AIR FORCE SCHOOL AMBALA CANTT

CHEMISTRY INVESTIGATORY PROJECT

PAPER CHROMATOGRAPHY

PREPARED BY : ASHWANI
CLASS : XII A
 Acknowledgment
I wish to express my deep gratitude and sincere thanks to the Principal, Mrs.
SUNITA SHARMA for her encouragement and for all the facilities that she
provided for this project work. I extend my hearty thanks to Mr. gurinder singh ,
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.
  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
 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.
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.
 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.
 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
 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
 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
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
 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.
 Stationary phase • Concentration of analyte
 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
 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
 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.
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.
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 ….
 BIBLIOGRAPHY

1. N.C.E.R.T text book

2. Block C (Wikipedia.com)
3. Richard J (encyclopedia Britannica)

4. Durrum K (Gold Book)

5. Emmett L (Slideshare.com)
6. Zweig (Botshop.co.in)
7. Gunter (Live.com)
Thank you