Nano-structured Materials in Chiral Chromatography

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A breakthrough in chiral materials technology simplifies chiral chromatography more than ever before, allowing chiral chromatography columns to behave more like familiar normal and reversed-phase columns. This breakthrough has immediate practical implications for many lab researchers.

Recently developed chiral materials separate chiral shape interactions from chromatographic chemical interactions using a breakthrough in chemical nanopatterning. In conventional approaches to chiral chromatography, the surface chemistry of the solid stationary phase particles interacts with analyte molecules at multiple locations on the analyte. On average, the net interaction between the surface chemistry and an analyte molecule is slightly different for different enantiomers, leading to a small difference in adsorption/desorption kinetics.

This small difference is amplified chromatographically, leading to a separation. Because these differences can be very small for chiral molecules, a large number of chromatographic theoretical plates is required to achieve a typical chiral separation, limiting the flexibility of many chiral chromatographic technologies.

Evolved Nanomaterial Sciences Inc. (ENS) (Cambridge, MA) has taken a different approach that uses nanotechnology to create a shape-based solution to an inherently shape-based problem. This novel approach enables new classes of chiral separations and dramatically reduces the complexity of methods development. Moreover, the nanotechnology utilizes a new chiral material that allows a wide range of formats for chiral separations, chromatography and extraction.

This is achieved by using solid stationary-phase nano-structured materials where the shape of the internal surface area of the stationary phase is as highly controlled as the surface chemistry. These chiral materials possess a unique physical interaction mechanism—a chiral sorting property—based on the unique shape of the interior microstructure and nanostructure, defined at the supermolecular level. Typical chiral stationary phases use multiple points of chemical interaction to indirectly sense chiral shape, but ENS nanostructured materials sense chiral shape directly using transport properties through and within the nanostructure.

Analyte molecules in the proprietary chiral stationary-phase experience a very uniform high curvature, high surface area chiral environment inside the channels of the chiral material. The size and chiral shape of the channels create a strong chiral preference when analyte is inside the channels. Extremely uniform pore sizes and shapes (see Figure 1) enhance the selectivity and efficiency of both chemical and physical mechanisms for chiral selection. This novel physical chiral selection mechanism can address chiral molecules in a simple and general shape-based manner. While other approaches require strongly interacting functional groups on the analyte to obtain enough "points" to indirectly sense chiral shape and achieve a separation, chiral nanochannels, used to sense and sort chiral shape directly, eliminate this need.

Because chiral chemistry and chiral resolution are growing rapidly, the implications of the new approach to chiral separations are potentially far reaching. In addition to the burgeoning interest in chiral chemistry, chiral chromatography is rapidly expanding. The pharmaceutical industry is a major driver of the surge of interest in chiral molecules. More than 80% of the drugs in pharmaceutical development pipelines are now chiral.
While chiral resolution technologies have improved, there is still room for simplification and ease-of-use advances, especially in the areas of batch chromatographic throughput and methods development. Although commercially available chiral HPLC columns address a large number of compounds, there remain entire classes of compounds that are challenging, as well as molecules that cannot be resolved using liquid chromatography.

The new chiral columns are a normal-phase column and a reverse-phase column, both capable of high generality in their ranges of chiral molecules separated. Chiral chromatographers have learned to address chiral separations as a separate and unique set of problems, with its own unique solutions and tools. The normal- and reverse-phase chiral columns offer a new approach to chiral separations that allows chromatographers to use the more predictable and well-understood chemical chromatography paradigm. Normal-phase analytes that are chiral can be separated into chiral enantiomers on the normal-phase chiral column. Reversed-phase analyte, which happen to be chiral, will chirally separate on the chiral reversed-phase column.

The ENS NP chiral column addresses the need for simplicity in chiral methods development and broadens the pool of possible chiral separations to include volatile compounds, low molecular weight compounds, hydrocarbons, and other chemical classes that are poorly addressed by conventional chiral LC column technologies. For example, one normal-phase column with ENS technology can separate chiral alcohols, amines, terpenes, propargylic alcohols, and carboxylic acids, and aliphatic alcohols, as well as more complex pharmaceutical molecules, such as propranolol and thalidomide.

In pre-release tests of the reversed-phase column, a similarly broad spectrum of chiral selectivity is observed. Highly polar and charged analytes have more symmetric traces on the reversed-phase column, and loading capacities are higher for analytes soluble in polar solvents on the reversed-phase column. This pair of columns — a normal-phase chiral column and a reversed-phase chiral column — simplifies chiral chromatographic method development. Rather than considering a large number of chemically distinct chiral selectors and interactions with an analyte, the chemist can choose a normal-phase column for analytes that would separate on a normal-phase non-chiral column. The reversed-phase chiral column works with analytes that would be chemically separated on a non-chiral reversed-phase column.

For example, chiral chromatographic traces are shown for several small chiral alcohols in Figure 2, all of which contain few functional groups. In alcohols such as 2-octanol (shown in Figure 2), there is some aliphatic character and an alcohol group – functionality insufficient for the three or more "points" of interaction required by other approaches to chiral chromatographic separation. Additionally, because the nanochannel-based mechanism does not rely on active sites and surface binding, the capacity is potentially much higher. This capacity is apparent in the per injection column loads used for the alcohol traces in Figure 2 – up to 7.9 milligrams per injection on a 4.6 x 250 mm analytical column.

Larger pharmaceutical molecules can also be separated on the ENS chiral NP column, although highly polar molecules and molecules with multiple amine groups typically separate better on the reversed-phase column. Examples of separations of pharmaceutical-like larger molecules include thalidomide and propranolol, as shown in Figure 3. In typical normal-phase, low-polarity solvents, many of these compounds are only slightly to moderately soluble. Analyte solubility in the mobile phase is a key driver for loading capacity on the ENS chiral NP column, as well as on the reversed-phase column).

As an example of this solubility driven capacity, the loading study of propranolol on the 4.6 x 250 mm NP chiral column (Figure 4) shows good baseline resolution across a range of loadings, but solubility of propranolol in the mobile phase becomes an issue long before the separation degrades significantly, at approximately 100 ug per 10 ul injection. In experiments with the reversed-phase column based on the same materials technology, mobile phases can be selected that offer better solubility for propranolol. Under conditions where propranolol is significantly more soluble in the reversed-phase mobile phase, higher loading capacities are obtained.

Today's conventional approaches to chiral separation require processes that optimize chiral enrichment, often at the expense of other process parameters such as throughput. In fact, conventional approaches force lab chemists to think about chirality first and chemistry second. ENS’ new suite of integrated solutions allow chiral chemists to put chemistry back at the front of the process in the lab, to simplify and speed up the discovery processes, and to scale the methods developed on up the drug-development pipeline.

Regina Valluzzi, PhD is founder and chief scientific officer at Evolved Nanomaterial Sciences Inc.