Normal-phase and Reverse-phase chromatography

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The two chromatographic modes known as the normal-phase and the reverse-phase chromatography lends chromatographic separation some unique, task-specific characteristics. A complex analyte mixture that we are aiming to separate certainly consist of individual components with varying polarities and a diverse nature. How can we then separate and identify these components? Is it possible to apply the principle of like-attracts-like and retain components of interest onto the stationary phase while flushing everything else with the mobile phase? If yes, How? This article addresses all these questions so keep reading.

The polarity of the stationary phase and the mobile phase can be altered as per chromatographic requirement. The distinction between the two phases on the basis of polarity gives birth to the two types of chromatography: normal-phase and reverse-phase as discussed below. But, before proceeding further, you may like to read some basics regarding the difference between a stationary phase and a mobile phase.

What is normal-phase chromatography

Normal-phase chromatography is a type of chromatographic separation in which the stationary phase is relatively more polar than the mobile phase. In other words, the mobile phase in normal-phase chromatography is non-polar or relatively less polar than the stationary phase.

Two of the most common examples of normal-phase chromatography includes paper chromatography and thin-layer chromatography (TLC). Water trapped in the cellulose layers of the chromatographic paper acts as the more polar stationary phase in paper chromatography as compared to the organic solvents used as mobile phases. For TLC, the highly polar silica gel is used as the stationary phase while non-polar and/or less polar organic solvents are employed as mobile phases against it.

We recommend our special articles on paper chromatography and TLC to read more interesting facts about these two chromatographic techniques.

High-performance liquid chromatography (HPLC) can also be performed in a normal-phase mode by using a column packed with a polar stationary phase material such as acid-activated bare silica. Pure, microporous, spherical silica particles act as a highly polar stationary phase due to the presence of hydroxyl (OH) functional groups on its surface. It possesses a high surface area; hundreds of square meters per gram which provides a large space for analyte retention. Different types of functional group moieties present on a silica surface in normal-phase chromatography is as shown in the figure below.

Polar analytes from a complex mixture gets retained onto this polar stationary phase primarily by hydrogen bonding. Other forces of attraction such as dipole-dipole interactions and weak intermolecular forces of attraction including the London dispersion force may also be involved in analyte retention on polar silica while performing normal-phase column chromatography. Non -polar components on the other hand elute out first, with the mobile phase.

Normal-phase chromatography

What is reverse-phase chromatography

Reverse-phase chromatography is a type of chromatographic separation in which the stationary phase is relatively less polar than the mobile phase. In other words, a hydrophobic/non-polar stationary phase is used in reverse-phase chromatography while the mobile phase is usually water-based or composed of highly polar solvents such as ethanol and methanol.

Column chromatographies such as HPLC are preferably performed in a reverse-phase mode as opposed to a normal-phase chromatographic separation. The silica we talked about in normal-phase HPLC, can be made non polar by a chemical treatment called silanization. Silanization refers to covering the bare silica surface with organofunctional alkoxysilane molecules. Long hydrocarbon chains are attached to the surface of silica to retain non-polar analyte components.

Silica can be treated with a propyl (C3H7), a butyl (C4H9), an octyl (C8H17) or an octadecyl (C18H37) functional group to prepare a non-polar stationary phase, valuable for reverse-phase chromatography. A silane coupling agent such as trimethyl silane is essential for the silanization reaction. Longer the hydrocarbon chain attached, less polar the silica surface. Octadecyl silane (ODS) columns are a popular choice for reverse-phase HPLC.

Low polarity analyte components get strongly retained on this non-polar surface by hydrophobic interactions, π-π stacking between alkyl chains of stationary phase and aromatic rings of the analyte etc. More polar components however elute out of the column with the mobile phase. The polarity of the mobile phase can consequently be decreased in order to overcome the force of attraction between analyte and the stationary phase thus eluting the non-polar components as well.

Reverse-phase chromatography

Hydrophobic interaction chromatography (HIC) is a special type of reverse-phase chromatographic technique.

Scope and Applications

In conclusion, both types of chromatographic modes; normal-phase and reverse-phase have their special applications in the chromatographic world. For instance, normal-phase is applied for separating amino acids, metal complexes and inorganic dyes. Reverse-phase chromatography on the other hand is important for isolating and purifying biomolecules, pharmaceutical drugs and plant metabolites etc.

Similarly, if we have to separate extremely non-polar components from an analytical mixture for example; lipids present as an interference in a biological sample, we will apply a normal-phase chromatographic mode. Lipids will at once pass out of the column with the non-polar mobile phase, making our route to separate other components easier.

Test Yourself!

Q) The analytical chemist, John whom we introduced to you in What is Chromatography has three components in another mixture M’ provided to him. Their polarities are as given below.

ComponentDipole Moment/Debye (D)
A5.76
B17.4
C16

John is asked to separate M’ into A, B and C via thin-layer chromatography. He obtained 3 spots on his TLC plate as shown below. Can you identify spots 1,2 and 3 into A, B and C. John wants to know on what basis are you making your judgment?  

Ans) TLC is a type of normal-phase chromatography. More polar solutes are strongly retained onto the polar stationary phase while relatively less polar components travel up the plate to a longer distance, with the non-polar mobile phase thus spot 1= B, spot 2=C and spot 3=A.

References

1. Dang, A., J. J. Pesek and M. T. Matyska (2013). “The use of aqueous normal phase chromatography as an analytical tool for food analysis: Determination of histamine as a model system.” Food Chemistry 141(4): 4226-4230.

2. Snyder, L. R., J. J. Kirkland and J. L. Glajch (2016). Practical HPLC Method Development.

Normal-phase and Reverse-phase chromatography

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