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