Introduction
Many pharmaceutically active molecules are chiral and their physiological effects are determined by enantioselective interactions with proteins in a biological organism. These effects can be very different for the enantiomers of a chiral substance. Hence, it is of utmost importance to produce enantiopure substances in the pharmaceutical industry.
The prerequisite for optimizing the production of enantiopure substances is the development and implementation of suitable analytical techniques for process monitoring. However, a major problem in this context is that the enantiomers are virtually identical in terms of molecular structure. This implies that they also exhibit identical physicochemical properties, which makes it very difficult to distinguish between them. In particular, methods that provide structural information and allow for enantioselective discrimination are rare. The list of available methods includes microwave and fluorescence spectroscopy, nuclear magnetic resonance (NMR), vibrational circular dichroism (VCD), Raman optical activity (ROA), cavity ringdown polarimetry, and surface enhanced Raman spectroscopy (SERS). However, all these methods have their particular disadvantages. Some require sampling and sample preparation, some require long measurement times, and some require adding tracers or nanoparticles. Therefore, the suitability of these methods for process monitoring in situ and in real time is limited. Hence, there is a need for new Process Analytical Technology (PAT) to enable inline measurements with high temporal resolution, good reproducibility, and high accuracy as well as precision.
Enantioselective Raman Spectroscopy
Although being a standard tool in many analytical labs, conventional Raman spectroscopy has not been considered as a possible enantioselective technique. Quite the contrary, many textbooks and research articles explicitly state that Raman spectroscopy is inherently incapable of discriminating between the enantiomers of a chiral substance. However, the recent introduction of enantioselective Raman (esR) spectroscopy has proven these statements wrong.1
The esR technique takes advantage of the optical activity of a sample containing chiral molecules. On the one hand, this optical activity results in a rotation of the polarization of the laser light as it travels through the sample. On the other hand, it also rotates the polarized and depolarized signal components. The latter is utilized for the enantioselective detection. However, before this is possible, the inherent symmetry of the scattered light in terms of polarization rotation has to be broken. This symmetry arises from the different enantiomers rotating the polarization plane by exactly the same angle but with opposite sign. Consequently, looking at the vertically and horizontally polarized signal components leads to identical Raman spectra for both enantiomers.
This problem can be overcome by inserting a simple optical component into the signal detection path before the vertically and horizontally polarized parts are separated from each other. This magic component is an achromatic half-wave plate, which rotates the signals of the two enantiomers by different angles and thus breaks the symmetry. The schematic experimental setup is illustrated in Figure 1. It is a conventional Raman spectroscopy setup with polarizationresolved signal detection in direction perpendicular to the laser beam. The only additional component is the half-wave plate inserted before the polarizing beam splitter. The principle of breaking the symmetry is shown in the inset polarization diagrams before and after the half-wave plate. It becomes clear that the polarization directions of the Raman signals are asymmetric with respect to the 0° axis, and thus they can be distinguished from each other.
Figure 1. Schematic experimental esR spectroscopy setup: L = lens, BD = beam dump, HWP = half-wave plate, PBS = polarizing beam splitter, SM1/SM2 = spectrometer. The polarization diagrams to the left indicate how the half-wave plate breaks the symmetry to enable enantioselective detection of the D- and L- enantiomer of a chiral substance.Opportunities and Challenges
A key benefit of the esR technique over a classical determination of the sole rotation angle is that it provides the same rich structural information as conventional Raman spectroscopy. Hence, the signal can be used for a qualitative analysis of the sample enabling the identification of all molecular species in the measurement volume. In addition, Raman spectroscopy is a quantitative tool as the signal intensity scales with the concentration of the molecules. Consequently, esR is technically capable of doing both a qualitative and quantitative measurement. This makes it a perfect tool for process analytical technology. A question that may arise, however, is whether or not the quantitative analysis can yield information about the ratio of the enantiomers in a mixture. Such capability would mean that the technique can be used to monitor the enantiomeric purification in a production process. The answer to this question is: yes, it can! Theoretical considerations have demonstrated that a careful and thoughtful alignment of the half-wave plate in the setup facilitates the determination of the overall concentration of the chiral substance and, in addition, the enantiomeric ratio.2
Another key feature in the context of process monitoring is the short acquisition time required to record an esR spectrum. If required, this measurement time can be on the order of nanoseconds and repetition rates up to kHz are basically possible. This is a major advantage over many other existing analytical methods for enantioselective measurements. Consequently, the new esR method will be a useful technology for both real-time process monitoring and fast screening of chiral samples.
A few challenges remain regarding the experimental realization and the data evaluation. First experiments have, on the one hand, demonstrated that the method works, but, on the other hand, they also revealed the need for high-quality achromatic half-wave plates. Otherwise, artifacts appear in the signal and must be taken into account in the data processing and evaluation. However, these challenges will certainly not prevent the method to be deployed in production processes in the future.
Conclusion
In conclusion, the question in the title can without a doubt be answered with “yes”. The recently developed enantioselective Raman spectroscopy approach can provide qualitative and quantitative measurements with high accuracy, short measurement times, and high repetition rates. Therefore, it is a very promising candidate for inline process monitoring in the pharmaceutical industry.
Acknowledgement
The author thanks Dr. Kristina Noack for helpful discussions.
Author Biography
Prof. Dr. Johannes Kiefer is Chair Professor and Head of the division Technische Thermodynamik at the University of Bremen, Germany. In addition, he is an Honorary Professor at the University of Aberdeen, Scotland, and he holds a guest professorship of the Erlangen Graduate School in Advanced Optical Technologies (SAOT) at the University Erlangen-Nuremberg, Germany. His research interests are the areas of developing and applying spectroscopic techniques for the characterization of advanced materials and processes.
References
- Kiefer J, Noack K. Universal enantioselective discrimination by Raman spectroscopy. Analyst 2015;140(6):1787-1790.
- Kiefer, J. Quantitative enantioselective Raman spectroscopy. Analyst 2015;140(15): 5012-5018.