Chiroptical spectroscopy and the validation of crystal structure stereochemical assignments

Tetrahedron: Asymmetry 28 (2017) 1192–1198

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Chiroptical spectroscopy and the validation of crystal structure stereochemical assignments George E. Tranter ⇑, Delphine D. Le Pevelen Chiralabs Ltd, Oxford University Begbroke Science Park, Begbroke Hill, Begbroke, Oxfordshire OX5 1PF, United Kingdom

a r t i c l e

i n f o

Article history: Received 21 August 2017 Accepted 21 August 2017 Available online 10 October 2017 Dedicated to the memory of Dr. Howard Flack

a b s t r a c t The absolute stereochemistry of chiral molecules is ideally established to atomic resolution by X-ray crystallographic analysis. However, chiroptical spectroscopies, namely electronic circular dichroism (ECD), optical rotatory dispersion (ORD), vibrational circular dichroism (VCD) and Raman optical activity (ROA), play important complementary roles in establishing relative and absolute sterochemistries as well as allowing determinations of optical purity. A brief summary of chiroptical spectroscopies is presented, along with guidance to their advantages and disadvantages. The application of ECD to verifying that single crystals selected for crystallographic analysis are indeed representative of bulk material is described. Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction The determination of the handedness of substances has been a challenge since the days of Pasteur.1 Nowadays, the assignment of absolute stereochemistry of chiral molecules by single crystal X-ray diffraction is generally considered the ‘‘gold-standard” approach, of which the absolute structure parameter (otherwise known as the Flack enantiopole parameter or Flack x parameter) has played a critical role since its inception in 1983 by Howard Flack.2 Meanwhile, chiroptical spectroscopies, which probe the interaction of light with chiral species, provide important complementary analyses that are often used for determining stereochemistry and optical purity as well as giving insights into chiral-related phenomena. Here we provide a concise résumé of the chiroptical techniques that can be employed for the study of chiral molecules. In addition, we describe a method based on electronic circular dichroism to validate crystallographic absolute stereochemical assignments of single crystals. Discussions with Howard Flack over the decades often led him to suggest we publish our approach; in fond memory of him we now do so. 2. Chiroptical spectroscopies Chiroptical observations have a long history; as early as 1811 Arago had discovered that quartz plates caused plane polarised ⇑ Corresponding author. E-mail addresses: [email protected] (G.E. Tranter), d.lepevelen@chiralabs. com (D.D. Le Pevelen). https://doi.org/10.1016/j.tetasy.2017.08.019 0957-4166/Ó 2017 Elsevier Ltd. All rights reserved.

light to be rotated by different degrees with varying wavelength of light, essentially the birth of the spectroscopic study of optical rotations. Nowadays there are a multitude of different chiroptical spectroscopies, detecting different aspects of the interaction of light with chiral species. Many in-depth reviews and authoritative works on chiroptical spectroscopies can be found in the literature;3–9 here we briefly review the main features of the most common as a quick aid to choosing the most appropriate technique and avoiding their pitfalls. All are primarily used to analyse solutions or liquids, however studies on solids, whether they be crystalline, amorphous, films or sheets, can be undertaken with the appropriate adaptations for presenting the sample and interpreting the resulting spectra. Historically, chiroptical spectroscopy was limited to either Optical Rotatory Dispersion (ORD) or, subsequently, Circular Dichroism (CD) in the visible and ultra-violet wavelength ranges. The former effectively monitors the differential speed of left and right-handed circularly polarised light10 (CPL) through a chiral sample, manifested as an optical rotation of plane polarised light,11 whereas the latter monitors the differential absorption of the CPL due to electronic excitations in chiral species instead.12 The two phenomena are implicitly linked with each other via a Kramers–Kronig transform and thus may be thought to embody similar information about a sample. However, the two techniques are not interchangeable, have very different sensitivities and advantages, although CD instruments can be adapted to measure ORD.11 Nowadays ORD is mostly out of fashion, but it has particular advantages for compounds that do not possess a chromophore, such as carbohydrates, which give significant ORD signals. There are also a number of variants of circular dichroism, such as

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fluorescence detected CD and circularly polarised luminescence,9 which have utility on occasions for fluorescent or luminescent species, although are not as commonly available. Polarimetry, which is simply ORD at a single wavelength, typically the NaD line(s) at 589.3 nm, may also be considered as a member of the chiroptical spectroscopy ‘‘club” and has a much wider usage due to the simplicity of its instrumentation and apparent ease of interpretation, although the interpretation can actually be misleading due to a number of confounding issues often overlooked.11 The relatively newer techniques of Vibrational Circular Dichroism7 (VCD) and Raman Optical Activity6 (ROA) have now also become more widely available, expanding the possibilities of chiroptical spectroscopy. VCD is the infra-red equivalent of UV–visible CD (now otherwise know as ‘‘electronic” ECD to distinguish itself from VCD), probing the vibrational transitions of a molecule rather than the electronic excitations. ROA is the analogous phenomenon for Raman scattering. All of these techniques give signed spectra with a series of positive and negative bands across their wavelength or wavenumber range (examples of ECD spectra are given later). All other things being equal, oppositely handed enantiomers should give spectra which are mirror images of each other, i.e. of equal magnitude but opposite signs to each other. Racemates necessarily therefore give a zero spectrum, as do achiral substances, unless a chiral or handed/orientating influence is imposed. This has led to optical rotations being used to define the stereochemical notation of chiral molecules. If a molecule exhibits a positive rotation it can be described as the ‘‘(+)” or ‘‘d” enantiomer, whereas if it exhibits a negative rotation it can be denoted as the ‘‘(
)” or ‘‘l” enantiomer; the d and l deriving from dextrorotatory (right rotation) and laevorotatory (left rotation) respectively. However, it should be remembered that optical rotation will change sign across the wavelengths, so such a description relies on the use of a specific wavelength; invariably the NaD line is chosen. In addition, optical rotations are sensitive to solvent and other environmental factors, therefore care has to be taken on using such assignments of stereochemistry without full consideration – one should not assume a ‘‘(+)” enantiomer will always give a positive rotation in all circumstances, even just changing the solvent may change the sign despite the actual molecular structure remaining unchanged.

Figure 1. Crystal of mandelic acid selected for crystallographic studies; plate of size ca. 0.8  0.6  0.18 mm.

It should be clear to the reader that these notations are a physical description of the optical properties of the enantiomers, they are not uniquely related to the Fischer L/D notation or the Cahn-Ingold-Prelog R/S notation, both of which are determined by molecular geometry albeit from different perspectives. The use of the terms ‘‘left” and ‘‘right” to describe enantiomers always needs qualification as to which notation system is in use and, if optical rotation is used, the conditions need stating. Table 1 Crystallographic data obtained for S-mandelic acid. Compound sample

S-Mandelic acid

Formula Molecular mass (g/mol) Temperature, K Crystal system Space group Z Cell parameters

C8H8O3 152.15 293 Monoclinic P21 4 a = 8.6241(1) Å b = 5.8592(1) Å c = 15.1762(2) Å b = 102.8018(14)° 747.796(19) 320 0.80  0.58  0.18 1.351 0.875 1.5415 0.60, 0.86 5.260–67.073 13192 2648 0.02 216 2640 R1(F2) = 0.030, wR2(F2) = 0.079 0.952
0.12, 0.14 0.01(17)

V (Å3) F(0 0 0) Crystal size (mm3) Dx (g/cm3) m (mm-1) k(Cu-Ka) (Å) Tmin,max h Range for Data Collection (°) Reflections measured Independent reflections Rint Number of parameters Reflections used Final R indices [I >
3r(I)] Goodness-of-fit on F2 Dqmin, Dqmax (e Å
3) Flack parameter

Figure 2. View along b of unit cell of S-mandelic acid.

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Chiroptical techniques are also widely used to determine enantiomeric ratios and optical purities since the sign and magnitude of the spectrum can be directly related to the enantiomeric excess of a compound. For example, from ECD spectra the enantiomeric excess (ee) of a mixture of both enantiomers (assuming the two enantiomers act as independent species) is given directly by the ratio of the observed CD to that expected of the pure single enantiomer (where it has been determined, or can be deduced from samples of known optical purity):

All the chiroptical spectroscopies are employed widely to assign relative stereochemistry so as to distinguish enantiomers from each other, although polarimetry and ECD still dominate currently. By the same means, the techniques can also give absolute stereochemistries by comparison of spectra with that of otherwise authenticated reference materials of known handedness. Alternatively, prediction of the expected chiroptical spectrum from a particular enantiomer and comparison with that observed provides a means of assignment. Historically, quadrant and octant rules based on molecular geometry for predicting the signs of ORD and ECD features were popular and employed to assign stereochemistry, but have generally fallen out of favour due to their lack of definitiveness, although do have utility on occasions.13 Fortunately, considerable progress has been made in predictive ab initio quantum mechanical calculations of chiroptical spectra, with advances being made for ECD, VCD and ROA. Alas, currently, the match to observed spectra is still sometimes far from perfect and the assignment of absolute stereochemistry by this means can require a degree of optimism (or, at worst, wishful thinking) that a correlation between the observed and predicted spectra is present. The discrepancies between calculated and observed spectra are often poorest for molecules with a high degree of flexibility, due to the issues of averaging the spectra over the various conformational states it can adopt. Nonetheless, in suitable cases, the absolute stereochemistry can now be determined a priori by the chiroptical methods. Numerous examples of the use of ab initio calculations of spectra being used for assigning absolute stereochemistry can now be found in the literature, for example using ECD,14 VCD15,16 and ROA17 spectra.

eeð%Þ ¼

DAobs;k  100% DApure;k

where DAobs,k is the observed CD and DApure,k is that of the pure enantiomer in question, both at a given wavelength k. In principle, if only the two enantiomers are present, the ratio should evaluate to the same result at every wavelength, although it will be ill-determined at wavelengths where little CD is observed and is best determined at the wavelength of maximum CD. Each of the chiroptical spectrocopies has its own peculiarities, pros and cons. These can be broadly summarised as follows, although it should be stressed that these are general guidelines rather than categorical rules, based on the authors’ experiences: 2.1. Electronic circular dichroism Relatively high sensitivity with low sample requirements (typically 0.1–1 mg in the uv region) and wide variety of solution conditions, concentrations and solvents accommodated (water being particularly good for far-ultra violet work). Sample must

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Wavelength (nm) Figure 3. Observed CD spectrum (top) and absorption spectrum (bottom) for the solution of the mandelic acid crystal selected for crystallographic analysis, acquired in a 1 cm pathlength cell; blue line multiplied by 10 for clarity.

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have a chromophore absorbing in wavelength region of interest. Spectra may take a few minutes to an hour to acquire faithfully. It is capable of discerning 1% differences (e.g. enantiomeric excess) in many cases, or 0.1% for samples with high CD magnitudes. It is mainly for relative stereochemistry or absolute assignment versus reference data; ab initio calculations beginning to make a priori absolute assignment possible.

temperature and impurities, including reversal of sign – literature data can have significant errors due to these factors. If consistency of measurement is maintained, it is capable of discerning 0.1% differences (e.g. enantiomeric excess) in many cases, or better for samples with high specific rotations. It is mainly for relative stereochemistry or absolute assignment versus reference data, but always with caveats as to misleading and confounding data.

2.2. Optical rotatory dispersion

2.4. Vibrational circular dichroism

Can have similar sensitivity and sample requirements to ECD in wavelength regions of absorptions (0.1–1 mg), but otherwise may need significantly higher sample quantity (1–10 mg). Wide variety of solution conditions accommodated (water being particularly good for far-ultra violet work). Compound need not have a chromophore absorbing in monitored wavelength region. Spectra may take a few minutes to an hour to acquire faithfully. It is capable of discerning 0.1% differences (e.g. enantiomeric excess) in many cases, though can be better for substances exhibiting high specific rotations. It is mainly for relative stereochemistry or absolute assignment versus reference data.

Sensitive, but can require relatively high concentrations of sample (e.g. 50 ll of 0.1 M, ca. 1 mg). Solvents must be IR transparent and can otherwise obscure some regions of spectra (water being particularly bad). Chiral molecules with vibrational spectra should be amenable to VCD; spectra are information rich (i.e. many peaks). Spectra may take several hours or more to acquire faithfully and thus may lead to stability issues. It can be capable of discerning 1% differences (e.g. enantiomeric excess) in good conditions. It is mainly for relative stereochemistry or absolute assignment versus reference data; ab initio calculations making a priori absolute assignment possible.

2.3. Polarimetry

2.5. Raman optical activity

Relatively low sensitivity for many compounds (1–100 mg) as measured at the NaD wavelength, where optical rotations are typically small compared to UV wavelengths. It can be misleading due to only a single wavelength being monitored and can give grossly different results with changing concentration, solvent,

Low sensitivity, requiring high concentrations of sample (e.g. 50 ll of 0.2 M, ca. 2 mg), but variety of solvents accommodated. Chiral molecules with vibrational spectra should be amenable to ROA; spectra are information rich. Spectra may take several hours or more to acquire faithfully and thus may lead to stability issues,

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crystal, which is often not a trivial task. As an example of Sod’s Law, it sometimes seems the compounds where there is the greatest desire to obtain crystals are the hardest to crystallise successfully, although rational crystallisation screening can ameliorate this challenge.22–24 Moreover, there invariably remains the question as to whether the crystal selected for analysis is truly representative of the bulk material, or just a chance event. Even if the bulk material has a high enantiomeric excess of one particular hand, with much smaller amounts of the opposing enantiomer, on growing crystals from it, it is still very plausible to obtain a chance crystal of the opposing enantiomer or a racemate. The chance of the latter is increased in the event of the racemate inherently crystallising more readily than either single enantiomer. As a consequence, analysis of such an unrepresentative crystal, inadvertently selected, would lead to a false assignment of the dominant stereochemistry of the bulk material. This problem has impact on the structural assignment not just of research compounds, where limited sample may be available, but through to the characterisation of commercial scale chemicals and pharmaceuticals where assignment by the ‘‘gold standard” technique would often be thought to take precedence over the results of other techniques. One obvious answer to this problem is to analyse multiple crystals and hopefully establish the consistency of the assignment statistically. However, this is often impractical, not least due to paucity of sample, poor crystallisation, lack of resources and/or time.

especially as they are exposed to a high power laser which may lead to photoreactions and heating. Fluorescence from samples, or impurities, can be an issue and often they are deliberately photobleached by the laser for 10–60 min prior to spectral acquisition. It can be capable of discerning some quantitative differences, but mostly qualitative. It is mainly for relative stereochemistry or absolute assignment versus reference data; ab initio calculations making a priori absolute assignment possible. In practice, it is often worth employing more than one of the techniques, as exemplified by the analysis of isoflavanones by Batista et al.18 and marine antibiotics by Hopmann et al.19 While the above short summary has focussed on the study of small chiral molecules in terms of their stereochemical assignment, it should be recalled that all of the chiroptical techniques have considerable utility in studying other features,12 including configurational analysis (e.g. a and b forms of nucleosides), conformational analysis (e.g. flexibility and conformational changes), interactions (binding etc.) and ECD can be hyphenated to chromatographic systems for online assignment.20 Moreover they are powerful techniques for investigating chiral macromolecules such as proteins and nucleic acids and are thus extensively used for studying secondary structure and interactions of biomacromolecules.21 3. Validation of crystallographic stereochemical assignments Without doubt, crystallography is the most definitive method of assigning stereochemistry in most cases. However, the obvious drawback of such analyses is the need for a suitably diffracting

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Wavelength (nm) Figure 5. Observed molar CD spectrum (top) and molar absorption spectrum (bottom) for the solution of the crystallographically analysed crystal of S-mandelic acid (red and green) overlaid with that of the solution of the bulk S-mandelic acid (black and blue); green and blue lines multiplied by 10 for clarity.

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CD and absorption spectra, giving a negative signed CD in this region. At shorter wavelength the more intense absorption from the phenyl is evident at ca. 217 nm, with an accompanying positive CD. At even shorter wavelength the beginnings of the absorption of the carboxylate and another band from the phenyl are evident. To validate that this crystal was representative of the bulk of the mandelic acid sample, a representative portion of this bulk sample was then taken, accurately weighed and made up to a 0.1 mg/ml solution in water (i.e. 0.656 mM). The UV absorption and CD spectra of this ‘‘bulk solution” is presented in Figure 4, normalised to molar absorbance and molar CD, as the concentration was known. It can be immediately seen that the signs of the bands in the CD spectrum of the ‘‘bulk solution” match those of the ‘‘crystal solution” presented in Figure 3, consistent with their sharing the same predominant stereochemistry, i.e. the single crystal is indeed representative of the bulk in these terms. On correcting for concentration, a further level of confirmation of consistency can be made. By comparing the molar absorbance of the ‘‘bulk solution” with the absorbance of the ‘‘crystal solution”, the concentration of the latter can be deduced. The spectra of the ‘‘crystal solution” can then also be normalised to molar absorbance and molar CD. Such normalised molar spectra of the ‘‘crystal solution” are depicted in Figure 5, with that of the ‘‘bulk solution” overlaid. It can be seen that there is a very close correspondence between the spectra of the two solutions both in terms of sign and magnitude, giving more confidence that assignment of the single crystal is representative and that their optical purity is similar. It may be noted that the residual oil used to mount the crystal for crystallography did not interfere with the CD analysis, not least

Here we present the approach used by one of the authors (GET) since the 1980’s to validate crystal assignments of numerous compounds, particularly small molecule pharmaceuticals, using circular dichroism of a solution of the actual crystal analysed crystallographically. It is applicable to virtually any chiral compound that possesses a chromophore in its molecular structure. 3.1. Example of stereochemical validation As an example of the approach of validating a crystal assignment, an enantiopure sample of mandelic acid was obtained, with its supposed handedness not initially revealed to the study. This was then recrystallised from water and a single crystal selected and subjected to crystallographic analysis. Figure 1 depicts the selected crystal, of plate morphology with approximate dimensions of 0.8  0.6  0.18 mm. Although the crystal was far from perfect, the resulting refined crystal structure was adequately determined and the Flack enantiopole parameter indicated that the crystal likely corresponded to the S-enantiomer, although not categorically (but a correct assignment when the original handedness of the sample was subsequently revealed). The resulting crystal parameters are tabulated in Table 1 and its unit cell depicted in Figure 2. The crystal parameters are consistent with previously reported data for S-mandelic acid.25 This single crystal was then dissolved in 1 ml of water to yield the ‘‘crystal solution” and its UV-absorption and CD spectra acquired; the resulting spectra are depicted in Figure 3. The relatively weak vibronic bands deriving from the phenyl ring of mandelic acid are apparent in the 250–270 nm region in both the

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Wavelength (nm) Figure 6. Observed molar CD spectrum (top) and molar absorption spectrum (bottom) for the solution of the crystallographically analysed crystal of R-mandelic acid (red and green) overlaid with that of the solution of the bulk R-mandelic acid (black and blue); green and blue lines multiplied by 10 for clarity.

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as it is not chiral. In the case here, the minor discrepancy in the CD spectra at the far-UV end of the wavelength range, below 240 nm, is know to be due to the occurrence of an impurity in the bulk mandelic acid, which is not present in the recrystallised material. For completeness, this exercise was repeated but with a sample of the opposite enantiomer of mandelic acid, with the conclusion that it was R-mandelic acid as expected. The resulting CD spectra of the solutions of the bulk and single crystal for the R-enantiomer are presented in Figure 6. It can be seen that there is correspondence in the CD signs, confirming that the single crystal as again representative of the bulk. This also illustrates the feature of chiroptical spectroscopies that, all other things being equal, enantiomers give equal magnitude but oppositely signed spectra, as can be seen by comparing the CD spectra of Figures 5 with 6. It is important to realise that this validation was possible without recourse to micro-CD apparatus, due to the relatively high sensitivity of CD spectroscopy and the relatively large crystal employed. In the example presented here, the crystallographically analysed crystal amounted to a mass of ca. 30 lg. Much smaller crystals are amenable to this approach simply by using micro-CD, with focussing optics to allow transmission through a low-volume microcell. By such means, solutions of crystals of a mass of ca. 1 lg, can often be analysed by far-UV CD, provided the molecular structure incorporates a suitable chromophoric group. Thus crystals of ca. 0.1  0.1  0.1 mm size can be verified as representative by CD in this way. 4. Experimental details Mandelic acid samples were purchased from Sigma Aldrich: S-(+)-mandelic acid (Reagent Plus grade >99%, Product M2004, Lot 04327JD); R-(
)-mandelic acid (Reagent Plus grade >99%, Product 154210, Lot 04305CJ). CD spectra were acquired on a Jasco J720 spectrometer, flushed with copious evaporated nitrogen to improve performance in the far UV wavelength region. Accompanying absorption spectra were simultaneously acquired as the second detection channel of the instrument. The instrument was calibrated for wavelength using Holmium and Didynium glass filter standards and for intensity using a standard aqueous solution of ammonium d-10-camphor sulphonate (De [290.5 nm] = 2.399 M
1 cm
1). Instrumental parameters were set as: spectral bandwidth 1 nm, scan speed 10 nm/min, response 4 s, accumulations 4, data interval 0.1 nm. Quartz Suprasil cylindrical cells, specially selected for CD studies, were employed of 1 cm and 0.1 cm pathlength as appropriate for maximal signal-to-noise and minimal stray light. The cells and the solutions therein were kept under temperature control throughout spectral acquisitions by a bespoke thermostatic cell holder within the spectrometer. In each case, a CD spectrum and accompanying UV absorption spectrum were simultaneously acquired, with a water baseline under the same conditions in the same cell also acquired at a proximal time. The spectra were corrected by baseline subtraction; no noise-reduction or Lorentz– Lorenz solvent correction was employed.

Single crystal X-ray diffraction crystal structures were acquired on an Oxford Diffraction Supernova instrument with Cu radiation at a temperature of 293 K. The crystal was mounted using a mounting oil. Acknowledgements The study was supported by Chiralabs Ltd. We acknowledge the help given by Richard Cooper of the Chemical Crystallography Department of the University of Oxford and of Alexandra Stanley of Rigaku Oxford Diffraction in acquiring the crystallographic data. References 1. Flack, H. D. Perspectives and Concepts: Chirality in Nineteenth Century Science. Tranter G. E., Ed. Vol. 8: Separations and Analysis, Carreira E.; Yamamoto H., Eds., Comprehensive Chirality; Elsevier, Oxford, 2012, pp. 1–10. 2. Flack, H. D. Acta Crystallogr. A 1983, 39, 876–881. 3. Encyclopedia of Spectroscopy & Spectrometry; Lindon, J. C., Tranter, G. E., Koppenaal, D. W., Eds., 3rd ed.; Academic Press Elsevier: Oxford, 2017. 4. Tranter, G. E. Separations and Analysis. In Carreira E., Yamamoto H., Eds., Comprehensive Chirality; Elsevier: Oxford, 2012. 5. Mason, S. F. Molecular Optical Activity & the Chiral Discriminations; Cambridge University Press: Cambridge, 1982. 6. Barron, L. D. Molecular Light Scattering & Optical Activity, 2nd ed.; Cambridge University Press: Cambridge, 2004. 7. Nafie, L. A. Vibrational Optical Activity: Principles and Applications; Wiley: UK, 2011. 8. Berova, N.; Nakanishi, K.; Woody, R. Circular Dichroism: Principles and Applications, 2nd ed.; Wiley VCH, 2000. 9. Brittain, H. G. J. Pharm. Biomed. Anal. 1998, 17, 933–940. 10. Tranter, G. E. Spectroscopic Analysis: Polarised Light and Optics, Tranter G. E., Eds. Vol. 8: Separations and Analysis, Carreira E.; Yamamoto H., Eds. Comprehensive Chirality; Elsevier: Oxford, 2012, pp. 406–410. 11. Tranter, G. E. Spectroscopic Analysis: Polarimetry and Optical Rotatory Dispersion, Tranter G. E., Eds. Vol. 8: Separations and Analysis, Carreira E.; Yamamoto H., Eds. Comprehensive Chirality; Elsevier: Oxford, 2012, pp. 411– 421. 12. Tranter, G. E. Spectroscopic Analysis: Electronic Circular Dichroism, Tranter G. E., Eds. Vol. 8: Separations and Analysis, Carreira E.; Yamamoto H., Eds. Comprehensive Chirality; Elsevier: Oxford, 2012, pp. 422–437. 13. Macleod, N. A.; Butz, P.; Simons, J. P.; Grant, G. H.; Baker, C. M.; Tranter, G. E. Isr. J. Chem. 2004, 44, 27–36. 14. Kłys, A.; Makai, A.; Zdzienicka, A. Tetrahedron: Asymmetry 2017, 28, 135–145. 15. Wesolowski, S. S.; Pivonka, D. E. Bioorg. Med. Chem. Lett. 2013, 23, 4019–4025. 16. Burgueño-Tapia, E. P. Tetrahedron: Asymmetry 2017, 28, 166–174. 17. Profant, V.; Jegorov, A.; Bour, P.; Baumruk, V. J. Phys. Chem. B 2017, 121, 1544– 1551. 18. Batista, J. M., Jr.; Wang, B.; Castelli, M. V.; Blanch, E. W.; López, S. N. Tetrahedron Lett. 2015, 56, 6142–6144. 19. Hopmann, K. H.; Šebestík, J.; Novotná, J.; Stensen, W.; Urbanová, M.; Svenson, J.; Svendsen, J. S.; Bourˇ, P.; Ruud, K. J. Org. Chem. 2012, 77, 858–869. 20. Mistry, N.; Roberts, A. D.; Tranter, G. E.; Francis, P.; Barylski, I.; Ismail, I. M.; Nicholson, J. K.; Lindon, J. C. Anal. Chem. 1999, 71, 2838–2843. 21. Tranter, G. E. Protein Structure Analysis by CD, FTIR and Raman Spectroscopies, Lindon, J. C.; Tranter, G. E.: Koppenaal, D. W., Eds. Encyclopedia of Spectroscopy & Spectrometry 3rd ed.; Academic Press Elsevier: Oxford, 2017, pp. 740–758. 22. Le Pevelen, D. D. Physical Separations: Solid-state Forms and Habits of Chiral Substances, Tranter G. E., Eds. Vol. 8: Separations and Analysis, Carreira E.; Yamamoto H., Eds. Comprehensive Chirality; Elsevier: Oxford, 2012, pp. 54–62. 23. Le Pevelen, D. D., Tranter, G. E. FT-IR and Raman Spectroscopies: Polymorphism Applications, Lindon, J. C.; Tranter, G. E.: Koppenaal, D. W., Eds. Encyclopedia of Spectroscopy & Spectrometry 3rd ed.; Academic Press Elsevier: Oxford, 2017, pp. 750–761. 24. Chemical Engineering News 2005; Vol. 83, pp. 41-51. 25. Patil, A. O.; Pennington, W. T.; Paul, I. C.; Curtin, D. Y.; Dykstra, C. E. J. Am. Chem. Soc. 1987, 109, 1529–1535.