Chiral Analysis Using Broadband Rotational Spectroscopy - The

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Chiral Analysis Using Broadband Rotational Spectroscopy V. Alvin Shubert,†,‡ David Schmitz,†,‡ Cristóbal Pérez,†,‡,¶ Chris Medcraft,†,‡ Anna Krin,†,‡ Sérgio R. Domingos,†,‡,¶ David Patterson,§ and Melanie Schnell*,†,‡,¶ †

Max-Planck-Institut für Struktur und Dynamik der Materie, Luruper Chaussee 149, D-22761 Hamburg, Germany Center for Free-Electron Laser Science, Luruper Chaussee 149, D-22761 Hamburg, Germany ¶ The Hamburg Centre for Ultrafast Imaging, Universität Hamburg, Luruper Chaussee 149, D-22761 Hamburg, Germany § Department of Physics, Harvard University, Cambridge, Massachusetts 02138, United States ‡

ABSTRACT: broadband microwave spectroscopy is a proven tool to precisely determine molecular properties of gas-phase molecules. Recent developments make it applicable to investigate chiral molecules. Enantiomers can be differentiated, and the enantiomeric excess and, indirectly, the absolute configuration can be determined in a molecule-selective manner. The resonant character and high resolution of rotational spectroscopy provide a unique mixture compatibility. Future directions, such as extending the technique to chemical analysis, are discussed.

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species are present. Another challenge can be the unambiguous differentiation between diastereomers and enantiomers for molecules with multiple stereogenic centers. One way to separate and differentiate them is to go via the solid phase using fractional crystallization. Furthermore, in some chiroptical techniques such as vibrational circular dichroism (VCD), the chiral effect can be very weak for vibrational modes that are distant from the stereogenic centers. As a consequence, reports of unambiguous differentiation between diastereomers are limited compared to the vast number of studies involving enantiomers.10 A third challenge concerns the a priori determination of the handedness of the chiral molecules involved, that is, their absolute configuration.

hiral molecules are omnipresent in our daily life. Sugars, amino acids, and pharmaceutical drugs can be chiral, and the basis behind the homochirality of life is still a mystery.1 Although Pasteur’s discovery of chirality was more than 150 years ago2 and Lord Kelvin defined chirality in 1884 in his Baltimore Lectures (“I call any geometrical figure, or group of points, chiral, and say it has chirality, if its image in a plane mirror, ideally realized, cannot be brought to coincide with itself.”),3 chemists remain fascinated by chiral molecules. When omitting the parityviolating character of the weak interaction, the two enantiomers of a chiral molecule are identical in many physical properties, such as their boiling and melting points. However, once in a “chiral environment”, they can show significant differences. One impressive example is the difference in odor for the two enantiomers of carvone; while the S enantiomer smells like caraway, the R enantiomer smells like spearmint, indicating an enantioselective character of our olfactory system for which a molecular-level understanding of olfaction has not yet been fully developed.4 For an introduction into the history of chiral molecules, please refer to ref 5 and references therein. Chirality also plays a major role in the development and function of pharmaceutical drugs, and thus, it comes as no surprise that a number of different analysis methods for chiral samples has been developed. These include polarimetry, circular dichroism, chiral chromatography, and X-ray diffraction approaches. Despite the success and broad applicability of these techniques (summarized, for example, in refs 6−9), a number of challenges in chiral molecule analysis remain. This includes the precise determination of the enantiomeric excess (ee) of either nearly racemic (i.e., having a very small ee) or nearly enantiopure samples, particularly when several chiral © 2015 American Chemical Society

Microwave three-wave mixing exploits the mirror-image character of the enantiomers of chiral molecules. It can be applied to differentiate enantiomers in chiral mixtures. It allows determination of their enantiomeric excess and absolute configuration.

Received: November 2, 2015 Accepted: December 29, 2015 Published: December 29, 2015 341

DOI: 10.1021/acs.jpclett.5b02443 J. Phys. Chem. Lett. 2016, 7, 341−350

The Journal of Physical Chemistry Letters

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Today, determination of the absolute configuration of uncharacterized chiral molecules is primarily achieved through X-ray diffraction of enantiopure crystalline samples as well as circular dichroism of solvated samples. The first method was developed by Bijvoet and co-workers in 1951.11 Purification and crystallization can be a challenging and time-consuming procedure and sometimes not even possible. Chiroptical methods, such as VCD and vibrational Raman optical activity, combined with sophisticated ab initio calculations, also provide information about the molecules’ absolute configuration and conformation and are widely used.12 These chiroptical techniques can be limited due to the intrinsically small effects on which they rely.13 Although significant advances have been made,14−17 such techniques sometimes lack the signal intensity and resolution to address important aspects of biomolecular systems, including mixture compatibility. A number of new techniques investigating chiral molecules in the gas phase have been developed recently.18 Using high-power circularly polarized synchrotron radiation or femtosecond laser pulses, the photoelectron circular dichroism of chiral molecules can be studied, which expresses itself in a forward−backward asymmetry of the photoelectron distributions with respect to the propagation direction of the radiation.19−24 With this technique, enantiomers can be differentiated and the ee determined.25 Another approach relies on Coulomb explosion of chiral molecules after multiple ionization of the molecules, for example, by using high-power femtosecond lasers. The charged fragments are recorded in coincidence on a detector, which allows the reconstruction of the respective molecular structures and thus their handedness.26,27 The requirement that many fragments must be detected simultaneously represents a significant challenge to extending this technique to larger molecules, including those containing several stereogenic centers. Recently, we demonstrated a new approach, based on rotational spectroscopy, to differentiate enantiomers and to determine ee, all within chiral mixtures.28−30 The method relies on a unique spectroscopic property of chiral molecules, which can have closed cycles involving three electric-dipole-allowed transitions of the form, for example, |1⟩ a |2⟩ b |3⟩ c |1⟩, due to ⃗ ⃗ ⃗ their lack of definite parity. Each electric-dipole-allowed transition involves another transition dipole moment component μa, μb, or μc. Furthermore, the scalar triple product of the three transition dipole moment components μ⃗ a · (μ⃗b × μ⃗ c) is of opposite sign for a pair of enantiomers. Hirota initially proposed that such cycles could be exploited to differentiate between enantiomers,31 and after experimental demonstration,28,29 Grabow gave a theoretical description based on the density matrix formalism.32 One key aspect of the technique is that in Fourier transform microwave (FTMW) spectroscopy, the molecular signal is recorded as a free-induction decay (FID) in the time domain, similar to FT-NMR spectroscopy. This detection is inherently phase-sensitive, which we exploit in our experiments. Enantiomeric pairs show a relative phase difference of π radians (180°) in the FID signals of their so-called listen transitions (i.e., transition c in the above scheme connecting |3⟩ and |1⟩; also see below). This phase difference arises from the opposite signs of the scalar triple products of the transition dipole moments μ⃗ a · (μ⃗ b × μ⃗ c) between the enantiomers (Figure 1). The obtained chiral molecular signal is of comparable strength to nonchiral FTMW spectroscopy involving two energy levels. It is the main purpose of this Perspective to give an overview of the latest developments in this

Figure 1. Two enantiomers are mirror images of each other as are their dipole moments. This property leads to opposite signs for the scalar triple products of the transition dipole moment components for a fixed coordinate system because the sign of the scalar triple product depends on the order of the vectors. It changes upon mirror reflection and is even under time-reversal symmetry, which makes this quantity a measure of true chirality.

new area of high-resolution rotational spectroscopy. For more technical details, we refer to ref 33, for example. One notable advantage of using rotational spectroscopy for the analysis of chiral molecules is its inherent mixture compatibility. Due to the high resolution and structural sensitivity of the technique, structural isomers, conformers, diastereomers, and also isotopologues all show unique rotational spectra. With the implementation of a novel spectrometer design based on a broadband microwave chirp, published by Pate and co-workers in 2008,34 this high structural selectivity can now be fully exploited. The chirp technique revolutionized the research field of high-resolution rotational spectroscopy, and significant extensions continue to be developed. It allows for the recording of broadband rotational spectra of increasingly complex and conformer-rich molecules and also mixtures thereof within a single acquisition.34−36 broadband operation over tens of GHz has transformed CP-FTMW spectroscopy into a fast, sensitive technique.37−40 For instrumental details, the reader is referred to the above-mentioned references, for example. In short, a broadband microwave chirp is generated using an arbitrary waveform generator (AWG), which is either coupled into a vacuum chamber directly or after multiplication and upconversion. The chirp is broadcast into the vacuum chamber using horn antennas, where it interacts with a packet of supersonically expanded, internally cold molecules. If the molecules are resonant with a frequency within the chirp, polarization of the sample is achieved, and a macroscopic dipole moment is formed. We subsequently record the decay of this macroscopic dipole moment as a FID in the time domain with a high-speed digital oscilloscope. Phase stability allows for averaging high numbers of acquisitions in the time domain. Using Fourier transformation (FT), the molecular response is transformed from the time to the frequency domain. The high acquisition speed, paired with the instrument’s sensitivity, also enables the study of the kinetics of isomerizations41 and pyrolysis reactions,42 as well as the precise structure determination of complex molecules and molecular clusters via their isotopologues, either in natural abundance or by using isotopically enriched samples. Examples are the recent works on the water nonamer and decamer43 as well as microsolvated camphor.44 In Figure 2, the broadband spectrum of menthone (2-isopropyl-5methylcyclohexanone) and its diastereomer isomenthone is shown, 342



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common level with the transition selectively excited by the RF pulse is depleted in the spectrum because the resonant RF pulse influences the signal coherence of the microwave transition. In addition to changes in the amplitude, we observed characteristic phase changes in the FID of the microwave signal transition when scanning the RF through resonance (see Figure 3). The phase was not extensively considered in previous experiments. The direction of the phase change allows for definitive information not only about which energy levels are connected with each other but also about how they are connected, that is, in a progressive or regressive arrangement (Figure 3b and d).49 This additional information goes clearly beyond what can be obtained from an amplitude analysis and can be key to facilitating the assignment of complex rotational spectra. The measured phase is typically substantially more stable than the amplitude, which can be affected, for example, by instabilities of the supersonic jet expansion. One can envision that in the near future, broadband doubleresonance microwave spectroscopy experiments will be able to support the spectral assignment and to pave the road toward automated assignment. These developments are inspired by methods in FT-NMR spectroscopy, which is technically quite similar. However, the lower frequencies (RF) involved in FT-NMR spectroscopy allowed the development of complex pulse sequences already some decades ago, such as those applied in 2D-NMR schemes.50 The newly developed microwave three-wave mixing technique for chiral molecule analysis can also be understood as a doubleresonance experiment but performed in a polarization-sensitive manner (see Figure 4).29,30 The implementation of microwave three-wave mixing relies on closed cycles of three rotational transitions (Figure 4). Each rotational transition solely depends on one molecular dipole moment component, that is, either on μa (a-type transition), on μb (b-type transition), or on μc (c-type transition). We modify the experimental setup of a CP-FTMW spectrometer such that we excite a drive transition (for example, a rotational c-type transition) and a twist transition (for example,

The chirp technique revolutionized the research field of high-resolution rotational spectroscopy. illustrating the high structural sensitivity of broadband rotational spectroscopy. The upper trace of Figure 2a shows the experimental spectrum, while the lower traces display results of successfully fitting four different asymmetric rotor Hamiltonians to the experimental spectrum. Three conformers for menthone were observed under the cold conditions of the molecular jet and one conformer for isomenthone. As described in ref 36, this observation can be explained with a difference in the barrier heights in the conformational landscape of the two diastereomers. It arises from the preferred configuration of the cyclohexane ring. The two diastereomers and their conformers can thus be unambiguously differentiated by their rotational spectra. The richness and complexity of rotational spectra often go hand in hand with the structural information contained therein. While the advent of the broadband chirp technique has greatly reduced the acquisition time of a complex spectrum, the analysis and assignment of the spectrum can still require many days or weeks of skilled work, in particular, when multiple species are involved. Different approaches were developed to support spectral assignment by exploiting the broadband capabilities. These can mainly be attributed to one of two categories: (i) from the theoretical side, the implementation of computer-assisted autoassignment routines36,45,46 and (ii) from an experimental side, the use of double-resonance schemes. The latter can significantly facilitate the analysis of complex high-resolution rotational spectra by identifying the connectivity of energy levels.35,47,48 For microwave radio frequency (MW-RF) doubleresonance experiments, as reported in ref 49 and illustrated in Figure 3, the amplitude of any microwave transitions that share a

Figure 2. (a) Part of the rotational spectrum of a menthone/isomenthone isomeric mixture (top trace), with simulations based on the fitted molecular parameters for the four assigned conformers (bottom traces). Due to the high line density, only the excerpt from 4.75 to 5.62 GHz of the 2−8.5 GHz broadband spectrum is displayed. Three conformers were assigned to menthone and one to isomenthone. (b) Schematic molecular structures of the four stereoisomers of menthone. Menthone and isomenthone are diastereomers, which can be differentiated by their rotational spectra. (−)-Menthone and (+)-menthone as well as (−)-isomenthone and (+)-isomenthone are enantiomers of each other. The stereogenic centers are marked with asterisks. Rotational constants for conformer A of menthone and for the isomenthone conformer obtained from fitting asymmetric rotor Hamiltonians to our broadband rotational spectra are also given to illustrate their clear difference. 343



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Figure 3. Double-resonance experiments on isomenthone to determine the ordering of the molecular energy levels. The phase of the FID of the microwave signal transition is monitored as a function of the pump frequency. Opposite phase behavior is observed for progressive and regressive energy level arrangement (b,d), as can be explained with the AC Stark effect (adapted from ref 49).

carvone, denoted as EQ2 and EQ1.30,52 Note that both conformers were simultaneously excited in the same experiment employing a stacked excitation scheme. The molecular response was then recorded as a broadband FID. The two plots show the same portion of the FID but filtered using the corresponding listen frequencies (5098.12 MHz for EQ2 and 2811.42 MHz for EQ1). It is intriguing to illustrate microwave three-wave mixing in the frequency domain. In the lower part of Figure 6, frequency domain spectra of 4-carvomenthenol are shown for the frequency range around the drive transition at 5459.28 MHz (left) and around the listen transition at 5624.47 MHz (right) for two different cases, with the radio frequency twist excitation pulse on or off. When there is no twist pulse, no signal is obtained for the listen transition because the excitation cycle is not closed. With the twist pulse, the drive transition is significantly depleted, accompanied by a strong amplitude increase for the listen transition. As described in Figure 4, the twist pulse transfers coherence from the drive transition to the listen transition, manifesting itself in the observed amplitude change. Maximum chiral signal will be obtained when the microwave drive excitation fulfills π/2 Bloch conditions, that is, maximum coherence between the energy levels a and b will be generated, and when the twist pulse fulfills π Bloch conditions, that is, maximum coherence will be transferred. The double-resonance experiments discussed above (Figure 3) are also used to optimize the excitation conditions for the twist. As discussed in more detail in refs 30 and 33, the phase of the three-wave mixing signal depends on the sign of the product of the transition dipole moment components, while the amplitude of the three-wave mixing signal depends directly on the chiral composition of the sample, that is, on the ee. For ee determination, calibration of the amplitudes with an internal reference, such as a sample of known ee, is required. Because the signals of S

a rotational a-type) of the chiral sample using orthogonally polarized excitation fields. Note that the excitation fields do not arrive in the interaction zone simultaneously, which is important for determining the absolute phase of the chiral signal.33 We then probe the molecular response on a third transition, the listen transition, which in this example has to be b-type, along the third, mutually orthogonal direction (Figure 4). Because the triple scalar products of the transition dipole moment components μ⃗ a · (μ⃗ b × μ⃗c) have opposite signs between the two enantiomers (Figure 1), we observe a characteristic phase difference in the FID of this listen transition.

One can envision that in the near future, broadband double-resonance microwave spectroscopy experiments will be able to support the spectral assignment and to pave the road toward automated assignment. Thus, three different transition frequencies are required for successful enantiomer differentiation using rotational spectroscopy. The experimental setup must thus be suited to excite and detect different microwave frequencies simultaneously, which is the case for broadband CP-FTMW spectrometers. Even simultaneous measurement of several three-wave mixing cycles becomes feasible by using coadded or stacked excitation schemes.30 Recently, extension to higher-frequency ranges was demonstrated.51 The characteristic phase shift of π radians is clearly visible in Figure 5 for the example of the two low-energy conformers of 344



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Figure 4. Scheme of microwave three-wave mixing as a polarization-sensitive double-resonance experiment. The upper right part shows an energy level diagram for microwave three-wave mixing of menthone. A triad of rotational energy levels connected by a-, b-, and c-type transitions is involved in the excitation (drive and twist) and detection (listen transition). The molecular sample is polarized using two mutually orthogonally polarized excitation pulses, the drive and the twist. In the scheme (upper left), the drive field expands along the Y axis and is linearly polarized along the X axis. The twist field expands along the Z axis and is polarized along the Y axis. As a result of this combined excitation, molecular emission can be recorded at the listen transition frequency in the X direction, linearly polarized along the Z axis. In the lower part, a timeline of the experimental procedure is given together with the evolution of the simulated coherences of the three transitions involved.

and R enantiomers exactly cancel for a racemic mixture, zero signal is obtained. This zero signal provides us with a high sensitivity for determining small ee values.29,30 Samples with very large ee close to enantiopurity are also accessible due to the rather high precision of ee determination. The basic property of microwave three-wave mixing, a sign change of the triple scalar product of the transition dipole moments for the two enantiomers within a pair, also applies to molecules including more than one stereogenic center, as we showed recently for menthone and isomenthone.53 The diastereomers menthone and isomenthone have different geometries and thus different rotational spectra (Figure 2), so that they and their conformers can be differentiated via rotational spectroscopy. The rotational spectra of their enantiomers, however, are identical, but they differ in their microwave threewave mixing phase, as shown in Figure 7 for the two enantiomers of menthone, (+)-menthone and (−)-menthone. Note that (+) and (−) describe the direction of rotation of linearly polarized light by the sample, such as in a polarimetry experiment.

(+)-menthone corresponds to (2R,5S)-2-isopropyl-5-methylcyclohexanone, and (−)-menthone corresponds to (2S,5R)-2isopropyl-5-methylcyclohexanone. The three-wave mixing signal of enantiopure (+)-menthone and (−)-menthone (99% purity) is shown along with results for a commercially available mixture of isomers containing both menthone and isomenthone. Again, the phases are clearly opposite for (+)-menthone and (−)-menthone. A comparison of the phases allows us to determine (−)-menthone as the excess enantiomer in the previously uncharacterized mixture. Note that even though the measured phases are not perfectly shifted by π radians (but within the accuracy of the phase measurements of about ±0.25), they were stable even through changes on the order of centimeters of the valve position and through breaking vacuum and refilling the sample. Due to the long wavelengths of the microwave radiation, environmental effects (vibrations, temperature changes) have only small influences at the resolution to which the phases were measured. Currently, we perform more studies on isomenthone to fully characterize the sample, which is a good test for our new experimental technique. 345



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Figure 5. Chirality-sensitive microwave three-wave mixing to distinguish the enantiomers for two conformers (EQ2 and EQ1) of carvone. In the upper part, the molecular structures and the applied microwave three-wave mixing schemes are shown. In the lower part, magnifications of the filtered FIDs are displayed. For both conformers, the clear phase difference of about π radians between the R and the S enantiomers can be seen.

Figure 6. Illustration of microwave three-wave mixing in the frequency domain using the example of the lowest-energy conformer of 4-carvomenthenol. In the upper part, the employed three-wave mixing cycle is displayed along with a scheme of the molecular structure of the conformer. In the lower part, frequency domain spectra obtained using the three-dimensional microwave three-wave mixing arrangement are shown for the drive (left) and for the listen transition (right) under two different experimental conditions. In the lower trace of the spectra, the molecular sample is solely excited by the drive pulse, while the twist pulse is off so that no coherence transfer from the drive to the listen can take place. In the upper trace, the twist pulse is added, leading to a depletion of the drive transition accompanied by an amplitude increase for the listen transition.

As discussed using the example of menthone, the handedness of the respective enantiomer, that is, its absolute configuration, can be identified in an indirect way using microwave three-wave

mixing. Generally, this task can be accomplished in several ways. One approach is to first take measurements of samples of a molecule with known excess enantiomer and compare the 346



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acquired phases with those from the unknown samples that contain the target molecule. Naturally, this technique is only applicable to samples that are available with known excess

enantiomer for calibration (which can be tabulated). This approach was successfully used to characterize the unknown mixture of isomers of menthone (vide supra).53 Note that for comparative measurements with known samples, one only needs a phase resolution of approximately 1 radian to confidently differentiate enantiomers (Figure 7). Another approach is based on combining a full characterization of the travel times of signals through the various electronic components and the chamber along with a theoretical determination of the transition dipole moment components of the enantiomers, which is discussed in more detail in ref 33, and allows absolute configuration determination in a single measurement. The transition dipole matrix elements of the involved transitions have to be evaluated for each enantiomer. We can then predict absolute phases of either 0 or ±π for the two enantiomers.33 The effects of the timing on the observed phases must be considered because the excitation pulses do not arrive at the interaction region at the same time and the FID recording does not begin when the molecular listen signal starts (t0,RF in Figure 4). When the travel times of the excitation pulses through the experiment (including dispersion of the amplifiers, switches, cables, and, most importantly, horns) are characterized for the frequencies of interest, the absolute phase and thus the absolute configuration can be determined via comparison with the predicted transition dipole matrix elements. It is worth noting once more that the π radian phase difference between enantiomers leads to an easy identification of the excess enantiomer in a mixture, even if the derived absolute phase from the unknown sample might have a large uncertainty (e.g., up to 1 radian). Potential Future Directions. One of the unique advantages of microwave three-wave mixing based on broadband rotational spectroscopy is its inherent mixture compatibility. The requirements for successful microwave three-wave mixing are that the

Figure 7. Excerpt of the microwave three-wave mixing signals for three different samples of menthone, an enantiopure sample of (−)-menthone (99% purity, blue trace), an enantiopure sample of (+)-menthone (99% purity, green trace), and an unpurified menthone/isomenthone mixture of isomers. As expected, (+)-menthone and (−)-menthone show a clear phase difference of about π radians in their time domain three-wave mixing signal, within the accuracy of the phase measurements (about ±0.25). The result for the mixture exhibits the same phase as (−)-menthone within the errors (−1.60(24) (mixture) versus −1.95(17) radians enantiopure (−)-menthone, respectively), demonstrating the robust character of microwave three-wave mixing.

Figure 8. Part of the broadband spectrum (300 000 averages) obtained for peppermint oil of natural origin. Seven different terpenes can be unambiguously identified by their rotational spectra. The two zoom-ins (bottom) also reveal the presence of less abundant molecules, such as pulegone and limonene. Note that the intensity axes between the two zoom-ins differ by 1 order of magnitude. 347



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development of the appropriate chiral stationary phase can be tedious and time-consuming, so that alternative approaches are of interest. For microwave three-wave mixing, chiral samples can be analyzed without preseparation. While mixture compatibility has recently been demonstrated for conformational mixtures30 and mixtures of different terpene species (menthone, isomenthone, and carvone with at least six different molecular species present in the sample53), an important next step toward a chemical analysis tool will be to apply microwave three-wave mixing to more complex samples, such as those of biological relevance. Examples include essential oils extracted from plants, such as peppermint oil. These essential oils often consist of a number of structurally related components, like menthol, menthone, neomenthol, and isomenthol in the case of peppermint oil. Because the oils are of natural origin, they are likely to be enantio-enriched. Analysis of their chiral composition and comparing microwave three-wave mixing results to those of other techniques will help to identify the present limitations and possible improvements of our technique. In Figure 8, a part of the broadband spectrum of commercially available peppermint oil of natural origin is shown. 300 000 single FIDs have been averaged. It reveals the presence of seven different terpenes, all identified by their high-resolution rotational spectra. Eucalyptol and menthol are the most dominant. Other interesting samples will be those of pharmaceutical relevance, such as ibuprofen. Only the S enantiomer shows the well-known anti-inflammatory effect, while the R enantiomer has no physiological effect. This chiral selectivity makes it particularly interesting for further studies. We could identify four conformers in the broadband rotational spectrum under the cold conditions of a molecular jet arising from different orientations of the isobutyl substituent with respect to the central phenyl ring.55 With the knowledge of the rotational constants, we can now move on to microwave three-wave mixing experiments and test their applicability to such molecular systems. Sample preparation is a critical step. Stringent requirements for microwave three-wave mixing are that the molecules can be brought into the gas phase and that they are polar. Laser desorption and ablation techniques are successfully applied to tackle this challenge.56−58 However, further research might be needed for the application to real-life samples. Microwave three-wave mixing should also be applicable to the study of molecular clusters of chiral molecules. Chiral preference in cluster formation is well-known, examples being the chiral recognition in dimers of menthol and neomenthol59 or dimers of glycidol.60 In the case of the menthol dimer, a remarkable chirality discrimination upon dimer formation is observed with a preference for forming homochiral clusters over heterochiral clusters. It will be interesting to reveal the underlying mechanisms of this preference, which might also be relevant for a better understanding of the origins of the homochirality of life. In summary, we discuss microwave three-wave mixing as a new technique to differentiate enantiomers and to determine ee and the absolute configuration of the excess enantiomer in the gas phase. The resonant character and the high resolution and sensitivity of the technique make it highly mixture-compatible, which can be particularly relevant for natural and pharmaceutical samples. In the future, the extension of this technique to chemical analysis and to samples of biological and pharmaceutical relevance will be an interesting challenge. Experiments aiming at manipulating and finally controlling the composition of a chiral sample using well-defined microwave pulses can open up a fascinating new area of molecular physics experiments.

molecules can be brought into the gas phase and that all three transition dipole moment components are nonzero. This latter condition is generally met for chiral molecules containing at least one stereogenic center due to their lack of symmetry, although there are a few examples, such as lactic acid and camphor, where one dipole moment component is very small due to their specific geometry. However, the required minimum dipole values for microwave three-wave mixing are not fully explored yet. There are also chiral molecules having C2 symmetry such as H2O2 or biphenyl, which do not necessarily fulfill the required conditions for the dipole moment components, but these represent only a small group. In the following, we discuss potential future directions of studying chiral molecules and mixtures thereof using microwave spectroscopy. One direction will focus on improved instrumentation and characterization of microwave three-wave mixing, such as the development of an elegant protocol for absolute configuration determination. Another direction focuses on extending the range of accessible molecular samples. Extension to Higher-Frequency Ranges. Our setup in Hamburg is currently limited to drive excitation pulses between 2 and 6 GHz and twist excitation pulses between 50 and 550 MHz. However, microwave three-wave mixing is not generally restricted to such low frequencies. Recently, Pate and co-workers showed an extension of the microwave three-wave mixing technique to higher-frequency ranges by employing a second set of horn antennas perpendicular to each other. In principle, they can use drive and twist pulses up to 18 GHz. In ref 51, they demonstrate enantiomer differentiation of solketal by employing excitation pulses at 6.4 and 1.8 GHz, respectively. Absolute phases were not recorded. We are currently modifying our existing setup in Hamburg to have greater flexibility in the frequency ranges that we can cover. Mapping Chirality to Rotation: Toward Enantiomer Separation. So far, microwave three-wave mixing yields chirality-sensitive microwave radiation associated with the listen transition that can be detected as a chiral signal. It is intriguing to try to reverse this situation, that is, to apply tailored microwave fields of a specific frequency, duration, and phase to manipulate a sample, for example, to prepare a rotational state with a specific ee. We are currently setting up an experiment in which we will selectively promote either S or R enantiomers from a racemic mixture to a specific excited rotational state. We will do this by adding a third, phase-controlled excitation pulse to the two excitation pulses used in the current version of the microwave three-wave mixture, following a similar method suggested by Kral and Shapiro in 2001.54 Application to Chemical Analysis. Due to its resonant character and inherently high resolution, microwave three-wave mixing and broadband rotational spectroscopy in general are well-suited for chemical analysis. As demonstrated above, the enantiomers can be easily distinguished based on their π radians signal phase difference, and the ee can be determined via the amplitude of the chiral signal by comparing to an internal standard. As such, microwave three-wave mixing is interesting for chemical analysis, for example, to complement chiral column chromatography. There, the stationary phase contains a single enantiomer of a chiral component. The two enantiomers of the same analyte compound differ in affinity to the chiral stationary phase and therefore exit the column at different times, allowing for their separation and identification. The chiral stationary phase can be prepared by attaching a suitable chiral compound to the surface of an achiral support such as silica gel. The identification and 348



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REFERENCES

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Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest. Biographies V. Alvin Shubert obtained his Ph.D. at Purdue University on probing potential energy landscapes of biomimetic molecules using infrared− ultraviolet spectroscopy in 2008. He then became a postdoctoral research associate at Argonne National Laboratories before moving to Hamburg in 2011, where he was working as a PostDoc until summer 2014 on microwave three-wave mixing experiments. David Schmitz is a researcher at the Max Planck Institute for the Structure and Dynamics of Matter in Hamburg, Germany. He earned his Ph.D. in Physics from the University of Hamburg. His research focuses on broadband microwave spectroscopy, in particular, on three-wave mixing techniques and autoassignment routines. Cristóbal Pérez received his Ph.D. from Universidad de Valladolid (Spain) in 2011. From 2011 to 2014, he did a postdoctoral stay with Prof. Brooks Pate at the University of Virginia. He then joined the group of Dr. Melanie Schnell in 2014, where he became an Alexander von Humboldt fellow. He is currently working on methods for chirality determination via microwave spectroscopy as well as on the structural characterization of biologically relevant molecules. Chris Medcraft obtained his Ph.D. from Monash University where he studied the infrared spectra of cold molecules using synchrotron radiation. He then moved to Hamburg to perform microwave spectroscopy on a variety of molecules. He is continuing his microwave spectroscopic work at Newcastle University using laser ablation techniques. Anna Krin received her M.Sc. in Molecular Life Sciences from the University of Lübeck, Germany, in 2014. She is currently working towards her Ph.D. in the group of Melanie Schnell in Hamburg. Her research field includes studies of biologically relevant chiral molecules using microwave spectroscopy. Sérgio R. Domingos obtained his M.Sc. in Physics Engineering in 2008 from the University of Coimbra. In 2013, he completed a Ph.D. in Physics at the University of Amsterdam. Since 2014, he has been a postdoctoral scientist in the group of Melanie Schnell doing research on high-precision spectroscopy of chiral molecules. David Patterson has been working with cold atoms and molecules at Harvard University since 2004 and specializes in buffer gas cooling and mixture analysis using microwave spectroscopy as well as chiral analysis. Melanie Schnell obtained her Ph.D. in physical chemistry from the Universität Hannover in 2004. After PostDocs at NIST, Gaithersburg, and the Fritz-Haber-Institut in Berlin, she became an independent group leader in Hamburg, Germany, in 2011. She is also Privatdozentin at the Leibniz Universität Hannover. Her group is involved in high-resolution spectroscopy of complex molecules and microwave three-wave mixing. Group website: http://www.mpsd.mpg.de/en/research/irg/ccm

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ACKNOWLEDGMENTS This work has been supported by the excellence cluster “The Hamburg Centre for Ultrafast Imaging - Structure, Dynamics and Control of Matter at the Atomic Scale” of the Deutsche Forschungsgemeinschaft. The authors acknowledge funding by the Fonds der Chemischen Industrie and the Deutsche Forschungsgemeinschaft (via Grant SCHN1280/1-1). 349



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Chiral Analysis Using Broadband Rotational Spectroscopy - The

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