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

664 Separation and Purification of Biochemicals

FIGURE 9 A chromatographic setup including buffer vessels and valve, pumps, mixer, a sample feed port, the column,
and a detector. The system can be controlled with adequate software; fractions can be collected after detection. [From
Freitag, R. and Giovannini, R. (2001). Biotechnology and Bioengineering, Wiley-Liss, Inc. a subsidiary of John Wiley
& Sons, Inc. With permission.]

interactions between the sample components and the chro- even though particularly biomacromolecules show some
matographic matrix. A standard setup for liquid chro- deviations from it, e.g., because of multipoint interac-
matography consisting of buffer reservoir(s) and pumps, a tion, aggregation or multiple retention mechanisms, and
mixer, a sample feeding loop (or pump), the column, and other secondary equilibria. At low sample concentration,
a detector is shown in Fig. 9. A fraction collector is also Henry’slawusuallyholdsandtherelationshipbetweenC m
useful, especially in preparative chromatography. and C s is linear. At higher concentrations, the isotherm
The pressure drop over the column may be calculated becomes nonlinear. Consequently, competition between
from the Hagen–Poiseuille equation, in which the void the various molecules for the adsorption sites takes place
fraction (or porosity) of the column, the velocity and the and the multicomponent isotherm of a given compound
viscosity of the mobile phase, and the particle size are will be suppressed as compared to the single-component
considered. Changing the particle size from 100 to 10 µm isotherm. Taking the isotherm into account, a chromato-
improves the plate number, but also increases the flow graphic separation can be mathematically described by
resistance by two orders of magnitude. The consequent solving the mass balance equation within the appropri-
need for high-pressure systems for small particles can be ate boundary conditions. According to the inlet bound-
a disadvantage in a production environment, where low- ary conditions, three modes of chromatography can be
pressure systems are often preferred. The viscosity is of distinguished—namely, elution, frontal, and displacement
special significance when applying viscous samples (e.g., chromatography.
certain culture supernatants, or solutions of high DNA
1. Gradient and Isocratic Elution Chromatography
contents) and when transferring methods to the cold room.
Linear elution chromatography (see Fig. 10a) is the pre-
ferred operational mode in analytical chromatography.
A. The Different Modes of Chromatography
The sample is introduced into the column approximately
Adsorption (interactive) chromatography as opposed to as a Dirac pulse. The components move through the col-
size-exclusion chromatography is based on the differen- umn with different velocities, due to differences in their
tial distribution of each substance between the stationary distribution between the mobile and the stationary phase,
and the mobile phase due to interactions between the com- and thus are separated. If the composition of the mobile
ponentsand the chromatographicsurface.Theequilibrium phase does not change throughout the separation, the pro-
relationship for the distribution of the components be- cess is referred to as isocratic elution. However, many
tween the mobile and stationary phases is given by the biologicals tend to show an all-or-nothing type of bind-
respective adsorption isotherms. In the case of biochro- ing under these circumstances, i.e., except for a narrow
matography, the Langmuir isotherm model is widely used, window of eluent strength, the molecules will either bind