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               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
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