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➢ Properties of adsorbent:
1. They should be insoluble in solvent.
2. They should be chemically inert.
3. They should be active but not so active that no movement of components occurs.
4. They should be colorless to facilitate the observation of zones (bands).
5. They should be allowing suitable flow of mobile phase.
6. They should have reproducible properties.
7. There particle size should be between 75-150 µ.
➢ Classification of adsorbents:
a. Weak affinity adsorbents
1. Sucrose
2. Starch
3. Insulin
4. Talc
5. Na CO
a. Medium affinity adsorbents
1. CaCO
2. MgCO
3. MgO
4. Ca(OH)
a. Strong affinity adsorbents
1. Activated alumina
2. Activated silica
3. Activated charcoal
4. Activated MgSiO
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➢ Advantages of Column chromatography
1. It can be used in both analytical and preparative applications.
2. It is used to determine the number of components of a mixture.
3. It is also used to separate and purify substantial quantities of those components for subsequent
analysis.
➢ Disadvantages of Column Chromatography
1. Properly setting up the column requires some technical skill and manual dexterity.
2. It is very time consuming and tedious, especially for large samples.
3. Collecting vessels must be frequently switched and solvent levels need to be topped up.
➢ Application of Column Chromatography
For analytical purpose, column chromatography has an unlimited value, although it is used to
determine amino acid contents of protein hydrolyte. Some other applications are----
1. Separation of----
a. 17-ketosteroid glucuronides
b. Urinary 17-ketosteroids
c. Plasma cortisols
d. Mixtures containing stereo isomers or related compounds
e. Geometrical isomers
1. Purification of technical products
2. Purification of homogeneity of colored compounds.
➢ Why silica is used as adsorbent?
Silica is used as adsorbent because------
1. Fine
2. Inert
3. Porous
➢ Solvents used in the Column Chromatography
Adsorption is most powerful from non-polar such as petroleum, ether or benzenes and a single
solvent may also often be effective in developing the chromatogram. The rate of movement of the
compounds down the column can be increased by the addition of a second solvent is usually to the
mobile phase. The second solvent is usually more polar then first.
List of solvents used in column chromatography is given below:-
➢ (Q) Suppose you want to separate a mixture of two colored compounds-one yellow, one blue. The
mixture looks green. The blue is more polar than the yellow one. Explaining what is happening? What
initiative should be taken if you want to collect the blue compound?
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Answer:
Suppose we wanted to separate a mixture of two colored compounds - one yellow, one blue. The mixture
looks green. Then we have to make a concentrated solution of the mixture preferably in the solvent used in
the column.
First I have to open the tap to allow the solvent already in the column to drain so that it is level with the top
of the packing material, and then add the solution carefully to the top of the column. Then I have to open
the tap again so that the colored mixture is all absorbed into the top of the packing material, so that it might
look like this:
Then I have to add fresh solvent to the top of the column, trying to disturb the packing material as little as
possible. Then open the tap so that the solvent can flow down through the column, collect it in a beaker or
flask at the bottom. As the solvent runs through, I have to keep adding fresh solvent to the top so that the
column never dries out.
The next set of diagrams shows what might happen over time.
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It is going to take ages to wash the blue compound through at the rate it is travelling at the moment! However,
there is no reason why I can't change the solvent during elution.
If I replace the solvent and I have been using by a more polar solvent once the yellow has all been collected.
That will have two effects, both of which will speed the blue compound through the column.
• The polar solvent will compete for space on the silica gel or alumina with the blue compound. Any
space temporarily occupied by solvent molecules on the surface of the stationary phase isn't available
for blue molecules to stick to and this will tend to keep them moving along in the solvent.
• There will be a greater attraction between the polar solvent molecules and the polar blue molecules.
This will tend to attract any blue molecules sticking to the stationary phase back into solution.
The net effect is that with a more polar solvent, the blue compound spends more time in solution, and so
moves faster.