cases, however, the absolute energetics of these interactions are quite small, and the
differences of importance are even smaller. But as separation methods are based on
weak interactions, they are uniquely well suited to the difficult measurement of such
interactions. Basically, a chiral selector is either immobilised on a surface support as
a stationary phase or present as an additive to the mobile phase. In all cases,
chromatographic parameters are determined for these enantioselective separations
and then related to, for example, thermodynamic parameters governing these
enantioselective interactions. The correlation of separation parameters to the energetics of solute/phase interactions is possible because LC retention processes are
well described by equilibrium thermodynamics (Giddings 1965). Each solute zone
proceeds through the column at a rate controlled by competing interactions of the
solute with the stationary phase and the solute with the mobile phase. This retention
process results in an increase in the solute migration time (t R ) relative to the
movement of a non-retained species (t 0 ) and is often described with the solute
capacity factor (k), which is equal to (t R – t 0 )/t 0 . Although mobile-phase flow creates
an inherently non-equilibrium condition, LC retention has been shown to be well
modelled as an equilibrium process. Ringo and Evans (1998) attribute this to the fact
that solute retention is assessed from movement at the centre of the zone profile,
where non-equilibrium effects from mobile-phase flow are minimised. As a result, a
capacity factor measurement in a flowing HPLC system may be directly correlated to
the equilibrium thermodynamics of solute interactions with the stationary phase and
mobile phase. A considerable range of thermodynamic parameters can be determined by using LC measurements (Giddings 1965). For an extensive discussion,
including a derivation of the respective equations for calculation of these parameters,
the reader should refer to the literature by Ringo and Evans (1998) and recent
literature on this topic (Han et al. 2018; Zhang et al. 2018). Recently, especially
the application of affinity chromatographic methods like molecular imprinting (MIP)
have been used specifically for the simulation of receptor-agent interactions and
effect elucidations (Ansell 2005; Sambe et al. 2005; Torres et al. 2012). However, no
MIP application in the determination of enantiomeric ratios for environmental
application is reported yet.
In addition to quantitative measurements of enantioselective binding, HPLC/
UHPLC can also be used for qualitative investigations of solute binding interactions
with the selector (Hage 2001; Andrisano et al. 2002; Sun et al. 2010; Alanazi et al.
2014). Many large chiral selectors may exhibit several distinct binding sites, with
proteins as the most prevalent example in the literature (Negi et al. 2006; Sun et al.
2010; Yu et al. 2012; Alanazi et al. 2014; Ma et al. 2016). The interactions of solutes
with different binding sites can be independent, competitive or allosteric, whereby
the binding of a solute at one site perturbs the binding of a solute at a different site.
The number and cooperativity of active sites involved in a complexation interaction
play an important role in the measurement and interpretation of all the thermodynamic parameters (Ringo and Evans 1998) shown in Table 3.3. This information
also has considerable practical impact in the prediction of competitive binding
between enantiomers, as well as competitive and allosteric interactions with other
solutes (Lienqueo et al. 2006; Nakashima et al. 2009; Gorynski et al. 2013).
46
3 Enantiomer-Selective High- and Ultra- High-Performance Liquid Chromatography
differences of importance are even smaller. But as separation methods are based on
weak interactions, they are uniquely well suited to the difficult measurement of such
interactions. Basically, a chiral selector is either immobilised on a surface support as
a stationary phase or present as an additive to the mobile phase. In all cases,
chromatographic parameters are determined for these enantioselective separations
and then related to, for example, thermodynamic parameters governing these
enantioselective interactions. The correlation of separation parameters to the energetics of solute/phase interactions is possible because LC retention processes are
well described by equilibrium thermodynamics (Giddings 1965). Each solute zone
proceeds through the column at a rate controlled by competing interactions of the
solute with the stationary phase and the solute with the mobile phase. This retention
process results in an increase in the solute migration time (t R ) relative to the
movement of a non-retained species (t 0 ) and is often described with the solute
capacity factor (k), which is equal to (t R – t 0 )/t 0 . Although mobile-phase flow creates
an inherently non-equilibrium condition, LC retention has been shown to be well
modelled as an equilibrium process. Ringo and Evans (1998) attribute this to the fact
that solute retention is assessed from movement at the centre of the zone profile,
where non-equilibrium effects from mobile-phase flow are minimised. As a result, a
capacity factor measurement in a flowing HPLC system may be directly correlated to
the equilibrium thermodynamics of solute interactions with the stationary phase and
mobile phase. A considerable range of thermodynamic parameters can be determined by using LC measurements (Giddings 1965). For an extensive discussion,
including a derivation of the respective equations for calculation of these parameters,
the reader should refer to the literature by Ringo and Evans (1998) and recent
literature on this topic (Han et al. 2018; Zhang et al. 2018). Recently, especially
the application of affinity chromatographic methods like molecular imprinting (MIP)
have been used specifically for the simulation of receptor-agent interactions and
effect elucidations (Ansell 2005; Sambe et al. 2005; Torres et al. 2012). However, no
MIP application in the determination of enantiomeric ratios for environmental
application is reported yet.
In addition to quantitative measurements of enantioselective binding, HPLC/
UHPLC can also be used for qualitative investigations of solute binding interactions
with the selector (Hage 2001; Andrisano et al. 2002; Sun et al. 2010; Alanazi et al.
2014). Many large chiral selectors may exhibit several distinct binding sites, with
proteins as the most prevalent example in the literature (Negi et al. 2006; Sun et al.
2010; Yu et al. 2012; Alanazi et al. 2014; Ma et al. 2016). The interactions of solutes
with different binding sites can be independent, competitive or allosteric, whereby
the binding of a solute at one site perturbs the binding of a solute at a different site.
The number and cooperativity of active sites involved in a complexation interaction
play an important role in the measurement and interpretation of all the thermodynamic parameters (Ringo and Evans 1998) shown in Table 3.3. This information
also has considerable practical impact in the prediction of competitive binding
between enantiomers, as well as competitive and allosteric interactions with other
solutes (Lienqueo et al. 2006; Nakashima et al. 2009; Gorynski et al. 2013).
46
3 Enantiomer-Selective High- and Ultra- High-Performance Liquid Chromatography