C&C Pharmacognosy By Dr.

Shahid Rasool Chromatography

CHROMATOGRAPHY

DEFINITION
Chromatography is derived from two Greek words; Chroma meaning “Color” and Graphos
meaning “To write”.
“It is the technique for separation of mixture of solutes in which separation is brought about by
differential movement of individual solutes through a porous medium under the influence of a
moving solvent.”
OR
Chromatography is a technique for separating mixtures into their components in order to analyze,
identify, purify, and/or quantify the mixture or components.

Analyze
Separation Identify
Chromatography Purify
Quantify

Mixture of Components Individual Components

Chromatography was invented by T.S.WETT; a Russian Biologist in 1906. He separated a

vegetable plant by using glass column under the influence of petroleum which acts as mobile
phase. He observed different color bands in column and named the process as chromatography.

PHASES OF CHROMATOGRAPHY
In chromatography, two phases are involved.
 Stationary phase
 Mobile phase
Stationary phase
The phase which is fixed and not movable is called stationary phase e.g. porous solid, finely
divided solid, a liquid that has been bound to some inert supporting material.
Mobile phase
The phase which is movable is called mobile phase e.g. gas, liquid, solution.

BASIC PRINCIPLE OF CHROMATOGRAPHY

Chromatography system achieves their ability to separate mixture of chemicals by selectively
retarding the passage of some components through stationary phase while permitting others to
move freely. So two forces are involved in chromatography
 Driving force
 Resistive force
Driving force
The mobile phase drives the solute from stationary phase and driving force is the flow of mobile
phase.
Resistive force
The stationary phase exerts more or less selective force on migrant solute which counter act the
driving force of mobile phase and tends to retard migration of individual components of sample
mixture. The resistive force of stationary phase in chromatography is termed as sorption and
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C&C Pharmacognosy By Dr. Shahid Rasool Chromatography

driving force of mobile phase in chromatography is termed as desorption. During

chromatography, the migrant solute molecules repeatedly undergo sorption and desorption.
Sorption selectively retard their flow with mobile phase while desorption allow them to be carry
along mobile phase. This continuous distribution process forms the basis of chromatographic
separation.

EFFECT OF POLARITY OF MOBILE PHASE IN CHROMATOGRAPHY

Polarity refers to separation of charges within a molecule or the ionic character of molecule.
Ionic compounds are therefore will be high polar because of more electro-negativity difference
of bonded atoms.
A general rule in chromatography is “to match the polarity of solvent with that of sample i.e.
mixture of solutes.”
 For a mixture of non-polar substances, if we choose polar mobile phase, then polar
solvent will be absorbed and sample will move rapidly through the system and separation
does not occur. For this kind of sample, we must use non-polar mobile phase.
 On the other hand, if we choose a non-polar solvent for polar sample, then solvent will
move rapidly through the system and polar sample will be absorbed at bottom of column
resulting in no separation.
 For samples of least polarity, least polar or non-polar mobile phase is used.
 For samples of intermediate polarity, mobile phase of intermediate polarity is used.
 For polar samples, mobile phase of high polarity is used.
 If the polarity of sample is unknown, then mixture of solvents is used i.e. polar,
intermediate polar and non-polar.
n-hexane Chloroform Methanol Water Acid
Polarity

SIGNIFICANCE OF CHROMATOGRAPHY
Most chemical operations including chemical analysis involved a separation of mixtures into
individual components e.g. Distillation, Filtration, Extraction and Electrolysis. The individual
component of a mixture gets more and more similar in physical and chemical properties so it
becomes very difficult to separate them by these conventional techniques. So for this purpose,
we use chromatographic techniques.
For example, when a protein is heated in the presence of HCl, it is converted in to mixture of
amino acids which are more and more similar in physical and chemical properties so it becomes
very difficult to separate them by conventional techniques. So for this purpose, we use
chromatographic techniques.

APPLICATIONS OF CHROMATOGRAPHY
Following are the applications of chromatography:
 It is used for separation and identification of biological substances e.g. Amino acids
 It is used for separation and identification of medicinal plant constituents e.g.
carbohydrates, alkaloids, tannins, resins, glycosides etc.
 It is helpful in the determination of structure of molecules using spectroscopic
techniques.
 It, also has forensic applications e.g. detection of poison and narcotic drugs in blood.

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C&C Pharmacognosy By Dr. Shahid Rasool Chromatography

 It is used for separation and identification of large no. of organic, inorganic, natural and
synthetic compounds.
 It is also used for estimation of pesticides on crops and vegetables.

CHOICE OF CHROMATOGRAPHY
 Nature and amount of sample
 Objective of separation
 Limitation of time
 Equipment
 Expenditure

CLASSIFICATION OF CHROMATOGRAPHY
Chromatography is classified on the following two basis
 Classification on the basis of purpose
 Classification on the basis of mechanism

Classification on the basis of purpose

 Analytical Chromatography
 Preparative Chromatography
Analytical Chromatography
This type of chromatography is used on small scale for identification and quantification of
compounds from sample of mixture of components.
Examples: Paper Chromatography, Thin Layer Chromatography (TLC), Gas Chromatography
Preparative Chromatography
This type of chromatography is used on large scale for isolation and purification of compounds
from sample of mixture of components.
Examples: Column Chromatography (CC), High Performance Liquid Chromatography (HPLC)

Classification on the basis of mechanism

 Adsorption chromatography
 Partition chromatography
 Ion exchange chromatography
 Size exclusion chromatography

ADSORPTION CHROMATOGRAPHY
In this type of chromatography, the stationary phase used is always solid. In adsorption
chromatography the mobile phase containing the dissolved solutes passes over the surface of the
stationary phase.
Principle of adsorption chromatography
In adsorption chromatography, small difference in sorption and desorption behavior of
substances between a moving solvent and the stationary phase are utilized to achieve separation.
Adsorption is a surface phenomenon denoting a higher concentration at interface than compared
to surrounding medium. In adsorption chromatography, mobile phase passes over the stationary
phase carrying the dissolved compound with it. The rate at which solute molecule moves or
separates depends upon various degrees of affinity for adsorbent.
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C&C Pharmacognosy By Dr. Shahid Rasool Chromatography

Adsorbent
It is a solid which has the property of holding the molecules at its surface particularly when it is
porous and finely divided and it acts as stationary phase in adsorption chromatography. The most
popular adsorbents used are Silica gel and Alumina. Others are Charcoal and Polyamides.
Types of adsorption chromatography
Adsorption chromatography is further classified into two types
Liquid-Solid chromatography
In this type of adsorption chromatography, mobile phase used is liquid e.g.
TLC (Thin Layer Chromatography)
CC (Column Chromatography)
HPLC (High Performance Liquid Chromatography)
Gas-Solid chromatography
In this type of chromatography mobile phase is gas e.g. GSC (gas solid chromatography)

THIN LAYER CHROMATOGRAPHY (TLC)

In this type of adsorption chromatography, the stationary phase used is always solid (Adsorbent)
while mobile phase used is liquid.
The technique of TLC was originated by German Pharmacognocist, Egon Stahl.
Procedure for TLC
It involves following steps.
Preparation of TLC plates
20×5 cm glass plates are used. Slurry of silica gel is spread uniformly on
glass plate with the help of spreader in the form of a thin layer of around
1-2 mm. The plates are dried at room temperature and activated in oven
at 1100C for 30 minutes (activation means the removal of moisture). Pre-
coated plates of size 20×20 cm are also available in the market on which
silica gel is spread over aluminum foil.
Application of sample to TLC plate
A line with lead pencil is marked at one edge of the plate at about 1-2
cm distance. A drop of sample is applied with capillary tube on line at
different positions.
Choice of solvent (mobile phase)
It depends upon the nature of substances present in the sample. If sample contains polar
substances, then mobile phase should be polar i.e. CH3OH (methanol), if the substances present
in a sample are of intermediate polarity then the mobile phase should be of intermediate polarity
i.e. CH3Cl (chloroform) and if the substances present in a sample are non-polar then the mobile
phase used should be non-polar i.e. Petroleum ether or n-hexane. If the polarity of sample is
unknown, then mixture of solvents is used i.e. polar, intermediate polar and non-polar. Suitable
solvent is poured in chromatographic jar.
Saturation of chromatographic jar
A filter paper is cut according to the size of chromatographic jar and placed along the inner walls
of chromatographic jar. After pouring mobile phase, close the jar and rotate the jar, so that filter
paper become wet with mobile phase. This is called saturation of chromatographic jar.
Placement of TLC plate in chromatographic jar

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C&C Pharmacognosy By Dr. Shahid Rasool Chromatography

TLC plate is placed inside the chromatographic jar at an angle of 45 0 so that the sample spots
should not be dipped in mobile phase. Mobile phase is allowed to travel on TLC plate till the
required distance ¾ th, is achieved.
Drying of TLC plate
TLC plate is removed from the chromatographic jar and solvent front (distance travel by mobile
phase) is noted. Plate is dried by fan or hair dryer.
Location of substances on chromatogram (TLC plate)
Different substances on TLC plate can be located by following methods.
 Ordinary light
 UV light by using UV lamp
 Iodine vapours
 Chemical detection, spraying of detecting agents e.g. Drangdroff reagent is used for
detection of alkaloids. Nin-hydrin solution is used for detection of amino acids.
Libermann’s reagent is used for detection of steroids and turpenoids. Bontrager’s reagent
is used for detection of glycosides.
Calculation of Rf and hRf values
Rf (Retention factor) value is calculated by following formula

Rf value should be between 0-1.

hRf value is calculated from Rf to avoid fractions by formula hRf = Rf × 100

TYPES OF DEVELOPMENT IN CHROMATOGRAPHY

Chromatography can be carried out by following methods
Ascending method: When mobile phase travel in upward direction by capillary action e.g. TLC
and paper chromatography
Descending method: When mobile phase travel in downward direction. The movement of
solvent in this method is faster than ascending method due to gravity rather than capillary action
e.g. column chromatography
One dimensional method: When mobile phase travel either upward or downward direction.
Two dimensional method: The plate or paper is first developed by one direction using a mobile
phase and then it is reversed at right angle and developed again with different mobile phase.
Radial or Circular method: The mobile phase moves from center towards periphery of circular
paper sheet.

TLC (Ascending) Paper (Ascending) Column (Descending) Radial (Circular)

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C&C Pharmacognosy By Dr. Shahid Rasool Chromatography

COLUMN CHROMATOGRAPHY (CC)

It is a type of preparative, descending adsorption chromatography in which the liquid mobile
phase passes through the solid stationary phase usually silica gel. It was invented by a Russian
Biologist, T.S.WETT in 1906.
Procedure
In this type of chromatography, a simple straight glass tube
tapered at bottom is used called Column. This is about 20-30
cm long and 2-3 cm in diameter. Glass wool or cotton plug is
used as a support for clumping of column. Column is
clumped vertically. Slurry of adsorbent (Silica gel, Alumina)
is added by portions in column till the desired height is
obtained and surface of adsorbent should remain covered
with solvent or mobile phase. Now sample is placed over the
adsorbent at the top of column. A suitable solvent (mobile
phase) is poured from a glass funnel on the top of the
column. After entire portion of solvent has passed through
the column, various color bands become well defined which
are collected separately is separate flasks and can be
identified by using TLC.

PARTITION CHROMATOGRAPHY
In this type of chromatography, stationary phase is a liquid. It may also be defined as “the
method which involves the separation of mixture of substances by means of their partition
coefficient between mobile phase and liquid stationary phase held on a suitable solid support”.
Principle of partition chromatography
It is based on the difference of partition co-efficient of a substances between liquid stationary
phase and mobile phase. The only factor which influences the movement of a component as the
solvent travel along the system, is the relative solubility of that component in the liquid
stationary phase and mobile phase.
Types of partition chromatography
Partition chromatography is divided into two types.
Liquid-Liquid chromatography
In this type of partition chromatography mobile phase used is a liquid e.g. Paper
Chromatography
Gas-Liquid chromatography
In this type of partition chromatography mobile phase used is a gas e.g. GLC (gas liquid
chromatography)

PAPER CHROMATOGRAPHY
It is a type of partition chromatography in which chromatographic paper is used as stationary
phase i.e. water entrapped in the cellulose network of paper serves as stationary phase.
Principle of paper chromatography
It is based on the difference of partition co-efficient of a substances between liquid stationary
phase i.e. filter paper with associated water and mobile phase. In paper chromatography, the only
factor which influences the movement of a component as the solvent travel along the paper is the

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C&C Pharmacognosy By Dr. Shahid Rasool Chromatography

relative solubility of that component in the water of

paper and mobile phase i.e. partition co-efficient of
that component between water and mobile phase.
Chromatographic paper
The chromatographic paper is made up of highly
purified cellulose fibers having spaces between these
fibers which naturally contain 5-7% of water.
Individual part of each fiber together with their
associated water contributes minute cells. It is the
distribution of substances between moisture of cell
and solvent flowing over it actually brings about
separation.
Procedure for paper chromatography
It involves following steps
Preparation of chromatographic paper
Filter paper of suitable size is cut according to the size of chromatographic jar so that it does not
touch the walls of chromatographic jar and dried in open air if there is any moisture on it.
Application of sample on paper
A line with lead pencil is marked at one edge of the paper at about 1-2 cm distance. A drop of
sample is applied with capillary tube on line at different positions. Suitable solvent is poured in
chromatographic jar and saturation of chromatographic jar is carried out.
Placement of paper in chromatographic jar
The paper is placed inside the chromatographic jar at an angle of 45 0 so that sample should not
be dip in mobile phase and mobile phase is allowed to travel on the paper (chromatographic
paper) till the required distance is achieved.
Drying of paper
Chromatographic paper is removed form chromatographic jar and solvent front is noted. Paper is
dried with the help of fan or hair dryer.
Location of substances on chromatogram (chromatographic paper)
Different substances on paper can be located by following methods.
 Ordinary light
 Iodine vapours
 Chemical detection, spraying of detecting agents e.g. Drangdroff reagent is used for
detection of alkaloids. Nin-hydrin solution is used for detection of amino acids.
Libermann’s reagent is used for detection of steroids and turpenoids. Bontrager’s reagent
is used for detection of glycosides.
Calculation of Rf and hRf values
Rf (Retention factor) value is calculated by following formula

Rf value should be between 0-1.

hRf value is calculated from Rf to avoid fractions by formula hRf = Rf × 100

GAS CHROMATOGRAPHY
The chromatography in which mobile phase used is a gas and stationary phase may be solid or
liquid depending upon the type and instrument being used.
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C&C Pharmacognosy By Dr. Shahid Rasool Chromatography

Classification of Gas Chromatography

Gas Solid Chromatography (GSC)
It is a type of adsorption chromatography in which solid stationary phase is used.
Gas Liquid Chromatography (GLC)
It is a type of partition chromatography in which liquid stationary phase is used.

Instrumentation of Gas Chromatography

The components of gas chromatography are as follows.
Carrier Gas
Flow control valve
Sample inlet system
Columns
Detectors

Figure: Flow Sheet Diagram of Gas chromatography

Carrier gas
Gases are available in pure form in steel cylinders and kept under pressure. For optimum result
gas is dried before use by passing it through drying tubes. Inert gases like H, He, Ne and Ar are
used so that they should not react with sample.
Flow-control valve
The rate of flow of carrier gas through apparatus is controlled by means of a special valve and is
measured by a device known as “flow meter”.
Sample inlet system
The actual amount of sample introduced will depend upon the nature of solutes, the size of
column and type of detectors. Gas and liquid samples are introduced into carrier gas from a
micro syringe or similar device. Solids and viscous liquids are introduced by weighing a small
amount in thin-walled glass ampoule, placing this ampoule into carrier gas stream and then
crushing it.

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C&C Pharmacognosy By Dr. Shahid Rasool Chromatography

Columns
Gas chromatographic separations are usually carried out at high temperature so that some form
of heating and thermostatic control of column is required. Columns are made up from a variety
of materials including glass, plastic, metals and stainless steel.
In gas chromatography, following two types of columns are used.
Partition Columns
These types of columns are packed with celite, which is a liquid coated on diatomaceous earth
after activation with acid or base and then calcinated. These types of columns are used in GLC in
which separation depends upon partition of sample molecules between a liquid supported on a
solid and a gas flowing through it i.e. distribution of constituents between gas and liquid phase.
Adsorption Columns
These types of columns are packed with adsorbent like silica gel or alumina. These types of
columns are used in GSC in which separation depends upon adsorption of sample molecules
between adsorbent and a gas flowing through it.
Detectors
They are externally important in gas chromatographic separations and great deal of efforts has
been expanded in their manufacture.
Two types of detectors are used in gas chromatography.
Differential type detectors
These are thermal conductivity detectors (TCD) in which hydrogen and helium are used as
carrier gas e.g. Katharometer
Integral type detectors
In these type of detectors, pure CO2 is used as carrier gas e.g. Nitrometer
Identification of compounds
The information from gas chromatography separation is in the form of chromatogram. There is
no chemical identification. The substance are identified by the time they take to emerge from
column called retention time. In some very sophisticated machines, compounds are analyzed
directly by mass spectrophotometry e.g. GCMS (Gas Chromatography mass spectrophotometry)
Operation of gas chromatography
A small amount of material being separated is injected into a stream of inert gas which carries it
into a column containing a suitable medium capable of retarding the flow of individual
components of sample as they flow through column. The separated components then emerge
from the column at discrete intervals and pass through some form of detectors which are
amplified and recorded in the form of chromatogram. Difference in adsorption or partition on
sample components in the column is the factor which makes separation possible.

HPLC
 High pressure liquid chromatography
 High performance liquid chromatography
 High priced liquid chromatography
 Hewlett-Packard liquid chromatography
Definition
It is type of adsorption chromatography which uses the
high-efficiency (high-performance) column and high inlet
pressure of liquid mobile phase for separation of
components through adsorbent.
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C&C Pharmacognosy By Dr. Shahid Rasool Chromatography

Mobile phase
Mixture of a polar solvent (such as alcohol) and a non-polar solvent such as hydrocarbons must
be used as mobile phase.
Stationary phase
Adsorbent like Silica-gel (for basic compounds), Alumina (adsorbs acidic compounds) or Nylon-
6 (is a polyamide) can be used as stationary phase.
In HPLC, separation depends upon the adsorption of solute molecule between adsorbent and a
pressurized solvent flowing through it.
Instrumentation of HPLC
Main components of HPLC are:
 Mobile Phase ((Solvent reservoir)
 Pump
 Injector
 Separation column
 Detector
 Amplifier and recorder

Figure: Flowsheet Diagram of HPLC

Applications of HPLC
Pharmaceutical
• Separation and identification of medicinal plant constituents
• Identification of spurious and counterfeit drug products
• Quality control of pharmaceuticals
• Shelf-life determinations of pharmaceuticals
Environmental
• Determination of phenols in drinking water
• Determination of estrogens in coastal waters
Forensic
• Identification of poison, narcotics in blood serum, urine, sweat and hair
• Identification of steroids in blood serum, urine, sweat, hair
Food and flavor
• Ensuring soft drink consistency and quality
• Sugar analysis in fruit juices
• Estimation of pesticides on fruits and crops
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Advantages of HPLC
• It allows columns to be reused a number of times.
• It allows analysis to be done in a shorter time.
• It achieves a higher degree of resolution.

ION EXCHANGE CHROMATOGRAPHY

In ion exchange chromatography, the stationary phase consists of a polymeric matrix (Resin)
which is filled in a glass column. Ionic functional groups e.g. carboxylic acid or quaternary
amines are chemically bounded on the surface of this matrix. As the mobile phase passes over
the resin surface, ionic solutes (sample) are retained on it by electrostatic forces. The mobile
phase used in ion exchange chromatography is always a liquid. This technique is useful for
inorganic cat-ions, amino acids etc.

Figure: Ion Exchange Chromatography

SIZE EXCLUSION CHRMATOGRAPHY

It is also known as gel filtration chromatography. The stationary phase is a polymeric gel
substance containing numerous pores of molecular dimensions. Solutes with small molecular
size can diffuse into the pores while larger molecules cannot diffuse through matrix and remain
in mobile phase. This method is used for the separation of mixture containing solute of different
molecular sizes. The mobile phase can be either liquid or a gas.

Figure: Size Exclusion Chromatography

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