8. How do I develop separation methods using the range of Daicel’s immobilised chiral columns?
As already mentioned, immobilised polysaccharide derived CSPs have a large number of benefits that should be considered in the screening process. Based on exhaustive investigations on immobilised columns, it was concluded that a group of solvents and their mixtures can lead to better selectivity values for most of the compounds tested. It could be considered as a primary set of mobile phases for screening with these CSPs. Primary solvent configurations for the analytical screening system are composed of heptane/ethanol, heptane/2 propanol, dichloromethane (DCM) based and methyl tert butyl ether (MtBE)-based mixtures (Table 1).
Chlorinated solvents are usually submitted to a more rigorous control than the rest, due to environmental reasons. Nevertheless, their use at analytical level may be allowed in most laboratories, provided that their waste residues are treated appropriately. If the use of DCM is constrained due to environmental restrictions, tetrahydrofuran (THF)-based mixtures could be implemented instead.
If additional screening of mobile phases was required, the use of the following mixtures as a secondary set was recommended: heptane/ethyl acetate (EtOAc), heptane/ THF, acetonitrile (ACN) and pure alcohols (ethanol (EtOH) or 2 propanol (2-PrOH)) or their mixtures (Table 1).
Table 1. Mobile phases for sample screening with immobilised CSPs (CHIRALPAK IA, IB, IC, ID, IE and IF columns)
| Solvent mixture | Starting condition | Typical optimization range | |
| Alkane/EtOH | 80/20 | 99/1 to 50/50 | |
| Primary | Alkane/2-PrOH | 80/20 | 99/1 to 50/50 |
| mobile phases | Alkane/DCM/EtOH | 50/50/2 | 85/15/0 to 0/80/20 |
| Alkane/MtBE/EtOH | 0/98/2 | 80/20/0 to 0/40/60 | |
| Alkane/EtOAc | 50/50 | 80/20 | |
| Secondary | Alkane/THF | 70/30 | 95/5 to 0/100 |
| mobile phases | MeOH | 100 | |
| ACN | 100 |
Different alcohols, with shorter or longer aliphatic chains, e.g. EtOH, n-propanol and 2-PrOH, can play a “dual” role to mix either with alkanes or with acetonitrile and methanol. However, the solvent nature varies little and hence the choice stays quite limited if the viscosity factor is considered. In contrast, solvents of such as THF, EtOAc, MtBE, DCM, etc. belong to different solvent families with significantly different properties. Unlike heptane, methanol and acetonitrile, those solvents are all of mid polarity and miscible with both apolar and polar solvents. This feature is beneficial for fine-tuning composition of mobile phases, as it allows easy adjustment in retention, selectivity and resolution.
Note that DCM, THF, ethyl acetate and MTBE will destroy conventional, coated polysaccharide-based chiral columns and should only be used with the new immobilised columns.
References:
Zhang T, Franco P, Analytical and preparative potential of immobilised polysaccharide-derived chiral stationary phases, in Chiral Separation Techniques – A Practical Approach, third ed., G. Subramanian (Editor), Wiley-VCH, Weinheim, Germany, 2006, 99-134
Common approaches for efficient method development with immobilised polysaccharide-derived chiral stationary phases, P. Franco, T. Zhang, J. Chromatogr. B, 875 (2008) 48
P. Franco, T. Zhang, Common screening approaches for efficient analytical method development in LC and SFC on columns packed with immobilised polysaccharide stationary phases, in Chiral Separations, Methods and Protocols, second ed., G. Scriba (Editor), Humana Press, 2013, 113-126
Enantiomer resolution screening strategy using multiple immobilised polysaccharide-based chiral stationary phases, T. Zhang, D. Nguyen, P. Franco, J. Chromatogr. A, 1191 (2008) 214-222
Solvent versatility of immobilised 3,5-dimethylphenylcarbamate of amylose in enantiomeric separations by HPLC, T. Zhang, C. Kientzy, P. Franco, A. Ohnishi, Y. Kagamihara, H. Kurosawa, J. Chromatogr. A, 1075 (2005) 65-75
Cellulose 3,5-dimethylphenylcarbamate immobilised on silica: A new chiral stationary phase for the analysis of enantiomers, T. Zhang, D. Nguyen, P. Franco, T. Murakami, A. Ohnishi, H. Kurosawa, Anal. Chim. Acta, 557 (2006) 221-228
Cellulose tris(3,5-dichlorophenylcarbamate) immobilised on silica: A novel chiral stationary phase for resolution of enantiomers, T. Zhang, D. Nguyen, P. Franco, Y. Isobe, T. Michishita, T. Murakami, J. Pharm. Biomed. Anal., 46 (2008) 882-891
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The approach of method development and optimization on the coated CSPs can be based on a similar strategy to the one used for the immobilised-type phases. However, the choice of solvents would be restricted to those compatible with these supports and all mobile phases that could potentially dissolve the polysaccharide derivative must be avoided. The screening set will then be composed of alkane/EtOH, alkane/2-PrOH, alcohol-based and ACN-based mixtures (Table 2). In practice, these mobile phase systems are normally set up using HPLC screening systems, but often ACN or its mixtures with alcohols will be screened separately (or with accurate intermediate rinsing), in order to avoid any potential accidental mixing between alkane and ACN. If such an event happens, the coated column could be irreversibly damaged.
Table 2. Mobile phases for sample screening with coated CSPs
| Solvent mixture | Starting condition | Typical optimization range |
| Alkane / EtOH | 80/20 | 99/1 to 50/50 |
| Alkane / 2-PrOH | 80/20 | 99/1 to 50/50 |
| MeOH | 100% | |
| ACN | 100% |
