nail polish remover)

Developing the Marker Chromatogram

 Felt tip marker, black
 Isopropyl alcohol; 10 ml

Additional Suggestions
 Green food color
 Food extract like carrot or broccoli
Procedure
1. Using the mortar and pestle, grind the colored leaves you gathered. Obtain at least 2 to 3 ml extract
and collect it in your watch glass. Set aside the pigment so that the solvent will evaporate partially
and thus concentrate the plant leaf pigments.
2. Create two filter paper circles and cut a wick in each.
3. Using a pencil, mark a short line at the center of each circle. This mark will serve as a "starting
point" or the point of application for your samples to be chromatographed. (Do not use a ball pen
in making a mark because the ink might dissolve in the solvent. The application point would move,
and the ink might mix with the sample and be chromatographed).
4. Cut a spare sheet of filter paper circle that would serve as an absorbent pad during the application
of each sample to the point of application.
5. Using one of your capillary tubes, dip into the leaf extract in the watch glass. By capillary action,
the solution will rise in the tube.
6. Quickly apply a drop of the extract to the extra sheet of filter paper with the capillary tube. Repeat
this spotting technique until you can make spots no larger than 5 mm in diameter.
7. Allow the spot to dry by waving the filter paper.
8. Repeat the process of spotting and drying the filter paper several times until a dark-colored spot is
formed. Make sure to let the sot dry between each application.
9. Make a chromatography mixture (acetone or clear nail polish remover) for about 10-15 ml and
place it into the top half of the petri dish.
10. Bend the wick of the filter paper circle downward and lay the circle on the dry rim, and make sure
that only the wick is in the solvent.
11. Carefully place the bottom half of the petri dish over the filter paper circle to serve as a cover; do
not push it down into the top half. The petri dish will allow a solvent-saturated atmosphere, which
results in less rapid evaporation of solvent from the paper. Thus, a more uniform movement of a
solvent across the paper which acts as a stationary phase.
12. Set aside the chromatogram and allow it to develop undisturbed until the solvent front has advanced
almost to the edge of the petri dish.
13. Prepare and develop the felt tip mark chromatogram. Using a black felt tip marker, make a spot
about 5 mm in diameter with its center at the point of application of your second filter paper circle.
Make the spot darker so that enough ink will be transferred to the paper for a good chromatogram.
14. Dry the spot completely by waving the filter paper circle.
15. Pour a mixture of 10 ml isopropyl alcohol and 5 ml water into the top half of a petri dish.
16. Set up your petri dish as before.

Read your Chromatogram

1. When the chromatogram has developed, remove it from the petri dish. Using a pencil,
IMMEDIATELY marks the solvent front (end of the wet area).
2. Air-dry the chromatogram.
3. When dried, quickly mark your chromatogram by doing the following:
a. Write a number on each colored spot you find.
b. Using a pencil, mark the center of each spot.
c. Note the color of each spot.
d. Measure the distance from the center of each spot to the point of application in mm.
4. Calculate the Rf value.
a. Begin at the point of application, measure the distance traveled by the solvent.
b. Calculate the Rf values for each of the components in ink and the leaf extract.
c. Record your data on your report sheet.
d. Attach the dry chromatograms to your report sheet.
Alternative procedure:
1. Use green food coloring as one of your mixtures to the chromatograph. Obtain Rf values by
following the procedure written above.
2. Use another food such as carrots or broccoli instead of leaf extract. Obtain the extract using mortar
and pestle and follow the same procedure written above.

Guide Questions
Instruction: Use the report sheet below in answering the questions. Write it in a short bond paper or
encode it using Microsoft Word Office. Send you laboratory report via gmail provided by your instructor.
1. Define what is the Rf value.
2. Why should a pencil be used to mark the chromatography paper?
3. What physical principle causes the components of a mixture to become separated during
chromatography?
4. Calculate the Rf value of a component whose center was 25 mm from the point of application if
the solvent front was 30 mm from the point of application.
5. Two components in a mixture have Rf values of 0.20 and 0.80, respectively. In the mobile phase,
which component is more soluble? How can you tell?

REPORT SHEET
NAME: ____________________________ Course, Yr. & Section: ______________
Exercise No: __________

Table 1 Data and Observations for your Paper Chromatogram

Distance Traveled
Spot Number Color Rf Value
Solute Solvent
Rf Calculations

Answers to the questions:

1.
2.
3.
4.
5.