Exercise No. 1 Paper Chromatography

Exercise 1: 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 wherein it is the
stationary phase, through which a solution is made to pass. It 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 chromatographic technique 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 an acceptable 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.
distance travelled by compound
Rf =
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 in 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:
For instructional purposes only • 1 st Semester SY 2024-2025
To detect the contaminants in drinks and foods.
1. To study the process of fermentation and ripening.

2. To determine dopes and drugs in humans and animals.
3. To examine the reaction mixtures in biochemical laboratories.
4. To inspect cosmetics.
5. To check the purity of pharmaceuticals.
6. 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 Rf values can be
resolved with this type of paper chromatography.
Learning Outcomes
At the end of the activity, students will be able to:
1. To be able to understand the significance of paper chromatography.
2. To demonstrate the separation of components of two mixtures using paper
chromatography.
Materials
- Oslo paper (3 pcs.)
- Pencil
- Scissors
- Ruler
- Leaves (any color; should have extract)
- Marker (black, blue and red colors)
- Acetone (60 ml)
- Isopropyl alcohol (60 ml)
- Paper clip
- Barbecue stick (6 pcs.)
- Rubber bands
- Mortar and Pestle
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and innovative technologies for sustainable communities and environment. No.
- Beaker (7 pcs. 400 ml & 110 ml)

- Capillary tube
- Graduated cylinder
Procedure
__________________________________________________________________________________
________
Preparation of Chromatograms
1. Using a mortar and pestle, grind 4-5 pcs. of any leaves and transfer the
extract to a small 100 ml beaker. Add more leaves until you can collect 3
ml of leaves extract. Set aside.
2. Cut a six (6) rectangular paper strips using oslo paper the same length as
your 400 ml beaker.
3. Using a pencil, draw a faint line approximately 1 cm away from one end of
the paper strip. Label this as “Line A”, indicating the point at which the
strip should be immersed in the solvent. Parallel to that line is “Line B”
approximately 1 cm away from line A, indicating the point at which the
sample (leaves extract or marker) will be placed.
4. For your leaf-extract chromatogram, use a capillary tube to make a dark
spot with the leaves’ solution. The spot should be on Line B. Do the
spotting several times, allowing the spot to dry between treatments.
5. For your marker chromatogram, make a dark spot using your marker. One
chromatogram using black marker, one for blue marker and another one
for red marker.
Developing the Leaf-extract Chromatogram
1. Make three (3) leaf-extract chromatograms by following step no. 4.

2. In a 400 ml beaker, mix 5 mL water and 5 mL acetone. Prepare three
beakers with this mixture and label them as “Trials 1, 2 and 3.”
3. Immerse the paper strip inside the beaker. Place the stick horizontally on
top of the beaker, and using a paper clip, inserting the leaf-extract
chromatogram and hang it inside the beaker.
4. The paper strip should come into contact with the solvent but not go over
the dark spot. Cover the beaker with a watch glass (so that the air inside is
saturated with solvent).
5. Remove the paper strip once the solvent has reached the upper edge of
the strip. You'll notice bands of various colors. Mark a point using a pencil
where the solvent and each pigment all traveled up the paper.
6. Obtain Rf values by following the calculation written on the introduction.
Write your observations and calculations to your laboratory notebook.
Developing the Marker Chromatogram
1. Make marker chromatograms by following step no. 5 on the preparation of

chromatogram.
2. In a 400 ml beaker, mix 5 mL water and 5 mL isopropyl alcohol. Prepare
three beakers with this mixture and label them as “Trials 4, 5 and 6”.
3. Immerse the black chromatogram in trial 4, blue chromatogram in trial 5
and red chromatogram in trial 6.
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4. Place the stick horizontally on top of the beaker, and using a paper clip,
inserting the leaf-extract chromatogram and hang it inside the beaker.
5. The paper strip should come into contact with the solvent but not go over
the dark spot. Cover the beaker with a watch glass (so that the air inside is
saturated with solvent).
6. Remove the paper strip once the solvent has reached the upper edge of
the strip. You'll notice bands of various colors. Mark a point using a pencil
where the solvent and each pigment all traveled up the paper.
7. Obtain Rf values by following the calculation written on the introduction.
Write your observations and calculations to your laboratory notebook.
Read your Chromatogram
1. When the chromatogram has developed, remove it from the beaker. Using
a pencil, IMMEDIATELY mark 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 (1,2,3 ….) on each colored spot you find.
b. Using a pencil, mark the end of each spot which travelled up the
paper strip.
c. Note the color of each spot.
d. Measure the distance (in centimeters) from the center of the spot
(leaves or ink) to the point where its travel comes to a halt.
Tabulate your results in your laboratory report under results and
discussion section.
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.
Questions
1. Why should a pencil be used to mark the chromatography paper?
2. What is the purpose of drawing "Line A" and "Line B" on the paper strips?
3. What role does the water and acetone mixture play in the chromatography
process?
4. How do you determine the Rf value for each component in the
chromatogram?
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?
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Exercise 1: Paper

Paper Chromatography was first proposed in Russia around 1903 by M.S.

All forms of chromatography work on the same principle. They have a

The distance of each compound moves, divided by the distance traveled

In paper chromatography, the principle involved can be partition

There are many applications of paper chromatography. Some of the uses

To detect the contaminants in drinks and foods.

1. To study the process of fermentation and ripening.

There are types of paper chromatography:

1. Ascending Paper Chromatography

- Beaker (7 pcs. 400 ml & 110 ml)

Developing the Leaf-extract Chromatogram

1. Make three (3) leaf-extract chromatograms by following step no. 4.

Developing the Marker Chromatogram

1. Make marker chromatograms by following step no. 5 on the preparation of

Read your Chromatogram

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