Chromatographic Separation Mechanisms

Chromatographic Separation Mechanisms are based on four different sorption mechanisms, surface adsorption, partition, ion exchange and size exclusion.

Chromatographic Separation Mechanisms 1: Surface Adsorption Chromatography

The separation mechanism depends upon differences in polarity between the different feed components. The more polar a molecule, the more strongly it will be adsorbed by a polar stationary phase. Similarly, the more non-polar a molecule, the more strongly it will be adsorbed by non-polar stationary phase.
During a surface adsorption chromatography process, there is competition for stationary phase adsorption sites, between the materials to be separated and the mobile phase. Feed molecules of low polarity spend proportionally more time in the mobile phase than those molecules that are highly polar, which are retained longer. Therefore the components of a mixture are eluted in order of increasing polarity.

Almost any polar solid can be employed as a polar stationary phase. The choice of stationary phase is governed by the polarity of the feed components. If the feed components are adsorbed too strongly, they may be difficult to remove. Weakly polar mixtures should be separated on highly active absorbents, or little or no separation will occur.

The choice of mobile phase is equally important. The polarity of the mobile phase should be chosen to compliment the choice of stationary phase. In general, good separation is achieved by using fairly polar stationary phases and low polarity mobile phases such as hexane. Water, it should be noted, is a very polar solvent.

The 2 most common adsorbents used in chromatography are porous alumina and porous silica gel. Of lesser importance are carbon, magnesium oxide, and various carbonates. Alumina is a polar adsorbent and is preferred for the separation of components that are weakly or moderately polar, with the more polar components retained more selectively by the adsorbent, and therefore eluted from the column last. In addition, alumina is a basic adsorbent, thus preferentially retaining acidic compounds. Silica gel is less polar than alumina and is an acidic adsorbent, thus preferentially retaining basic compounds. Carbon is a non-polar (apolar) stationary phase with the highest attraction for larger non-polar molecules.

Adsorbent-type sorbents are better suited for the separation of a mixture on the basis of chemical type (e.g. olefins, esters, acids, aldehydes, alcohols) than for separation of individual members of a homologous series. Partition chromatography is often preferred for the latter, wherein an inert solid (often silica gel) is coated with a liquid phase

Hydrophobic interaction chromatography (HIC) is a special form of surface adsorption chromatography. The materials to be separated should be at least partially hydrophobic in nature. Separation is facilitated by differences in the relative strength of interaction between these materials and a matrix substituted with suitably hydrophobic groups. This type of process is extensively used for the preparative-scale separation of proteins.

Chromatographic Separation Mechanisms 2: Partition Chromatography

Unique to chromatography is the liquid-supported or liquid-bonded solids, where the mechanism is absorption into the liquid, also referred to as a partition mode of separation or partition chromatography. With mobile liquid phases, there is a tendency for the stationary liquid phase to be stripped or dissolved. Therefore, the stationary liquid phase has to be chemically bonded to the solid bonding support.

In partition chromatography, the stationary liquid phase is coated onto a solid support such as silica gel, cellulose powder, or kieselguhr (hydrated silica). Assuming that there is no adsorption by the solid support, the feed components move through the system at rates determined by their relative solubilities in the stationary and mobile phases.

In general, it is not necessary for the stationary and mobile phases to be totally immiscible, but a low degree of mutual solubility is desirable. Hydrophilic stationary phase liquids are generally used in conjunction with hydrophobic mobile phases (referred to as “normal-phase chromatography”), or vice versa (referred to as a ‘”reverse- phase chromatography”).

Suitable hydrophilic mobile phases include water, aqueous buffers and alcohols. Hydrophobic mobile phases include hydrocarbons in combination with ethers, esters and chlorinated solvents.

 Ion Exchange Chromatography (IEC)

In this process, the stationary phase consists of an insoluble porous resinous material containing fixed charge-carrying groups. Counter-ions of opposite charge are loosely complexed with these groups.

Passage of a liquid mobile phase, containing ionised or partially ionised molecules of the same charge as the counter-ions through the system, results in the reversible exchange of these ions.

The degree of affinity between the stationary phase and feed ions dictates the rate of migration and hence degree of separation between the different solute species.

The most widely used type of stationary phase is a synthetic copolymer of styrene and divinyl benzene (DVB), produced as very small beads in the micrometer range. Careful control over the amount of DVB added dictates the degree of cross-linking and hence the porosity of the resinous structure.

Resins with a low degree of cross-linking have large pores that allow the diffusion of large ions into the resin beads and facilitate rapid ion exchange. Highly cross- linked resins have pores of sizes similar to those of small ions.

The choice of a particular resin will very much be dependent upon a given application. Cation (+) or anion (-) exchange properties can be introduced by chemical modification of the resin.

Ion exchange chromatography has found widespread uses in industrial processes. This technique is used in the separation of transition metals, the removal of trace metals from industrial effluents and in the purification of a wide range of organic compounds and pharmaceuticals. The resin matrix is usually relatively inexpensive when compared with other types of stationary phase. Ion exchange chromatography is probably the most widely used large-scale chromatographic process, but is limited to ionisable, water soluble molecules.

Size Exclusion Chromatography (SEC)

In this process, also known as gel permeation chromatography, molecules of a feed material are separated according to their size or molecular weight. The stationary phase consists of a porous cross-linked polymeric gel.

The pores of the gel vary in size and shape such that large molecules tend to be excluded by the smaller pores and move preferentially with the mobile phase. The smaller molecules are able to diffuse into and out of the smaller pores and will thus be retarded in the system.

The very smallest molecules will permeate the gel pores to the greatest extent and will thus be most retarded by the system.

The components of a mixture therefore elute in order of decreasing size or molecular weight.

The stationary phase gels can either be hydrophilic for separations in aqueous or polar solvents, or hydrophobic for use with non-polar or weakly-polar solvents. Sephadex, a cross-linked polysaccharide material available in bead form, is widely used with polar/hydrophilic mobile phases. The degree of cross-linking can be varied to produce beads with a range of pore sizes to fractionate samples over different molecular weight ranges. Hydrophobic gels are made by cross-linking polystyrene with DVB and are therefore similar to ion exchange resins but without the ionic groups.

SEC is used extensively in the biochemical industry to remove small molecules and inorganic salts from valuable higher molecular weight products such as peptides, proteins and enzymes.