Dr Regina Valluzzi, Liya Liu and Dr Christopher Sprout at ENS reveal how nanotechnology has expanded the chemical range of chiral chromatography
Chirality presents an exciting opportunity and a difficult challenge for industries that use speciality chemicals. Formulations with controlled chirality are forming the bases of new pharmaceuticals and other important chemical classes. Evolved Nanomaterial Sciences (ENS) is developing a new approach to chiral separations and chemistry: by using a novel suite of chiral materials, it is assembling a chiral toolkit to reduce method development time and risk for high-throughput chromatography at scales ranging from the bench to pilot plant.
Since chiral enantiomers are chemically identical and differ only in their 3D geometry, selectively eliminating one or more enantiomers can be challenging. ENS technology approaches chiral separations in a radically different way, using a "smart" nano-structured material with very strong and general chiral shape recognition to address a broad spectrum of molecules and challenges in chiral chemistry.
The structure of the "smart" material allows it to act as a metamaterial for chemical processing. In contrast to chiral materials that select for specific molecules or classes of molecules, each material in the ENS family of nano-technology-enhanced sorbents has an extremely broad spectrum of chiral selectivity. For example, the identical stationary phase made from ENS material, used in a packed HPLC column, can separate chiral alcohols, free amines, chiral acids, terpenes, alkaloids, amino acid derivatives and a number of compounds within each of these classes that are difficult to separate using competing state-of-the-art HPLC column technology.
In a series of rapid screens for selectivity, the ENS HPLC column was shown to be selective for clenbutarol HCl; propranolol; catechin hydrate; hydroxymandelic acid; hydroxy-methylmandelic acid; camphor; ionone; 2-methyl-2,4-pentane diol; ethyl lactate; 2-octanol; 2-pentanol; 2-methyl-2; 4 -pentane diol; 2-methyl -1-propanol; 2-butanol; 3-butyn-2-ol; 1-hexyn-3-ol; a-methyl benzyl-amine; 2-heptanol; 2-methly-1-butanol; thalidomide; 3-aminopentane nitrile; sec-butyl acetate; benzoin; lysine; histidine; nicotine; salbutamol (albuterol); a-terpineol; a-tocopherol; Troger's base and tryphtophan - all using the identical column and stationary phase, and using primarily mild solvents such as ethanol, methanol and hexane mixtures without peak enhancing additives.
promising approach
Similar generality is observed in supercritical fluid chromatography (SFC), which uses liquid CO2, an alkane-like solvent, to replace a significant volume of the liquid solvent used in HPLC, and is a promising approach to enhancing chiral chromatographic throughput. Selectivity for DL-tryptophan; DL-phenylalanine; DL-histidine; DL-lysine; Ionone; vanilmendelic acid; DL-phenyl-glycine HCl; thalidomide; hydroxymandelicacid; 2-methyl-2; 4-pentane diol; camphor; 2-butanol has been seen using the identical column and ENS material stationary phase.
ENS technology has several unique features as a platform for chiral resolution. The nanomaterials are powerful and selective enough to be used in non-chromatographic formats, since the very high theoretical plate numbers achieved in a chromatographic column are frequently unnecessary. The materials are strongly chirally selective when used in a diffusion kinetics-based format, such as chiral chromatography.
However, strong selectivity is also observed in the absence of chromatographic flows, when enantiomers are allowed to diffuse into solvent swollen media in a batch format. Furthermore, convection through the material is also strongly selective, resulting in double digit ees for a single pass through a filter made from the ENS material. The nano-enhanced functionality of these materials can thus be used in "batch sorbent" mode.
For many analytes, the sorbent possesses a high enough selectivity and capacity in a single batch sorbent step to be substituted for a chiral crystallisation reagent. ENS materials used as batch sorbents have not yet been developed to the same degree as chromatographic approaches; however, batch sorbent mode selectivities of greater than 50% ee and high capacities for ENS materials used as sorbents have been demonstrated for limonene; carvone; phellandrene; linalool; several amino alcohols; alpha-methyl benzyl-amine; lactic acid; malic acid; a number of basic (net basic) amino acids and derivatives, and a number of acidic amino acids and derivatives. In general these separations are performed neat or using concentrated solutions of the analyte in an ethanol-water mixture.
ENS technology also has attractive features when used as an HPLC stationary phase. Chiral HPLC columns packed with ENS materials in the stationary phase can be used to separate very small chiral molecules such as 3-aminopentane nitrile, 1-hexyn-3-ol, 2-methyl-1-pentanol and other molecules with low molecular weight and few strongly interacting functional groups. These molecules represent an important class of chiral compounds because they are widely used as intermediates and starting points in the synthesis of many products, including chiral pharmaceuticals.
Since these starting materials are relatively inexpensive in comparison with the final products, it can be far more economical to address chirality in an early precursor or intermediate than through chiral resolution of a valuable product molecule.
small molecule
In some cases the technology is enabling LC separation of compounds not previously possible. This is especially evident in separations of small aliphatic molecules such as aliphatic chiral alcohols. Our analytical scale results indicate that ENS materials have a high capacity for the molecules resolved, including many of these enabled separations. While capacity alone does not predict throughput at the production scale, the high capacity of these sorbents is very promising.
The concentration of analyte injected onto an HPLC column containing ENS chiral material as a stationary phase is limited by the solubility of the analyte in the solvents comprising the mobile phase and the maximum viscosity capabilities of the injector, rather than by the availability of sufficient active sites on the stationary phase. In demonstrations with proprietary customer samples, loading capacities of 10x - 100x conventional chiral columns of the same volume have been observed routinely.
Enabling resolutions, high capacity chromatographic resolutions and high selectivity and capacity per batch sorbent stage have been observed for a number of compound classes. Of these, the terpenes and chiral alcohols are especially interesting because of their widespread application in diverse areas of the chemical industry.
As with many chiral compounds, chiral alcohols are used as chiral intermediates and synthetic building blocks for pharmaceutical and other compounds. Both of the enantiomers are valuable, while the racemic mixtures are inexpensive commodities. A number of chiral alcohols are also interesting as chiral products. Chiral alcohols are used as chiral solvents in separations and analyses, and as reagents in chiral crystallisation. Despite this wide applicability there are currently only a limited number of chiral alcohols readily available.
Many of the chiral alcohols are produced through asymmetric synthesis, using chiral catalysts to obtain a chiral alcohol from a closely related form. The enantiomeric purity obtained depends to a large degree on the efficacy of the catalyst used to produce a particular alcohol. Catalysts can be highly specific, providing a reasonable approach only for limited groups of closely related alcohols.
Some of the larger alcohols, especially those with aromatic functional groups and other interaction sites, are well resolved chromato-graphically. However, a number of low molecular weight alcohols, aliphatic and other alcohols present a challenge for chromatographic resolution.
Often these types of alcohols can be resolved only using gas chromatography, a technique that typically lacks the throughput to be a viable production method. In many cases derivitisation or functionalisation of the chiral alcohol is required to obtain a resolution.
ENS technology provides a general high capacity chromatographic approach to liquid chromatographic resolution of chiral aromatic alcohols and aliphatic alcohols. Using a general purpose ENS chiral column containing approximately 2g of ENS stationary phase, it is even feasible to resolve alcohols with simple chemical structures and low molecular weights such as 2-pentanol and 2-methyl-1-pentanol.
These small alcohols contain few sites for binding interactions or other interactions typically used in liquid chromatographic chiral selection. The difference in the chiral shape of the two enantiomers is small and the molecules are volatile. Any of these factors can cause difficulties in a liquid chromatographic chiral resolution, and consequently, these molecules are considered challenging for chiral liquid chromatographic resolution.
improved resolution
Neither molecule contains a strong UV chromophore, making detection difficult and necessitating a relatively high loading of the chiral alcohol analyte to detect the chromatographic peaks from the resolved enantiomers. As a consequence, chiral alcohol solutions for HPLC are prepared in a 50% chiral alcohol/50% lower viscosity solvent solution prior to injection into the HPLC. Sometimes lower chiral alcohols can be injected neat (2-butanol is an example).
The resolution of ENS general purpose analytical columns is currently in the process of being improved, focusing on a narrow distribution of 5-10µm spheroidal particles, rather than the larger particle sizes and broader distributions used in the early prototypes and beta release columns.
Several alcohols have been tested in a batch sorbent mode, which consists of the addition to a measured amount of sorbent to a stirred vessel containing a solution of the analyte, or a neat analyte oil. Most of the analyte absorption into the sorbent occurs in less than five minutes and is essentially complete in less than 15 minutes.
Several chiral alcohols have been tested for selectivity in "batch sorbent" mode, and alcohols such as 2-butanol and 2-amino-1-butanol can be fully resolved in fewer than five batch sorbent stages. Additional alcohols are still being investigated.
ENS is currently preparing to release analytical and semi-preparatory scale chromatographic columns in the early summer of 2006. The company is also considering pilot scale opportunities for use of its revolutionary materials, licensing, and collaborative opportunities.