Selection of the Liquid Chromatographic (LC) / HPLC Separation Mode

The column is the heart of the chromatograph. The column is where the separation occurs. The selection of the correct column packing material is the most important step in the development of an analytical method. If no separation of components occurs the chromatogram is useless. Even though attempts have been made to develop a scientific approach to column selection (packing material) for each type of mixture of analytes  the experimental results obtained were not promising. The process of packing material selection (column selection) is largely empirical.

In general, three primary characteristics of chemical compounds can be used to create LC / HPLC separations:

Polarity

Electrical Charge

Molecular Size

Which are the main liquid chromatography / HPLC separation modes?

The character of the stationary phase determines the LC or HPLC mode. There are four main modes (Fig 1): Liquid-Solid, Liquid-Liquid, Ion Exchange, Steric Exclusion chromatography. It should be also mentioned that there is another separation mode that can be used in conjunction with the above mentioned called reversed phase chromatography.

The predominant mechanism of each mode, describing the interaction between the solute and the stationary phase is also shown in. Fig. 1

It should be also mentioned that mixed separation mechanisms frequently occur.

Fig 1: The main modes of liquid chromatography (LC)

What is the Liquid-Solid Chromatography mode (LSC)?

Liquid/solid chromatography is adsorption chromatography. The full nature of adsorption is not completely understood. Selection of column packings and operating conditions requires much experimentation. Silica gel, charcoal and alumina are the more commonly used adsorbents.

Adsorption is the accumulation of atoms or molecules on the surface of a material. This process creates a film of the the molecules or atoms being accumulated on the adsorbent's surface. Adsorbents are used usually in the form of spherical pellets, rods, moldings with hydrodynamic diameters between 0.5 and 10 mm. They must have high abrasion resistance, high thermal stability and small pore diameters, which results in higher exposed surface area and hence high surface capacity for adsorption. The adsorbents must also have a distinct pore structure which enables fast transport of targeted molecules.

The interaction between the solute molecule and the stationary phase (adsorbent) occurs on the surface of the stationary phase. This interaction is a competitive phenomenon in which the molecules of the mobile phase and the solute are in competition for the surface.

Most adsorbents used in LSC are polar solids and the order of elution follows sample polarity. Generally speaking the polarity of compounds increases in the order:

fluorocarbons < hydrocarbons < halogenated compounds < ethers < esters < ketones ~ aldehydes < alcohols ~ amines < acids.

The Liquid Solid separation mode is based on the polarity of the molecules within the sample  analyzed. The functional group(s) in a molecule often determine whether the molecule is polar or non-polar. Generally, the most polar group of a polyfunctional compound governs its adsorption behavior.

Molecules with similar chromatographic polarity tend to be attracted to each other; those with dissimilar polarity exhibit much weaker attraction, if any, and may even repel each other. This behavior (“like attracts like”) becomes the basis for chromatographic separation modes based on polarity.

The surface hydroxyl group is the predominating group on silica and alumina and governs their adsorption characteristics. The number and their arrangement in space determine the activity of the solid adsorbent. The surface hydroxyls interact with the functional groups on the solute molecules and depending on the strength of this interaction preferentially adsorb one solute relative to another.

For example a hydroxyl functional group of an alcohol would interact more strongly than an ester group with a silica surface hydroxyl group (see comparison of the polarity of compounds above). Fig. 2 shows such an LSC separation obtained on an actual mixture of the above compounds. Methyl benzoate is the least retained and elutes second while benzyl alcohol is strongly retained and elutes close to the end (compound #9). 

 

Fig. 2: Separation of a mixture of compounds that have attached different functional groups by liquid-solid chromatography.

In general, isomeric and multifunctional compounds are best separated by LSC.

Liquid-liquid chromatography  (LLC) is another separation mode used by chromatographers. Please see the post entitled "Which are the main liquid chromatography separation modes: Liquid-Liquid Chromatography (LLC)"

References

1. C.A. Dorschel, J.L. Ekmanis, J.E. Oberholtzer  et al. “LC Detectors” Anal. Chem., 61, 951A–968A, 1989 
2.  Nina Hadden et al., “Basic Liquid Chromatography”, Varian Aerograph, 1971
3. C. F. Simpson, “Techniques in Liquid Chromatography” Wiley-Hayden: Chichester, England, 1982