Chromatography

Chromatography from Greek Chroma which means "color" and graphein "to
write") is the collective term for a set of laboratory techniques for the separation of
mixtures.
Chromatographic techniques are based on four different sorption mechanisms,
surface adsorption, partition, ion exchange and size exclusion.
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.
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.