The reverse phase chromatography is a type of HPLC chromatography; it is working on the principle of hydrophobic interactions and is one of the most popular techniques, as it applies to a wide variety of analytes. The RP- chromatography is a commonly used separation technique, in which the molecules are separated based on polarity. The polar mobile phase and a non-polar stationary phase are used in RP-chromatography. The more hydrophobic molecules are the more strongly attaches to the column and the more volume of solvent required to elute the molecule. Hence, the retention times are longer for non-polar components, while polar compounds are more readily elute from the stationary phase.
The advantages of reversed-phase chromatography are as follows.
- It is an economical method compared to other chromatographic techniques.
- RP-HPLC allows water to be used in the composition of the mobile phase with other solvents.
- Another advantage of using reversed-phase chromatography is that it provides accurate results with small amounts of sample.
- RP-chromatography also has the advantage of being able to use pH selectivity to improve the separation.
- The hydrophobic stationary phase in reverse-phase columns works well for the retention of most organic molecules.
- In RP-chromatography we can use pH selectivity to get better separations.
- About 75 percent of all HPLC methods use reversed-phase chromatography.
The disadvantages of reversed-phase chromatography are as follows.
- Water-insoluble compounds and amines can be more difficult to analyze.
- In RP-HPLC need to create pressure.
- It requires technical capability and skill to handle the system.
- The silica of the reversed-phase column can be dissolution at pH > ~7.5.
- The eluted sample from the column cannot be recovered.
- Additional techniques are needed to confirm the identity of the analytes.