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Column

Column chromatography is a technique used for separating components in a mixture based on their adsorption to a stationary phase, typically silica or alumina, and a mobile phase, which is a liquid solvent. The process involves packing the column, adding the sample and mobile phase, and then allowing the components to separate based on their affinities to the stationary phase. Factors such as column length and packing technique can affect the efficiency of the separation, and various elution techniques can be employed to optimize the process.

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5 views31 pages

Column

Column chromatography is a technique used for separating components in a mixture based on their adsorption to a stationary phase, typically silica or alumina, and a mobile phase, which is a liquid solvent. The process involves packing the column, adding the sample and mobile phase, and then allowing the components to separate based on their affinities to the stationary phase. Factors such as column length and packing technique can affect the efficiency of the separation, and various elution techniques can be employed to optimize the process.

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Adsorption chromatography

Solid-liquid chromatography
What is column chromatography?
Components
1. Column (made up of glass)

2. Stationary phase/ adsorbent

3. Mobile phase (liquid)


Pure solvent or mixture of solvents.
column
Stationary phase
• Silica or Alumina commonly used.
• Both adsorb the polar components present in the
mixture/sample.
• Silica polar & acidic in nature. (most widely used)
• Alumina polar & basic in nature.
silica
Silica

1 Chemical Formula SiO2

2 it is acidic in nature

3 Higher Surface area 350-550 m2/gm

5 Pore diameter 20,60,100,300, 1000A0


Mobile phase

Petroleum ether (pet ether)


Obtained from petroleum refineries as the
portion of the distillate. • Chemically,
petroleum ether is not an ether; but mixture of
various volatile, highly flammable, liquid
hydrocarbon mixtures e.g., pentane, hexane
and heptane. Contains no aromatics. Because
of its lower cost, used instead of pentane,
hexane and heptane.
Factors affecting column efficiency

Column efficiency depends on column length width ratio.


Steps involved in column chromatography

1. Packing of column
2. Addition of sample
3. Addition of mobile phase
4. Development/ separation
5. Collection of components
6. detection
1. Packing of column
• Addition of stationary phase in column is called
packing of column.
There are two types of packing of the column, they
are

• 1. dry packing
• 2. wet packing (most commonly used)
Demerits/disadvantage:
Air bubbles are entrapped between mobile phase
and stationary phase and the column may not be
uniformly packed.
 Cracks appear in the stationary phase present in
the column.
Wet packing
This is the ideal technique.

• Slurry formation;
In a beaker, the required amount of stationary phase, is
added to the solvent, use a glass rod to mix the
slurry. Pour slurry into column.

Keep in mind that


There should be
No bubble in the column otherwise Not give
proper
Don’t let the column dry . Proper Separation.
2. Introduction of Sample in column
3. Introduction of mobile phase in column
the mobile phase is introduced from the top of the
column.
Elution techniques
1. Isocratic elution technique: 2. Gradient elution technique
• A single solvent • Solvents of gradually
composition or solvent of increasing polarity are used
same polarity is used during the process of
throughout the process of separation e.g. initially
separation. hexane, then chloroform,
• It may be polar or non polar then ethyl acetate.
solvent.
e.g. Use of chloroform alone.

A single solvent with increasing polarity or Mixture of different solvents


( polar & non polar) of increasing polarity can be used in Gradient elution
technique.
It may be polar or non polar.
4. How does separation take place?

• Sample is pushed down the column by


gravitational force.
How does separation take place?
How does separation take place?
• When the mobile phase is introduced from the
top of the column, the movement of the
individual components of the mixture is at
different rates.
• The components with lower adsorption to
stationary phase travel faster while the
component with greater adsorption and affinity
with the stationary phase will move slow.
• The components that move fast are removed first
whereas the components that move slow are
eluted out last.
Remember

• Higher the adsorption to the stationary phase,


the slower the molecule will move through
the column.

• Higher the solubility in the mobile phase, the


faster the molecule will move through the
column.
5. How to identify the separated component

Thin layer chromatography .


For structure determination Nuclear magnetic
resonance (NMR), IR spectroscopy is used.
Important Terms
Term Definition

1 Mobile phase or carrier solvent moving through the column


Stationary phase or
2
adsorbent substance that stays fixed inside the column

3 Eluent fluid entering the column


fluid exiting the column (that is collected in
4
flasks/test tubes). it is mixture of solvent &
Eluate component.
the process of washing out a compound
5
Elution through a column using a suitable solvent
mixture whose individual components have to
6
Analyte be separated and analyzed
Separating the polar component

B component is non polar , has no attraction


with silica (which is polar) . It has affinity or
solubility in mobile phase i.e. hexane (non
polar) so will move along with mobile phase &
elute first from the column.
Separating the polar component

To separate the polar component A , a polar mobile phase


will be used e.g ethyl acetate or acetonitrile etc.
Advantages & disadvantages of column chromatography
More silica is used.
Rotary evaporator

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