INTRODUCTION……………………………………………………….

History and Development of Chromatography………..…………….1

Types of Chromatography………………………..…………………...2

Uses of Chromatography…………………………….………………..2

Application of Chromatography……………………….……………...2

PAPER CHROMATOGRAPHY……………………………………….3

Principle of Paper Chromatography………………………………….3

Types of Paper Chromatography……………………..………………4

Modes of Paper Chromatography…………………………………….4

EXPRIMENT…………………………………..……………………......6

MATERIALS AND METHODOLOGY………………….……………..6

Aims and Objectives of the Project…………………….……………. 6

Apparatus/Equipment………………………………..………………...7

Materials………………………………………………….……………..7

Methodology…………………………………………..………………..7

Precautionary Measures……………………………………..………..8

RESULTS AND DISCUSSIONS…………………………………......8

CONCLUSION………………………………………………………..12

RECOMMENDATION………………………………………………..12

BIBLIOGRAPHY……………………………………………………...13
 ABSTRACT

The technique helps in analyzing, identifying, purifying and quantifying unknown separable
mixtures. The mobile phase is either a liquid or gas which moves the solvent through the
stationary phase during the process. The stationary phase is a liquid or solid component that is
fixed in a place for the procedure. Paper chromatography works majorly on capillary attractions.
The capillary attraction which depends on adhesive and cohesive forces allows the mobile phase
to move up the stationary phase due to created surface tension interaction from the forces. The
major types are the paper chromatography, thin layer, gas chromatography, column
chromatography, high performance liquid chromatography, paper chromatography and thin layer
chromatography. There are several applications of paper chromatography and other main types
of chromatography techniques. This technique is applicable in pharmaceutical industries,
hospitals, forensic science, environmental science and manufacturing plants. This report
describes the experiment conducted using paper chromatography to identify an unknown
mixture. This will be done by comparing four known amino acids with the two unknown
mixtures to identify the unknown mixtures. The experiment will also help to master the
technique and analyze the movements made by both unknown mixtures and the known amino
acids. Materials gloves, goggles, lab coat, filter paper, toothpick, ninhydrin solution, mixtures are
to be identified. The laboratory procedures entail different steps that eventually lead to
identification of the unknown mixtures. This procedure is divided majorly into stationary phase
preparation, mobile phase preparation and chromatograph development. For the stationary phase
preparation, the required markings are made on the paper for identification and creation of
baseline. The baseline marks are the 1.7 cm from the shorter left edge and 1.0 cm from the
bottom of longer edge. Known amino acid symbols are mark on the paper. Spotting of the known
four amino acids and two unknown mixtures are then done using separate toothpicks which will
help to prevent contamination. Mobile phase preparation was done by pouring 10 ml of solvent
mixture in a 400 ml of Berzelius beaker while the chromatography development was done after
the filter paper is already dried.
 INTRODUCTION

Chromatography involves the separation process that is due to different rate of migration
of constituent mixtures that interact in a polar or nonpolar medium. Most samples are mixtures,
which are made of two or more substances that is constantly interacting with each other.
It is a technique for separating the components, or solutes, of a mixture on the basis of the
relative amounts of each solute distributed between a moving fluid stream, called the mobile
phase, and a contiguous stationary phase. The mobile phase may be either a liquid or a gas, while
the stationary phase is either a solid or a liquid.

HISTORY ON DEVELOPMENT OF CHROMATOGRAPHY

There has being several controversies on who invented chromatography. In early 1850,
Schönbein used filter paper to partially separate substances in solution. He found that not all
solutions reach the same height when set to rise in filter paper. In 1861, Goppelsröder (in
Switzerland) found relations between the height to which a solution climbs in paper and its
chemical composition. He wrote ‘I am convinced that this method will prove to be very practical
for the rapid determination of the nature of a mixture of dyes, especially if appropriately chosen
and characterised reagents are used’. Even though both of them did valuable work towards the
progress of paper chromatography, the invention of modern chromatography is assigned to
Michael S. Tswett, shortly after 1900. Through his successive publications, one can indeed
reconstitute his thought processes, which makes of him a pioneer, even if not the inventor, of this
significant separation method. His field of research was involved with the biochemistry of
plants. At that time, one could extract chlorophyll and other pigments from houseplants,
usually from the leaves, easily with ethanol. By evaporating this solvent, there remained a
blackish extract, which could be re-dissolved in many other solvents, and in
particular in petroleum ether, (now one would say polar or non-polar solvents).
Tswett put forth the assumption that in plants chlorophyll was retained by some molecular forces
binding on the leaf substrate, thus preventing extraction by petroleum ether. He foresaw the
principle of adsorption here. After drawing this conclusion, and to test this assumption he had
the idea to dissolve the pigment extract in petroleum ether and to add filter paper (cellulose), as a
substitute for leaf tissue. He realized that paper collected the colour and that by adding ethanol to
the mixture one could re-extract these same pigments.In 1931 chromatography emerged from its
relative obscurity when the German chemist Richard Kuhn and his student, the French chemist
Edgar Lederer, reported the use of this method in the resolution of a number of biologically
important materials. In 1941 two British chemists, Archer J.P. Martin and Richard L.M. Synge
contributed greatly to the development of paper chromatography and the pioneering use of
chromatography to separate gases breakthroughs that changed the way basic research in organic
chemistry is conducted. For this work, they shared the 1952 Nobel Prize in Chemistry for
Partition Chromatography. In 1956, two Soviet Pharmacist, Nikolayy A. Izmaylov and Maria S.
Shrayber worked extensively on thin layer chromatography that could be manipulated in
similar fashion like the paper chromatography, which was reported in 1938 Soviet Studies but
was not realized until 1952. In 1944, Austrian Chemist, Erika Cremer developed Gas
Chromatography. Martin and James made the first extensive exploitation of the method in
1952, when they reported the elution gas chromatography of organic acids and amines. In
this work, small particles of support material were coated with a non-volatile liquid and packed
into a heated glass tube. Mixtures injected into the inlet of the tube and driven through by
compressed gas appeared in well-separated zones. Petroleum chemist recognized this
development as simple and rapid methods of analysis of hydrocarbon mixtures in Petroleum
Products. From 1957 until date, more researchers are developing more chromatographic
equipment while minimizing size and increasing speed of Chromatography Analysis.

TYPES OF CHROMATOGRAPHY

There are various types of Chromatography. They includes:

1.Liquid Chromatography is used in the world to test water samples to look for pollution in
lakes and rivers. It is used to analyze metal ions and organic compounds in solutions. Liquid
chromatography uses liquids which may incorporate hydrophilic, insoluble molecules.

2.Gas Chromatography is used in airports to detect bombs and is used is forensics in many
different ways. It is used to analyse fibers on a persons body and also analyze blood found at
a crime scene. In gas chromatography helium is used to move a gaseous mixture through a
column of absorbent material.

3.Thin-layer Chromatography uses an absorbent material on flat glass or plastic plates. This
is a simple and rapid method to check the purity of an organic compound. It is used to detect
pesticide or insecticide residues in food. Thin-layer chromatography is also used in forensics
to analyse the dye composition of fibres.

4.Paper Chromatography is one of the most common types of chromatography. It uses a strip
of paper as the stationary phase. Capillary action is used to pull the solvents up through the
paper and separate the solutes.

USES OF CHROMATOGRAPHY

1.It is widely used in biochemical research for the separation and identification of chemical
compounds of biological origin.

2.In the petroleum industry, the technique is employed to analyse complex mixtures of
hydrocarbons.

3.In Forensic Analysis, it is used to assess presence of prohibited substances in a Banned

Products in Food and Drugs.

APPLICATION OF CHROMATOGRAPHY

1. It is capable of separating all the components of a multicomponent chemical mixture

without requiring an extensive foreknowledge of the identity, number, or relative
amounts of the substances present.
 2. It is versatile in that it can deal with molecular species ranging in size from viruses
composed of millions of atoms to the smallest of all molecules—hydrogen—which
contains only two; furthermore,

3. It can analyse large or small amounts of material. Some forms

of chromatography can detect substances present at
the pictogram (10−12 gram) level, thus making the method a
superb trace analytical technique extensively used in the
detection of chlorinated pesticides in biological materials
and the environment,

4. It can analyse large or small amounts of material. Some forms

of chromatography can detect substances present at
the pictogram (10−12 gram) level, thus making the method a
superb trace analytical technique extensively used in the
detection of chlorinated pesticides in biological materials
and the environment,

PAPER CHROMATOGRAPHY

Paper chromatography is one of the types of chromatography procedures, which runs on a piece
of specialized paper. It is a planar chromatography system wherein a cellulose filter paper acts as
a stationary phase on which the separation of compounds occurs.

PRINCIPLE OF PAPER CHROMATOGRAPHY

The principle involved is partition chromatography wherein the substances are distributed
or partitioned between liquid phases. One phase is the Solvent, which acts as mobile medium,
which is held in the pores of the filter paper used (stationary medium); and other is the mobile
phase, which moves over the paper. The compounds in the mixture get separated due to
differences in their affinity towards Solvent (in stationary phase) and mobile phase solvents
during the movement of mobile phase under the capillary action of pores in the paper.
The principle can also be adsorption chromatography between solid and liquid phases, wherein
the stationary phase is the solid surface of paper and the liquid phase is of mobile phase.
However, most of the applications of paper chromatography work on the principle of partition
chromatography, i.e. partitioned between to liquid phases.
TYPES OF PAPER CHROMATOGRAPHY
Paper Chromatography is divided into two types:

1.Paper Partition Chromatography: In this process, chromatographic paper acts as an

inert support in which one solvent (usually water present in the cellulose of the filter paper)
acts as a stationary phase and the other solvent (usually an organic solvent) acts as a mobile
phase. Thus, the substances in a mixture move in two non-mixing solvents and partition
levels.

2.Adsorption Paper Chromatography: In this process, chromatographic paper is

impregnated with an adsorbent such alumina or silica and a single solvent is allowed to
flow over the unknown components. Thus, some of the component may be adsorbed more
strongly than the others causing in the variation for substance which tends to flow with
the solvent. This causes the separation of the components

MODES OF PAPER CHROMATOGRAPHY

Based on the way the development of chromatogram on paper there are five modes of
chromatography:

1.Ascending chromatography: As the name indicates, the chromatogram ascends. Here, the
development of paper occurs due the solvent movement or upward travel on the paper. The
solvent reservoir is at the bottom of the beaker. The paper tip with sample spots just dips into
the solvent at the bottom so that spots remain well above the solvent.
2.Descending chromatography: Here the development of paper occurs due to solvent travel
downwards on the paper. The solvent reservoir is at the top. The movement of solvent is
assisted by gravity besides the capillary action.

3.Ascending- descending mode: Here, solvent first travels upwards and then downwards on
the paper

4.Circular or Radial mode: Here the solvent travels from centre (mid-point) towards the
periphery of circular chromatography paper. The entire system is kept in a covered Petri dish
for the development of the chromatogram. The wick at the centre of paper dips into mobile
phase in a petri dish, by which the solvent drains on to the paper and moves the sample radially
to form the sample spots of different compounds as concentric rings
5.Two-dimensional chromatography: Here the chromatogram development occurs in two
directions at right angles. In this mode, the samples are spotted to one corner of rectangular
paper and allowed for first development. Then the paper is again immersed in mobile phase
at a right angle to the previous development for second chromatogram

Expriment
The aim is the Determination of colour pigments from selected flowering plants
The objectives is:
a)To separate and identify colour pigments present in flower by Ascending Paper
Chromatography.
values from the Paper Chromatogram
b)To determine the retention factor  RF

APPARATUS/ EQUIPMENT

1. Ruler
2.Stapler
3.Scissor
4.Calculator
5.Pencil
6.Whatman Filter Paper
7.Capillary tube
8.250mL beaker
9.100ml beaker
10. Flat Bottom Flask
11. Laboratory Gypsum Mortar and Pestle
MATERIALS AND METHODOLOGY

MATERIALS

1.Distilled Water
2.Solvent (Acetone, n-hexane, Ethyl Alcohol etc.)
3.Flower used (Alternanthera Sp., Acacia Sp., Hibiscus Sp.)

METHODOL
OGY

1. Preparation of Paper Chromatogram: For the sake of the Project, we choose the
Ascending Paper chromatography process. Filter Paper No 42 was collected and cut into
strips to produce paper strips with dimension of 12cm by 2cm. Pencil was used to draw 2cm line
for spotting.

2.Preparation of Solvent: Three solvent solution was utilized for separation process:
Acetone, Ethyl Acetate, N-Hexane and a separate mixture of N-Hexane and Ethyl Acetate in a
ratio of 7:3 respectively.

3. Sourcing of Flower/ Leafs: The Purple knight leaf (Alternanthera Dentata) were
collected from a garden in Imo State while Acacia flower (Acacia Mearnsii) and the Zobo/
Roselle flower (Hibiscus Sabdariffa) was sourced from Ifite, Anambra State. The name of the
flower was authenticated with the help of Chemistry Chief Technologist, Department of
Chemistry, Nnamdi Azikiwe University, Akwa, Anambra State.

4.Preparation/ Extraction of Flower: The Flower was cut into small pieces and
crushed using a laboratory mortar and pestle to produce a paste. 30 ml of Acetone Solvent was
added to extract the colour pigment from the flower or leaf. The mixture was transferred to
100ml beaker sealed with cellophane and allowed for 10 minutes to allow for extraction process.
5.Experimental Procedure: The different Plant Extract Samples was spotted twice at
centre position of drawn line of three paper strips respectively using a capillary tube. The spotted
paper was immersed in the solvent solution (Acetone, Ethyl Acetate, N-Hexane and N-Hexane-
Ethyl Acetate in 7:3 ratio) in a 250ml beaker sealed to rise on the chromatographic sheet by
capillary action. The paper chromatogram developed into various colour level, removed once it
has attained sufficient solvent height and allowed to dry under open air. The chromatogram was
observed and measured directly to reveal the location of colour pigments

PRECAUTIONARY MEASURES

1. Safety Eye Google, Hand gloves and Laboratory Coat was worn to prevent laboratory
hazards.

2. The paper chromatogram strip was not allowed to touch the wall of the Flask to prevent
interference.

3. The Paper chromatogram was allowed to separate sufficiently to its different colour
pigments.

RESULTS AND DISCUSSION

Table:4.0 Observed Value for Knight Purple (Alternanthera Sp.) Extract using Acetone Solvent

Table :4.1

Table :4.2

From Figure 4.0, the separation of Knight Purple (Alternanthera Sp.) and Acacia Sp. showed
seven colour fragmentation due to variation of colour pigment. Roselle/Zobo (Hibiscus
Sp.) showed presence of four colour fragments respectively. It implies a high-level of separation
due to affinity of non-polar colour pigment to Acetone solvent.
CONCLUSION

The study compared the efficiency of Solvent used for the paper chromatography of Knight
Purple (Alternanthera Sp.), Acacia Sp. and Roselle/Zobo (Hibiscus Sp.) as Acetone Solvent
showed better separation into various colours than other solvent solutions due to high affinity,
surface tension, and rate of adsorption by the paper. The various colour fragments shows
presence of Carotene (Yellow, Red, Purple), Xanthophyll (Brown), Chlorophyll a (Blue-Green,
Blue, Green), Chlorophyll b (Light green); and Pheopythin a, b (Gray)

RECOMMENDATION

The Paper Chromatography can be utilized in separation process. In addition, Iodine Vapour
or Ultraviolet Lamp should be used to increases the visibility of paper chromatogram, as human
eye cannot identify colour spectra correctly.

USES AND APPLICATIONS OF PAPER CHROMATOGRAPHY

1.It is used for the separation of a mixture having polar and non-polar compounds.
2.It is used to determine organic compounds, biochemicals in urine, blood etc.
3.In the pharmaceutical sector it is used for the determination of hormones, drugs, etc.
4.Sometimes, it is used for evaluation of inorganic compounds like salts and complexes.
5.To check the control of purity of pharmaceuticals,
6.For detection of adulterants,
7.Detect the contaminants in foods and drinks,
8.In the study of ripening and fermentation,
9.For the detection of drugs and dopes in animals & humans
10. In analysis of cosmetics
11. Analysis of the reaction mixtures in biochemical labs.
 Bibliography

Archer J. P. Martin (2009). Microsoft® Encarta® [DVD]. Redmond, WA: Microsoft

Corporation.

Chromatography. (2014). Encyclopædia Britannica. Encyclopædia Britannica Ultimate

Reference Suite. Chicago: Encyclopædia Britannica.

Forensic Science Activities. (2014). Using Paper Chromatography. University of Colorado

Boulder Hughes Initiative. http://www.colorado.edu/Research/hughes/web.forensics.html.
Retrieved 01 August, 2019

Mahadev kammar. (2017) Types of Paper Chromatography https://www.

slideshare.net/MahadevKammar/types-of-paper-chromatography . Linkedin Slide-share.
Retrieved 01 August, 2019

Nedungaadi P., Raman R., & McGregor M. (2013). Enhanced STEM learning with Online
Labs: Empirical study comparing physical labs, tablets and desktops. In Frontiers in
Education Conference. IEEE. Pp 1585-1590.

Paper Chromatgraphy (2008). https://www.slideshare.net/shaisejacob/paper-

chromatography-ppt-new Linkedin Slide Share. Retrieved 01 August, 2019

Paper Chromatography (2009). Microsoft® Encarta® [DVD]. Redmond, WA: Microsoft

Corporation.

Paper Chromatography (2019). https://byjus.com/chemistry/paper-chromatography/ BYJU'S.

Retrieved 01 August, 2019

Paper Chromatography Report (2017). http://www.biologyjunction.com/paper

chromatography_report.htm Biology Junction. Retrieved 01 August, 2019

Paper Chromatography. http://namrataheda.blogspot.in/search/label/types%20of%20paper

%20chromatography. Retrieved 01 August, 2019

Paper Chromatography. https://www.biochemden.com/paper-chromatography/. Retrieved 01

August, 2019

Paper Chromatogrpahy (2013). http://amrita.olabs.edu.in. Retrieved 01 August, 2019

Sagar Aryal (2018). Paper Chromatography. https://microbenotes.com/paper-

chromatography/ Microbiology Notes. Retrieved 01 August, 2019

Separate Colors in a Green Leaf Using Chromatography. http://photoscience.la.asu.edu/

photosyn/education/colorchange.html. Retrieved 01 August, 2019