Unit 3 separation techniques Column & Short Column Chromatography 1. Basic Principles 2. Column packaging 3. Column development 4. Detection

1. COLUMN & SHORT
COLUMN CHROMATOGRAPHY
UNIT 3, SEPARATION TECHNIQUES (NP- 510)
PRIYANSHA SINGH (PC2021-14/226)
M.S. PHARM- PHARMACOLOGY & TOXICOLOGY
NIPER- GUWAHATI

2. CONTENTS
• BASIC PRINCIPLES
• COLUMN PACKING
• COLUMN DEVELOPMENT
• DETECTION

3. HISTORY &
BASIC
CONCEPT

4. TYPES OF COLUMN CHROMATOGRAPHY
ADSORPTION CHROMATOGRAPHY
PARTITION CHROMATOGRAPHY
GEL CHROMATOGRAPHY
ION EXCHANGE CHROMATOGRAPHY

5. • STATIONERY PHASE- aka adsorbent/
solid.
• MOBILE PHASE- aka solvent &
eluent/ liquid.
• SAMPLE- aka adsorbate which gets
adsorbed.
• ELUTION- process of removing the
components from column.
• ELUATE- separated component.
TERMINOLOGY

6. Adsorption Chromatography
• Stationery phase- solid
• Mobile phase- liq./ gas
PRINCIPLE OF ADSORPTION CHROMATOGRAPHY

7. Rate of movement of component in
adsorption chromatography

8. STATIONERY
PHASE

9. TYPES OF
ADSORBENTS

10. SELECTION
OF
ADSORBENT

11. PREPARATION OF STATIONERY PHASE

12. DEVELOPERS
e.g.- HYDROGEN SULFIDE. POTASSIUM FERROCYANIDE
THERE ARE COMPOUNDS/ REAGENTS WHICH ARE USED FOR THE
PRODUCTION OF COLOURS FOR THE COLOURLESS SUBSTANCES

13. MOBILE PHASE
• To introduce mixture into the column as solvent.
• To develop the zones for separation as developing agent.
• To remove pure component out of the column as the Eluant.
• Different mobile phases uses for e.g.- in increasing order of polarity/
elution strength
• Cyclohexane< Carbondisulfide< ether< benzene< toluene< esters< alcohols< chloroform< acetone< water<
pyridine< organic acids.

14. COLUMN CHARACTERISTICS

15. PACKAGING OF COLUMN
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16. Importance of packing
THE COLUMN MUST
PACKED AS UNIFORMLY AS
POSSSIBLE TO MINIMIZE
THE DISTORTION OF THE
CHROMATOGRAPHIC
BOUNDARIES.
CHANNELING IS USUALLY
CAUSED BY INCLUSION OF
AIR BUBBLES DURING
PACKAGING.

17. TYPES OF PACKAGING
DRY PACKAGING WET PACKAGING

18. INTRODUCTION OF SAMPLE

19. ELUTION
• GRADIENT/ STEPWISE ELUTION
• The solvents of increasing polarity/
elution strength are used during the
process of separation. Initially low
polar solvent is used followed by
the increasing polarity
• e.g.- initially benzene then
chloroform, ethyl acetate,
methanol etc.
• ISOCRATIC GRADIENT ELUTION
• In this elution technique, the same
solvent/ solvent system of same
polarity is used throughout
the process of separation.
• e.g.- chloroform only, petroleum:
ether= 1:1

20. Elution contd.....
ONE CARTIRIDGE CAN SEPARATE ALL 3 DYES

21. DETECTION OF COMPONENTS IN SAMPLE
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22. RECOVERY OF COMPONENTS IN SAMPLE

23. APPLICATIONS
SEPARATION OF MIXTURE OF
COMPONENTS
REMOVAL OF IMPURITIES/
PURIFIACTION
ISOLATION OF BIOACTIVE
COMPONENTS/ METABOLITES FROM
BIOLOGICAL FLUIDS
ESTIMATION OF DRUGS FROM CRUDE
EXTRACTS OR FROM FORMULATION.

24. FACTORS
AFFECTING
COLUMN
EFFICIENCY
1. DIMENSIONS
OF THE COLUMN
2. PARTICLE
SIZE OF THE
ADSORBENT
3. NATURE OF
THE SOLVENT
4.
TEMPERATURE
OF THE
COLUMN
5. PRESSURE
APPLIED.

25. ADVANTAGES v/s DISADVANTAGES of COLUMN
CHROMATOGRAPHY
ADVANTAGES
• Any type of mixture can be separated by
this method.
• Any quantity of mixture can also
be separated.
• Wider choice of mobile phase
• In preparative type sample can be
separated & reused.
• Automation is possible
DISADVANTAGES
• Time consuming
• More amount of solvents are required
which are expensive
• Automation makes the technique
more complicated.

26. Partition Column
Chromatography
• GRADIENT ELUTION MEANS TO
MAINTAIN A VARIABLE CONCENTRATION
IN THE MOBILE PHASE.

27. SHORT COLUMN CHROMATOGRAPHY
• A chromatography method where the stationary bed is within a
tube (of less than the standard length of 25 cm). The particles of
the solid stationary phase or 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).