Page 65 - Subyek Encyclopedia - Encyclopedia of Separation Science

60 I / CHROMATOGRAPHY/ Derivatization

separation of polar molecules that are either insoluble ized the analysis of inorganic and organic ions in
in organic solvents or bind too strongly to inorganic industrial and environmental laboratories. As well as
oxide adsorbents for normal elution. RPC employing electrostatic interactions, retention in IEC is in-
acidic, low ionic strength eluents is a widely estab- Suenced by hydrophobic sorptive interactions
lished technique for the puriRcation and characteriza- between the sample and stationary phase similar
tion of biopolymers. Other favourable attributes in- to those in RPC, and size and ionic exclusion
clude the possibility of simultaneous separation of effects. Resolution is optimized by choice of the
neutral and ionic solutes; rapid equilibrium between mobile-phase counterion, the ionic strength, pH,
phases facilitating the use of gradient elution; and the temperature, Sow rate, and addition of organic
manipulation of secondary chemical equilibria in the modiRers.
mobile phase (e.g. ion suppression, ion pair forma- In size exclusion chromatography (SEC) retention
tion, metal complexation and micelle formation) to differences are controlled by the extent to which
optimize separation selectivity in addition to vari- sample components can diffuse through the pore
ation in solvent type and composition of the mobile structure of the stationary phase, as indicated by the
phase. A large number of chemically bonded station- ratio of sample molecular dimensions to the distribu-
ary phases of different chain length, polarity and tion of stationary-phase pore size diameters. Since no
bonding density are available to complement mobile- separation will result under conditions where the
phase optimization strategies. About 70% of all sep- sample is completely excluded from the pore volume
arations performed in modern LC are by RPC, which or can completely permeate the pore volume, the zone
gives an indication of its Sexibility, applicability and capacity of SEC is small compared with that of the
ease of use. The main driving force for retention in other LC techniques. The separation time is predict-
RPC is solute size because of the high cohesive energy able for all separations, corresponding (ideally) to
of the mobile phase compared to the stationary a volume of eluent equivalent to the column void
phase, with solute polar interactions, particularly sol- volume. No solvent optimization beyond Rnding
ute hydrogen bond basicity, reducing retention. These a solvent for the sample that is compatible with the
Rndings strongly reSect the properties of water, stationary phase is required. For synthetic polymers
which is the most cohesive of the solvents normally this can result in the use of exotic solvents and high
used in LC, as well as a strong hydrogen bond acid. temperatures. SEC is a powerful exploratory method
Ion exchange chromatography (IEC) is used for the for the separation of unknown samples, since it pro-
separation of ions or substances easily ionized by vides an overall view of sample composition within
manipulation of pH. Stationary phases are character- a predictable time, and is also commonly employed in
ized as weak or strong ion exchangers based on the sample fractionation to isolate components belonging
extent of ionization of the immobile ionic centres, to a deRned molecular size range. Analytical separ-
and as anion or cation exchangers based on the ations employ small particles of rigid, polymeric or
charge type associated with the ionic centres. Thus, silica-based gels of controlled pore size to separate
sulfonic acid groups are strong, and carboxylic acid samples of different molecular size and to obtain
groups are weak, cation exchangers. Most of the average molecular weights and molecular weight dis-
metal cations in the Periodic Table have been separ- tribution information for polymers.
ated by IEC with acids or complexing agents as elu- Fundamentally the retention mechanisms for LC
ents. In clinical laboratories ion exchange has long and TLC are identical. TLC is selected over LC when
been employed as the basis for the routine, automated advantage can be taken of the attributes of employing
separation of amino acids and other physiologically a planar format for the separation. Examples include
important amines involved in metabolic disorders when a large number of samples requiring minimum
and to sequence the structure of biopolymers. Soft, sample preparation are to be separated, when post-
nondenaturing, ion exchange gels are widely used in chromatographic reactions are usually required for
the large-scale isolation, puriRcation and separation detection, or if sample integrity is in question. The use
of peptides, proteins, nucleosides and other biological of a disposable stationary phase for TLC allows
polymers. Metal-loaded ion exchangers and anion sample clean-up and separation to be performed si-
exchange chromatography of complexed carbohy- multaneously. Reasons for preferring LC over TLC
drates are well-established separation techniques in are its greater separation capacity for mixtures con-
carbohydrate chemistry. The combination of pellicu- taining more components than can be adequately
lar ion exchange columns of low capacity, low con- resolved by TLC; a wider range of stationary phases
centration eluents with a high afRnity for the ion are available for methods development; a wider selec-
exchange packing, and universal, online detection tion of detection techniques exist; and automation for
with a Sow-through conductivity detector revolution- unattended operation is more straightforward.