3.1 Separation Principles
All types of enantiomer-selective chromatographic separation methods need a certain type of selective interaction between the chiral target substance and the stationary phase or chiral selector that must be different for the respective target
enantiomer. In general, two types of enantiomer selectivity can be described for a
direct chromatographic separation (Rocco et al. 2013):
1. A chiral component as a selector added to the mobile phase (solvent additive)
2. An enantiomer-selective chiral stationary phase (CSP) or an additive to an achiral
standard stationary phase (ASP)
3.1.1 Derivatisation and Mobile-Phase Additives
When discussing stereoselective separation, indirect actions are also possible to
provide a sufficient enantioselective separation. As a standard method for the
pre-chromatographic preparation, a diastereomeric derivatisation can be employed
allowing direct chromatographic separation on non-enantiomer-selective SPs. This
approach belongs to the oldest methods used for the enantiomer-selective separation
by HPLC (Burden et al. 1987; Feitsma and Drenth 1988; Euerby et al. 1989; Nation
1989; Srinivas and Igwemezie 1992; Bhushan and Martens 1998; Hayamizu et al.
1998; Waksmundzka-Hajnos 1998). The principle of diastereomeric derivatisation is
simple but effective. Basically, two stereogenic centres are bound together, thus
creating a diastereomeric complex. Underivatised enantiomers are normally not
separated by standard chromatographic methods. However, diastereomers that
were formed by derivatisation exhibit different physical characteristics and thus
standard chromatographic methods can be employed for such a separation. Various
types of complex-building derivatives such as crown-ethers and binding groups like
amino-, carboxyl- and thiol-groups are still used for different applications (Burden
et al. 1987; Srinivas and Igwemezie 1992; Hayamizu et al. 1998; Habel et al. 2007).
The advantages of such derivatisation methods can be described as follows:
1. Enantioselectivity is achieved in combination with standard stationary phases
(RP and NP columns).
2. By using suitable derivatives, the detectability of the compound can be substantially improved (e.g. using a chromophore as derivatising agent and fluorescencedetection after HPLC separation).
A further potential approach to obtain enantiomeric resolution is to add chiral
additives into the mobile phase. Various applications are described for drug analysis
where reversible diastereomeric complexes were formed by adding chiral compounds
into the mobile phase (Karnes and Sarkar 1987; Arai 1998; Shamsi and Danielson
2007; Kapnissi-Christodoulou et al. 2014; Patel et al. 2016). These additives form
32
3 Enantiomer-Selective High- and Ultra- High-Performance Liquid Chromatography
All types of enantiomer-selective chromatographic separation methods need a certain type of selective interaction between the chiral target substance and the stationary phase or chiral selector that must be different for the respective target
enantiomer. In general, two types of enantiomer selectivity can be described for a
direct chromatographic separation (Rocco et al. 2013):
1. A chiral component as a selector added to the mobile phase (solvent additive)
2. An enantiomer-selective chiral stationary phase (CSP) or an additive to an achiral
standard stationary phase (ASP)
3.1.1 Derivatisation and Mobile-Phase Additives
When discussing stereoselective separation, indirect actions are also possible to
provide a sufficient enantioselective separation. As a standard method for the
pre-chromatographic preparation, a diastereomeric derivatisation can be employed
allowing direct chromatographic separation on non-enantiomer-selective SPs. This
approach belongs to the oldest methods used for the enantiomer-selective separation
by HPLC (Burden et al. 1987; Feitsma and Drenth 1988; Euerby et al. 1989; Nation
1989; Srinivas and Igwemezie 1992; Bhushan and Martens 1998; Hayamizu et al.
1998; Waksmundzka-Hajnos 1998). The principle of diastereomeric derivatisation is
simple but effective. Basically, two stereogenic centres are bound together, thus
creating a diastereomeric complex. Underivatised enantiomers are normally not
separated by standard chromatographic methods. However, diastereomers that
were formed by derivatisation exhibit different physical characteristics and thus
standard chromatographic methods can be employed for such a separation. Various
types of complex-building derivatives such as crown-ethers and binding groups like
amino-, carboxyl- and thiol-groups are still used for different applications (Burden
et al. 1987; Srinivas and Igwemezie 1992; Hayamizu et al. 1998; Habel et al. 2007).
The advantages of such derivatisation methods can be described as follows:
1. Enantioselectivity is achieved in combination with standard stationary phases
(RP and NP columns).
2. By using suitable derivatives, the detectability of the compound can be substantially improved (e.g. using a chromophore as derivatising agent and fluorescencedetection after HPLC separation).
A further potential approach to obtain enantiomeric resolution is to add chiral
additives into the mobile phase. Various applications are described for drug analysis
where reversible diastereomeric complexes were formed by adding chiral compounds
into the mobile phase (Karnes and Sarkar 1987; Arai 1998; Shamsi and Danielson
2007; Kapnissi-Christodoulou et al. 2014; Patel et al. 2016). These additives form
32
3 Enantiomer-Selective High- and Ultra- High-Performance Liquid Chromatography