Normal phase chromatography uses a polar stationary phase and a nonpolar mobile phase.
You might be aware that, chromatography is a separation technique that uses two phases, i.e., a stationary phase and a mobile phase.
The stationary phase is water-soluble and polar in nature. In contrast, the mobile phase is water-insoluble and less polar in nature.
In the initial phases of the discovery and development of chromatography, silica was widely used as the stationary phase.
This silica gel is polar in nature and hence this type of chromatography is a normal type.
While the other ones which do not employ silica gel or any polar stationary phase are considered to be reverse phase-type.
The mobile phase used in this type is some organic solvents that are nonpolar.
But interestingly, this method of chromatography finds less use in research and industry.
Instead, reverse phase chromatography is widely used. This is because of the wide advantages that the method offers.
Normal Phase Chromatography principle
In routine chromatography, sand or silica gel which is polar in nature, is used as a stationary phase.
The mobile phase used is mostly nonpolar and organic in nature.
When compounds like plant extracts are to be separated, those with polar nature tend to bind to the stationary phase.
While the compounds with nonpolar nature tend to move fast with the nonpolar mobile phase as they have less affinity to the silica phase
Hence the nonpolar compounds are eluted out first, leaving the polar ones inside. These compounds retained can be seen as colored bands called chromatograms.
This method avoids solvents like water, alcohol, and other highly polar ones being used as mobile phases.
Instead, nonpolar solvents like petroleum ether, hexane, etc., are widely used.
Normal phase chromatography elution order
Here the stationary phase in polar so, the components that are non-polar elute out first as they come out along with the mobile phase.
The polar like inorganic salts, acids, bases elute out last as they are adsobed to the stationary phase in the column.
So the order of elution for the different compound is as
- 1st Highly non-polar or fat soluble compounds
- 2nd Medially non-polar compounds
- 3rd Less non-polar compound
- 4th Less polar compounds
- 5th medially polar compounds
- Lastly highly polar compounds elute out.
Applications of normal phase chromatography
Though this method is less widely used, it is still a preferred method for
1. Column chromatography.
2. For the separation of nonpolar compounds in organic chemistry.
3. To separate plant extracts as the plant material has a lot of pigments, lipids, and nonpolar in nature.
4. When the mixture’s components are damaged by water, they can be separated by this method.
5. Also, isomers and compounds which are too hydrophilic and very hydrophobic can be separated well.
Disadvantages of this method
- Less range of compounds can be analyzed by this method as most of the compounds we use as medicine, food, and household chemical are polar in nature. They can not be analyzed by this method. Instead, reverse phase chromatography is preferred.
- In this method’s precise control of variables like solvent composition and pH is not easy.