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Encyclopedia of Physical Science and Technology En001f25 May 7, 2001 13:58
562 Analytical Chemistry
FIGURE 15 Schematic representation of a high-performance ion chromatograph.
5. Supercritical Fluid Chromatography 6. Chromatographic Methods
in Biochemical Science
Supercritical fluids exist at temperatures and pressures
above the supercritical point of a compound (e.g., where Biochemists address a chemically limited, yet extremely
a gas at STP exists as a fluid). As mobile phases for chro- complex area of organic chemistry. The complexity has re-
matography, supercritical fluids provide properties that are sultedintheevolutionofspecializedseparationtechniques
intermediate to liquid and gas chromatography. The sol- suitable for macromolecules that are difficult to determine
vent strength is closely related to density and offers an- by gas and high-performance liquid chromatographic sys-
other operating parameter in the form of pressure–density tems. Though many specialized separation methods are
programming. The low densities and high diffusivities of employed by biochemists, two techniques have found
gasesgivegaschromatographybetterresolutionperunitof widespread use in many areas of chemistry.
time than liquid or supercritical chromatography, but gas Gel permeation (or size-exclusion) chromatography
chromatography is limited to the analysis of low molecular is a technique based on molecular separation partially
weight, volatile, and thermally stable compounds. High by size rather than chemical interaction with a station-
liquid densities provide liquid chromatography with an ary phase. A water-swollen polymeric gel acts as the
excellent range of solvating power, but this technique is stationary phase and is designed by polymeric cross-
relatively show and is limited by a lack of sensitive univer- linking to contain a certain size distribution of pores
sal detectors. The ability of supercritical fluids to solvate or cavities. As a sample passes through the polymeric
compounds at lower temperatures by controlling the mo- sieve in a water-based mobile phase, smaller molecules
bile phase density makes it possible to readily analyze diffuse into the pores more readily than larger macro-
thermally labile and nonvolatile compounds. The mobile molecules, so that the latter elute first while the progress
phase is pumped as a liquid and heated to above the crit- of the smaller molecules is retarded. The most popu-
ical temperature before passing through the column as a lar polymer for this type of separation is prepared by
supercritical fluid via an injection loop valve. A pressure cross-linking epichlorohydrin with the polysaccharide
restrictor is incorporated after the column to ensure that dextran.
conditions throughout the column remain supercritical. Electrophoresis is a second specialized technique, em-
The technique uses either packed liquid chromatography ploying for analysis the migration of ionic molecules or
columns or open-tubular fused-silica columns, with a va- aggregates in an applied electric field. Commonly, a sta-
riety of nonpolar, polar, or novel chiral-bonded stationary tionary phase such as a water-swollen gel saturated with an
phases. electrolyte is placed between two do high-voltage (hun-
The most commonly used mobile phase in SFC is car- dreds to thousands of volts) electrodes. The sample mi-
bon dioxide (CO 2 ) because it has a critical temperature grates through the gel as a function of its charge-to-mass
◦
(T c ) of 31.05 C and a critical pressure (P c ) of 72.9 atm, ratio, as well as being influenced by all the factors previ-
which are easy to work with; it is nontoxic and non- ously described in Section II.C. Staining of the gel after
flammable; and it is readily available at low cost. Mod- the application of the voltage for a defined time allows vi-
ifiers such as methanol are sometimes added to change sualization of sample species. It is possible to arrange the
solute elution characteristics. instrumentation to use standard high-performance liquid