Chromatography is a primary separation technique which is used in chemical analysis. It isolates the sample mixture by continuous partitioning between the mobile and stationary phases. The principle of chromatography involves the isolation obtained by dissolving compounds in a mobile phase and traveling over a stationary phase. Each compound of sample mixture has different retention time, as the compound with strongly interacting with the stationary phase appear to travel very slowly, while compounds that have less affinity tend to elute rapidly.
The high-performance liquid chromatography is one of the most powerful separation techniques since it can apply different types of separation techniques for different types of molecules. The reversed-phase chromatography is a type of HPLC that is currently the most broadly used method of isolation in HPLC since it is versatile, simple, separate the analytes of diverse polarity and molecular mass, and has more scope of the technique.
The principle of reversed-phase chromatography depends on the interaction of the solutes with the hydrophobic group. It has analytical and preparative applications in the field of biochemical isolation and purification. It is typified by a mobile phase of polar properties and a stationary phase with non-polar properties. The reversed-phase chromatography does works in contrast to normal phase chromatography.
Separation mechanism of reversed-phase chromatography:
The separation mechanism in reversed-phase HPLC generally depends on the distribution of components between the mobile phase and the stationary phase. This process can also be interpreted as a type of component extraction between the stationary phase and the mobile phase. In the stationary phase, a non-polar substance dissolves preferentially, remains there preferentially, and is thus gradually eluted from the chromatographic column. Polar substances are easily eluted from the column, as they tend to remain in the solvent system. Subsequently, the retention time of the solutes varies as their distinct affinity for the stationary phase.
The stationary phase in reverse phase chromatography:
The stationary phase of reversed-phase HPLC is usually composed of hydrophobic alkyl chains that interact with the solutes. Octadecyl carbon chain (C18) bonded silica is the most popular column used in RP-HPLC as it has a longer carbon chain compared with the C4 and C8.
The mobile phase of reversed-phase chromatography:
The mobile phase used in reversed-phase HPLC has a certain amount of water or aqueous buffers and organic solvents are used to isolate as well as to obtain a symmetrical peak shape. The water-miscible solvents such as methanol, acetonitrile, and tetrahydrofuran are most commonly used organic solvents. The retention of ionizable compounds is very sensitive hence the pH of the mobile phase or buffer can play an important role.
Commonly asked questions on chromatography is as follows.
Why is reversed-phase chromatography useful?
The RP_HPLC is most widely used technique amongst the different types of HPLC chromatography since it has a hydrophobic stationary phase and can be used with the polar (hydrophilic), non-polar (hydrophobic), ionic and ionizable molecules to isolate their different molecules, which is depends on the procedure being used.
Which compound will elute first in a reversed-phase HPLC separation?
Polar compounds will elute first in a reversed-phase HPLC separation. The polar (aqueous) mobile phase is used in the reversed-phase HPLC As a consequence, in the polar solvent system hydrophobic solutes begin to adsorb into the hydrophobic stationary phase, and in the mobile phase, hydrophilic solutes pass through the column and are first eluted.
What is the difference between the normal phase and reversed-phase chromatography?
The major difference between reversed-phase chromatography and normal phase chromatography is that reverse phase chromatography has a non-polar stationary phase and a polar mobile phase while normal phase chromatography has a very polar stationary phase and a non-polar phase.
What is the major advantage of reversed-phase chromatography?
The major advantage of reversed-phase HPLC is that its hydrophobic stationary phase works well for the retention of most organic analytes. This allows the aqueous mobile phase to be used that includes water and the type of buffer solution such as phosphate, acetate, etc. RP-chromatography also has the advantage of being able to use pH according to the pKa of the molecule to improve the separation.
What are the major types of chromatography?
There are various types of chromatography has, such as high-performance liquid chromatography (HPLC), gas chromatography (GC), column chromatography, paper chromatography, thin-layer chromatography (TLC), and mass spectroscopy (LC-MS), etc.
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