Normal phase liquid chromatography (LC) / HPLC?

In normal-phase chromatography a polar stationary phase is used in conjunction with a less polar mobile phase for elution of the analytes (in contrast to reversed phase chromatography where a nonpolar stationary phase is used with a more polar mobile phase). Neutral solutes in the mobile phase are separated on the basis of their polarity. The more polar the solute  the more it is retained on the stationary phase. The mobile phase that it is used is normally less polar than the stationary phase. If the polarity of the mobile phase continuously increases (in a gradient elution scheme) the solute retention is decreased.

The stationary phases used in normal phase chromatography are usually silica or alumina and they are polar as a result of hydroxyl groups (-OH) (Fig. 1). The surface hydroxyl groups interact with the functional groups on the solute molecules and depending on the strength of this interaction preferentially absorb one solute relative to another (absorb the more polar compound relative to the less polar). In case the solute is a neutral molecule that has a permanent dipole or if a dipole can be induced on it, then it will be attracted by dipole-dipole interaction to the stationary phase.

A mechanism that describes the adsorption process in normal phase liquid chromatography is shown in Fig. 1.

Fig. 1: The mechanism of retention of the solute Ph-OH is shown in a typical normal phase chromatography separation. An equilibrium is established between the charged and the protonated form of the solute. The charged form binds to the surface of the polar stationary phase and competes for the same positions with solvent molecules. If the polarity of the solvent is increased then more solvent molecules bind to the surface of the stationary phase and the solute (PhOH) elutes faster since it remains relatively unretained.

 

Silica is the preferred stationary phase mainly because its availability, known performance and low cost. For basic compounds such as amines, which are very strongly retained on silica, alumina can be used as an alternative. In addition to the above stationary phases, a variety of polar bonded phases are used with functional groups such as cyano [-(CH₂)₃CN], amino [-(CH2)_nNH₂], diol, bonded to the silica stationary phase.

The mobile phases used in normal phase chromatography are nonpolar hydrocarbons such as hexane, heptane, octane (Table I.1) to which is added a small amount of a more polar solvent such as 2-propanol.

Solvent selectivity is controlled by the nature of the added solvent. For example:

• solvents that are good proton donors such as water and chloroform interact preferentially with basic solutes such as amines

• solvents that are good proton acceptors such as alcohols, ethers and amines tent to interact best with acids and phenols

• solvents with large dipole moments such as methylene chloride interact preferentially with solutes that have large dipole moments such as nitriles, amines, sulfoxides and nitro compounds.

Mobile Phases used in Normal Phase Chromatography
Solvent strength ε○
Solvent	Silica	Alumina	Solvent Properties
n-hexane	0.01	0.01	nonpolar
n-heptane	0.01	0.01	nonpolar
isooctane	0.01	0.01	nonpolar
chloroform	0.26	0.40	proton acceptor
methylene chloride	0.32	0.42	large dipole
ethyl acetate	0.38	0.58	proton donor
THF	0.44	0.57	proton acceptor
propylamine	~0.5	-	proton acceptor
acetonitrile	0.5	0.65	dipole
methanol	~0.7	0.95	proton acceptor

 

Table I.1: Solvents that are used as mobile phase in normal phase chromatography. The solvent strength parameter ε^○ defines the strength of the solvent. In normal phase chromatography a solvent with a low ε^○ is chosen and quantities of a second solvent with a greater ε^○ is added until the desired separation is achieved.

In general, the order of elution for species separated by normal phase chromatography is the following:

Saturated hydrocarbons < olefins < aromatic hydrocarbons ≈ organic halides < sulfides < ethers < nitro compounds < esters, aldehydes, ketones < alcohols , amines < sulfones < amides < carboxylic acids.

The more polar compounds are retained longer than the nonpolar compounds such as satutated hydrocarbons.

Normal phase chromatography is mainly applied to the analysis of samples that are soluble in non-polar  solvents. Amongst them isomeric and multifunctional compounds are best separated by this method. It is also possible to separate species on the basis of the number of electronegative atoms such as oxygen or nitrogen. Fat and water-soluble vitamins, pesticides and hydrocarbons have all been separated using hexane as the mobile phase.

The major disadvantages of normal phase chromatography are the following:

• Lack of selectivity of the stationary phase (all compounds are eluted in the same order regardless of the stationary phase selected. The mobile phase is therefore used to achieve any change in selectivity).

• Absorption of water (water absorbed from the atmosphere is adsorbed onto the strongest adsorption sites on the stationary phase leading to a decrease in solute retention – deactivation of the stationary phase).

 

References

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

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

3) A. Braithwaite and F.J. Smith, “Chromatographic Methods”, 4^th Ed., Chapman & Hall, 1990