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Mechanism of Reversed Phase HPLC

Reversed phase HPLC is characterized by a situation in which the mobile phase used is MORE POLAR than the
stationary phase

The name ‘Reversed Phase’ arises as this was the second, (chronologically), mode of chromatography after Normal (or ‘Straight’) phase in which a polar stationary phase is used in conjunction with a less polar mobile phase.

Typical reversed phase stationary phases are hydrophobic and chemically bonded to the surface
of a silica support particle (Figure 1). 

Figure 1: Schematic representation of Reversed Phase HPLC

The most commonly used stationary phases are shown (Figure 2). Other support materials and bonded phases are available.

Figure 2: Common reversed phase HPLC stationary phases.

For more information on bonded phases see the Column Chemistry module


For neutral analytes, the mobile phase consists of water (the more polar component) and an organic modifier that is used to vary the retention of analytes by lowering the polarity of the mobile phase.

Increasing the water content will repel (‘squeeze’) hydrophobic (non-polar) analytes out of the mobile phase and onto the non-polar stationary phase where they will reside for a time until ‘partitioning’ out into the mobile phase again. Each ‘on-off’ partition is called a ‘Theoretical Plate’.

When ionizable (or ionic) analytes are present, other additives such as buffers or ion pairing reagents can be added to the mobile phase to control retention and reproducibility.

Figure 3: Representative reversed phase chromatogram detailing analyte retention order based on hydrophobicity or hydrophilicity.

The Chromatogram shown (Figure 3) illustrates the general elution order of hydrophilic and hydrophobic analytes.  When working with ionizable analytes the hydrophobicity and, hence, retention characteristics of the analyte will be affected depending on its ionization state (ionized or non-ionized), this will be discussed later in the module.
 
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