Defragmenting Chiral Chromatography

Chiral molecules continue to drive growth across a number of industries as new chemistries, active ingredients, and products are developed and commercialized. These industries include pharmaceuticals, fine chemicals and intermediates, agrichemicals, and flavors and fragrances, as well as emerging nutraceutical and cosmeceutical areas. Due to significant benefits provided by single-enantiomer therapeutics and by FDA requirements, the pharmaceutical industry in particular has been actively driving chiral chemistry and technology.

But for all the advantages of chiral compounds, isolating and purifying them has remained a difficult, inefficient, and costly proposition. The technology discussed here approaches chiral separations in a radically different way. A new class of “smart” nano structured materials have been developed that are highly effective at separating a broad spectrum of chiral chemical compounds. This technology, called EnantioSelective Polymer (ESP, Evolved Nanomaterial Sciences [ENS], Cambridge, MA), comprises a suite of novel chiral-selective materials that can be used in a variety of industry formats to deliver chiral separation solutions.

Chiral drugs are one of the fastest-growing categories of pharmaceuticals. Each molecule of a chiral drug has a mirror image. These mirror image molecules, or enantiomers, are identical in terms of their chemical structure and physical properties. While enantiomers can be very hard to distinguish using chemistry, they can have very different biological effects, since the physiological environment is also predominantly chiral. A chiral drug containing a mixture of enantiomers may contain two or more distinct biological ingredients, each with different biological activities. This can cause problems with dosing, lower effectiveness for the chiral drug, unpredictable pharmacokinetics, or harmful side effects. For some chiral drugs to be effective, the mirror image isomer must be eliminated. Since the enantiomers are chemically identical, and differ only in their three-dimensional geometry, selectively eliminating the undesirable enantiomers can be very challenging.

Several different conventional avenues exist to separate enantiomers from a chiral mixture. Chiral crystallization methods, including diastereomeric crystallization, have been around for decades, but method development is tedious and success is often “hit or miss.” On the other hand, chiral chromatography has a higher rate of success and is growing in popularity.

The difficulties that exist in chiral separations have spurred a continued interest in chiral-selective materials that can be used as stationary phases. Most of the current chiral materials select for a particular enantiomer by weakly binding it to another chiral molecule that is immobilized on a support within a column. The chiral molecule, or selector, plus the support, make up the chromatographic stationary phase. Different enantiomers are retained by the stationary phase with different kinetic time scales, resulting in a separation. In most cases, the stationary phase incorporates a large, complex molecule that can stick to the analyte enantiomers at multiple points, leading to chiral selectivity. In other cases, a folded protein is stuck to the support, and analytes are captured in the chiral-shaped pockets of the protein and are held through hydrogen bonds and other complexation interactions. Still other stationary phases rely on beads comprised entirely of polymers of chiral-selective molecules, eliminating the need for a support. All of these approaches rely on a set of chemical interactions and binding events to detect the difference between chiral enantiomers.

EnantioSelective Polymer uses nanotechnology to directly sense the three-dimensional chiral geometry of a molecule by using physical interactions rather than chemical binding. The phenomenon is somewhat analogous to the shape effects observed in chiral-imprinted polymers, where chiral molecules in a polymer are dissolved away, leaving chirally shaped cavities that can select for enantiomers. Polymer imprinting does not work well in the absence of binding interactions, because the shape of the polymer molecules includes nonchiral voids or free volume that “washes out” the imprinted shape recognition. By using nanoscale engineering techniques, the company has created a nanostructured polymer that realizes the true capability of a physical, “chirally imprinted” approach. ESP incorporates a very large number of interconnected chiral voids. The walls of the voids are very dense, preventing the polymer free volume from degrading the chiral selectivity of the chiral voids. Interconnecting the voids makes much more of the material accessible to analyte molecules. ESP medium has an ultrahigh interior surface area comprising minute channels larger than the size of a large molecule. The channels are tightly packed with chiral molecular chains that protrude like dense brushes. Diffusing molecules are forced to interact with these brushes because the interstices are too small to allow any other pathway through the material. The chiral brush and chiral shapes of the channels select for the enantiomers.

Figure 1 - Using ESP technology, the same stationary phase and identical column can separate enantiomers of two alcohols (a and b), an alkaloid (c) that causes adsorption problems in conventional HPLC media, and a small acetate (d). Other compound classes such as fatty chain molecules, acids, and amines have also been separated using the same stationary phase. Small molecules such as the acetates and alcohols shown (a, b, and d) often present challenges for chromatographic separation.

In contrast to chiral materials that select for specific molecules or classes of molecules, ESP has a broadly selective spectrum. For example, the identical ESP-packed HPLC column can separate chiral alcohols, free amines, chiral acids, terpenes, alkaloids, and amino acid derivatives, as illustrated in Figure 1. A number of compounds within these classes, as well as entire classes such as small alcohols and hydrocarbons, have proven very difficult to separate employing conventional HPLC column technology. These compounds and compound classes can be successfully resolved with ESP, and are represented in Figure 1. Several HPLC traces, all using the identical column without repacking or a change of stationary phase, are shown in the figure. It is important to note that the solvents used for ESP HPLC separations are mild and environmentally friendly: typically, ethanol, isopropanol, and hexane.

In addition to chromatographic separations, the polymer can be used as a batch sorbent or as a chiral molecular sieve. This characteristic is unusual and extremely powerful; ESP is indeed a “smart” material and allows for different separation techniques to be utilized in order to obtain a pure enantiomer. The same material used in a columnlike device can also be used as a sorbent. In a number of cases, the polymer has been used to achieve high enantiomeric excess (EE) in a single batch sorbent step using a breathtakingly simple approach. The enantiomeric mixture is poured into a container as a neat liquid or greater than 5 wt % solution. ESP with a 150–250 μm particle size is added to the jar. The ratio of ESP used to undiluted enantiomers ranges from 1:1 to 5:1 by weight. Once the powdered ESP has been added to the container holding the enantiomers (as neat liquid or a reasonably concentrated solution), the container is sealed and agitated for roughly 3–5 min. The ESP is filtered off, leaving predominantly one enantiomer behind. To avoid confusion with the theoretical plates in a chromatography column, the company refers to this procedure as a single sorbent stage. In many cases, greater than 95% EE can be attained in one to five sorbent stages.

This type of simple, highly scaleable separation has been demonstrated for phellandrene, nicotine, several amino acids and their derivatives, lactic and malic acid, and a number of terpenes. The terpenes make a very useful demonstration because chiral compounds such as limonene are inexpensive, not dangerous, and have commercially available enantiomers for comparison. In a typical demonstration, observers are invited to smell the racemate, which is subsequently added to two vials of ESP. One ESP has been designed to exclude the (+) enantiomers; the other excludes the (–) enantiomers. Once the racemate has been added to the two vials containing the two ESP types, the vial contents are mixed by tapping. Observers are then invited to smell the individual enantiomers. Typically, a high EE, greater than 70%, is required to begin to distinguish enantiomers of a fragrance. A distinct difference requires a higher EE. In order to perform a successful smell test demonstration, the liquids must also be added neat, since solvents would interfere with the enantiomers’ individual fragrances. A high loading of neat racemate is also required in order to wet the ESP enough to allow mixing. This simple demonstration establishes the enantio selective power, speed, and capacity of this technology.

The manufacturer is applying this technology to deliver novel tools for the analytical, synthetic, or process chemist working with chiral compounds in a variety of applications, across many industries. The ESP suite of technologies provides breakthrough performance addressing the many challenges posed by chiral compounds.

Dr. Valluzzi is Chief Scientific Officer, and Dr. Chaloner-Gill is V.P. Chromatography, Evolved Nanomaterial Sciences (ENS), 675 Massachusetts Ave., 14th floor, Cambridge, MA 02139-3309, U.S.A.; tel.: 617-441-5107; fax: 617-902-2700; e-mail: