Using Mid-IR Vibrational CD Spectroscopy
This convenient, quantitative approach needs no additional chiral reagents or reactions.
P r a s a d L . Po l a v a r a p u Jiangtao He VA N D E R B I LT U N I V E R S I T Y © 2004 AMERICAN CHEMICAL SOCIETY
Chirality plays a central role in the existence and continuation of life. Vastly different biological and pharmacological effects imparted by the enantiomers of a chiral species attest to the importance of developing a knowledge base at the molecular level, where researchers seek to understand the 3-D molecular structures (absolute configuration and conformation) of chiral species or the reactions that are specific to chiral structures. Manufacturing enantiomeric drugs is a major source of revenue for pharamaceutical companies. Determining the purity of enantiomeric drugs has now become a regulatory requirement (1). The pharmacological and toxicological activities of both enantiomers of a chiral drug must be established before the drug is released for public consumption. F E B R U A R Y 1 , 2 0 0 4 / A N A LY T I C A L C H E M I S T R Y
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are realized when the region of measurement is switched from visible to IR detector the mid-IR, although the advantages outweigh the disadvantages. Light source Lens One advantage is the large amount PEM Interferometer of information. Because every molecule has 3N – 6 vibrations, where N Lock-in Filter is the number of atoms in the molamplifier ecule, many vibrational transitions, FT FT in principle, are available for CD investigations. More stringent requiredc ac ments for obtaining the final 3-D molecular structure satisfy a larger amount of information and hence minimize the probability for a wrong FIGURE 1. Schematic of an FT instrument for VCD measurements in the mid-IR. conclusion. Another advantage is reliable theoretical support. Quantum mechanical predictions of VCD spectra are quite successful in replicating Chiral chromatography is well developed and routinely used the corresponding experimental spectra in the mid-IR region. for separating the enantiomers of a given chiral species and de- Such reliability in the quantum mechanical predictions of midtermining their enantiomeric purity (2). However, chromatog- IR VCD has been a crucial factor for successful chiral structural raphy offers little information about the absolute configuration analysis. In practice, if quantum mechanically predicted spectra or 3-D molecular structure of the species. On the other hand, for different possible structures are compared to the experimenoptical rotation (OR) and electronic circular dichroism (ECD), tal spectra for a molecule with an unknown structure, then the which are inherently sensitive to molecular chirality and are re- predicted spectrum that best matches the experimental spectrum ferred to as chiroptical techniques, can be used independently to reveals the structure of that molecule. One disadvantage is that longer wavelengths associated with determine 3-D molecular structures (3, 4). These methods have certain limitations that originate from underlying phenomena the mid-IR region probe molecular chirality less efficiently comthat involve excited electronic states. OR and ECD methods are pared to the shorter wavelengths associated with the visible rewidely used in combination with chromatography as chiral de- gion. As a result, CD signals in the mid-IR region are ~3 orders tection techniques. NMR and MS are inherently insensitive to of magnitude smaller than those in the visible. Another disadchirality (5, 6) but can be made sensitive by using supplemen- vantage is that the detectors used for mid-IR are inherently less tary chiral reagents that form diastereomeric complexes with the sensitive than the corresponding detectors for visible detection, chiral species of interest. These techniques have their own advan- resulting in a S/N that is lower than that in the visible region. tages and disadvantages, but they offer limited or no information These disadvantages translate into the need for longer data collection times and larger sample concentrations. Typical data about 3-D molecular structure. Some of the driving forces for developing new techniques in- collection times vary from 30 min to several hours per sample clude a need for improved accuracy, better understanding, and for sample concentrations of ~1–50 mg/mL. Within these time ease in arriving at the end result. Vibrational circular dichroism and sample concentration limitations, VCD in the mid-IR is a (VCD) spectroscopy in the mid-IR region is an emerging tech- reliable and convenient chiral analysis tool. nique that meets some of these qualifications for determining the 3-D structure of chiral molecules and serves as an alternate tool Instrumentation and experimental considerations for determining the enantiomeric purity of chiral species. In the early developmental stages, instruments for measuring VCD in the mid-IR were built in individual laboratories (7, 8). They were based on dispersive mid-IR spectrometers, where polyVCD in the mid-IR The principles of measuring CD in the visible spectral region by chromatic light from a mid-IR source is dispersed by using a probing electronic transitions are well known and fairly routine in grating in the monochromator. One drawback of dispersive inthe chemical and biochemical communities. The same principles struments is the significant light loss at the entrance and exit apply to measuring CD in the mid-IR region, except that funda- slits of the monochromator; FTIR spectrometers, on the other mental vibrational transitions are probed. The successful emergence hand, do not have this problem. Furthermore, in FTIR specof VCD in the mid-IR is a consequence of advances made in both trometers, the nature of interference phenomena permits the deinstrumentation and quantum mechanical predictions of vibrational tection of light at all wavelengths simultaneously. These two adproperties. Neither of these two developments alone would have vantages, referred to as throughput and multiplex advantages, made this research area attractive, but together they brought this have led to the adoption of FTIR spectrometers for VCD mearesearch to the forefront. Both the advantages and disadvantages surements in the mid-IR (9). A N A LY T I C A L C H E M I S T R Y / F E B R U A R Y 1 , 2 0 0 4
During the initial period of these developments, VCD spec- oratories have adopted this technique. (Examples of commercial tra for single enantiomers could not be obtained without mea- instruments include the ChiralIR from BioTools, www.btools. suring the same for the opposite enantiomer or racemic mixture, com; the JV2001 from Jasco, www.jascoinc.com; and the PMA-37 because of artifacts. Minimization of artifacts in the VCD mea- accessory from Bruker Optics, www.brukeroptics.com.) The experimental measurements themselves are not difficult, surements has been the primary focus for several years. With the minimization of artifacts (10–13) and the ability to record mid- and a technical person can be trained in a relatively short time IR VCD spectra for single enantiomers, plans to commercialize to make routine measurements. The solvent should have few mid-IR absorption bands in the region of measurement. Inert the instruments began to emerge. solvents such as CCl4 and CS2 are ideal Most current instruments are based on the FT method (9, 14, 15; Figure 1). Light from a choices. However, polar solvents such as polychromatic mid-IR source is passed through CH3OH, CHCl3, and CH2Cl2 can also be a Michelson interferometer, which modulates used, but to avoid interference with sample different wavelengths at different frequencies. absorption bands, their deuterated analogues These Fourier frequencies are usually less than may also be needed. The sample concena few kilohertz and depend on the velocity of tration is, to some extent, dictated by the a moving mirror in the interferometer. The strength of the sample absorption bands. At light exiting the interferometer is circularly the same time, to avoid the complexities in polarized through the use of a linear polarizinterpreting the resulting spectra, concener and photoelastic modulator (PEM), which trations that lead to sample aggregation or converts the linearly polarized light into alintermolecular hydrogen bonding should ternating left and right circularly polarized not be used. As a rule of thumb, the ablight components. The modulation between sorbance of a band of interest needs to be circularly polarized components takes place kept to
