What is reversed phase liquid chromatography (LC) / HPLC?

Reversed phase chromatography is the most commonly used LC or HPLC separation mode. It is used to separate nonpolar molecules in solution. In reversed phase the stationary phase is nonpolar and the mobile phase is polar. The name “reversed phase” is derived from the opposite technique of “normal phase” chromatography which involves the separation of molecules based upon their interaction with a polar matrix (silica beads) in the presence of a nonpolar mobile phase (nonpolar solvent).

This chromatographic technique is far superior to the other modes of separation in the variety of target compounds it can analyze.

The mechanism in reversed phase separation is shown in Fig. 1 and  has to do with the binding of a solute - having both hydrophobic and hydrophilic parts (groups) -  to a stationary hydrophobic molecule (stationary phase) in a polar solvent. This partitioning occurs as a result of the solute molecule tending to have hydrophobic groups, and binding via those groups to the hydrophobic stationary phase. A buffer of increasing hydrophobicity is used to dissociate the bound molecule at a point at which the hydrophobic interaction between the exposed groups and the stationary phase is less favorable than the interaction between the bound molecule and the solvent. The molecule releases from the stationary phase and elutes.

 

Fig. 1: Reversed phase chromatography mechanism. The hydrophobic solute (nonpolar solute) binds to the surface of the hydrophobic (nonpolar) C-18 chain. A more polar mobile phase is used for solute elution to occur. The polarity of the mobile phase is decreased by changing its composition as elution progresses (gradient elution) to speed up the process.

 Two types of reversed-phase chromatography column packing are used:

Silica gel with chemically bonded alkyl-chains

Resin-based packing

Typical alkyl groups that are chemically bonded to the silica gel are the octadecyl group (C18), the octyl group (C8), the trimethyl group, phenyl groups. The cyanopropyl group is also used. Since modern reversed phase chromatography typically refers to the use of chemically bonded stationary phases, where a functional group is bonded to silica, it is also known as bonded-phase chromatography.

In case more hydrophobic packing is required or resistant to extreme pH values (<2 or >8)  then resins/polymers (i.e. copolymers of styrene-divinylbenzene) or graphitized carbon can be applied.

The mobile phase composition used in reversed phase chromatography is based on a polar solvent, typically water, to which a less polar organic solvent such as acetonitrile or methanol is added. The three major factors that influence separation are: 

• type of organic solvent

• proportion of organic solvent

• pH of the solvent

Acetonitrile and methanol are the most commonly used organic solvents.

Solvent selectivity is controlled by the nature of the added solvent as shown below:

• Solvents with large dipole moments (methylene chloride) interact with solutes that have large dipole moments (nitriles, amines, nitro-compounds).

• Solvents that are good proton donors (water, chloroform) interact best with basic solutes (amines, sulfoxides).

Reversed phase chromatography is the most popular technique for the separation of low molecular weight (<2500), neutral species that are soluble in water or other polar solvents. It is widely used in the following areas:

• in the pharmaceutical industry for separation of compounds such as vitamins, steroids, β-blockers.

• in the food and beverage industry for analysis of sweeteners, food additives and carbohydrates. 

• in the chemical industry is used for analysis of polymer additives.

• in the field of environmental monitoring for analysis of pesticidesand herbicides.

• in clinical analysis for the determination of catecholamines.

 Because of the fact that the mobile phase in reverse phase chromatography is polar, reverse phase chromatography is applied to the separation and determination of polar molecules that either are insoluble in organic solvents or are attached too strongly to the polar stationary phase in normal phase chromatography. A large number of biological molecules fall into this category such as amino acids, peptides, proteins, nucleic acids and oligosaccharides.

 

References

 L.R. Snyder. J.J. Kirkland, “Introduction to Modern Liquid Chromatography”, 2^nd edition, Wiley, 1979

A. Weston and P. Brown, “HPLC and CE Principles and Practice”, Academic Press, 1997

C.F. Poole, S.K. Poole, “Chromatography Today”, Elsevier, New York, 1991