About column chromatography.

1. Column Chromatography
history: The Russian botanist Mikhail Tsweet (1872-1919) regarded as the father of
chromatography. In 1906,Tsweet used to chromatography to separate plant pigments. He called the new
technique chromatography because the result of the analysis was “written in color” along the length of
the adsorbent column. The Greek word chrome means ‘color’ & graphein means to ‘write’. It plays a
very important role in chemistry & biological science because 12 Nobel Prizes have awarded between
1937 & 1972 alone for work in which chromatography played a vital role.
Column Chromatography: It may be defined as the selective adsorption & separation of a
mixture of chemical substance on a column of adsorbent through which a suitable solvent has been
passed.
types of Column Chromatography:
S.NO. Types of column
chromatography
Mobile phase Stationary phase Sample phase
01. Adsorption Chromatography Liquid Solid adsorbent solution
02. Partition Chromatography Liquid Immiscible solvent
on solid matrix
solution
03. Ion-Exchange
Chromatography
Liquid Ion exchange resin solution
04. Gel Chromatography Liquid Solvent held in the
interstices of a
polymetric solvent
solution
BasiC prinCiple: when a mixture of compounds dissolved in the mobile phase is introduced into
the column, the individual components move with different rates depending upon their relative affinities.
The compound with lesser affinity towards stationary phase moves fasters & it is eluted out of the column
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Mikhail Tsweet (1872-1919)

2. first. The one with the greater affinity towards stationary phase moves slower down the column & hence
it is eluted latter. Thus the compounds are separated.
terminology:
Stationary phase: It is also called as adsorbent. It is a solid.
Mobile phase: It is also known as solvent & eluent. It is a liquid.
Sample: It is also known as adsorbate which get adsorbs.
Elution: Process of removing the components from column.
Eluate: Separated compound.
Developers: This are the compounds/reagents which are used for the production of color for coloe
less substances.
praCtiCal requirements:
Stationary phase;
Mobile phase;
Column characteristics;
Preparation of the column;
Introduction of sample;
Development techniques;
Detection of components;
Recovery of components.
types of stationary phase:
Weak Medium Strong
Sucrose;
Starch;
Inulin;
Talc;
Sodium carbonate.
CaCO3;
Ca3(PO4);
MgCO3;
MgO.
Activated silica gel;
Activated alumina;
Activated charcoal;
Activated magnesia;
Fuller’s earth.
Fig: Column.

3. Different types moBile phases: In increasing order of Polarity or elution strength:
Cyclohexane<Carbondisulphide<Ether<Benzene<Toluene<Esters<Alcohols<Chloroform<Acetone<Wat
er<Pyridien<Organic acid.
paCking of the Columns: The adsorbent is applied to the column in two ways:
1. Slurry packing (Wet method): The adsorbent is suspended in to the mobile phase &stirred very
well to drive all air bubbles. The resulted slurry is then poured into the column. At the top end of the
column a pice of glass wool or cotton must be added before the slurry application. Sand may be added
after the slurry. After slurry application the column must be allowed to settle overnight.
2. Dry packing: In this method the adsorbent is poured to the column directly. Vibration is applied to
get ride of air bubbles then the mobile phase as passed through the adsorbent.
preparation of the Column:
It consists of a glass tube with the bottom portion of the column packed with glass wool/cotton
wool or may contain asbestos pad;
Above this adsorbent is packed;
After packing a paper disc is kept on the top, so that the adsorbent layer is not distributed during
the introduction of the sample;
Slurry is introduced into the column using funnels;
The level of the solvent must never be allowed to fall below the level of adsorebent to prevent
cracks.
Development teChniques: The developments techniques are categorized in to three types:
Elution analysis:
 Isocratic elution technique: In this elution technique, the same solvent or solvent
system of same polarity is used throughout the process of separation.
Fig: Column Preparation.

4.  Gradient/Stepwise elution technique: the solvents of gradually increasing
polarity or increasing elution strength are used during the process of separation.
Frontal analysis &
Displacement analysis.
DeteCtion:
For color compounds: Separated as different band & identified visually.
For color less compounds:
Absorption of light(UV/Visible);
Fluorescence or light emission characteristic;
Flame ionization detector;
Refractive index detector;
Evaporate the solvent & collect residue.
faCtors affeCting effiCienCy of a Column:
S.NO. Factor’s Effect’s
01. Particle size of the solid
stationary phase
Decrease in size improves separation.
02. Column dimensions Efficiency increases as ratio length.
03. Column temperature Increase in column temperature results in speed of
elution but does not improved separation.
04. Solvent It should be of low viscosity & high volatility.
05. Solvent flow rate Uniform & low follow rate gives better resulation.
Commonly useD aDsorBents for separation of ChemiCal Constituents
in Column Chromatography:
S.NO. Adsorbent Separable chemical constituents
01. Alumina, Magnesia Alkaloids, sterols, Vitamins.
02. Aluminium Chloride Sterols.
03. Calcium Carbonate Caratenoids, Xanthophylls.
04. Carbon Amino acids, Carbohydrates, Peptides.
05. Starch Enzymes.
06. Silica Gel Amino acids, sterols.
07. Magnesium silicate Alkaloids, Glycerides, Sterols.
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5. appliCation:
Separation of mixture of compounds;
Removal of impurities;
Isolation of active constituent;
Isolation of metabolites from biological fluids;
Estimation of drugs in formulation.
aDvantages:
Any type of mixture can be separated by column chromatography;
Wider choice of mobile phase;
Automation possible;
Any quantity of mixture can also be separated;
DisaDvantages:
Time consuming;
More amount of mobile phase are required;
Automation makes the technique more complicated & expensive.
referenCes:
01. A.H.BECKETI & J.B.STENLAKE, Practical Pharmaceutical Chemistry, 4th
edition, Part two, Page
No: 86-105.
02. ASHUTOSH KAR, Pharmaceutical Analysis Two, Page No: 162-181.
03. Dr. S.RAVI SHANKAR, Pharmaceutical Analysis, 3rd
edition, Page No: 13-4 to 13-13.
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