1. International Symposium on Molecular Spectroscopy, 71st Meeting
Champaign-Urbana, June 20-24, 2016
Chiral analysis of isopulegol by Fourier Transform Molecular Rotational Spectroscopy
Luca Evangelistsi1, Nathan Seifert2, Lorenzo Spada1, Brooks H. Pate
Department of Chemistry
University of Virginia
1 Dipartmento di Chimica “Giacomo Ciamician” Bologna
2Department of Chemistry, University of Alberta

2. The Problem from the Physical Chemistry Perspective:
Rotational Spectroscopy of Single Molecules
The Problem from the Physical Chemistry Perspective:
Modern computational chemistry produces structures and conformational potential energy surfaces that provide accurate estimates of the spectroscopic constants and relative energies.
Is there a reason to perform the measurement?
The Dream of Analytical Chemistry:
A rapid spectroscopy technique where molecules can be unambiguously identified (especially in a mixture) by direct comparison of experimental spectroscopic constants to theoretical estimates.
Library-free detection (the ability to perform analysis without a previously prepared, high purity reference sample)

3. Rotational Spectroscopy in Analytical Chemistry
Can’t compete with “gold standard” methods that use mass spectrometry/ion detection: GC/MS (HPLC methods)
Reduction in the cost of consumables and technical expertise required
Samples that can’t use chromatography (reactive) – direct mixture analysis
Monitoring applications requiring “real-time” quantification (~1 min)
Analysis of isomers
Isotopologues: Site-specific stable isotope analysis
Chiral Analysis: Diastereomers and Enantiomers
Isomer ratios are required
Major Strength: Potential for Library-Free Analysis
(Molecule identification by theory alone)

4. Chiral Analysis for Molecules with Multiple Chiral Centers
1) Quantitative Analysis of Diastereomers
Resolution for molecules with multiple chiral centers
2) Absolute Configuration
Determination of left-handed or right-handed form
3) Enantiomeric Excess Measurements
Internal calibration for accurate, linear ee measurement
Dihydroartemisinic Acid
Image Credit: R S

5. Chiral Analysis of Isopulegol:
Three chiral centers: 23 isomers
Four diastereomers each with enantiomers
Goal: 99.5% purity levels (regulatory)
Isopulegol
Diasteromers: OH Iso CH3 E(cm-1)*
Isopulegol: Eq Eq Eq
Neoisopulegol: Ax Eq Eq
Isoisopulegol: Eq Eq Ax
Neoisoisopulegol: Ax Eq Ax
* B2PLYP D G**
M06-2X/ g(d,p)

6. Low Frequency (2-8 GHz) Chirped-Pulse
Fourier Transform Microwave Spectrometer
General Spectral Properties:
Measurement Bandwidth: 6000 MHz
FWHM Resolution: 60 kHz (105 data channels)
Transitions in a Spectrum: ( % of range)
RMS Frequency Error in Fit: 6-10 kHz (~10% of FWHM)
AUTOFIT
C. Perez, S. Lobsiger, N. A. Seifert, D. P. Zaleski, B. Temelso, G.C. Shields, Z. Kisiel, B. H. Pate, Chem. Phys. Lett. 571, 1 (2013).
N.A. Seifert et al., “Autofit, an Automated Fitting Tool for Broadband Rotational Spectra, and Applications to 1-Hexanal”, J. Mol. Spectrosc. 312, (2015).

7. Isopulegol 5% of bandwidth
Isopulegol has three chiral centers with four diastereomers.
Isopulegol is an intermediate in the synthesis of menthol which is produced at 3,000 tons per year.
A single diastereomer is needed because only a single diastereomer of menthol has the desired flavor.
Ryōji Noyori shared the 2001 Nobel Prize in Chemistry for the stereoselective synthesis of menthol (94% ee).
5% of bandwidth
Carlos Kleber Z. Andrade*, Otilie E. Vercillo, Juliana P. Rodrigues and Denise P. Silveira, J. Braz. Chem. Soc., 15, , 2004.

8. Identification of Conformers and Diastereomers:
Computational Chemistry and Structure Determination
Species
Theory
Experiment
Percent Error
Isopulegol
1948.4
(299)
0.08
700.5
(113)
-0.04
591.1
(111)
-0.08
Neo isopulegol
2136.0
(207)
0.10
675.2
(76)
0.21
626.8
(73)
-0.13
Neo isoisopulegol
1706.2
0.44
783.3
-0.46
674.2
-0.23
Iso isopulegol
1941.5
0.05
746.1
0.14
706.7
-0.28
Theory: B2PLYP D G**
Structure Determination using 13C and 18O Isotopologue Spectra Directly Confirms Conformer and Diastereomer Geometry
Isopropenyl Dihedral Angle
Hydroxyl Dihedral Angle

9. Diastereomer Ratio in Aldrich Sample
Molecule identification is made through high accuracy matching of the rotational transition frequencies – relative intensity information is quantitative but less accurate.
A rich rotational spectrum is produced through a small set of spectroscopic parameters giving many detection windows.
Because the measurement is made under ideal gas conditions, the spectroscopic signature is independent of the sample matrix.
0.33% of bandwidth
Spectrum Simulation for Neoisopulegol at 0.1% Diastereomer Abundance

10. Physical Principles for FT-MRR Analysis of Enantiomers
D. Patterson, M. Schnell, and J.M Doyle, Nature 497, (2013).
D. Patterson and J.M. Doyle, Phys. Rev. Lett. 111, (2013).
The Sign of the Product of the Dipole Moment Vector Components is Unique to an Enantiomer
J.U. Grabow, Angew. Chem. 52, (2013).
V.A Shubert, D. Schmitz, D. Patterson, J.M Doyle, and M. Schnell, Angew. Chem. 52, (2013).
The Excitation Pulses and Chiral Signal have Mutual Orthogonal Polarization
Energy
000
110
101
µa : MHz
µb : MHz
µc : MHz
Simon Lobsiger, Cristobal Perez, Luca Evangelisti, Kevin K. Lehmann, Brooks H. Pate, “Molecular Structure and Chirality Detection by Fourier Transform Microwave Spectroscopy”, J. Phys. Chem. Lett. 6, (2015).

11. Three Wave Mixing Measurements
303
404
414
MHz
MHz
844.3 MHz
μ a
μ b μc
Optimal Pulse Sequence: π/2 - π
Simon Lobsiger, Cristobal Perez, Luca Evangelisti, Kevin K. Lehmann, Brooks H. Pate, “Molecular Structure and Chirality Detection by Fourier Transform Microwave Spectroscopy”, J. Phys. Chem. Lett. 6, (2015).

12. Features of Microwave Three Wave Mixing Spectroscopy
The chiral signal has an amplitude comparable to regular rotational transitions.
The chiral signal is background free.
The chiral signal is triply resonant and compatible with complex sample mixtures.
Actual digitized data points are shown along with sine wave fit.
Simon Lobsiger, Cristobal Perez, Luca Evangelisti, Kevin K. Lehmann, Brooks H. Pate, “Molecular Structure and Chirality Detection by Fourier Transform Microwave Spectroscopy”, J. Phys. Chem. Lett. 6, (2015).

13. Normalized Enantiomeric Excess Measurements
Three Wave Mixing Measurements on Several Isopulegol Samples
Optimal Pulse Sequence: π/2 - π
𝑒𝑒= ( 𝑁 𝑅 − 𝑁 𝑆 ) ( 𝑁 𝑅 + 𝑁 𝑆 )
303
404
414
MHz
MHz
844.3 MHz
Normalized ee Measurement Results
Aldrich Analytical Reference Sample: (Certificate of Analysis: 99.9: 0.1)
Aldrich Sample: : 0.4
Alfa Aesar Sample: : 43.3

14. Comparison with GC Analysis for the Alfa Aesar Sample
Diastereomer Analysis (GC/MS): Sample consumption 1µg
GC/MS CP-FTMW*
Isopulegol 66% 60%
Neoisopulegol 23% 33%
Isoisopulegol 6% 4%
Neoisoisopulegol % 3%
enantiomer Analysis
Chiral GC: ~50:50 (not fully resolved)
Three Wave Mixing: 57(-):43(+) (Isopulegol only)
Retention Time (min)
* Different lot number; similar CoA
Retention Time (min)

15. Conclusions BROOKS PATE  WK04
Spectroscopy expected to allow confident structural analysis for molecules up to 500 Da even for direct analysis of samples that are complex mixtures
Instruments for broadband FT-MRR spectroscopy can approach 10,000:1 dynamic range in a measurement (12 hours, ~200 mg) but require advances in source technology for volatilizing solids
Analytical chemistry is still in early stage of development with technique advances and measurement validation needed in phase calibration (absolute configuration), quantitative ee measurements, pulsed jet characterization (clusters), and spectroscopic library development
BROOKS PATE  WK04
DHAA

16. Acknowledgements
Pate Group
BrightSpec
NSF
MC-IOF
RSC