Chromatography is used to separate the individual components of a mixture
on the basis of differences in their physical characteristics, e.g. molecular size,
shape, charge, volatility, solubility and/or adsorption properties.
components of a chromatographic system are:
- A stationary phase, where a solid, a gel or an immobilized liquid is held
by a support matrix.
- A chromatographic bed: the stationary phase may be packed into a glass
or metal column, spread as a thin layer on a sheet of glass or plastic, or
adsorbed on cellulose fibres (paper).
- A mobile phase, either a liquid or a gas which acts as a solvent, carrying
the sample through the stationary phase and eluting from the
- A delivery system to pass the mobile phase through the chromatographic
- A detection system to visualize the test substances.
The individual substances in the mixture interact with the stationary phase to
different extents, as they are carried through the system, enabling separation
to be achieved.
In a chromatographic system, those substances which
interact strongly with the stationary phase will be retarded to the greatest
extent, while those which show little interaction will pass through with
minimal delay, leading to differences in distances travelled or elution
Chromatography is sub-divided according to the mechanism of interaction of
the solute with the stationary phase.
This is a form of solid-liquid chromatography. The stationary phase is a
porous, finely divided solid which adsorbs molecules of the mixture on its
surface by dipole-dipole interactions, hydrogen bonding and/or van der
Waals' interactions (Fig. 31.1). The range of adsorbents is limited to
polystyrene-based resins for non-polar molecules and silica, aluminium oxide
and calcium phosphate for polar molecules. Most adsorbents must be
activated by heating to 110-120°Cbefore use, since their adsorptive capacity
is significantly decreased if water is adsorbed on the surface. Adsorption
chromatography can be carried out in column or thin-layer form, using a wide range of organic solvents.
|Fig. 31.1 Adsorption chromatography (polar stationary phase).
This is based on the partitioning of a substance between two liquid phases, in
this instance the stationary and mobile phases. Substances which are more
soluble in the mobile phase will pass rapidly through the system while those
which favour the stationary phase will be retarded (Fig. 31.2). In normal
phase partition chromatography the stationary phase is a polar solvent,
usually water, supported by a solid matrix (e.g. cellulose fibres in paper
chromatography) and the mobile phase is an immiscible, non-polar organic
solvent. For reversed-phase partition chromatography the stationary phase is a non-polar solvent (e.g. a C18 hydrocarbon, such as octadecylsilane) which is
chemically bonded to a porous support matrix (e.g. silica), while the mobile
phase can be chosen from a wide range of polar solvents, usually water or an
aqueous buffered solution containing one or more organic solvents, e.g.
acetonitrile. Solutes interact with the stationary phase through non-polar
interactions and so the least polar solutes elute last from the column. Solute retention and
|Fig. 31.2 Liquid-liquid partition chromatography, e.g. reversed-phase HPLC.
separation are controlled by changing the composition of the mobile phase (e.g. % v/v acetonitrile), Reverse-phase high-performance liquid chromatography is used to separate a broad range of nonpolar, polar and ionic molecules, including environmental compounds (e.g. phenols) and pharmaceutical compounds (e.g. steroids).
Ion-exchange chromatography (IEC)
Here, separations are carried out using a column packed with a porous
matrix which has a large number of ionized groups on its surfaces, i.e. the
stationary phase is an ion-exchange resin. The groups may be cation or anion
exchangers, depending upon their affinity for positive or negative ions. The
net charge on a particular resin depends on the pKa of the ionizable groups
and the pH of the solution, in accordance with the Henderson-Hasselbalch
For most practical applications, you should select the ion-exchange resin
and buffer pH so that the test substances are strongly bound by electrostatic
attraction to the ion-exchange resin on passage through the system, while the
other components of the sample are rapidly eluted (Fig. 31.3). You can then
elute the bound
|Fig.31.3 Ion-exchange chromatography (cation exchanger).
components by raising the salt concentration of the mobile
phase, either step wise or as a continuous gradient, so that exchange of ions of
the same charge occurs at oppositely charged sites on the stationary phase.
Weakly bound sample molecules will elute first, while more strongly bound
molecules will elute at a higher concentration.
Computer-controlled gradient formers are available: if two or more
components cannot be resolved using a linear salt gradient, an adapted
gradient can be used in which the rate of change in salt concentration is
decreased over the range where these components are expected to elute. IEC
can be used to separate mixtures of a wide range of anionic and cationic
compounds. Electrophoresis is an alternative means of
separating charged molecules.
Gel permeation chromatography (GPC) or gel filtration
Here, the stationary phase is in the form of beads of a cross-linked gel
containing pores of a discrete size (Fig. 31.4). The size of the pores is
controlled so that at the molecular level, the pores act as 'gates' that will
exclude large molecules and admit smaller ones (Table 31.1). However, this
gating effect is not an all-or-nothing phenomenon: molecules of intermediate
size partly enter the pores. A column packed with such beads will have within
it two effective volumes that are potentially available to sample molecules in
the mobile phase, i.e. Vi, the volume surrounding the beads and Vii, the
volume within the pores. If a sample is placed at the top of such a column,
the mobile phase will carry the sample components down the column, but at
different rates according to their molecular size. A very large molecule will
have access to all of Vi but to none of Vii, and will therefore elute in the
minimum possible volume (the 'void volume', or V0, equivalent to VI)' A very small molecule will have access to all of Vi and all of Vii, and therefore it has to pass through the total liquid volume of the column (Vt equivalent to Vi + Vii) before it emerges. Molecules of intermediate size have access to all
of Vi but only part of Vii, and will elute at a volume between Vo and Vb in
order of decreasing size depending on their access to Vii.
Cross-linked dextrans (e.g. Sephadex®), agarose (e.g. Sepharose®) and
polyacrylamide (e.g. Bio-gel®) can be used to separate mixtures of macromolecules,
particularly enzymes, antibodies and other globular proteins. Selectivity in GPC is solely dependent on the stationary phase, with the mobile
phase being used solely to transport the sample components through
|Fig. 31.4 Gel permeation chromatography.
|Table 31.1 Fractionation ranges of selected
column. Thus, it is possible to estimate the molecular mass of a sample
component by calibrating a given column using molecules of known molecular
mass and similar shape. A plot of elution volume (Ve
) against log10
mass is approximately linear. A further application of GPC is the general
separation of components of low molecular mass and high molecular mass,
e.g. 'desalting' a protein extract by passage through a Sephadex®
is faster and more efficient than dialysis.
Affinity chromatography allows biomolecules to be purified on the basis of
their biological specificity rather than by differences in physico-chemical
properties, and a high degree of purification (more than l000-fold) can be
expected. It is especially useful for isolating small quantities of material from
large amounts of contaminating substances. The technique involves the
immobilization of a complementary binding substance (the ligand) onto a
solid matrix in such a way that the specific binding affinity of the ligand is
preserved. When a biological sample is applied to a column packed with this
affinity support matrix, the molecule of interest will bind specifically to the
ligand, while contaminating substances will be washed through with the
buffer (Fig. 31.5). Elution of the desired molecule can be achieved by
changing the pH or ionic strength of the buffer, to weaken the non-covalent
interactions between the molecule and the ligand, or by addition of other
substances that have greater affinity for the ligand.
|Fig. 31.5 Affinity chromatography.