CHROMATOGRAPHY

A Project submitted to

Kendrapara Autonomous College , Kendrapara

In the partial fulfilment for the award of Degree of
BACHELOR OF SCIENCE IN CHEMISTRY

Submitted By

SHUBHASMITA ROUT
ROLL NO :- BS22-243
Regd No :
UndeR THe GUidAnCe Of

[Link] Parida

2022-2025
DEPARTMENT OF CHEMISTRY
KendRAPARA AUTOnOMOUS COLLeGe
KendRAPARA , OdiSHA, 754211
 DECLARATION

I hereby declare that investigatory project entitled

“CHROMATOGRAPHY” has been carried out by own
efforts and fact arrived at my observation under the guidance
and motivation of subject teacher Mrs. Nirupama Parida,
lecturer in the Department of Chemistry.

PLACE – KENDRAPARA SHUBHASMITA ROUT

DATE - ROLL NO. – BS22-243

2
 CERTIFICATE

This is to certify that the project entitled “Chromatography” is done

by Shubhasmita Rout, 6th semester student of [Link]. degree in
chemistry under my direct supervision and guidance during the
academic year 2024-2025, as partial fulfilment of [Link]. degree in
chemistry from Kendrapara Autonomous College. On the basis of
declaration made by her I recommend the project report for
evaluation.

Mrs. Nirupama Parida

Department Of Chemistry
Kendrapara Autonomous College

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 ACKNOWLEDGEMENT

I would like to thank my supervisor Mrs. Nirupama Parida lecturer

in chemistry for her enthusiasm, wise words and continuing support
throughout the research. I would also like to thank the Head of the
department Dr. Girija Prasad Mishra for his support in
completing my project. I am also thankful to my friends for providing
their wholehearted support and cooperation in completing the
project.

Last but not the least I feel very fortunate and thankful to my college
for giving me this wonderful opportunity to work on such project and
do the research.

SHUBHASMITA ROUT
Roll No. : BS22-243

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Contents. Page No.

Introduction……………………………………………………………………..6
Historical Development………………………………………………………...7
Basic Principles of Chromatography………………………………………….8
Basic Terminology……………………………………………………………...10
Classification of Chromatography…………………………………………….11
Chromatography techniques on the basis of interaction mechanism……….12
Chromatography techniques on the basis of state of mobile phase…………16
Principle, process, instrumentation, advantage and disadvantage of
Thin layer Chromatography……………………………………………18
Paper Chromatography………………………………………………....24
Column Chromatography……………………………………………....29
Applications of Chromatography in Different Fields………………………..33
Conclusion……………………………………………………………………...35
Reference…………………………………………………………………….....36

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Introduction
Chromatography is a nondestructive technique for separating mixtures into their components in
order to analyze, identify, purify or quantify the mixture or components. So it can be defined as
“A technique by which a mixture is separated into its components on the basis of relative ability
of each component to be move along a stationary phase by mobile phase”. The technique of
chromatography is based on the differences in the rate at which the components of a mixture
move through a porous medium (stationary phase) under the influence of some solvent or gas
(mobile phase). The term chromatography is derived from two Greek word ―chroma meaning
color and ―grapho meaning to write or record. The stationary phase can be solid or liquid and
the mobile phase can be liquid, gas or a supercritical fluid.

Chromatography operates on the basis of differential migration of the components in the mixture due
to their differing interactions with the stationary phase and mobile phase. Components that interact
slowly with the stationary phase will move slower, while those that have a stronger attraction to the
mobile phase will move faster. This difference in movement causes the components to be separated
over time, allowing each to be identified and quantified. As a result, chromatography is an essential
tool in chemical analysis, quality control and environmental monitoring.

One of the primary advantages of chromatography is its versatility. It can be used for a wide range
of applications, including the purification of components, the analysis of complex mixture and the
identification of unknown substances. It is a very effective separation technique in pharmaceutical
industry, food and beverage industry, forensic science, biochemical researches etc. It is a very useful
separation method for both micro and macromolecular components in a mixture. Various types
of chromatography methods is used to separate complex compounds like amino acids, inorganic
polymers etc.

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By examining chromatography in detail, this project aims to offer a deeper understanding of some
essential chromatographic methods, its history, principle, instrumentation and
application including the factors that influence separation efficiency.

Historical Development
The method was developed by botanist Mikhail Tsvet in 1901- 1905 in universities of Kazan and
Warsaw. He developed the technique and coined the term chromatography in the first decade of
the 20th century, primarily for the separation of plant pigments such as chlorophyll, carotenes
and xanthophylls. Since these components separates in bands of different colors (green, orange
and yellow respectively) they directly inspired the name of the technique. Color (in Greek
chroma) and to write or record (in Greek grapho), hence the name chromatography was derived.
New types of chromatography developed during the 1930s and 1940s made the technique useful
for many separation process.
In the 1940s, Richard Synge and Archibald Martin further advanced
the technique by developing partition chromatography. This method involved using a liquid
stationary phase and a mobile phase for separation. It was crucial for separating complex mixtures,
such as amino acids and other biochemicals. For this Synge and Martin were awarded the Noble prize
in chemistry in 1952.
In 1950s , A.J.P Martin and R.L Smith further developed the technique of gas chromatography, which
involves a gaseous mobile phase. This method was particularly useful for analyzing the volatile
compounds, leading to its widespread use in environmental analysis, pharmaceuticals industries.
In 1960s the development of high performance liquid chromatography marked a significant step
forward in chromatography. This innovation allowed for more efficient separation and quantification
of complex mixtures, especially in fields like biotechnology and pharmaceuticals.

Continuous evolution of chromatography developed modern chromatographic techniques such as ion

exchange chromatography, affinity chromatography, and other various detectors like mass
spectrometer that are often coupled with chromatography methods. A new field was originated which
was supercritical fluid chromatography. Supercritical fluid chromatography is a hybrid of gas and
liquid chromatography and cost advantageous feature of both gas and liquid chromatography. It will
not be wrong to say that the entire twentieth-century can be named as the century of chromatography.

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Basic Principles Of Chromatography
Chromatography is an important biophysical technique that enables the separation, identification
and purification of the components of a mixture for qualitative and quantitative analysis. The
mixture is dissolved in a fluid called the mobile phase, which carries it through a structure
holding another material called the stationary phase. The various constituents of the mixture
travel at different speeds, causing them to separate. The separation is based on the differential
portioning between the mobile and stationary phases. This separation depends upon the specific
properties of the substance involved, such as their polarity, size or charge. The two phases are
immiscible with each other. When a solute in a solvent (mobile phase) is passed through or
around the outside of a matrix (stationary phase), interactions occur between the solute and
stationary phase. Solutes with different properties are separated based on differences in these
interactions. The basic principles behind the chromatography are discussed below-

1. Separation principle

The heart of chromatography lies in the principles of partitioning (or differential migration).
Chromatography works by exploiting the differences in how individual components of a mixture
interact with two phase: the stationary phase and the mobile phase.

(a) Stationary phase :

This is the phase that remains fixed in place during the chromatography process. It could be a
solid or a viscous liquid. The stationary phase interacts with the components of the mixture,
causing them to move at different rates. Stationary phase is non moving material that the mobile
phase passes through, facilitating the separation of a mixture based on the different affinities of
its components for the stationary phase. It provides a surface or medium for the components of
a mixture to interact with, allowing for separation based on their interactions with the stationary
phase. The stationary phase can be solid or liquid. Examples of solid stationary phase include silica
gel, alumina, or other porous solids. Liquid stationary phase include a layer of liquid coated on a
solid support.

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(b) Mobile phase :

The mobile phase is a liquid or gas that moves through or over the stationary phase. It carries the
components of mixture with it, but each component moves at a different rate, leading to separation.
Common mobile phases in liquid chromatography include water, methanol, acetonitrile and various
mixture of these solvents. In gas chromatography, common mobile phases are inert gases like helium,
argon or nitrogen. The choice of mobile phase is crucial for achieving effective separation, as it
influences the interaction between the sample components and the stationary phase.

2. Interaction mechanism
Chromatography relies on several key interaction mechanisms that govern the behaviour of
substances as they move through the stationary and mobile phases.
These mechanisms include:
Partition
In partition mechanism, substances are separated based on their distribution between two immiscible
phases: the stationary phase and the mobile phase. A solute in the mobile phase distributes itself
between the two phases based on its affinity for each phase.
A common example of chromatography based on partition principle is liquid-liquid , where a liquid
stationary phase interacts with the mobile phase and compound partition themselves between the two
phases based on their solubility.
Adsorption
In adsorption mechanism the separation occurs based on the differing degrees to which components
in a mixture adsorb to the stationary phase. The stationary phase, often a solid like silica gel or
alumina, provides surface sites for adsorption. More strongly adsorbed compounds will move slowly
through the column, while less adsorbed compounds will move faster. This principle is used in
techniques like thin layer chromatography and column chromatography.
Ion Exchange
Ion exchange principles involve the reversible exchange of ions between the stationary phase and the
mobile phase. This technique is particularly useful for separating ions based on their charge. In ion
exchange chromatography, ions from the sample mixture are exchanged with ions that are attached
to the stationary phase. The rate at which ions are exchanged depends on factors such as the ion‘s
charge, size, and affinity for the resin.

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Size Exclusion
According to this principle, separation occurs based on the size of the molecules in the sample. A
porous stationary phase allows smaller molecules to enters its pores, which causes them to travel
more slowly through the system. Larger molecules are excluded from the pores and therefore, travel
more quickly. This technique is widely used for the separation of proteins, nucleic acids and
polymers.

Basic Terminology
Besides these principles, there are some other chromatographic terms which are essential to
understand to proceed the chromatographic technique and identify the components. Such terms are :

Solute –the sample components in chromatography

Solvent – any substance capable of solubilizing another substances, and especially the liquid mobile
phase in liquid chromatography.
Analyte – the substances to be separated during chromatography i.e the components which is needed
from the mixture.
Eluent – is the solvent or mixture of solvents (mobile phase) used to carry the sample through the
column and separate its components.

Eluate – is the mixture of solvent or carrier gas and the analytes.

Eluotropic series – a list of solvents ranked according to their eluting power. Solvents with higher
eluting power can displace more strongly adsorbed analytes from the stationary phase, allowing them
to move through the column more quickly.
Retention Time – the characteristic time it takes for a particular analyte to pass through the system
under set conditions.
Detector – the instrument used for qualitative and quantitative detection of analytes after separation.
Chromatogram – the visual output of the chromatograph. In the case of an optimal separation,
different peaks or patterns on the chromatogram correspond to different components of the separated
mixture.

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Chromatograph – an instrument that enables a sophisticated separation, e.g. gas chromatographic
or liquid chromatographic separation.

Retention Factor - is also known as retardation factor, is a crucial parameter to measure how far
a compound travels relative to the solvent front, calculated as the distance travelled by the
compound divided by the distance travelled by the solvent front. A higher Rf value means
compound interacts less strongly and lower Rf value means compound interacts more strongly.
Rf values are always between 0 and 1.

Rf = (distance travelled by the sample) / (distance travelled by the solvent)

Classification of Chromatographic Techniques

Classifying chromatography techniques is crucial for understanding their principles, selecting
the appropriate method for a specific separation, and interpreting results, as different types rely
on varying mechanisms and stationary or mobile phase. The choice of chromatography method
depends on the nature of the sample, the desired separation and the available equipment.
Classification helps us identify the most appropriate technique for a specific application. A
standardized classification system helps in better understanding of all the techniques involved.

Mainly classification of chromatography techniques is based on three different parameters as

discussed below.

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So on the basis of how solute interact with the stationary phase , chromatography is divided into four
types. They are -

(a) Adsorption Chromatography

It is the oldest type of chromatographic method. Actually Tsvet’s work was kind of adsorption. The
stationary phase in adsorption chromatography is called ―adsorbent‖. In this chromatography,
separation of a components of a mixture takes place as the adsorption efficiency of the sample. The
most strongly adsorbed component forms the topmost band. The least adsorbed components forms
the lowermost band on the adsorbent column. Degree of separation depends upon the separation of
surface area of adsorbent. There are number of adsorbents commercially used.

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The most commonly used adsorbents are – powdered charcoal, silica gel, alumina oxide etc.
Examples of this chromatography include column chromatography, thin layer chromatography, gas
solid chromatography.

(b) Partition Chromatography

Partition chromatography is process of separation whereby the components of the mixture get
distributed into two liquid phases due to differences in partition coefficient during the flow of
mobile phase in the chromatographic column. This mode of partition chromatography applies to
liquid-liquid , liquid-gas chromatography. Because partition is the phenomenon in between a
liquid and liquid or liquid and gas or gas and gas. But not in solid involvement. The stationary
phase immobilizes the liquid surface layer, which becomes stationary phase. Mobile phase passes
over the coated adsorbent and depending upon relative solubility in the coated liquid, separation
occurs. The component of sample mixture appear separated because of differences in their
partition coefficient. Examples of this chromatography include liquid-liquid chromatography, gas-
liquid chromatography etc.

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(c) Ion Exchange Chromatography
Ion exchange chromatography is the separation technique for charged molecules by their interaction
with the oppositely charged stationary phase in the form of ion exchange resin. This chromatography
based on the isoelectrostatic interaction between charged protein groups, and solid support
materials(matrix). Matrix has an ion load opposite to that of protein to the column rated and the
affinity of the protein to the column achieved with ionic ties. Proteins are separated from the column
either by changing pH concentration of the ion salts or ionic strengths of the buffer solution.

Ion exchange chromatography is of 2 types – cation exchange chromatography and anion

exchange chromatography. In cation exchange chromatography, solute cations are attached to
the negatively charged sites covalently bond to the stationary phase. In anion exchange
chromatography, solute anions are attached to the positively charged sites covalently bond to the
stationary phase.

(d)Size Exclusion Chromatography

Size exclusion chromatography (SEC) is a chromatography method in which molecule in a

solution are separated by their size, and in some cases molecular weight. It is usually applied to
large molecules or molecular complexes such as proteins and industrial polymers. Typically,
when an aqueous solution is used to transport the sample through the column, the technique is

14
known as gel filtration chromatography, versus the name gel permeation chromatography,
which is used when an organic solvent is used as a mobile phase. A mixture of molecules
dissolved in liquid (mobile phase) is applied to a chromatography column which contains a solid
support in the form of microscopic spheres, or ―beads‖ (stationary phase).

The mass of beads within the column is often referred to as the column bed. The beads act as
―traps" or ―sieves" and function to filter small molecules which become temporarily trapped
within the pores. Larger molecules are excluded from the beads. Large sample molecules cannot
or can only partially penetrate the pores, whereas smaller molecules can access most or all pores.
Thus, large molecules elute first, smaller molecules elute later, while molecules that can access
all the pores elute last from the column. Particles of different sizes will elute through a stationary
phase at different rates.

On the basis of physical state of mobile phase, chromatography is divided into three types: liquid
chromatography, gas chromatography, and super critical fluid chromatography.

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(a) Liquid Chromatography
Liquid chromatography(LC) is used to separate the components of a mixture using a liquid
mobile phase that flows through a stationary phase. It separates molecules based on their
differing affinities for the stationary phase (a solid or a liquid adsorbed on a solid) versus the
mobile phase (a liquid solvent). A sample containing the mixture to be separated is injected
into the LC system. The mobile phase carries the sample through a column packed with the
stationary phase. As the sample flows through the column, different components interact with
the stationary phase to varying degrees. Components that interact more strongly with the
stationary phase will move more slowly through the column, while those that interact less
strongly will move faster. As the components elute the column, they are detected and
quantified.
Liquid chromatography is of four types :
Normal phase chromatography : In normal phase chromatography, the stationary phase is
more polar, and so the more polar solutes being separated more to the stationary adsorbent
phase.
Reverse phase chromatography : In this chromatography, the polarities of the mobile and
stationary phases are opposite to what they are performing in normal phase chromatography.
Ion exchange chromatography : In this technique molecules are separated on the basis of
their charge.
Size exclusion chromatography : in this chromatography molecules are separated on the
basis of charge.

Fig : schematic diagram of liquid chromatography

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(b) Gas Chromatography

Gas chromatography is a chromatographic technique of separating a volatile or gaseous

substances by using gas as a mobile phase. Presence of components like carbon monoxide,
hydrocarbon etc. in very small concentration can be detected by gas chromatography. In this
technique sample is converted to vapour state and injected on stationary phase of
chromatography, inert gas can act as mobile phase. When stationary phase is non volatile
liquid coated on inner support and the mobile phase is gas, then the technique is called as gas
liquid chromatography. GLC is a partition type of chromatography. When stationary phase
is solid of a large surface area and the mobile phase is gas, then the technique is called as gas
solid chromatography. Silica gel, alumina, charcoal etc. are used as a stationary phase.

Fig : schematic diagram of gas chromatography

(c) Super critical Fluid Chromatography

Super critical fluid chromatography (SFC) is a chromatographic technique in which the mobile
phase is a super critical fluid. The use of a supercritical mobile phase in chromatography was
first proposed in 1958 by [Link]. SFC is of importance because it permits the separation
and determination of the group of compounds that are not conveniently handled by either GC
or LC. These compounds are either nonvolatile or thermally labile so that the GC are in-
applicable, and contain no functional groups that make impossible detection by spectroscopic
or electrochemical techniques employed in LC. It can be used with the universal flame
ionization detector. It is important to producing narrower peaks due to rapid diffusion.
Supercritical fluids are suitable as a substitute for organic solvents in a range of industrial and
laboratory processes

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 Fig : schematic diagram of super critical fluid chromatography

On the basis of chromatographic bed shape, it is divided into 2 types – two dimensional and three
dimensional. Two dimensional chromatography includes two major chromatographic techniques
are Thin layer chromatography (TLC) and Paper chromatography (PC). Three dimensional
chromatography include Column chromatography. Detailed study of these three chromatographic
techniques are discussed below including their principle, instrumentation, procedure,
applications, advantage and disadvantages.

[Link] Layer Chromatography

Introduction
Thin Layer Chromatography (TLC) is a type of chromatography which is based upon the
distribution of biomolecules between two immiscible phases. TLC was originally developed to
separate lipid molecules and can be used to identify components in a sample, and for preparative
purposes. In TLC the stationary phase is a polar absorbent, like finely ground alumina (Al2O3)
or silica (SiO2) particles which are coated on a glass slide or plastic sheet to create a thin layer

18
of the particular stationary phase. Silica contains some free – OH groups which form hydrogen
bonds or other Van-der-Waals interactions with the analyte components and as a result adsorption
takes place. Sometimes a small amount of a binder such as plaster of Paris is mixed with the
absorbent to facilitate the coating.

Principle
The mixture to be separated is dissolved in a solvent and the solution is spotted at one end of the
coated TLC plate next to the reference material. In order to determine whether an unknown
substance is the same as a substance of known structure, it is necessary to run the two substances
side by side in the same chromatogram, preferably at the same concentration. The plate is placed
with spotted end down in a covered jar containing a shallow layer of suitable solvent. The solvent
(mobile phase) is allowed to move up the plate by capillary action through the adsorbent at its
own rate and as a result differential partitioning occurs between the components of the mixture
dissolved in the solvent and the stationary adsorbent phase. The more strongly a given
component of a mixture is adsorbed onto the stationary phase, the less time it will spend in the
mobile phase and the more slowly it will migrate up the plate. When the solvent front has moved
to within about 1 cm of the top end of the adsorbent, the plate should be removed from the
developing chamber.

If the components of the sample are coloured, they can be

observed directly. If not, they can sometimes be visualized by shining ultraviolet light on the
plate or by spraying the plate with a reagent (e.g. ninhydrin) that will react with one or more of
the components of the sample. Sometimes the spots can be visualized by allowing the plate to
stand for a few minutes in a closed container in which the atmosphere is saturated with iodine
vapour. The ninhydrin reaction is used to detect the presence of amino acids. Amino acids contain
a free amino and carboxyl group which reacts together with ninhydrin to produce a characteristic
blue colour (or occasionally pale yellow). In this reaction first an amino group is attached to the
first or alpha carbon of the amino acid‘s carbon chain and then the nitrogen atom of the amino
group reacts with ninhydrin to give a blue-purple product known as Ruhemann‘s purple as shown
in figure .

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In addition to qualitative results, TLC can also provide chromatographic separation of
biomolecules because the distance travelled by a substance relative to the distance travelled by
the solvent front depends upon the molecular structure of the substance. The relationship between
the distance travelled by the solvent front and the substance is usually expressed as the Rf value
which is also called “retardation factor” and expressed as following:

Rf = distance travelled by substance/ distance travelled by solvent front

The Rf values strongly depend upon the nature of the adsorbent and solvent. Therefore, for the
separation and subsequent identification of amino acids in a given mixture, Rf values of
individual amino acids have to be calculated by performing Thin Layer Chromatography.

Instrumentation Of TLC
Instruments used in TLC are : stationary phase, mobile phase, TLC plate, filter paper,
micropipette, developing chamber, analyte, detecting agents.

Stationary phase –

It is the phase which mobile phase passes over. The stationary phase is made of coated with
adsorbents. This is used to provide facility for the adsorption of analyte on TLC film. Some
commercially used adsorbents are silica gel, alumina, cellulose powder etc.

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Mobile phase

Mobile phase is a solvent or solvent mixture which moves on the stationary phase. The solvent
travels through the stationary phase by the capillary action. The ability of mobile phase to move
up is depend on the polarity itself. Commonly used mobile phase are ethyl acetate, hexane, water
etc.

Preparation of TLC plate

These are stable and chemically inert plates, where a thin layer of stationary phase is applied on
its whole surface layer. The stationary phase on the plates is of uniform thickness and is in a fine
particle size. The mixture is spreaded on an unreactive carrier sheet, usually glass, thick
aluminium or plastic. The resultant plate is dried and activated by heating in an oven for 30
minutes.

Developing chamber

This is used for the development of TLC plate. The chamber maintains a uniform environment
inside for proper development of spots. It also prevents the evaporation of solvents, and keeps
the process dust free. To equilibrate the atmosphere of empty space in chamber, it is kept closed
with lid with the mobile solvent. Filter paper has to be placed inside the chamber. After placing
the plate in the chamber, close it with lid. Use of forceps is a must for handling the plate. The
spotting area should not be immersed in mobile phase.

Detecting agents

For colorless solutes, visualization or detection techniques are required such as :

Non-specific : UV chamber for fluorescent compounds, iodine chamber method.

Specific : Ninhydrin, bromocresol green, iodine vapour, aniline phthalate etc. are used.

Let‘s understand the technique by separating amino acids mixture by following the procedure
given below.

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Procedure
1. Take a TLC plate and with the help of pencil draw two straight lines on the white surface
of the plate: one 2 cm from the bottom of the plate and another 1 cm from top of the plate.
Never use a pen as dyes (used in ink) may interfere with the results by developing spots
on the plate.
2. Mark 10 equidistant points on the bottom line for loading of amino acids samples and test
sample. While marking the lines and points do not make a trough with the pencil.
3. Allow all the amino acid samples and test sample to come to room temperature. Then
spot 1 μl of each amino acid and test sample along the bottom line on the TLC plate.
While spotting use separate tips for each sample.
4. Allow the plates to air dry (~ 10 minutes). Further drying should be done by keeping the
TLC plate at 70⁰C in a hot air oven or incubator for 2 – 3 minutes.
5. Take 10 ml of solvent system in the TLC chamber (with lid) and keep for 10 minutes at
room temperature.
6. Place the TLC plate inside the chamber with clean forceps. While keeping the plate make
sure that the spotted samples are near the solvent. Furthermore, the TLC plate should be
in a straight position so that the solvent phase can move uniformly along the plate.
7. Allow the solvent front to reach the top line of the plate. After that take it out with the
help of clean forceps and air dry the plate for 15 – 20 minutes. Keep the plates at 70⁰C
for 2 minutes for further drying.
8. Add 1 ml of the Developing Reagent on the plate and swirl the plate very carefully. Look
for the development of the coloured spots of different amino acids and the test sample.

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Applications
i. TLC is extremely useful in biochemical analysis such as separation or isolation
of biochemical metabolites from its blood plasma, urine, body fluids etc.
ii. TLC can be used to identify natural products like essential oils or volatile oil,
waxes, glycosides, alkaloids etc.
iii. It is widely used in separating multicomponent pharmaceutical formulations.
iv. It is used in food industry, to separate and identify colours, sweetening agent
and preservatives.
v. It is used in cosmetic industry. Etc.

Advantages And Disadvantages

i. It is a simple method of component separation.
ii. Fewer types of equipment are used in this technique. As the components elute rapidly, the
separation is achieved in a very short time.
iii. It is possible to visualize all elements of UV light.
iv. By this process, non volatile compounds can be isolate.
v. Easy isolation and recovery of the components of the compound mixture.

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Some limitations of this technique includes –

i. TLC plates do not have longer stationary phase.

ii. The results generate from TLC are difficult to reproduce.

iii. It is only a qualitative analysis technique and not quantitative.

So, TLC is a valuable, quick and cost effective separation method. It provides a simple way to
assess the purity and identify the compounds.

[Link] Chromatography

Introduction
Paper chromatography is a fundamental analytical technique for separating and identifying
components in mixtures. It separates substances based on their interactions with stationary and
mobile phases by utilizing partitioning or adsorption mechanisms. The technique is valued for
its affordability, simplicity, and minimal sample requirements, making it suitable for applications
such as amino acid analysis, plant pigment identification, and pharmaceutical testing. This
technique uses a specialized paper as the stationary phase to separate components based on their
differential affinities for the paper and the solvent, or mobile phase, that moves through it. The
paper acts as a porous medium where substances partition according to their relative solubility
and interaction with the paper. It is extensively employed to analyze and separate amino acids,
metal ions, and other organic and inorganic compounds. Overall, paper chromatography remains
a crucial tool in analytical chemistry, continually evolving to meet modern scientific and
industrial needs.

Principle
The principle underlying paper chromatography is partitioning; wherein different components of
a mixture are distributed between two liquid phases. In this technique, the filter paper serves as
the stationary phase, holding an aqueous solvent within its pores, while the mobile phase moves
over the paper. This setup enables the separation of chemicals in the mixture based on their

24
varying affinities for the solvents in the mobile phase and water in the stationary phase. The
separation process occurs primarily due to capillary action within the paper's pores, which causes
the components to migrate at different rates depending on their solubility and interaction with
the solvents. Alternatively, separation can also be achieved through adsorption, where the paper‘s
solid surface acts as the stationary phase and the liquid solvent as the mobile phase. Although
partitioning is the main principle, adsorption may also play a role, especially in specific
applications. After the separation the components are identified by their respective Rf values.

Instrumentation
In paper chromatography following apparatus are used:

Chromatography paper – it act as the stationary phase. It is usually made up of cellulose (e.g.
whatman filter paper)

Mobile phase – a liquid solvent or mixture that moves through the paper, carrying the
components of the sample within it.
Sample applicator – a capillary tube, micropipette, or syringe is used to spot the sample on the
chromatography paper.

Developing chamber – a closed container, that holds the solvent and allow solvent saturation,
which improve the separation.

Drying apparatus – an air dryer or oven to dry the chromatogram after development.

Procedure
The procedure for performing paper chromatography involves several key steps to ensure
effective separation and analysis of sample components. Here is the detailed steps :

1. Choosing the Development Type The selection of the development type depends on
various factors such as the composition of the sample, the complexity of the solvent
system, and the type of paper used. Radial chromatography is often preferred for its
excellent resolution, ease of use, and reproducibility. It also requires less time and is
straightforward to execute.

25
2. Selecting the Filter Paper The choice of filter paper is influenced by the quality of the
sample and the pore size of the paper. Generally, thin-layer filter papers like Whatman
No. 1 are used, which provide adequate resolution for most analyses. The paper‘s
characteristics should be compatible with the sample and solvent system.
3. Preparing the Sample The sample must be dissolved in a suitable solvent that does not
react with the analytes. The solvent, often referred to as the mobile phase, should be
chosen based on its ability to dissolve the sample and its compatibility with the stationary
phase.
4. Applying the Sample to the Paper The sample is carefully applied to the paper using a
capillary tube. It is crucial to place the sample accurately and consistently in a small spot
or line at the appropriate location on the paper, usually near one edge or in the center
depending on the chromatographic method used.
5. Developing the Chromatogram The paper is then submerged in the mobile phase, which
moves through the paper by capillary action. The components of the sample will migrate
at different rates depending on their affinity for the mobile phase and stationary phase,
forming a chromatogram.
6. Detecting and Drying the Components After development, the paper is initially dried at
room temperature to remove any residual solvent. It is then further dried using an air
dryer to ensure complete evaporation. The separated components are detected using
appropriate detection reagents or visualization techniques.
Ninhydrin is usually spread in case of amino acid mixture.

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Modes Of Paper Chromatography
Paper chromatography can be classified based on the method used to develop the chromatogram.
The primary modes are as follows:

1. Ascending Chromatography -In this method, a piece of paper with a sample spot is dipped
into a solvent reservoir at the bottom of the beaker. The solvent rises by capillary action,
carrying the sample components with it. It is important to ensure that the sample area remains
above the solvent level and to use an appropriately sized paper to prevent bending and
crumpling.
2. Descending Chromatography - In this method, the solvent moves down the paper due to
gravity. The solvent reservoir is placed at the top, allowing gravity to facilitate the solvent
flow. This method is advantageous for separating solutes with low Rf values and generally
requires less time to complete compared to ascending chromatography.
3. Ascending-Descending Chromatography: This technique involves the solvent moving
both upward and downward on the same sheet of paper. It combines the advantages of both
ascending and descending methods, including reduced operation time, the ability to separate
components with higher Rf values, and a longer flow distance for improved resolution.

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Application
i. Due to its simplicity, it is used in student‘s labrotory for simple analysis.
ii. It is used for the separation of mixture having polar and non polar compounds.
iii. It is commonly used for separation of amino acids, monosaccharides, other organic
compounds.
iv. It is used for separation of different plant pigments.

Advantages and disadvantages

i. Minimal Sample Requirements: Paper chromatography requires only a small amount of
sample, making it suitable for limited quantities of material.
ii. Cost-Effective|: Compared to other chromatographic techniques, paper chromatography
is relatively inexpensive, both in terms of equipment and consumables.
iii. Identification of Unknown Substances: The technique is effective for identifying
unknown chemical and inorganic substances, providing qualitative information about the
sample components.
iv. Space Efficiency: Paper chromatography requires less space than many other analytical
techniques or instruments, making it suitable for use in limited laboratory settings.

Limitations include –

i. Limited Sample Capacity: Paper chromatography is not suitable for handling large
quantities of samples, which can limit its use in certain applications.
ii. Quantitative Analysis Limitations: The technique is less effective for quantitative
analysis compared to more advanced methods such as High-Performance Thin Layer
Chromatography (HPTLC) or High-Performance Liquid Chromatography (HPLC).
iii. Reduced Accuracy: Paper chromatography is generally less accurate than HPTLC or
HPLC, particularly in terms of resolution and reproducibility.

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[Link] Chromatography
Column chromatography was developed by the American chemist D.T Day in 1900, M.S Tswett,
the polish botanist, in 1906 used adsorption columns in his investigations of plant pigments. It is
one of the most useful methods for the separation and purification of both solids and liquids.
This is a solid-liquid technique in which the stationary phase is a solid and mobile phase is a
liquid. It is used to separate a single chemical compound from a mixture dissolved in a fluid. It
separates substances based on differential adsorption of compounds to the adsorbent as the
compound move through the column at different rates which allow them to get separated. This
technique can be used on small scale as well as large scale to purify materials that can be used
in future experiments.

Principle
When the mobile phase along with the mixture that needs to be separated is introduced from the
top of the column, the movement of the individual components is at different rates. The
compounds with lower adsorption and affinity to stationary phase travel faster when compared
to the greater adsorption and affinity with the stationary phase. The components that move fast
are removed first whereas the components which move slow are eluted out last. The adsorption
of solute molecules to the column occurs in a reversible manner. The rate of the movement of
the components is expressed as :

Rf = (distance travelled by the solute) / (distance travelled by the solvent)

In column chromatography the two phases involved are

Mobile phase : The phase is made up of solvents. It act as a solvent – sample mixture can be
introduced in the column. It act as a developing agent – helps in the separation of components in the
sample to form bands. It act as an eluting agent – the components that are separated during the
experiment are removed from the column.

Stationary phase : It is a solid material which should have good adsorption property. Particles should
have uniform shape and size in the range of 60 – 200μm in diameter. Particles should be high
mechanically stable and chemically inert. It should not react with acids or base or any other solvents

29
used during the experiment. It should be colourless, inexpensive and readily available. It should allow
free flow of mobile phase. It should be suitable for the separation of mixtures of various compounds.

Instrumentation Of Column Chromatography

A typical column chromatographic system using a gas or liquid mobile phase consist of the following
components :

A stationary phase :- solid materials inside the column that interacts with the sample.
Commonly used materials include silica gel(polar) or reverse phase silica(non polar).

Mobile phase :- A liquid that moves through the column. It is chosen to complement the
stationary phase and hence to discriminate between the sample analytes and deliver a constant
rate of flow into the column.

Column :- the main component of the setup, typically a glass or metal tube. They filled with the
stationary phase.

Sample injector :- The sample is introduced into the column, either as a solution or directly
loaded onto the stationary phase.

Elution system :- A reservoir containing the mobile phase, usually connected to a pump for
controlled flow. Gravity or pressure driven elution method are used.

Detector :- In preparative chromatography fractions are collected manually. In analytical

chromatography, a detector (such as UV-Vis, refractive index, or mass spectrometry) monitors
compound elution.

Fraction collector :- Collects different separated fractions of the sample as they elute from the
column.

Procedure

Preparation of the column

1. The column mostly consists of a glass tube packed with a suitable stationary phase.

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2. A glass wool / cotton wool or an asbestos pad is placed at the bottom of the column before
packing with the stationary phase.
3. After packing, a paper disc kept on the top, so that the stationary layer is not disturbed by the
introduction of sample or mobile phase.
4. Before using column, it should be washed properly and dried.
5. The column should also be free from impurity and uniformly filled with the stationary phase.
There are two methods of preparing the column, they are –
(a) Dry packing : In this the required quantity of adsorbent is poured as fine dry powder in
the column and the solvent allowed to flow through the column till equilibrium is reached.
(b) Wet packing : In this, the slurry of adsorbent with the mobile phase is prepared and is
poured into the column. It considered as the ideal technique for packing. Stationary phase
settles uniformly and no crack in the column of adsorbent.

Introduction Of The Sample

1. The sample which is usually a mixture of components is dissolved in minimum quantity of

the mobile phase.
2. The entire sample is introduced into the column at once and get adsorbed on the top portion
of the column.
3. Then the solvent or a suitable solvent mixture is added at first touching the side of the glass
column slowly and carefully so that the top level of the stationary phase is not disturbed.
The solvent is repeatedly added as many times as needed throughout the process.
4. When the tap, is on the compounds in the compound mixture move along with the eluent
depending upon the polarity of the sample. The non polar components travel faster than the
polar components.

Elution Of The Sample Components

Continuous flow of the elutant through the packed column separates the components in the
sample. This is column development. There are two techniques of column development (a)
isocratic elution and (b) gradient elution. In isocratic elution only a single solvent is used for
the column development but in gradient elution the composition of the mobile phase is changed

31
giving rise to gradient. So gradient elution is a better approach as it gives good resolution
comparison to single elutant.

Detection Of Components

if the compounds separated in column chromatography are coloured, the progress of the
separation can simply be monitored visually. If the compounds are colourless, the small fractions
of the eluent are collected sequentically in labeled tubes and the composition of each fraction is
analyzed by TLC.

Suppose if a compound mixture contains three compounds blue, red and

green. According to the polarity the order of these compounds are blue>red>green. That means
blue is the more polar compound and thus will have less tendency to move along with the mobile
phase. So, the green colored compound will move first , then the red colored compound followed
by the blue color compounds.

Application
Column chromatography has a wide application in different fields. Its major application includes-

I. Separation of mixture of compounds.

II. Removal of impurities or purification process.
III. Isolation of active components.
IV. Estimation of drugs in formulation or crude extracts.

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 V. Separation of diastereomers.
VI. Determination of primary and secondary glycosides in plants.

Advantages And Disadvantages

Column chromatography has the following advantages –

I. Any type of mixture can be separated by this method.

II. Any quantity of the mixture can be separated.
III. Wider range of mobile phases can be used.
IV. Automation is possible.

However there are some limitations –

I. It is a time taking process.

II. It requires more amount of mobile phase.
III. Analysis of components is also time taking and automation needs more technical
knowledge and is expensive.

However, column chromatography is a very good process and can be used for any mixture
separation and in any principle of chromatography.

These are the different chromatography based on various principles, which

plays a crucial role in the separation, identification and purification of the components from the
mixture. Hence, chromatography is a cost effective, easy and simple method of separation.

Applications of Chromatography in various fields

1. In Pharmaceutical Industry

Chromatography plays a vital role in the pharmaceutical industry due to its precision,
sensitivity, and versatility. One of its primary applications is in purity testing, where it ensures
that pharmaceutical products are free from harmful impurities and that the active ingredients
are present in the correct concentrations. During drug development, chromatography aids in

33
 the separation and identification of complex chemical mixtures, which is essential for
formulating new medications and detecting degradation products. Additionally, it plays a key
role in pharmacokinetics by analyzing how drugs are absorbed, distributed, metabolized, and
excreted in the body, often through the measurement of drug levels in biological samples like
blood and urine.

2. In Chemical Industry

In the chemical industry, chromatography is essential for analyzing, separating, and purifying
chemical compounds. It is widely used in quality control to detect impurities and verify the
composition of raw materials and finished products. Chromatography also supports process
monitoring by tracking chemical reactions and optimizing production efficiency. Additionally,
it plays a key role in research and development by helping identify and characterize new
compounds and formulations.

3. In Food and Beverage Industry

Chromatography plays a crucial role in the food and beverage industry by ensuring product
safety, quality, and compliance with regulatory standards. One of its primary applications is in
the detection and quantification of contaminants such as pesticides, herbicides, toxins, and
heavy metals in food products. It is also used to analyze food additives, preservatives, artificial
colors, and flavoring agents to ensure they are within safe and acceptable limits. In quality
control, chromatography helps monitor the consistency of ingredients and the overall
composition of food and beverages, contributing to product uniformity and shelf-life stability.
Overall, chromatography is an essential tool in maintaining the safety, integrity, and innovation
of food and beverage products.

4. In Forensic Science

Chromatography is a valuable tool in forensic science for analyzing evidence from crime
scenes. It is commonly used to identify and separate substances such as drugs, alcohol, toxins,
and explosives in biological samples like blood, urine, or tissue. It also helps in examining

34
 trace evidence such as ink, dyes, fibers, and residues, which can link suspects to crime scenes.
By providing accurate and detailed chemical analysis, chromatography plays a critical role in
criminal investigations and legal proceedings.

5. In Environmental Analysis

Chromatography is widely used in environmental analysis to detect and measure pollutants in

air, water, and soil. It helps identify toxic substances such as pesticides, heavy metals, and
industrial chemicals, ensuring compliance with environmental regulations. This technique is
also used in monitoring water quality, assessing contamination levels, and studying the impact
of pollutants on ecosystems. Its high sensitivity and accuracy make chromatography a vital
tool for protecting environmental health.

Conclusion
Initially chromatographic techniques were used to separate substances based on their color as
was the case with herbal pigments. With time its application area was extended considerably.
Nowadays, chromatography is accepted as an extremely sensitive, and effective separation
method. Column chromatography is one of the useful separation, and determination methods.
Column chromatography is a protein purification method realized especially based on one of the
characteristic features of proteins. Besides, these methods are used to control purity of a protein.
HPLC technique which has many superior features including especially its higher sensitivity,
rapid turnover rate, its use as a quantitative method, can purify amino acids, proteins, nucleic
acids, hydrocarbons, carbohydrates, drugs, antibiotics, and steroids. It is very useful yet
inexpensive method, so largely used in pharmaceutical industry, environmental analysis, food
industry etc. In conclusion, chromatography is a powerful and versatile analytical technique used
across a wide range of industries. Its ability to separate, identify, and quantify components in
complex mixtures makes it essential for ensuring quality, safety, and regulatory compliance.
chromatography plays a crucial role in advancing science, improving products, and protecting
public health.

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Reference

1. Cassidy, H. G. (1952). Investigation of paper chromatography. Analytical Chemistry, 24(9),

1415- 1421.
2. Zweig, G., & Sherma, J. (1973). Paper Chromatography—Past, Present and Future.
Journal of Chromatographic Science, 11(6), 279-283.
3. Coskun, O. (2016). Separation techniques: chromatography. Northern Clinics of Istanbul,
3(2), 156.
4. Borders, C. Jr. (1972). Descending paper chromatography of oligosaccharides: A experiment.
Journal of Chemical Education, 49(6), 437.
5. Pramod, S. K., & Navnath, K. A. (2017). A Brief Review on Ultra Performance Liquid
Chromatography.
6. Principles of adsorption Chromatography : the separation of nonionic organic compound.
Lloyd R. Snyder.
7. Quantitative Analysis of gas chromatography : Josef Novak
8. [Link]
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10. [Link]

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.

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