1. COLUMN
CHROMATOGRAPHY
Presented By-
SK SAFIUR RAHAMAN
M.Pharm,Pharmacology,1st year
Department Of Pharmaceutical Technology
Maulana Abul Kalam Azad University Of Technology(WB)

2. CONTENTS
• Introduction
• Principle
• Classification
• Packing technique
• Factors affecting column chromatography
• Advantages
• Disadvantages
• Applications
• Conclusions
• References

3. Introduction
• The word chromatography comes from Greek words “Chroma” means colour and
“graphein” means write.so chromatography means ‘to write with colour’.
• Chromatography is a physical method of separation of a mixture in which the
mixture is dissolved in a fluid solvent called the mobile phase, which carries it
through a system called the stationary phase.
• There are a number of different kinds of chromatography, which differ in the
mobile and the stationary phase used.

4. Mobile phase
• Ideal properties:
It acts as a solvent
It acts as a developing agents
It acts as an eluting agents
Examples: Ethanol, Acetone, Water, Acetic acid, Pyridine, etc.
• Different mobile phases used:
Ex: In increasing order of polarity or elution strength.
Cyclohexane < carbon disulphide < ether < Benzene < toluene <ester < alcohol < chloroform <
acetone < water < pyridine <organic acids.

5. Stationary phase
• Ideal properties:
 High mechanical stability and chemically inert.
 Particle should have uniform shape and size(60-200 microns).
 It should be colourless, inexpensive and readily available.
 It Should allow free flow of mobile phase.
 It should be suitable for the separation of mixtures of various compounds.
• Ex: In increasing order of adsorbing strength-
sucrose < starch < inulin < talc < calcium carbonate < calcium phosphate < magnesia <
silica gel < magnesium silicate < alumina < charcoal.

6. Principle
• When a mixture of components dissolved in the mobile phase is
introduced into the column, the individual components move with
different rates depending upon their relative affinities.
• Which components have lesser affinity towards stationary phase
moves faster and eluted out first.
• The component which have greater affinity towards stationary
phase moves slower down the column and eluted out latter.
• The adsorption of solute molecules to the column occurs in a
reversible manner. Fig: 1
Ref:
https://www.sciencedirect.com/topics/biochemistr
y-genetics-and-molecular-biology/column-
chromatography [Accessed on: 31.01.2023;16:30]

7.  The rate of movement of component (R) is given by,
R= Rate of movement of component
Rate of movement of mobile phase
When a liquid mobile phase is used, equation is written as
R= Am/Am+ αAs
Where α = Partition coefficient
Am = Average cross section of mobile phase
As = Average cross section of stationary phase

8. Classification
These classifications are based upon stationary phase:
Types of column
chromatography
Mobile phase Stationary phase Sample phase
Adsorption
Chromatography
Liquid Solid adsorbent Solution
Partition
Chromatography
Liquid Immiscible solvent on
solid matrix
Solution
Ion exchange
chromatography
Liquid Ion exchange resin Solution
Gel chromatography Liquid Solvent held in the
interstices of a
polymeric solvent
Solution

9. Classification
Its depending on how the solvent flows down the column:
• Gravity column chromatography:- If the solvent is allowed to flow down the
column by gravity.
• Flash chromatography:- If the solvent is forced down the column by positive air
pressure. Nitrogen and argon is used.

10. Packing techniques
• There are two types of preparing the column:
Dry packing:
Adsorbent is packed in the column in fine dry powder form.
Fill the solvent till equilibrium is reached.
After filling tapping can be done to remove void spaces.
Ref: https://www.sciencedirect.com/topics/agricultural-and-
biological-sciences/column-chromatography
[Accessed on 29.01.23;22:50]
Wet packing:
The slurry of adsorbent with the mobile phase is prepared and is poured into the column.
Stationary phase settles uniformly and no crack and air bubbles present in the column of
adsorbent.

11. Factors Affecting column efficiency
Dimension of the column: column efficiency has been improved by increasing length/width ratio
of the column.
Particle size of column packing: separation to be improved by decreasing the particle size of the
adsorbent.
Temperature of the column: the speed of the elution increases at higher temperatures.
Quality of solvents: solvents having low viscosities is giving better results.

12. Advantages:
• Any type of mixture can be separated by column chromatography.
• Any quantity of mixture can also be.
• Wider choice of mobile phase.
Disadvantages:
• Time consuming method.
• More amount of solvent are required which are expensive.
• Drying and bubble presence are possible.

13. Applications
• Separation of mixture of compound or purify natural compound mixtures
like alkaloid, glycosides.
• Crude drug extraction.
• It is used for the separation of amino acid, proteins & carbohydrates.
• Isolation and purification of vitamins and hormones.
• Isolation of metabolites from biological fluids.
• It is used for the estimation of drugs in formulations.
• Examination of vegetable oil and pharmaceutical preparations.

14. Conclusions
We conclude that column chromatography is an effective way of
determining the components present in a compound such as a mixture
of pigments as well as in identifying the type of amino acid present.

15. References
• Yu Liu, Alec S. Lininger, Lauren N. McCaskey, Rachel M. Thomas. Separation of Fluorescent Protochlorophyllide from
Green Pumpkin Seed Using Column Chromatography. Journal of Chemical Education 2023, 100 (1) , 312-315.
• Knockaert, M. et al. Intracellular targets of cyclin-dependent kinase inhibitors: identification by affinity chromatography
using immobilised inhibitors. Chem. Biol.2000, 7, 411–422.
• Takeshi Tanaka, Yasuko Urabe, Takuya Hirakawa, and Hiromichi Kataura . Simultaneous Chirality and Enantiomer
Separation of Metallic Single-Wall Carbon Nanotubes by Gel Column Chromatography. Analytical
Chemistry 2015, 87 (18) , 9467-9472.
• Huaping Li, Lili Zhou, Tao Wu. Sodium dodecyl benzene sulfonate for single-walled carbon nanotubes separation in gel
chromatography. Diamond and Related Materials 2018, 88 , 189-192.
• Davies, Don R.; Johnson, Todd M. J. Isolation of Three Components from Spearmint Oil: An Exercise in Column and Thin-
Layer Chromatography. Chem. Educ. 2007, 84, 318.
• Figure 1: https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/column-
chromatography [Accessed on: 31.01.2023;16:30]
• Figure 2: https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/column-
chromatography [Accessed on 29.01.2023;22:50]

16. Thank You