CHROMATOGRAPHY

GROUP 3

• Hafiza Aiman Ikram

• Soha Akbar
• Mehreen Nasir
• Khaula Habib
• Mahum Shabir
• Ali Raza
 CONTENTS
• Introduction
• History
• Purpose
• Principle
• Experiment
• Terms/Phases or Chromatogram
• Factors
 CONTINUE…

• Types
• Applications
• Advantages and Disadvantages
• Conclusion
 INTRODUCTION

Chromatography mean color writing.

It is separation of a mixture by distribution of its
components between a mobile and stationary
phase over time.
 COMPONENTS
• Mobile phase: a solvent that flows through the
supporting medium
• Stationary phase: a layer or coating on the
supporting medium that interacts with the analytes
• supporting medium: a solid surface on which the
stationary phase is bound or coated
 Mobile phase Stationary phase
The solvent moving The substance which stays
through the column. fixed inside the column
Can be liquid or gas Solid or liquid supported
on a solid
The mobile phase The stationary phase may
completely dissolves the or may not have
sample. interactions with the
components in the sample.
 HISTORY
First devised in Russia by the
Italian-born scientist Mikhail
Tsvet in 1900
PURPOSE OF CHROMATOGRAPHY

•Chromatography is basically the

separation of a complex mixture of
components into separate entities.
 PRINCIPLE OF
CHROMATOGRAPHY
• Based on the principle where molecules in mixture
applied onto the surface or into the solid, and
fluid stationary phase (stable phase) is separating
from each other while moving with the aid of a
mobile phase.
• Basic principle- components in a mixture have
different tendencies to adsorb onto a surface or
dissolve in a solvent.
 EXPERIMENT
• The first true chromatography is usually attributed to
the Russian-Italian botanist Mikhail Tsvet. Tsvet
applied his observations with filter paper extraction to
the new methods of column fractionation that had
been developed in the 1890s for separating the
components of petroleum
• Thin layer chromatography
is used to separate the
colorful components of a
plant extract
 CHROMATOGRAM
“The pattern that formed on an absorbent medium by
the layers of component separated by chromatography ”
OR
“The colour pattern obtained on a paper is called
chromatogram”
 Data represented by chromatogram are used to help
identify and quantify the solute.
• Visual output of chromatograph.
• Separation- different peak or pattern on
chromatogram correspond to different
component of the separate mixture.
 CHROMATOGRAPHY TERMS
• Stationary phase-immobilized phase
• Mobile phase
• Sample (analyte)
• solvent
• Eluent
• Eluate
• Retention time
• Stationary phase
Immobilized on the support particles or in the inner wall of the
column tubing.
Example: Silica layer- Thin Layer Chromatography.

• Mobile phase
Moves in definite direction. Liquid (LC) or Gas (GC).
It moves through chromatography column where the sample attract
with stationary phase and is separated.

• Sample(analytes)
substance analyzed in chromatography.
• Solvent:
Any substance capable solubilizing into another substance

• Eluent:
Fluid entering the column/ solvent that carries the analyte.

• Eluate:
Mobile phase leaving the column.

• Retention time:
Time takes for the particular analyte to pass through system under
the set conditions
 FACTORS AFFECTING THE
SEPARATION
• Intermolecular interaction between the two
phases.
• Extent of dispersion of solute molecules over the
stationary phase.
TYPES
• Paper Chromatography
• Thin Layer Chromatography
• Gel Chromatography
• Column Chromatography
 PAPER CHROMATOGRAPHY
• Technique which is used to separate low-molecular-mass compounds
based on their distribution between stationary phase and mobile
phase.
•  Discovered by Synge and Martin in 1943
• Mobile Phase:
      air, alcohol
• Stationary Phase:
      Filter paper, cellulose
• Principle
       a)Adsorption Chromatography
       b)Partition Chromatography
• Types
i.     Ascending paper Chromatography
ii.     Descending paper Chromatography
iii.     Ascending-Descending Chromatography
iv.     Radial or circular Chromatography
v.     Two Dimensional Chromatography
THIN LAYER CHROMATOGRAPHY
• A technique used to isolate non-volatile mixtures.
• conducted on a sheet of aluminum foil, plastic, or glass which is coated
with a thin layer of adsorbent material. The material usually used is
aluminum oxide, cellulose, or silica gel.
• Mobile Phase:
   Hexane, ether petroleum, alcohol
• Stationary Phase
   Silica gel, alumina, polyamide
• Principle
   Separation principle
• Separation relies on relative affinity of compounds over phases. The
compounds on mobile phase move over the surface of stationary
phase.
• Rf =distance travelled by sample / distance travelled by solvent
 GEL CHROMATOGRAPHY
• Also known as gel permeation, molecular sieving or size exclusion
chromatography
• It’s a technique in which separation of components is based on their molecular
weight and size
• One of the effective method to isolate and analyze biomacromolecules
• Gel permeation chromatography
When organic solvent is used as mobile phase
• Gel filtration chromatography
When aqueous solution is used as mobile phase
 PRINCIPLE
In this method, Stationary phase is an open network of polymers
which are cross linked to each other to form pores of consistent size
• Stationary phase
Polysaccharide gel
• Mobile phase
Liquid solvent
• When mobile phase containing mixture of solutes is passed through
column, molecules that are large to enter the pores get eluted with
mobile phase fastly
• Molecules that are small in size diffuse in and out of the pores, thus
elute later
• Theory of separation
Vt = Vo + Vi + Vm
 COLUMN CHROMATOGRAPHY
• One of the most useful methods for separation and purification of
both solids and liquids
• Adsorption chromatography
When stationary phase is solid
• Partition chromatography
When stationary phase is liquid
 PRINCIPLE
Mixture of components dissolved in mobile phase is introduced into the column.
Components move depending upon their relative affinities
• Stationary phase
Silica gel, alumina, cellulose etc.
• Mobile phase (eluent)
Benzene, ester, water etc.
Compound attracted more strongly by the mobile phase will move rapidly
through the column
Compound strongly attracted to stationary phase will move slowly through the
column
 APPLICATIONS
• Pharmaceuticals Industry
To identify the drugs, avoid impurities, determine stability, doses
form and to find the quantity of drugs
Heavy metal analysis of drugs such as cadmium, arsenic, mercery
Pesticidal residue and micro chemical analysis of drugs
• Analysis of natural contamination
Phenol and mercery from sea water
• Forensic test
Determination of steroid in blood, urine and sweat
Detection of psychotropic drug in plasma
 CONTINUE...
• Clinical test
monitoring of hepatic cirrhosis patient through aquaporin
in the urine
• Food and assence manufacture
sweetener analysis in fruit juice
preservative analysis
 ADVANTAGES
• Simplest method for separation
• Controlled by single person
• Detect small amount of sample(grams, nanograms, milligrams)
• Rapid and precise method
• Very few sample quantity is required
• Works on broad range of samples
 DISADVANTAGES
• Required a trained person to operate the equipment
• Very expensive
• Errors due to overloading of sample and so
• Requires more amount of solvent
• Requires high power consumptions
• High operational pressure is required for efficient sample
 CONCLUSION
• Complex mixture of herbal drug can be separated by
chromatography passing through mobile to stationary phase
• Compulsory in the field of herbal drug standardization,
authentication and manufacturing
• Paper Ch. Is used to identify and separate simple
compounds of herbal drugs
 CONTINUE…
• High performance liquid chromatography and high
performance thin layered Ch. Are backbone of ayurveda for
drug standardization and authentication
• Gas Ch. Technique is widely used to detect and quantify
volatiles and aromatics of herbal drugs