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Encyclopedia of Physical Science and Technology EN014J-683 July 30, 2001 20:3

Separation and Purification of Biochemicals 665

FIGURE 10a Separation of a mixture of biomolecules achieved in a linear (A) or stepwise (B) gradient. [From Protein
Purification Handbook. Reproduced by kind permission of Amersham Pharmacia Biotech Limited.]

strongly to the surface of the stationary phase or show matographicsurfacethantheproduct,andtheothereluting
no tendency to bind at all. Since the elution windows of exclusively the product.
all sample components will rarely overlap, their separa-
tion by isocratic elution is often not practical. Instead, the 2. Frontal Chromatography
eluent strength is increased gradually or stepwise (differ-
Frontal chromatography is a binary separation process in
ential elution) during the chromatographic run, to bring
which only the least-retained component is isolated from
about the separation of a multicomponent mixture. If the
the others. The mixture to be separated is fed continuously
eluent strength is increased gradually, the migration veloc-
into the column under conditions that favor the binding of
ity of the eluates increases with the eluent strength, until
all the components but one. This component is obtained in
they move with the velocity of the mobile phase. Since
pure form at the column outlet, until the dynamic capacity
the molecules at the rear of a peak are moving in a zone
of the stationary phase is exhausted and the other sam-
of higher eluent strength than those at the peak maximum,
ple components break through; a typical chromatogram
they are sped up in relation to the bulk sample, while the
is shown in Fig. 10b. Frontal chromatography is the first
opposite effect is operative at the front of each sample
step in many biopolymer purification schemes involving
zone. Therefore elution with an appropriate gradient in-
differential elution. The method per se is applicable when
volves focusing, and results in relatively sharp peaks and a
the product to be purified has much lower affinity for the
reduction of peak “tailing.” At the same time, gradient elu-
stationary phase than the other feed components and there-
tion is much more demanding than isocratic elution as far
fore breaks through far ahead of the impurities.
as instrumentation and theoretical treatment of the process
are concerned. Step gradient elution is more commonly
3. Displacement Chromatography
used than linear gradient elution in preparative/process
chromatography, but also in affinity chromatography. Ide- This nonlinear multicomponent separation technique is
ally, two well-chosen elution steps suffice, one for recov- eminently suitable for preparative/process scale applica-
ering all components that bind less strongly to the chro- tions. In displacement chromatography, the competition