Different Types of HPLC Columns Used in Analysis

Columns are the main component in HPLC because the column is responsible for the separation of the sample components. The sample passes through the column with the mobile phase and separates in its components when it comes out from the column.

Generally, silica gel is filled in the high-performance liquid chromatography columns because of its particle size and porosity that helps in separation of components and silica gel is also an inert material that does not react with mobile phases. Therefore silica columns can be used to analyze the compounds of different chemical natures. The material filled in the HPLC columns is known as a stationary phase.

There are different types of chromatography columns on the basis of their composition and method of separation. Some of them are described here.

1) Normal Phase Columns

2) Reverse Phase Columns

3) Ion Exchange Columns

4) Size Exclusion Columns

1) Normal Phase HPLC Columns:

This type of columns has more polar stationary phase than the mobile phase. The packing material of the column should be more polar than the mobile phase and this condition is fulfilled by the silica that is polar material. But water is more polar than the silica, therefore, water is not used and methylene chloride, hexane and chloroform or a mixture of these with diethyl ether is used as mobile phase.

Separation of the sample components occurs on the basis of the polarity of the sample components. Sample components having more polarity interact more with polar stationary phase resulting in separation from the less polar component that interacts with less polar mobile phase. Silica columns are widely used in the pharmaceutical analysis. The chromatography column packing in which normal phase columns are used is known as Normal Phase Chromatography.

Related: Working and Principle of HPLC

2) Reverse Phase HPLC Columns:

In reverse phase columns as its name states, it is reverse of the normal phase columns. It has a non-polar or less polar stationary phase than the more polar mobile phase. Bonded hydrocarbons like C8 and C18 and other non-polar hydrocarbons are used as stationary phase in reverse phase columns while aqueous organic solution like water-methanol or water-acetonitrile mixture is used as mobile phase.

Separation of sample components in reverse phase columns also occurs on the basis on the polarity of the sample components but it happens just opposite of the normal phase HPLC columns, therefore, this type of chromatography is known as Reverse Phase Chromatography.

3) Ion Exchange HPLC Columns:

The compounds those can easily ionize are analyzed using these columns. Stationary phase in these columns remains acidic or basic having negative or positive charge while mobile phase is a polar liquid as the salt solution in water. Separation of molecules occurs on the basis of the attractive ionic force between molecules and the charged stationary phase. Due to the exchange of ions during the separation of sample components, it is known as Ion Exchange Chromatography.

Related: Determination of Theoretical Plates in Columns

4) Size Exclusion HPLC Columns:

Porous stationary phase in these columns allows the separation of the components according to their size. Combination of polymers like polysaccharides and silica is used as stationary phase in these columns. Small sample molecules penetrate in the pores of stationary phase while the large molecules penetrate partially into the pores. Therefore the large molecules of the sample elute first than the small molecules and this chromatography is called Size Exclusion Chromatography. These columns are generally not used in the analysis of pharmaceutical compounds.

HPLC columns have a different length varying from 30 mm to 250 mm and their particle size or porosity from 3µ to 5µ. These factors affect the analysis of sample, therefore, these are considered important during the HPLC analytical method development. Columns are selected according to the nature of the compound to be analyzed and the mobile phase. Column performance should also be evaluated time to time generally after 1000 runs or as required.