Cavity Supported HPLC of Cis/Trans Isomers of Proline
201
can be achieved considering the fact that the time scale of the chromatographic
run is in a faster/similar scale compared to the relaxation time of the cis/trans
isomerization. The relaxation rate is affected by temperature (low temperatures
delay the conformer interconversion), mineral acids and organic solvents which
increase the rate of isomerization. Based on these fundamental studies general
principles can be deduced for the chromatographic resolution of peptide isomers:
- short columns with high resolution packings;
- high flow rates of mobile phase or steeply rising gradients, respectively;
- low working temperatures;
- pH values stabilizing the zwitterionic state of the peptide.
However, low temperatures and high flow rates of mobile phase result in a diminished permeability of the column and in poorer diffusion coefficients leading to
insufficient mass transfers between mobile and stationary phases. These characteristic features can be overcome for instance by the application of unporous micropellicular supports and solvent additives which diminish the viscosity of the
mobile phase.
A simple re-chromatography of the fractionated isomer peaks should be performed to check that the split peaks are really conformers and not peptide impurities. After a certain time of re-equilibration the two isomer peaks can be monitored again.
On-line and off-line NMR measurements of the fractionated isomers from several prolyl peptides confirmed the higher hydrophobicity of cis isomers leading
to an elution order trans before cis (Kalman et al. 1996, Gebauer et al. 1998).
Table 14.1 summarizes some examples for the RP-HPLC of biologically active
peptides bearing one or more Xaa-Pro bonds in their sequence, including the
opioid pep tides 6-casomorphin and morphiceptin, the blood pressure controlling hormones angiotensin and bradykinin, dermorphin and tryptophilin. Of
special interest are present studies on the immunosupressiva cyclosporin A, C,
and D and FK 506 (Joshua 1991).
2.2
Cyclodextrin Bonded Stationary Phases
In recent years macrocyclic compounds including cyclodextrins (CDs), crown
ethers and calixarenes gained increasing interest in host -guest chemistry due to
their extraordinary potential for the molecular recognition of neutral and ionic
molecules. These compounds are characterized by well defined cavities, referring
to size and shape as well as ionic and hydrophobic properties. Therefore, they are
preferentially used as selectors in chromatography and electrophoresis for separations exploiting stereochemical interactions. Cyclodextrin bonded silicas as
stationary phases in HPLC were developed for separating enantiomers by forming diastereomers with the chiral centers of the CDs, but they were used too for
the steric discrimination of geometric and positional isomers.
Cyclodextrins of different ring sizes (a-, ~-, y-CD) offer the opportunity to
exploit a variety of selective interactions with peptides, such as inclusion com-
201
can be achieved considering the fact that the time scale of the chromatographic
run is in a faster/similar scale compared to the relaxation time of the cis/trans
isomerization. The relaxation rate is affected by temperature (low temperatures
delay the conformer interconversion), mineral acids and organic solvents which
increase the rate of isomerization. Based on these fundamental studies general
principles can be deduced for the chromatographic resolution of peptide isomers:
- short columns with high resolution packings;
- high flow rates of mobile phase or steeply rising gradients, respectively;
- low working temperatures;
- pH values stabilizing the zwitterionic state of the peptide.
However, low temperatures and high flow rates of mobile phase result in a diminished permeability of the column and in poorer diffusion coefficients leading to
insufficient mass transfers between mobile and stationary phases. These characteristic features can be overcome for instance by the application of unporous micropellicular supports and solvent additives which diminish the viscosity of the
mobile phase.
A simple re-chromatography of the fractionated isomer peaks should be performed to check that the split peaks are really conformers and not peptide impurities. After a certain time of re-equilibration the two isomer peaks can be monitored again.
On-line and off-line NMR measurements of the fractionated isomers from several prolyl peptides confirmed the higher hydrophobicity of cis isomers leading
to an elution order trans before cis (Kalman et al. 1996, Gebauer et al. 1998).
Table 14.1 summarizes some examples for the RP-HPLC of biologically active
peptides bearing one or more Xaa-Pro bonds in their sequence, including the
opioid pep tides 6-casomorphin and morphiceptin, the blood pressure controlling hormones angiotensin and bradykinin, dermorphin and tryptophilin. Of
special interest are present studies on the immunosupressiva cyclosporin A, C,
and D and FK 506 (Joshua 1991).
2.2
Cyclodextrin Bonded Stationary Phases
In recent years macrocyclic compounds including cyclodextrins (CDs), crown
ethers and calixarenes gained increasing interest in host -guest chemistry due to
their extraordinary potential for the molecular recognition of neutral and ionic
molecules. These compounds are characterized by well defined cavities, referring
to size and shape as well as ionic and hydrophobic properties. Therefore, they are
preferentially used as selectors in chromatography and electrophoresis for separations exploiting stereochemical interactions. Cyclodextrin bonded silicas as
stationary phases in HPLC were developed for separating enantiomers by forming diastereomers with the chiral centers of the CDs, but they were used too for
the steric discrimination of geometric and positional isomers.
Cyclodextrins of different ring sizes (a-, ~-, y-CD) offer the opportunity to
exploit a variety of selective interactions with peptides, such as inclusion com-