340
C. Sarzanini
18.2
Retention Mechanisms in Liquid Chromatography
18.2.1
Normal Phase Chromatography
Adsorption or straight phase or normal phase chromatography (NPC) is characterized by the use of an inorganic adsorbent or chemically bonded stationary phase
with polar functional groups (e.g. silica, cyanoalkyl-silica) and a non aqueous mobile
phase (one or more polar solvents diluted to the desired eluting power). A layer of
adsorbed mobile phase molecules is formed at the surface of the stationary phase.
The simplest model for analyte separation assumes the stationary phase covered by a
monolayer of mobile phase and solute molecules; a competition between analytes
and eluent for the surface is the driving force. Secondary solvent effects must also
be taken into account for polar compounds (e.g. hydrogen bonding) and conversely
to the competition model, a solvent bilayer adsorbed onto the stationary phase is assumed.
Retention and selectivity in NPC are dramatically influenced by the presence of polar additives (water) in the mobile phase.
18.2.2
Reversed Phase Chromatography
Reversed phase chromatography (RPC) employs as a stationary phase non polar solids of high-surface area (usually alkyl-bonded silica packing, e.g. C8 or CI8 groups
grafted to the silica surface), and an aqueous-organic solvent mixture is used as the
mobile phase.
Retention in RPC occurs by non-specific hydrophobic interactions of the solute with
the stationary phase. Secondary chemical equilibria (see below) optimize separation
selectivity varying the mobile phase composition. Solute retention is attributed to both
adsorption and partition phenomena; neutral and ionic solutes can be separated simultaneously.
18.2.3
Ion Chromatography
The term ion chromatography (IC) in the past included only ion-exchange chromatography. This technique is based on a reversible interchange of ions or ionized substances (e.g. by pH manipulation or complexation) between a solution and a solid,
inorganic or polymeric insoluble material, containing fixed ions and exchangeable
counter-ions. Analytes are separated on the basis of their different relative affinity for
the ionic centers of the stationary phase. Ion exchange chromatography is based on
electrostatic interactions between the ions to be exchanged but other kinds of reactions may occur, e.g. hydrophobic interactions between the sample and non-ionic regions of the stationary phase.
C. Sarzanini
18.2
Retention Mechanisms in Liquid Chromatography
18.2.1
Normal Phase Chromatography
Adsorption or straight phase or normal phase chromatography (NPC) is characterized by the use of an inorganic adsorbent or chemically bonded stationary phase
with polar functional groups (e.g. silica, cyanoalkyl-silica) and a non aqueous mobile
phase (one or more polar solvents diluted to the desired eluting power). A layer of
adsorbed mobile phase molecules is formed at the surface of the stationary phase.
The simplest model for analyte separation assumes the stationary phase covered by a
monolayer of mobile phase and solute molecules; a competition between analytes
and eluent for the surface is the driving force. Secondary solvent effects must also
be taken into account for polar compounds (e.g. hydrogen bonding) and conversely
to the competition model, a solvent bilayer adsorbed onto the stationary phase is assumed.
Retention and selectivity in NPC are dramatically influenced by the presence of polar additives (water) in the mobile phase.
18.2.2
Reversed Phase Chromatography
Reversed phase chromatography (RPC) employs as a stationary phase non polar solids of high-surface area (usually alkyl-bonded silica packing, e.g. C8 or CI8 groups
grafted to the silica surface), and an aqueous-organic solvent mixture is used as the
mobile phase.
Retention in RPC occurs by non-specific hydrophobic interactions of the solute with
the stationary phase. Secondary chemical equilibria (see below) optimize separation
selectivity varying the mobile phase composition. Solute retention is attributed to both
adsorption and partition phenomena; neutral and ionic solutes can be separated simultaneously.
18.2.3
Ion Chromatography
The term ion chromatography (IC) in the past included only ion-exchange chromatography. This technique is based on a reversible interchange of ions or ionized substances (e.g. by pH manipulation or complexation) between a solution and a solid,
inorganic or polymeric insoluble material, containing fixed ions and exchangeable
counter-ions. Analytes are separated on the basis of their different relative affinity for
the ionic centers of the stationary phase. Ion exchange chromatography is based on
electrostatic interactions between the ions to be exchanged but other kinds of reactions may occur, e.g. hydrophobic interactions between the sample and non-ionic regions of the stationary phase.