Chiral HPLC (Part 2 of 3)
Separation based on cyclodextrins, bonded to silica gel
Especially for chiral separations under reversed phase conditions, phases based on cyclodextrins covalently bonded to a silica matrix via a spacer, have been successful. Cyclodextrins are cyclic oligosaccharides of glucose units. The cyclic structure of a cyclodextrin ring can be described as a hollow truncated cone. The inner diameter of this truncated cone depends from the number of glucose units. Hence it increases from α-cyclodextrins (6 glucose units) to β-cyclodextrins (7 glucose units) to γ-cyclodextrins (8 glucose units), thus influencing the separation in relation to the molecular size of the analyte. The inner surface of the cavity is hydrophobic; thus a nonpolar part of the sample molecule can penetrate into the cyclodextrin ring. This forms so-called inclusion complexes, the stability of which is responsible for the retention of a compound. The chiral sugar units of the cyclodextrins allow enantioselective interactions and thus racemate separations.
Phases NUCLEODEX α-PM, NUCLEODEX β-PM / β-OH and NUCLEODEX γ-PM feature different ring sizes. NUCLEODEX β-OH contains as chiral selector β-cyclodextrin with free hydroxy groups, which can operate as proton acceptor and donor by forming hydrogen bonds. On the contrary, the phases NUCLEODEX α-PM, β-PM and γ-PM contain permethylated cyclodextrins, which can only act as proton acceptor.
Because of its properties the phase α-PM is successfully used for small molecules (e.g. styrene oxide). Due to their size, steroids (e.g. estrone) can be better separated with NUCLEODEX γ-PM. In addition to numerous enantiomer separations (e.g. dopamine besides R- and S-salsolinol), also positional isomers (e.g. o-, m-, p-nitroanilines) can be separated with NUCLEODEX β-OH. The permethylated NUCLEODEX β-PM is preferred for applications, where the free hydroxy groups result in unnecessary interactions and consequently in longer retention times. It also shows a good selectivity for some compounds which cannot be separated on β-OH (e.g. the pesticide mecoprop-methyl). An important requirement for a good separation on cyclodextrins is a sterically bulky group in the α-position of the target molecule. This increases the chance for a base-line separation enormously. Further applications of substances, which meet these requirements, can be found in the Sorbtech application database.
NUCLEODEX phases are normally operated under reversed phase conditions (water or phosphate buffer or triethylammonium acetate buffer / methanol or acetonitrile). But they can also be used under normal phase conditions (heptane with small amounts of alcohol). The pH value of the eluent should be between 3 and 8. The optimum column temperature should be under 50 °C, because the selectivity decreases with increasing temperature.
Protein phase for the separation of optical isomers
Several proteins can undergo enantioselective interactions with pharmacologically active compounds. This effect was first used for chromatographic separation by Stewart and Doherty . Allenmark et al.  applied it to HPLC with BSA (bovine serum albumin) covalently bonded to silica gel.
The protein phase RESOLVOSIL BSA-7 is based on wide-pore silica, to which BSA is covalently bonded. The separation mechanism of protein columns is not known in detail, although it is based on the principle of bioaffinity. Hydrophobic interactions, interactions of polar groups and steric effects are important for it.
The column is compatible with aqueous buffer systems (e.g. phosphate and borate buffers) of pH range 5 – 8. Retention and optical resolution can be regulated by pH, buffer strength (0.01 – 0.20 M) and surface tension via small amounts of n-propanol (0 – 5 %) added as a co-solvent. Already 1 to 2 % n-propanol drastically reduce the retention.
The advantages of the RESOLVOSIL column are the high flexibility to improve the separation by small changes in the mobile phase composition, its high selectivity and its high sensibility, due to its low capacity. Especially with high-sensitivity detectors (fluorescence, electrochemical), but also with UV detectors, only very low amounts (sample concentration <0.2 µmol per injection) need to be injected on the column. Thus, the enantiomeric purity of the sample can be determined very accurately.
From the numerous applications (Sorbtech application database) we wish to mention separation of the barbiturate benzonal and the enantioselective, microbial degradation of racemates.