05/09

05/09

Actual Topic

HPLC columns for the enantiomer separation

Molecules, which are like mirror images, are called “chiral” (from the Greek word for “hand”), because they are mirror-symmetrical like hands. They cannot be separated with conventional HPLC columns, because these enantiomers are chemically identical. Chirality is of vital importance in biological processes. Thus, one of the most important drugs against stomach and intestinal ulcers esomeprazol – the (S)-enantiomer of omeprazol – shows an enhanced activity in comparison with the racemate. This enhancement results from a slightly lower metabolism rate and thus from an increased availability of the enantiomeric agent.

Regulatory authorities for pharmaceutical products also demand unambiguous separations of racemates. Thus, enantiomer separation by HPLC has become indispensable for the development and analysis of chiral drugs. But also in biological and chemical, especially in catalytic research, as well as for foodstuffs, chiral HPLC is important.

This article describes several concepts of HPLC columns for enantiomer separation with reference to the racemates to be separated. Finally a flow chart is presented, which supports the – quite demanding – selection of a suitable column.

Enantiomer separation with ligand exchange phases

Ligand exchange phases for enantiomer separation in liquid chromatography were first described by Davankov et al. [1]. They are silica gels with covalently bonded copper(II)-chelate complexing agents as chiral selectors. The most important interaction between the enantiomers to be separated and the chiral selector is the formation of a transition metal complex with analyte and selector as ligands.

An L-hydroxyproline / Cu2+ complex serves as chiral selector for NUCLEOSIL® Chiral-1. Due to the asymmetry centers of hydroxyproline, diastereomeric complexes with the enantiomeric isomers of the analytes are formed. Differences in complex stability result in chromatographic separation.

A successful enantiomer separation can be expected, if the sample molecule contains two polar functional groups, which can form a chelate complex with copper ions, such as α-amino acids (e.g. phenylglycine, alanine, threonine etc.) and α-hydroxycarboxylic acids (e.g. lactic acid).
Aqueous CuSO4 solutions are used as eluents. By increasing the concentration of copper ions (0.2 – 3.0 mmol/l), the retention times can be reduced. A shortening of retention times, as well as an improvement of the peak symmetry can also be reached by an addition of organic solvents, like acetonitrile or methanol. An increase in temperature – we recommend 60 °C – will lead to better separation efficiency with shorter retention times.
Preparative applications are also possible. Thomas and Surber describe the preparative separation of enantiomers of an HIV antiinfective nucleoside [2].

Chiral columns with π-donor/acceptor systems

This type of chiral phase, which is used for the control of the optical purity of compounds under normal phase conditions, was first applied for the separation of racemic helicenes by Mikes et al. [3]. It is termed „brush type phase“, or also „Pirkle phase“, because the working group of Pirkle synthesized numerous versions of this type.

As chiral selector for NUCLEOSIL® Chiral-2, dinitrobenzoyl-D-phenylglycine is covalently bonded to silica gel via a spacer, the optical antipode dinitrobenzoyl-L-phenylglycine is used for NUCLEOSIL® Chiral-3.

There are numerous applications for the separation of enantiomers and diastereomers, especially for the control of optical purity of herbicides (e.g. fenoprop methyl), insecticides (e.g. cypermethrin) or drugs (e.g. the anticoagulant phenprocoumon).
For the determination of the optical purity of mecoprop methyl the less concentrated impurity can be eluted before the main peak by using NUCLEOSIL® Chiral-3, which contains the optical antipode of Chiral-2. Thus, a reliable quantification is possible.

The separation mechanism could not yet be clarified in all details. But besides π-donor/acceptor interactions hydrogen bonds, dipole/dipole interactions and steric effects determine a separation.
Nonpolar organic mobile phases, such as n-heptane, iso-octane with polar additives like tetrahydrofuran, alcohols or chlorinated hydrocarbons are used for the application of these chiral columns. Small amounts of strong acids (e.g. trifluoroacetic acid) can considerably improve the separation of the enantiomers. The solubility of basic compounds can often be enhanced by derivatisation (e.g. with benzoyl chloride or 3,5-dinitrobenzoyl chloride).

Separation based on cyclodextrins, bonded to silica gel

Especially for chiral separation 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) via 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 position 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 interaction 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. That increases the chance for a base-line separation enormously. Further applications of substances, which meet these requirements, can be found in the MN 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 [4]. Allenmark et al. [5] 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 1-propanol (0 – 5 %) added as a co-solvent. Already 1 to 2 % 1-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 (MN application database) we wish to mention separation of the barbiturate benzonal and the enantioselective, microbial degradation of racemates.

Chiral separation with efficient polysaccharide/silica phases

The chirality of polysaccharides was first used for paper chromatographic separation of racemic amino acid derivatives by Kotake et al. [6]. The cellulose structure of the paper functions as selector. Further studies used cellulose or potato starch with its ingredient amylose for thin layer and liquid chromatography [7, 8]. A line of cellulose-tris(phenylcarbamate) derivatives was synthezised by reaction of microcrystalline cellulose with substituted phenyl isocyanates by Okamoto et al. [9]. These derivatives can separate numerous racemates with different functional groups, their separation capability being influenced by the substituents on the phenyl group. Also amylose-tris(phenylcarbamate) compounds were successfully researched with regard to their enantioselectivity [10]. From the synthesized tris(phenylcarbamates) of cellulose and amylose, 3,5-disubstituted derivatives (e.g. tris(3,5-dimethylphenylcarbamates) show a remarkable enantioselectivity for numerous racemic separations.

This high enantioselectivity can be used with NUCLEOCEL Alpha (amylose-tris(3,5-dimethylphenylcarbamate adsorbed on high purity silica gel) and NUCLEOCEL Delta (cellulose-tris(3,5-dimethylphenylcarbamate/silica gel) under normal phase and under reversed phase conditions.

amylose cellulose

Thus, hexane or alternatively n-heptane with alcohols (propanol, ethanol) or even alcohol mixtures and acetonitrile/alcohol mixtures in any ratios can be used in NP mode. Addition of acetic acid or trifluoroacetic acid (0.1 – 1 %) for acidic analytes or diethylamine (0.1 – 1 %) for basic analytes is possible. In RP mode eluent mixtures of methanol or acetonitrile with water or buffer (acidic or basic phosphate and borate buffer) are used. Aggressive solvents (e.g. THF, dioxane, chloroform, DMSO, DMF) should be avoided, because the chiral selector is not bonded, but adsorbed to the silica.

Due to their high enantioselectivity NUCLEOCEL phases have a broad range of applications in chiral HPLC. Thus, numerous pharmaceutically active compounds, such as omeprazol, hexobarbital, verapamil, metoprolol or under RP conditions indapamide and warfarin can be successfully separated. Today the active compound thalidomid, which is historically known from its tragic side effects, can be reliably determined from the hypnotic Contergan.
Furthermore chiral environmental pollutants (e.g. the herbicide fenoxaprop ethyl), plant ingredients (e.g. benzoin from oil of bitter almond, linalool from oil of lavender) or fine chemicals (e.g. trans-stilbene oxide), which are inter alia used as chiral catalysts in synthesis, can be effectively separated.

Selection criteria for a suitable chiral column

In particular without any application note, the selection of a suitable chiral column for a successful enantiomer separation is not easy.
The following flow chart (modified according to Ahuja [11]) is meant as a tool for the selection of a column with an increased chance of separation.

If a compound can be dissolved in an NP eluent and can form π-donor/acceptor interactions, hydrogen bonds and dipole/dipole interactions with the chiral selector, then NUCLEOSIL Chiral-2 or Chiral-3 can be tried. If there is no acidic or basic group next to the chiral carbon, then derivatisation with a reagent of this constitution can be useful.
But a successful separation without derivatisation and without this constitution is often possible with NUCLEOCEL Alpha and/or Delta. Depending on the presence of aromatic carbonyl, tertiary nitrogen or hydroxy groups in the analyte, NUCLEOCEL Alpha or Delta may be applicable.
If a copper-chelate complex with a chiral compound can be formed, then NUCLEOSIL Chiral-1 has good chances of success.
If a complexation is not possible, RESOLVOSIL can be tried, if the molecule contains near the chiral centre a function ionizable under RP conditions.
If this is not the case, NUCLEODEX columns can be successful for the target separation by inclusion of hydrophobic groups into cyclodextrin. And if here no success is achieved, then NUCLEOCEL Alpha-RP or Delta-RP may provide a solution.

Conclusion

For the selection of a suitable HPLC column for enantiomer separation MACHEREY-NAGEL offers several concepts of chiral columns, which have the required enantioselectivity. Especially the polysaccharide/silica gel phases NUCLEOCEL Alpha and NUCLEOCEL Delta are often successfully applicable.
The MN application database provides numerous chiral application notes, which simplify the search for a successful separation.

Literature

[1] Davankov, V.A., Rogozhin, S.V., Semechkin, A.V., Sachkova, T.P., J. Chromatogr. 82 (1973), 359
[2] Thomas, S.B., Surber, B.W., J. Chromatogr. 586 (1991) 265-270
[3] Mikes, F. Boshart, G., Gil-Av, E., J. Chromatogr. 122 (1976), 205
[4] Stewart, K.K., Doherty, R.F., Proc. Natl. Acad. Sci. USA 70 (1973), 2850
[5] Allenmark, S., Bomgren, B., Boren, H., J. Chromatogr. 264 (1983), 63-68
[6] Kotake, M., Sakan, T., Nakamura, N., Senoh, S., J. Am. Chem. Soc. 73 (1951), 2973-2974
[7] Contractor, S.F., Wragg, J.A., Nature 208 (1965), 71-72
[8] Taylor, L.T., Busch, D.H., J. Am. Chem. Soc. 89 (1967), 5372-5376
[9] Okamoto, Y., Kawashima, M., Hatada, K., J. Chromatogr. 363 (1986), 173-186
[10] Okamoto, Y., Aburatani, R., Fukumuto, T., Hatada, K., Chem. Lett. (1987), 1857-1860
[11] Ahuja, S., LCGC North America, The Application Notebook (February 2008), 70-79

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