Chromatography

Chiral HPLC (Part 3 of 3)

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
Amylose
Cellulose
Cellulose
3-5-dimethylphenylcarbamate
3-5-dimethylphenylcarbamate

 

 

 

 

 

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 omeprazolehexobarbitalverapamilmetoprolol 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.

Flowchart

 

 

 

 

 

 

 

 

 

 

 

 

 

 

If a substance can be dissolved in an NP eluent and can form π-donor/acceptor interactions, hydrogen bonds and/or 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 SorbTech offers several types of chiral columns, which have the required enantioselectivity. Especially the polysaccharide/silica gel phases NUCLEOCEL Alpha and NUCLEOCEL Delta are often successful.
The Sorbtech 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

Product Highlight

NUCLEOCEL chiral stationary phase columns

Macherey-Nagel offers NUCLEOCEL as a new column family of chiral stationary phases (CSPs). The chiral selector is based on a modified polycaccharide derivative, which has been coated on high performance spherical silica particles. These columns allow due to their excellent enantioselective properties baseline separations with superior alpha values for the majority of all known racemic compounds.

The chiral selector of NUCLEOCEL DELTA is cellulose-tris (3,5-dimethylphenyl)-carbamate and together with the supramolecular helical polysaccharide surface structure mainly responsible for the chiral recognition mechanism. NUCLEOCEL ALPHA is the modified amylose based counterpart and offers an amplification of chiral selectivity.

Recommended range of applications:nucleodur

  • pharmaceutical active compounds (in accordance with USP column designations L40 and L51)
  • chiral pollutants (e.g. herbicides, PCBs)
  • chiral compounds in food (dyes, preservatives)
  • chiral catalysts and bioorganic molecules

NUCLEOCEL CSPs offer high loading capacities and thus they are recommended for scale-up methods. The stable silica support protects the coated polysaccharide and tolerates high flow rates with a maximum operating pressure up to 2000 psi. Whether for method development or for routine analysis NUCLEOCEL CSPs are the best choice for the successful separation of enantiomeric compounds according to their outstanding performance, flexibility and robustness.

More INFORMATION about NUCLEOCEL chiral columns – Please click here!

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Chiral HPLC (Part 2 of 3)

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Chiral GC (Part 1 of 2)

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