Page 246 - Academic Press Encyclopedia of Physical Science and Technology 3rd BioTechnology

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

654 Separation and Purification of Biochemicals

to facilitate the following purification steps. The next steps and later, with the introduction of paper chromatogra-
often involve highly selective methods to remove sub- phy, for the microanalysis of biological samples. Various
stances of similar physical properties and biochemical characteristics of biomolecules ranging from their gen-
functions from the product. At this point, chromatography eral physicochemical properties (size, charge, hydropho-
plays the major role. The fourth step is polishing, and may bicity) to biospecific interactions have been exploited
include gel filtration, crystallization, and lyophilization of for their chromatographic separation. Although several
the final product. Chromatography has an important place modes of operation have been recognized in chromatog-
in the methodologies used for downstream processing at raphy, most effort has been placed on the development of
nearly all levels. Electrophoretic methods have also been (linear) elution (syn.: desorption) chromatography. The
used in the past for the isolation of such target molecules, development of high performance liquid chromatogra-
but they have been outmoded, replaced by the modern, phy (HPLC) in the 1970s allowed high-speed analysis,
faster chromatographic methods. Electrophoresis is still and set new standards of precision and resolution in the
used nowadays, but mainly on the analytical scale in qual- liquid chromatography of small molecules and begin-
ity control. Biospecificaffinity beads or magnetic beads, ning in the 1980s also of larger biologicals (proteins,
and affinity precipitation, are timidly entering the field on DNA).
a preparative scale, for scavenging product in culture su- The following relationships are fundamental to all types
pernatant or sometimes directly in the culture media, by of chromatography. A certain number of basic parameters
one-step adsorption processes, and will not be detailed allow the description and evaluation of a chromatographic
here. separation, e.g., the calculation of the resolution from pa-
rameters such as the retention time and the zone widths.
A complete chromatographic process can be described by
I. PRINCIPLES OF CHROMATOGRAPHIC the mass balance of the system, also allowing modeling
SEPARATIONS of such processes. In addition, the number and kind of
separation principles used in size-exclusion and interac-
Chromatography has been the primary preparative sepa- tive chromatography is limited and common to all modes
ration method in biology and biochemistry since 1906, of biochromatography. An overview of the different chro-
when the Italo-Russian botanist Tswett separated the pig- matographic modes and the set-up conditions such as col-
ments of chlorophyll by passing petroleum ether extracts umn and mobile phase type is given in Table II. Table III
of plant material through a column packed with powdered compares the modes in terms of their suitability for a given
chalk. In the 1930s, column chromatography became application mode and some important factors to be con-
an important tool for the separation of natural products sidered during optimization.

TABLE II Main Characteristics of the Different Chromatographic Modes and Their Applications
Chromatographic mode Stationary phase Mobile phase Applications
Size exclusion (SEC) or Particles of well-defined size, of Aqueous buffer Desalting, buffer exchange
Gel filtration (GF) different sized pores Determination of molecular weight
Final polishing
Ion exchange (IEC) Particles coated with anion or Aqueous buffers, containing Separation of charged molecules
cation exchanger functionalities salts for elution Good choice for protein separation on
preparative scale
Affinity (AC) Covalently immobilized affinity Aqueous buffers Good choice for capture of target
ligand on particles molecule at low concentrations
in sample
Hydrophobic interaction (HIC) Particles of well defined size, Saline aqueous solutions Well developed for protein separation
coated with small hydrophobic No prior desalting necessary,
ligands C 2 —C 4 good after IEC
Reversed phase (RPC) Particles of well-defined size, Water, buffers of low Indicated for neutral and uncharged
coated with hydrophobic molarity, and organic molecules, soluble in
ligands C 4 —C 18 solvents for elution aqueous/organic mixtures
Presence of salts problematic Excellent for analytical HPLC, seldom
above 10 mM preparative (proteins denatured
by organic solvents)