11.3.4 Perfusion chromatography
The high resolution achieved by HPLC is based on the use of small diameter particles
for the stationary phase. However, this high resolution is achieved at the cost of the
generation of high pressures, relatively low flow rates and the constraints the high
pressure imposes on the instrumentation.Perfusion chromatographyovercomes
some of these limitations by the use of small particles (10 50 mm diameter) that have
channels of approximately 1mm diameter running through them that allow the use of
high flow rates without the generation of high pressures. The high flow rates result in
small plate heights (Section 11.2.4) and hence high resolution in very short separation
times. The particles are made of polystyrene-divinylbenzene and are available under
the trade name POROS. Two types of pore are available:through poresthat are long
(up to 8000 A ̊) anddiffusive poresthat are shorter (up to 1000 A ̊). The stationary
phase is coated onto the particles, including the surface of the pores. The eluent
perfuses through the pores allowing the analyte to equilibrate rapidly with the station-
ary phase. By comparison, the microporous particles used in HPLC have a much
smaller diameter pore, hence the greater back-pressure. All the forms of stationary
phase used for the various forms of chromatography are available for perfusion
chromatography. The technique uses the same type of instrumentation as HPLC.
Protein separations in as short a time as 1 min can be achieved.11.4 Adsorption chromatography
11.4.1 Principle
This is the classic form of chromatography, which is based upon the principle that
certain solid materials, collectively known asadsorbents, have the ability to hold
molecules at their surface. This adsorption process, which involves weak, non-ionic
attractive forces of the van der Waals’ and hydrogen-bonding type, occur at specific
adsorption sites. These sites have the ability to discriminate between types of mol-
ecules and are occupied by molecules of either the eluent or of the analytes in
proportions determined by their relative strength of interaction. As eluent is con-
stantly passed down the column, differences in these binding strengths eventually
lead to the separation of the analytes.
Silica is a typical adsorbent. It has silanol (Si-OH) groups on its surface, which are
slightly acidic, and can interact with polar functional groups of the analyte or eluent.
Thetopology(arrangement) of these silanol groups in different commercial prepar-
ations of silica explains their different separation properties. Other commonly used
adsorbents are alumina and carbon.
In general, an eluent with a polarity comparable to that of the most polar analyte in the
mixture is chosen. Thus, alcohols would be selected if the analytes contained hydroxyl
groups, acetone or esters would be selected for analytes containing carbonyl groups,
and hydrocarbons such as hexane, heptane and toluene for analytes that are predominantly
non-polar. Mixtures of solvents are commonly used in the context of gradient elution.453 11.4 Adsorption chromatography