Sandesh Walke completed a chemistry investigatory project on paper chromatography. He thanks his teacher Ms. Dhruva Pardeshi for guiding the project and his principal for providing facilities. Paper chromatography is a technique that separates mixtures by their different migration rates on chromatographic paper. Key aspects covered include sample application, development with a mobile solvent phase, and calculation of Rf values to quantify separation. Paper chromatography has various applications like separation of mixtures, purity testing, and analysis of foods, drugs and other samples.

1. Prepared by: -
Sandesh Walke
Class XII
2019 - 2020
Chemistry Investigatory Project
Paper Chromatography

2. Acknowledgment
I wish to express my deep gratitude and sincere thanks to the Principal,
Mrs. Aradhana Rana for her encouragement and for all the facilities that
she provided for this project work.
I extend my hearty thanks to Ms. Dhruva Pardeshi, our Chemistry
Teacher, who guided me to the successful completion of this project. I
take this opportunity to express my deep sense of gratitude for his
invaluable guidance, constant encouragement, immense motivation,
which has sustained my efforts at all stages of this Project work.
I can’t forget to offer my sincere thanks to my parents and also to my
classmates who helped me to carry out this project work successful and
for their valuable advice and support, which I received from them time to
time.
Sandesh Walke
Class 12th

3. Content
• Certificate
• Acknowledgement
• Introduction
• Principal of Chromatography
• Preparation of Chromatographic Paper
• Working of Chromatographic Paper
• Types of Paper Chromatography
• Different types of development Techniques.
• Rf values (retention factor)
• Factors affecting Rf Value
• Sources of error
• Separation of colored pigments
• Sample Spot Detection and development of colour
• Applications of Paper Chromatography
• Bibliography

4. Introduction
Paper chromatography is a chromatography technique used to separate mixture of
chemical substances into its individual compounds.
It is an inexpensive method of separating dissolved chemical substances by their
different migration rates across the sheets of paper. It is a powerful analytical tool that
uses very small quantities of material. It is used to determine organic compounds.
In the beginning of 1860, a scientific concept with the work of FRIEDRICH
GOPPELSROEDER who was a pioneer of paper chromatography he developed the
theory of capillary analysis by using paper strips while examining wine, milk, alkaloids,
dyes and oils among other.
Paper chromatography was discovered by Synge and Martin in the year 1943.

5. Principal of Paper Chromatography
The principle of separation is mainly “Partition” rather than adsorption. Cellulose
layers in filter paper contains moisture which acts as stationary phase & organic
solvents/buffers are used as mobile phase.
Preparation of Chromatographic Paper
• Cut the paper into desired shape and size depending upon work to be carried
out.
• The starting line is marked on the paper with an ordinary pencil 5cm from the
bottom edge.
• On the starting line marks are made 2cm apart from each other.

6. Working of Chromatographic paper
• A horizontal line is drawn near one end (about 1.5 cm from the bottom edge) of the
paper.
• The sample needs to be separated is placed as a small drop or line on to the paper
using capillary tube. Labelling the drop by a pencil with an alphabet or number help
to identify the compound later. The drops are then soaked on the paper and dried.
• The paper is then placed into a sealed container with a swallow layer of suitable
solvent (M.P). The solvent level must be lower than the pencil line or drop on it.
The container needs to be covered to stop the solvent to evaporate.
• The solvent rises up the paper chromatography taking each component of the
sample with it. The components travel with the solvent depends on three things:
✓ The polarity of the sample molecule. The non-polar components travel faster
than the polar component.
✓ The attraction between the sample molecule and the solvent or solvent mixture.
✓ The attraction between the sample and the silica.
• When the solvent rises near the end of the paper then the paper should be taken
out from sealed container and air dried. The paper with separated bands of
components are then observed under UV-light.
• This is what causes the different liquids within our original liquid mixture to spread
out on the solid.
• The key thing to remember is that chromatography is a surface effect.

7. Types of Paper Chromatography
• Paper adsorption chromatography: -
➢ It is based on the differential adsorption of the components on the adsorbed
(stationary phase).
➢ This means that different components are absorbed on adsorbent at
different degrees.
➢ Migration of the substance id due to difference in “PARTITION Coefficient “.
✓ Column Chromatography: -
✓ Thin layer Chromatography (TLC)
✓ Gas Chromatography
• Paper partition chromatography: -
➢ The basic principal of partition chromatography is the continuous differential
partitioning of components of mixture between stationary phase and mobile
phase.
➢ Separation of similar substances by repeated extraction by two immiscible
liquids.
➢ Migration of the substance is due to “ADSORPTIVE” Power.
Paper Chromatography
Paper adsorption
chromatograph
y
Paper partition
chromatography

8. Different types of Development Techniques
• Ascending Development (go up): -
➢ Here the solvent travels up the chromatographic paper. Both descending and
ascending paper chromatography are used for the separation of organic and
inorganic substances. The sample and solvent move upward.
➢ Like conventional type, the solvent flows against gravity. The spots are kept at
the bottom portion of paper and kept in a chamber with mobile phase solvent
at the bottom.
• Descending Development (go down): -
➢ This is carried out in a special chamber where the solvent holder is at the top.
The spot is kept at the top and the solvent flows down the paper.
➢ Development of the chromatogram is done by allowing the solvent to travel
down the paper. Here, mobile phase is placed in solvent holder at the top. The
spot is kept at the top and solvent flows down the paper from above.
➢ Advantages is that development is faster.

9. Rf Values
The retention factor (Rƒ) may be defined as the ratio of the distance traveled by the
solute to the distance traveled by the solvent. It is used in chromatography to quantify
the amount of retardation of a sample in a stationary phase relative to a mobile phase.
Rƒ values are usually expressed as a fraction of two decimal places.
• If Rƒ value of a solution is zero, the solute remains in the stationary phase and
thus it is immobile.
• The ratio of the distance moved up or travelled by the components from the origin
or point of application to the distance moved up by the solvent from the same
point.
Rƒ = Distance travelled by the solute from the original line
For example, if a compound travels 9.9 cm and the solvent front travels 12.7 cm, the
Rƒ value = (9.9/12.7) = 0.779 or 0.78. Rƒ value depends on temperature and the
solvent used in experiment, so several solvents offer several Rƒ values for the same
mixture of compound. A solvent in chromatography is the liquid the paper is placed in,
and the solute is the ink which is being separated.
Distance travelled by the solvent from the original line

10. Factors affecting Rf Value
• The temperature
• The purity of the solvents used
• The quality of the paper, adsorbents & impurities
• present n the adsorbents
• Chamber saturation techniques, method of drying and development
• The distance travelled by the solute & solvent
• Chemical reaction between the substances being partitioned.
• pH of the solution
• In many cases it has been observed that the solvent front is run off the end of
the paper. Rx value is thus used,
• It is the ratio of distance travelled by the sample and the distance travelled by
the standard. Rx value is always closer to 1.
• Mobile phase composition
• Stationary phase
• Concentration of analyte
• Particle size
• In liquid chromatography, in general, the mobile phase pH is an important
parameter
• In reversed-phase liquid chromatography (hydrophobic retention
chromatography) the solvent type and concentration are important factors

11. Sources of error
• Error during application of the spots: -
➢ Apply minimum volume of the concentrated solution in order to avoid
diffusion through the paper which leads to poor separation.
➢ Spots should be approximately of the same diameter.
• Development: -
➢ Improper adjustment of the paper in the tank leads to this error so the
paper should be held vertically.
➢ Do chamber saturation.
• Detection: -
➢ The spraying methods affect the final result

12. Separating Colored Pigments
• Paper chromatography is an effective technique for separating colored
pigments from a mixture.
• A few drops of the mixture of colored pigments are placed on the filter paper
(stationary phase) and it is then slowly submerged into a jar of solvent (mobile
phase).
• As the solvent rises up the paper, it dissolves the molecules present in the
mixture, their solubility depending on their polarity.
• That is Because of different polarity, molecules of each pigment leave the
solution at different places, as the solvent continues to rise up the stationary
phase.
• Thus, each pigment rises up to a particular level on the chromatography paper,
and gets separated in the process.

13. Sample Spot Detection and development of colour
• Spotting of the sample is done with the help of a capillary tube or automated
applicator.
• The sample is applied as a neat spot on a horizontal line drawn with a pencil
close to one edge.
• Allow the spot to dry and then immerse the paper in the developing chamber
as per the selected technique with the marked spot above the solvent level.
• The solvent begins to move and draws the sample components differentially
along with it.
• At the end of the development take out the paper and mark the solvent front with
another line.

14. Applications of Paper Chromatography: -
• Separation of mixtures of drugs
• To study the process of fermentation and ripening.
• To check the purity of pharmaceuticals.
• To inspect cosmetics.
• To detect the adulterant.
• To detect the contaminants in drinks and foods.
• To examine the reaction mixtures in biochemical laboratories.
• To determine dopes and drugs in humans and animals.
• Separation of carbohydrates, vitamins, antibiotics, proteins, etc.
• Analysis of metabolites of drugs in blood, urine ….

15. BIBLIOGRAPHY
• N.C.E.R.T text book
• Block C (Wikipedia.com)
• Richard J (encyclopedia Britannica)
• Durrum K (Gold Book)
• Emmett L (Slideshare.com)
• Zweig (Botshop.co.in)
• Gunter (Live.com)