Different types of HPLC columns are used to separate the analytes according to nature, composition, and separation modes of HPLC.
The column is the basic component in the analysis of the high-performance liquid chromatography as it is responsible for the separation of compounds. The sample solution moves through the column along with the mobile phase and it comes out of the column as the separate into its components. In general, silica gel is filled in columns due to its porosity and size of particle which helps to separate the compounds, and it is also inert solid support which does not react with solvents. Thus the silica columns are used in the analysis of compounds. Liquid chromatography can be classified into several modes. If the classification of chromatography depends on the stationary phase and the nature of the separation method, then it may be adsorption chromatography, size exclusion chromatography, and ion-exchange chromatography. With respect to the first type, there are two separation modes are based on polarity which is reverse-phase chromatography and normal-phase chromatography, these modes cover about 90% of all chromatography applications.
There are various types of HPLC columns are available for liquid chromatography depending on their composition and separation system, let’s check it.
Reversed-phase HPLC columns:
The reversed-phase chromatography has a non-polar stationary phase and a polar mobile phase. Bonded hydrocarbons such as C8 and C18 and other non-polar hydrocarbons are used in reverse phase columns as a stationary phase, while an aqueous organic solvent such as acid, buffer, methanol, acetonitrile, or a suitable mixture of it is used as a mobile phase. The isolation of molecules in reversed-phase columns often takes place based on the sample polarity, but this is unlike the normal phase HPLC column so this type of chromatography is known as reverse phase chromatography.
The commonly used columns in reversed-phase are:
- C18 RP-HPLC columns
- C8 RP-HPLC columns
- Alkyl reversed-phase columns
- Phenyl reversed-phase columns
- C4 RP-HPLC columns
- C1 RP-HPLC columns
- C30 RP-HPLC columns
Normal phase HPLC columns:
Normal-phase chromatography is a type of liquid chromatography it is used for the isolation of positional isomers which are complex to separate in RP-HPLC. The normal-phase chromatography has a more-polar stationary phase and a non-polar mobile phase. The solid support of the column is more polar hence rather than the water hexane, methylene chloride chloroform, diethyl ether, or a mixture of these used as mobile phase. Separation in normal-phase chromatography is based on the polarity of the analytes; more polar analytes interact more with the stationary phase.
The commonly used columns in normal-phase are:
Silica normal phase columns: these are potent and efficient column for isolating non-polar and moderately polar isomeric molecules.
Amino normal phase columns: These types of columns are mainly useful for the isolation of carbohydrates.
Cyano normal phase columns: These types of columns have an optional selectivity and are more rapidly equilibrated, providing better suitability for normal phase gradient isolation than unbonded silica columns
Ion-exchange HPLC columns:
Using these columns, the molecules which can quickly ionize are analyzed. In these columns, the stationary phase remains acidic or basic for a positive or negative charge, whereas a polar mobile phase used in the form of a salt solution in water. The isolation of components takes place between the components and the charged stationary phase based on the attractive ionic force. For stationary phase different mediums are used, the most generally used immobilized charged groups are trimethylaminoethyl, diethyl-2-hydroxypropylaminoethyl, triethylaminoethyl, diethylaminoethyl, aminoethyl, sulphomethyl, sulphopropyl, and sulpho. Successful column packing is a significant aspect of ion-exchange chromatography. Polystyrene is used for the separation of small molecules in amino acids as a medium. The cellulose medium can be used to isolate large solutes since they have large pores.
Size-exclusion HPLC columns:
Size exclusion chromatography (SEC) is also known as gel filtration chromatography. The size exclusion chromatography columns are typically filled with porous polystyrene divinylbenzene or silica particles. These columns are generally used for the analysis of synthetic polymers in organic solvents, whereas to separate the biopolymers the silica-based columns are used. The porous stationary phase in these columns allows the analytes to be separated as per their size. Small sample components enter the pores of the stationary phase, whereas larger molecules partially penetrate the pores. Thus the bigger sample components are eluted rapidly than the small molecules. Generally, these columns are not used for the analysis of pharmaceuticals.
Different types of gels in HPLC columns:
Silica gel: Silica gel is the most commonly used packing material. There are two types of silica gels are available such as spherical and irregular shape, out of these the spherical shaped gels are most commonly used. The silica gel used in liquid chromatography has a pore on the surface; it gives a bigger surface area than without holes. The 5μm is the most widely used size, although smaller sized 1.5 to 3μm gels is also used in HPLC columns.
Polymer gel: Silica gels are commonly used in HPLC development, although, polymer-based columns are also well-liked. Polyethylene and polypropylene are commonly known polymers for column packing. Columns of polymer gel are available in very small particles, like the silica gel.
Other gel: In addition to silica and polymer gels, the gels used include natural substances such as cellulose, chitosan, dextrin, and ceramics are used in liquid chromatography to isolate analyses but have very limited use.
The column is the most important part in the HPLC system, the actual isolation of molecules of the sample mixture happens in the column. A column is located after the injector where the mobile phase is in contact with the stationary phase, forming an interface with the enormous surface. In recent years, the development of most chromatography has contributed to the creation of several different methods for through this interfacial contact. Usually, RP-HPLC columns are available in lengths of 30 to 250 mm, the diameter of 01 to 05 mm, and pore size of 03, 05, and 10 μm.
Commonly asked questions on chromatography are as follows.
How many types of HPLC are there?
Depending on the stationary phase in the process, there are 4 types of HPLC, such as normal Phase HPLC, reverse phase HPLC, size-exclusion HPLC, and ion-exchange HPLC.
What is the difference between C8 and C18 HPLC columns?
The main difference between the C8 and C18 columns is their carbon atoms. C8 has 8 carbon atoms, whereas C18 has 18 carbon atoms. C8 has a small carbon chain, whereas C18 has a longer carbon chain. C8 has a short, whereas C18 has longer retention time (RT).
Which type of HPLC detector is most widely used?
The UV/VIS and PDA detectors are the most commonly used detector in HPLC as they give superior sensitivity for light-absorbing compounds.
Which is better GC or HPLC?
The gas chromatography (GC) is better for the separation of volatile compounds, whereas the high-performance liquid chromatography (HPLC) is better for the separation of less volatile samples.
What is the basic principle of chromatography?
There are different types of chromatography, although they operate on the same principle. They have a stationary phase and a mobile phase. The mobile phase has a sample mixture moves on the stationary phase, some solutes have an affinity for the stationary phase will travel slowly, While solutes that do not interact with the stationary phase will move faster.
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