Principle and Overview of Chromatographic Procedures: Techniques on the basis of the state of mobile and stationary phase.

1. PRINCIPLE AND OVERVIEW
OF CHROMATOGRAPHIC
PROCEDURES
Presented by
Hadia Khadija (Mphil 1st Semester)
Presented to
Prof. Dr. M. W. Akhter
School of Biological Sciences, University of Punjab, Lahore

2. INTRODUCTION
 Technique for isolation, purification, and
characterization of biomolecules
 Developed by Mikhail Tswett, an
Italian-born, Russian botanist (1906)
 Twelve Nobel Prizes were awarded
between 1937 and 1972 for work related
to chromatography
Mikhail Tswett
(1872-1919)

3. MIKHAIL’S CHROMATOGRAPHY
 Separate plant pigments in
chloroplast
 Separating columns: glass tubes
packed with fine powders like sucrose
and chalk
 Poured petroleum ether-derived plant
extracts through the columns -
formation of yellow and green zones
 Result of the analysis was “written in
color”. Chroma means “color” and
graphein means to “write”.
Image source:
https://images.app.goo.gl/L8rR8
krUfv7smqxQ6

4. COMPONENTS OF CHROMATOGRAPHY
 Analytes: Molecules targeted for analysis
 Mobile phase: may be a liquid or gas, moves the sample components
through stationary phase
 Stationary phase/Sorbent: may be the solid or liquid, having the
ability to “bind” with analytes
 Supporting Medium: a solid surface on which the stationary phase is
bound or coated
 Effluent: mobile phase containing the purified analytes
 Chromatogram: the visual output of the chromatograph.

5. GENERAL PRINCIPLE
• Molecules get separated because they differ in the extent to
which they are distributed between the mobile phase and
the stationary phase.
• Distribution Coefficient or Partition Coefficient (Kd)
Kd =
𝒄𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏 𝒐𝒇 𝒂𝒏𝒂𝒍𝒚𝒕𝒆 𝒊𝒏 𝒔𝒐𝒓𝒃𝒆𝒏𝒕
𝒄𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏 𝒐𝒇 𝒂𝒏𝒂𝒍𝒚𝒕𝒆 𝒊𝒏 𝒎𝒐𝒃𝒊𝒍𝒆 𝒑𝒉𝒂𝒔𝒆

6. Fig. A
representation of
the principle of
chromatography.

7. PARTITION VS ADSORPTION CHROMATOGRAPHY
Partition Chromatography
 The distribution of analytes between the
two phases is based primarily on
solubility differences.
 Used for the separation and
identification of amino acids,
carbohydrates, and fatty acids.
 Paper, thin layer, and gas
chromatography.
Adsorption Chromatography
 Relies on relatively specific interactions
between the analytes and binding sites
on the surface of the sorbent.
 Most effective when applied to the
separation of macromolecules including
proteins and nucleic acids.
 Ion exchange chromatography

8. TECHNIQUES BY
CHROMATOGRAPHIC BED
SHAPE
COLUMN CHROMATOGRAPHY
PLANAR CHROMATOGRAPHY

9. COLUMN CHROMATOGRAPHY
 Stationary bed is within a tube
 The particles of the solid
stationary phase or the support
coated with a liquid stationary
phase may fill the whole inside
volume of the tube (packed
column) or be concentrated on or
along the inside tube wall leaving
an open, unrestricted path for the
mobile phase in the middle part of
the tube (open tubular column).
Image Source:
https://en.wikipedia.org/wiki/Chrom
atography

10. PLANAR
CHROMATOGRAPHY
 Stationary phase is present as or on a
plane
 Driving Forces – Capillary Forces
 Paper Chromatography – Cellulose Paper
 Thin Layer Chromatography – Thin
layer of adsorbent like silica gel,
alumina, or cellulose on a flat, inert
substrate

11. Image Source: Principles and Techniques of Biochemistry and
Molecular Biology, Seventh edition, EDITED BY KEITH WILSON
AND JOHN WALKER

12. TECHNIQUES BY PHYSICAL
STATE OF MOBILE PHASE
GAS CHROMATOGRAPHY
LIQUID CHROMATOGRAPHY

13. GAS CHROMATOGRAPHY
 Mobile Phase: Gas (e.g., Helium, Nitrogen)
 Stationary Phase: Solid/Liquid
 Driving Force: Gas Pressure/Flow
 Chromatographic separation is always carried out in a column,
which is typically "packed" or "capillary".

14. GAS CHROMATOGRAPHY
Image Source: Principles and Techniques of Biochemistry and Molecular
Biology, Seventh edition, EDITED BY KEITH WILSON AND JOHN
WALKER

15. LIQUID CHROMATOGRAPHY
 Mobile Phase – Liquid
 Stationary Phase – Solvated solids
 Driving Force – Pump flow
 High Pressure Liquid Chromatography (HPLC)
 The sample is forced by a liquid at high pressure (the
mobile phase) through a column that is packed with a
stationary phase composed of irregularly or spherically
shaped particles.

16. HPLC
Image Source: Principles and Techniques of Biochemistry and Molecular Biology, Seventh
edition, EDITED BY KEITH WILSON AND JOHN WALKER

17. TECHNIQUES BY
SEPARATION MECHANISM
ION EXCHANGE CHROMATOGRAPHY
SIZE-EXCLUSION CHROMATOGRAPHY

18. ION EXCHANGE CHROMATOGRAPHY
 Uses an ion exchange mechanism to separate analytes
based on their respective charges.
 Charged stationary phase – Ion-exchange resin
 Driving Force – Electrostatic interactions with opposite
charges

19. ION EXCHANGE CHROMATOGRAPHY
Anion-Exchange
Chromatography
 stationary phase has positive
charge
 exchangeable ion is an anion
Cation-Exchange
Chromatography
 stationary phase has negative
charge
 exchangeable ion is a cation
Image Source:
https://www.google.com/search?q=ion+exchange+chr
omatography
Sample
Molecule
Sample
Molecule

20. SIZE-EXCLUSION CHROMATOGRAPHY
 Separates molecules according to their size
 Smaller molecules enter the pores of the media and, therefore,
molecules are trapped and removed from the flow of the mobile
phase.
 Mobile Phase – Liquid
 Stationary Phase – Micelles
 Driving Force – Electric Field
https://www.google.com/search?q=size+exclusion+chro
matography

21. SIZE-EXCLUSION CHROMATOGRAPHY
Image Source: Principles and Techniques of Biochemistry and Molecular
Biology, Seventh edition, EDITED BY KEITH WILSON AND JOHN
WALKER

22. CHROMATOGRAM
 Typical response
obtained by
chromatography
 Concentration versus
elution time
Where:
tR = retention time
tM = void time
Wb= baseline width of the peak in time units
Wh= half-height width of the peak in time
units

23. RETENTION FACTOR
Rf = distance moved by substance
distance moved by solvent front
 For substances that are very soluble in the liquid Rf will be
close to 1.
 For substances that are rather insoluble in the liquid Rf
will be close to 0.

24. QUESTION ANSWER SESSION
1. In Column chromatography, the stationary phase is made of _________ and
the mobile phase is made of _________
a) Solid, liquid
b) Liquid, liquid
c) Liquid, gas
d) Solid, gas
2. Liquid chromatography can be performed in which of the following ways?
a) Only in columns
b) Only on plane surfaces
c) Either in columns or on plane surfaces
d) Neither in columns nor on plane surfaces

25. 3. Which of the following types of chromatography involves the process,
where the mobile phase moves through the stationary phase by the
influence of gravity or capillary action?
a) Column Chromatography
b) High Pressure Liquid Chromatography
c) Gas Chromatography
d) Planar Chromatography
4. The process of passing a mobile phase through a chromatography
column is called which one of the following?
a) Flushing
b) Washing
c) Elution
d) Partitioning

26. 5. Characteristic feature of any form of chromatography is the ___
a) Use of molecules that are soluble in water.
b) Use of an inert carrier gas.
c) Calculation of an Rf value for the molecules separated.
d) Use of a mobile and a stationary phase.
6. In Anion-Exchange Chromatography stationary phase has ______
charge and the exchangeable ion is _______ .
a) Positive, anion
b) Negative, cation
c) Positive, cation
d) Negative, anion

27. REARRANGE THE 2ND COLUMN IN 3RD COLUMN
Chromatographic Procedure Driving Force (incorrect
order)
Driving Force (correct order)
Paper Chromatography Electrical Field
Gas Chromatography Pump Flow
HPLC Capillary Force
Ion Exchange Chromatography Gas Pressure/Flow
Size Exclusion Chromatography Electrostatic Interactions
Thin Layer Chromatography

28. THANK YOU