Page 263 - 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

Separation and Purification of Biochemicals 671

A. Particle-Based Stationary Phases ally show poor mechanical stability; therefore low pres-
sure and low flow rates are indicated, and their scale-up
1. Conventional Porous Particles
potential is limited. These stationary phases do, on the
The choice of conventional particle-based stationary other hand, show excellent biocompatibility due to their
phases for (bio)chromatography is vast and details cannot pronounced hydrophilicity. More recently, highly cross-
be given here. Table IV attempts to group the main types linked polysaccharide gels have been introduced, which
of stationary phases into categories, such as inorganic and are mechanically stable, with a rigidity almost equal to
organic (polymeric) materials, which may be of natural that of silica-based materials. Rigid porous microparticles
or (semi)synthetic nature. The final choice of a stationary (average diameter between 2 and 10 µm) with narrow par-
phase is governed as much by personal preference and ticle size distribution yield very high column efficiencies,
past experience as by considerations such as but need high pressure at the inlet to reach reasonable
flow rates. While such small particles are eminently suit-
The characteristics of the target molecule (size, able for analytical separations, bigger particles are usu-

isoelectric point, hydrophobicity, possible biospecific ally preferred for preparative separations, because of eco-
interaction) nomic or operating (pressure-drop) considerations. Many
The chosen chromatographic mode (stability of the preparative applications use columns packed with irregu-

stationary phase toward the mobile phase, required larly shaped particles showing a comparatively broad par-
cleaning procedures) ticle size distribution.

In this context the following advantages and limitation
2. Particles with Reduced Mass
have been noted for the different materials. Conven-
Transfer Limitation
tional silica is sensitive to elevated pH, making san-
itizing with alkaline solutions difficult. Polymer-based Several possibilities have been proposed to improve the
synthetic supports are more stable in this regard. Sil- performance of porous particle-based stationary phases.
ica is, on the other hand, superior in terms of mechan- Most of these approaches attempt to reduce the problem of
ical stability to most if not all other currently existing intraparticular mass transfer and the related loss in column
stationary phases. Polysaccharide-based supports gener- efficiency at higher flow rates. However, due to the high

TABLE IV Particulate Stationary Phases a
Inorganic Organic
Natural Chalk Polysaccharide-based materials
Charcoal Cellulose
Kieselguhr Starch
Glass Agarose
Sand Dextran
Aluminium oxide
Magnesium silicates
Hydroxyapatite
Synthetic Silica beads and monoliths Methacrylates
Glass beads of controlled shape, Polyacrylamides
size, and porosity Polystyrenes
Polyamides
Modified cellulose and various copolymers
of controlled shape, size, and porosity
The surface of most of the synthetic supports can be modified, functionalized for each
chromatographic mode. For example:
• A hydrophilic layer is added for SEC
• A hydrophobic layer is added for HIC and RPC: C 2 ,C 4 ,C 6 ,C 8 ,C 12 ,C 18
• Ionic groups are added for IEC: strong (QA, SO 3 ) and weak (DEAE, CM)
• Common affinity ligands such as Protein A and G, lectins
a
Beads are made of a core material, and their surface is modified accordingly for each chromatographic
mode.