26/11/21, 12:43 https://pediaa.

com/difference-between-normal-phase-and-reverse-phase-chromatography/

Difference Between Normal Phase And

Reverse Phase Chromatography
The main difference between normal phase and reverse phase chromatography is

that normal phase chromatography has a very polar stationary phase and a non-

polar mobile phase whereas reverse phase chromatography has a non-

polar stationary phase and a polar mobile phase. Furthermore, the stationary phase

of the normal phase chromatography is mainly pure silica, and the mobile phase is
a non-aqueous solvent such as chloroform while the stationary phase of the reverse

phase chromatography is a modified silica substrate with long hydrophobic long

chains and the mobile phase is mainly water, methanol or acetonitrile.  

Normal and reverse phase chromatography are two types of HPLC (high-performance

liquid chromatography) methods which operate under high pressure. Generally, they
have a higher resolution power when compared to the regular liquid chromatography. 

Key Areas Covered 

1. What is Normal Phase Chromatography

     – Definition, Process, Importance

2. What is Reverse Phase Chromatography

     – Definition, Process, Importance

3. What are the Similarities Between Normal Phase and Reverse Phase
Chromatography

     – Outline of Common Features

4. What is the Difference Between Normal Phase and Reverse Phase

Chromatography

     – Comparison of Key Differences

Key Terms 

https://pediaa.com/difference-between-normal-phase-and-reverse-phase-chromatography/ 1/8
26/11/21, 12:43 https://pediaa.com/difference-between-normal-phase-and-reverse-phase-chromatography/

HPLC, Liquid Chromatography, Mobile Phase, Normal Phase Chromatography, Reverse

Phase Chromatography, Stationary Phase

https://pediaa.com/difference-between-normal-phase-and-reverse-phase-chromatography/ 2/8
26/11/21, 12:43 https://pediaa.com/difference-between-normal-phase-and-reverse-phase-chromatography/

What is Normal Phase Chromatography 

Normal phase chromatography is a type of HPLC technique. It separates analytes
based on the degree of interaction towards the absorbent, which is polar silica.

Therefore, the stationary phase of this type of chromatography is hydrophilic. It can

also make hydrophilic interactions with the hydrophilic molecules in the sample
mixture. Generally, these interactions include hydrogen bonding, dipole-dipole
interactions, etc. Therefore, more non-polar analytes stay longer in the stationary
phase, increasing the retention time.

Figure 1: Normal Phase and Reverse Phase Chromatography – Properties

Furthermore, the mobile phase in the normal phase chromatography is non-polar and

non-aqueous. Therefore, non-polar or hydrophobic analytes in the mixture wash out

effectively with the mobile phase at the beginning of the process. Meanwhile, the

retention time of analytes reduces with the increasing polarity of the mobile phase.
https://pediaa.com/difference-between-normal-phase-and-reverse-phase-chromatography/ 3/8
26/11/21, 12:43 https://pediaa.com/difference-between-normal-phase-and-reverse-phase-chromatography/

Furthermore, the poor reproducibility of the retention time is the major drawback of

normal phase chromatography. Basically, this occurs due to the presence of a layer of

water or protic organic solvents on the surface of silica. However, this is eliminated in
the reverse phase chromatography. 

What is Reverse Phase Chromatography 

Reverse-phase chromatography is a type of recent HPLC. It has an increased

reproducibility of the retention time when compared to normal phase

chromatography. Basically, this increase of the reproducibility is achieved by making

the stationary phase non-polar. To do that, the surface of the silica stationary phase

is modified as RMe2SiCl, where R is a straight-chain alkyl group such as C18H37 or

C8H17.  However, due to the non-polar nature of the stationary phase, less polar

analytes in the sample mixture tend to have a higher retention time in contrast to
the normal phase chromatography.

Figure 2: Reverse Phase Chromatography – Elution

https://pediaa.com/difference-between-normal-phase-and-reverse-phase-chromatography/ 4/8
26/11/21, 12:43 https://pediaa.com/difference-between-normal-phase-and-reverse-phase-chromatography/

Moreover, one can increase the retention time by adding more water to the mobile

phase, which, in turn, increases the hydrophobic interactions between the non-polar
analytes and the stationary phase. Also, the mobile phase of the reverse phase

chromatography is polar, washing out polar analytes in the sample mixture. This
facilitates the separation of the non-polar analytes in the sample mixture.

Furthermore, the surface tension of the mobile phase, as well as its pH, have effects

on the retention time.  

Similarities Between Normal Phase and Reverse Phase

Chromatography  
Normal and reverse phase chromatography are two types of chromatographic

techniques of HPLC. 

Their schematic instrumentation includes a  degasser,  sampler, pumps, and a

detector.     

Both operate under high pressure.  

Moreover, their typical column dimensions are  2.1–4.6 mm in diameter, and 30–

250 mm in length.     
Both separate a small volume of a  sample.  

The separation is based on the different degrees of interactions of the

components of the sample with the adsorbent particles. These

interactions depend on the temperature.  

The smaller adsorbent particles (2–50 μm in average particle size) give a high-
resolution power to both types of chromatography.    

Furthermore, both types of chromatography give a quantitative analysis of the

sample components.    

They take around 2-60 mins per sample but, do not allow parallel analysis.  
The retention time of the chromatography can be increased by increasing the

interactions of the analytes with the column. 

 Analytes can be eluted by making the polarity of the mobile phase more similar
to the polarity of the stationary phase. 

https://pediaa.com/difference-between-normal-phase-and-reverse-phase-chromatography/ 5/8
26/11/21, 12:43 https://pediaa.com/difference-between-normal-phase-and-reverse-phase-chromatography/

Difference Between Normal Phase and Reverse Phase

Chromatography 

Definition 

Normal phase chromatography refers to a separation method which allows the

distribution of components of a mixture between two phases, one of which is a polar

stationary phase while the mobile phase is non-polar. In contrast, reverse phase

chromatography refers to the separation method, whose mobile phase is more polar
than the stationary phase. 

Evolution 

Normal phase chromatography was evolved in the 1970s in the form of liquid
chromatography. But, reverse phase chromatography is a recently evolved form of
HPLC. 

Stationary Phase 

Furthermore, normal phase chromatography uses a polar stationary phase, which is

mainly pure silica, while reverse phase chromatography uses a non-polar stationary
phase, which is a modified silica substrate with long hydrophobic long chains. 

Mobile Phase 

Normal phase chromatography uses a non-polar, non-aqueous solvent as the mobile

phase, which is mainly chloroform while reverse phase chromatography uses a polar
mobile phase, which is mainly water, methanol or acetonitrile.  

Types of Separation 

Moreover, normal phase chromatography separates polar analytes with high retention

time in the column, while reverse phase chromatography separates less polar
analytes, which have a high retention time in the column. 

Analytes in the Mobile Phase 

https://pediaa.com/difference-between-normal-phase-and-reverse-phase-chromatography/ 6/8
26/11/21, 12:43 https://pediaa.com/difference-between-normal-phase-and-reverse-phase-chromatography/

In the normal phase chromatography, the mobile phase carries non-polar analytes at

the beginning of the separation while in the reverse phase chromatography, the
mobile phase carries polar analytes.  

Increasing the Retention Time 

A non-polar mobile phase increases the retention time of normal phase

chromatography while a polar mobile phase increases the retention time of reverse
phase chromatography.  

Elution 

Analytes can be eluted by increasing the polarity of the mobile phase in the normal

phase chromatography while the analytes can be eluted by decreasing the polarity of
the mobile phase in the reverse phase chromatography. 

Stationary Phase Characteristics 

The stationary phase of the normal phase chromatography contains a layer of water

or protic organic solvent while the stationary phase of the reverse phase
chromatography does not contain water or a layer of protic solvent. 

Reproducibility of the Retention Time 

Moreover, normal phase chromatography has a poor reproducibility of the retention

time while reverse phase chromatography has a higher reproducibility of the

retention time.  

Damage to the Column 

The column of normal phase chromatography is easy to damage while the column of

the reverse phase chromatography is difficult to damage. 

Conclusion 

Normal phase chromatography is a type of HPLC which uses a polar stationary phase
and a non-polar mobile phase. As a result of this, non-polar analytes of the mixture

https://pediaa.com/difference-between-normal-phase-and-reverse-phase-chromatography/ 7/8
26/11/21, 12:43 https://pediaa.com/difference-between-normal-phase-and-reverse-phase-chromatography/

move out of the column easily while allowing the separation of polar analytes based
on the degree of interaction towards the absorbent of the stationary phase. On the
other hand, reverse phase chromatography is a type of recent HPLC, which uses

a non-polar stationary phase and a polar mobile phase. Therefore, polar analytes
move out of the column along with the mobile phase, allowing the separation of non-
polar analytes based on the degree of interaction with the stationary phase. Hence,
the main difference between normal and reverse phase chromatography is the type of

stationary and mobile phases.

References:

1. “HPLC Separation Modes.” Waters, Available Here.

Image Courtesy:

1. “HILIC Uses and Benefits” By Chem461S16Group4 – Own work (CC BY-SA 4.0) via
Commons Wikimedia  

2. “Reverse Phase Gradient Elution Schematic” By Nategm – Own work (CC BY-SA 4.0)

via Commons Wikimedia  

Salva

https://pediaa.com/difference-between-normal-phase-and-reverse-phase-chromatography/ 8/8