Introduction, types, mechanism and application of chromatography

1. Introduction of
Chromatography
By Shubhrat Maheshwari
Subject- Instrumental methods of analysis
Subject code- BP701T

2. Introduction
• Chromatography is a physical process where the components (solutes) of a sample
mixture are separated as a result of their differential distribution between
stationary and mobile phases.
Greek chroma meaning ‘color’ and
graphein meaning ‘writing’
ADVANTAGES OF CHROMATOGRAPHY OVER OTHER METHODS
• Separation of mixtures of chemicals having closer physical or chemical properties.
• Gentle method.
• Separation in even micrograms is possible.
• Simple, rapid and efficient technique.

3. Introduction
• Chromatography is a technique for separating mixtures into their
components in order to analyze, identify, purify, and/or quantify the
mixture or components.

4. Stationary Phase

5. Introduction
• Chromatography is used by scientists to:
Analyze – examine a mixture, its components, and their relations to one
another
Identify – determine the identity of a mixture or components based on
known components
Purify – separate components in order to isolate one of interest for
further study
Quantify – determine the amount of the a mixture and/or the
components present in the sample

6. Definition
• ‘A technique by which a mixture is separated into its components on
the basis of relative ability of each component to be moved
along/through 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 (called stationary phase) under the influence of some solvent
or gas (called moving/mobile phase).
• Chromatography is a nondestructive procedure.
• Applied for both qualitative and quantitative studies

7. History
• The subject of Chromatography was introduced into scientific world in a very
modest way by M. Tswett in 1906.
• He employed a technique to separate various pigments such as chlorophylls and
xanthophylls by passing the solution of these compounds into the glass column
which was packed with finely divided calcium carbonate.
• After the later, Thompson and Way had realized the Ion-Exchange properties of
soils.
• Almost after three decades, in 1935 Adams and Holmes observed the Ion
Exchange characteristics in crushed phonograph. This observation opened the
field for preparation of Ion Exchanged resins.
• The concept of Gas-Liquid Chromatography was first introduced by Martin and
Synge in 1941.

8. History
• They were also responsible for the development in Liquid-Liquid chromatography.
• In 1944, from Martin laboratory, the separation of amino acid by paper chromatography was
reported.
• In 1952, the importance of the chromatography was observed when both Synge and Martin were
awarded with Nobel Prize.
• In 1959, a technique known as Gel Filtration chromatography was observed which is used to
separate low molecular weight substances from high molecular substances.
• In 1960, further improvement in liquid chromatography led to the development of High
Performance Liquid Chromatography.
• The following decade of 1970 improvement in the field of adsorption chromatography in the form
of Affinity chromatography which was mainly based on biological interactions.

9. History
• A new field was originated which was supercritical fluid chromatography.
• Supercritical fluid chromatography is a hybrid of gas and liquid chromatography and combine
advantageous feature of the 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.

10. Principle
• Chromatography is usually based on principle of partition of solute
between two phases. It usually consists of a Mobile Phase and a
Stationary Phase.
• The Mobile Phase usually refers to the mixture of the substances to be
separated dissolved in a liquid or a gas .
• The Stationary Phase is a porous solid matrix through which the
sample contained in the mobile phase percolates.

11. Principle
• Like-dissolve-like or like-prefer-like.
• The basis-partition or distribution coefficient ‘K’ which describes the
way in which a compound distributes itself between two immiscible
phases.
• Defined as the molar concentration of analyte in the stationary phase
divided by the molar concentration of the analyte in the mobile phase
𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑐𝑜𝑚𝑝𝑜𝑛𝑒𝑛𝑡 𝑖𝑛 𝑠𝑡𝑎𝑡𝑖𝑜𝑛𝑎𝑟𝑦 𝑝ℎ𝑎𝑠𝑒
𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑐𝑜𝑚𝑝𝑜𝑛𝑒𝑛𝑡 𝑖𝑛 𝑚𝑜𝑏𝑖𝑙𝑒 𝑝ℎ𝑎𝑠𝑒
K =

12. Commonly used terms in chromatography
• The analyte is the substance to be separated during chromatography.
• Analytical chromatography is used to determine the existence and
the concentration of analyte(s) in a sample.
• A chromatogram is the visual output of the chromatograph. In the
case of an ideal separation, different peaks or patterns on the
chromatogram represent different components of the separated
mixture.

13. Commonly used terms in chromatography
• A chromatograph is an equipment that enables the separation e.g. gas
chromatographic or liquid chromatographic separation.
• The eluate is the mobile phase leaving the column.
• The eluent is the solvent that carries the analyte.
• An eluotropic series is a list of solvents ranked according to their eluting power.
• Elution is the process of extracting a substance that is adsorbed to another by
washing it with a solvent.
• An immobilized phase is a stationary phase that is immobilized on the support
particles, or on the inner wall of the column tubing.
• The mobile phase is the phase that moves over the stationary phase. It may be a
liquid (LC) or a gas (GC). The mobile phase moves through the stationary phase
where the sample interacts with the stationary phase and is separated.

14. Commonly used terms in chromatography
• The retention time is the time required for the mobile phase to sweep a
component from the stationary phase.
• The retention volume is the volume of the mobile phase required to sweep a
component through stationary phase.
• The sample is the matter analyzed in chromatography. It may consist of a single
component or it may be a mixture of components. When the sample is treated, the
phase or the phases containing the analytes of interest is/are referred to as the
sample whereas everything else separated from the sample before or during
analysis is referred to as waste.
• The solute refers to the sample components in partition chromatography.
• The solvent refers to any substance capable of solubilizing another substance, and
especially the liquid mobile phase in liquid chromatography.

15. Commonly used terms in chromatography
• The stationary phase is the substance fixed in place for the
chromatography procedure.
• It may be solid, gel or a liquid. e.g ; silica, alumina, cellulose
• The detector refers to the instrument used for qualitative and
quantitative detection of analytes after separation.
• Rf value or Retention factor (Rf) is defined as the ratio of the
distance traveled by the center of a spot (solute) to the distance
traveled by the solvent front (solvent)
𝑅𝑒𝑡𝑒𝑛𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟(𝑅𝑓) = 𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑠𝑜𝑙𝑢𝑡𝑒
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑠𝑜𝑙𝑣𝑒𝑛𝑡

16. Chromatography Mechanisms
• Adsorption
• Partition
• Ion exchange
• Size-exclusion
• Affinity Chromatography

17. Adsorption
• Based on relative polarities .
• Compounds having high affinity towards the stationary phase travel
slower
• Compounds having lesser affinity towards the stationary phase travel
faster
• No two components have same affinity for a combination of stationary
phase, mobile phase and other conditions

18. Partition
• Based on relative solubility.
• Solutes will be distributed according to their partition coefficients.
• Components which are more soluble in the stationary phase – Travel
slower
• Components which are less soluble in the stationary phase –Travel
faster
• No two components have same partition coefficient for a particular
combination of stationary phase, mobile phase and other conditions.

19. Ion exchange
• Stationary phase contains fixed charged groups and mobile counter
ions
• Counter ion exchange with ions of solute
• Reversible exchange of ions takes place between similar charged ions
of solute in the mobile phase and that of an ion exchange resin.

20. Size exclusion
• Retention depends on the extend to which the solute molecules are
trapped in the pores of inert stationary phase.
• This depends on size of molecules.
• Smaller molecules diffuses into the pores of the stationary phase –
Travel slower.
• Larger molecules do not enter the pores of stationary phase – Travel
faster.

21. Affinity
• Affinity chromatography is a method of separating a biomolecule from a mixture,
based on a highly specific macromolecular binding interaction between the biomolecule
and another substance.
• The specific type of binding interaction depends on the biomolecule of
interest; antigen and antibody, enzyme and substrate, receptor and ligand,
or protein and nucleic acid binding interactions are frequently exploited for isolation of
various biomolecules.
• Affinity chromatography is useful for its high selectivity and resolution of separation,
compared to other chromatographic methods.

22. Classification of Chromatography

23. Classification of Chromatography

24. Classification of Chromatography
• Adsorption Chromatography
1. Thin Layer Chromatography (TLC)
2. Column Chromatography
3. Ion Exchange Chromatography
4. High Performance (pressure) Liquid Chromatography (HPLC)
5. Gel Permeation Chromatography
6. Gas Solid Chromatography (GSC)
• Partition chromatography
1. Paper Chromatography
2. Gas Liquid Chromatography (GLC)

25. HOW TO CHOOSE A METHOD
• Polarity of the sample.
• Solubility and volatility of sample.
• Resolution required.
• Concentration of analyte.
• Detection limit.
• Physical and chemical properties of sample.

26. Uses of Chromatography
Real-life examples of uses for chromatography:
Pharmaceutical Company – determine amount of each chemical found
in new product
Hospital – detect blood or alcohol levels in a patient’s blood stream
Law Enforcement – to compare a sample found at a crime scene to
samples from suspects.
Environmental Agency – determine the level of pollutants in the water
supply
Manufacturing Plant – to purify a chemical needed to make a product

27. Applications
• Applications of Chromatography in the Pharmaceutical Industry
• Applications of Chromatography in the Food Industry
• Applications of Chromatography in the Chemical Industry
• Applications of Chromatography in the Field of Molecular Biology