Now that we know about various types of stereoisomers, we also need to know how to identify and separate them. In many applications, identifying or obtaining a pure sample of one isomer is essential.  

The first technique is the usual Fourier transform infrared spectroscopy. Sadly, FTIR is based on the changes in dipole moment due to the vibration of the bonds.  As a result, FTIR is unable to distinguish enantiomers from each other [5, 8]. However, it is still very useful when it comes to cis-trans isomers. We now usher back our good old butenedioic acid into the picture. 

In orange, we have the FTIR spectrum of the fumaric acid, while in blue we have the spectrum for the maleic acid. While the two spectra share some similarities like the carbon-carbon double bond absorption band and the hydroxyl stretching, the differences are also quite major. The hydroxyl stretching in fumaric acid is significantly ‘deeper’ than that of maleic acid, and this is not surprising given that maleic acid can form intramolecular hydrogen bond after the first protonation, damping the vibration. This is supported by the fact that maleic acid also has a carboxylate anion band at 1567-1587 cm^-1 which is absent in fumaric acid.  

Hence, for stereoisomers whose geometries result in quite a fair bit of differences in bonding, especially that of polar bonds, FTIR can be a useful tool to identify the isomers.  

Another common method that is used to identify chemical compounds is nuclear magnetic resonance spectroscopy (NMR). As it is with FTIR, NMR can distinguish diastereomers but cannot distinguish enantiomers [10]. To overcome this issue, a common method to distinguish enantiomers using NMR is to use a chiral derivatizing agent. Chiral derivatizing agent (CDA) is an enantiomerically pure agent that converts each enantiomer into different diastereomers. This then allows NMR spectroscopy to identify the two diastereomers then trace back to the enantiomers [11].  

One very versatile CDA is Mosher’s acid which is chiral and can exist as an (S) or an (R) enantiomer. Mosher’s acid can form esters or amides with enantiomeric alcohols or amines1. Since Mosher’s acid is extremely resistant to racemization, NMR can then be run on the esters or amides and use the slight to moderate differences in chemical shifts to trace the enantiomers. Otherwise, the esters or amides can also undergo chiral resolution to separate them before converting them back to enantiomerically pure alcohols or amines [4]. 

However, using CDA can be quite troublesome. Another method to separate the two enantiomers without reacting the sample is via chiral column chromatography. Chiral column chromatography is a high-performance liquid chromatography (HPLC) technique. The chiral HPLC column which is the stationary phase contains only one enantiomer of a chiral compound. Since the two enantiomers in the sample have different affinity to the stationary phase enantiomer, they move up the chiral HPLC column and different rates and exit the column at different times. This allows two separate, enantiomerically pure samples to be obtained [2, 7]. A popular chiral stationary phase is β-cyclodextrin a seven-ring sugar as it has a much better chiral recognition property than α-cyclodextrin or γ-cyclodextrin [3, 13].  

Another method of chiral resolution is via crystallization. Some of the racemic mixtures are known to be able to crystalize on its own to form to a mixture of enantiopure crystals. They are known as conglomerates. This is an example of spontaneous resolution. By applying two enantiopure crystals of each enantiomer on different sides of the reactor, it is possible to induce the enantiopure crystals of the same enantiomer to form on one side of the reactor and the enantiopure crystals of the other enantiomer on the other side. On the other hand, preferential crystallization can be done if the seed of only one enantiomer is added. Only that enantiomer will then crystallize out [1]. This is called resolution by entrainment. Using modern equipment, the purity of the crystals obtained can be close to 100%. 

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 References

1. Chaaban, J. H., Dam-Johansen, K., Skovby, T., and Kiil, S. (2014) Separation of Enantiomers by Preferential Crystallization: Mathematical Modeling of a Coupled Crystallizer Configuration. Organic Process Research & Development 18, 601–612. 
2. (2018, May 25) Chiral resolution. Wikipedia. Wikimedia Foundation. 
3. (2018, September 7) Cyclodextrin. Wikipedia. Wikimedia Foundation. 
4. Dale, J. A., Dull, D. L., and Mosher, H. S. (1969) .alpha.-Methoxy-.alpha.-trifluoromethylphenylacetic acid, a versatile reagent for the determination of enantiomeric composition of alcohols and amines. The Journal of Organic Chemistry 34, 2543–2549. 
5. (2018, October 17) Fourier-transform infrared spectroscopy. Wikipedia. Wikimedia Foundation. 
6. Haynes, R. D. (2009) Equilibrium coefficients for the adsorption of colloidal stickies onto mineral suspension particulates to improve paper recycling. Nordic Pulp and Paper Research Journal26, 421–428. 
7. (2018, October 20) High-performance liquid chromatography. Wikipedia. Wikimedia Foundation. 
8. (2008, April) IR Applied to Isomer Analysis. Spectra Analysis. 
9. Liu, Y. M.; Gordon, P.; Green, S.; Sweedler, J. V., Determination of salsolinol enantiomers by gas chromatography-mass spectrometry with cyclodextrin chiral columns. Anal Chim Acta 2000, 420 (1), 81-88. 
10. Luy, B. (2010) Distinction of Enantiomers by NMR Spectroscopy Using Chiral Orienting Media. Chem Inform 41. 
11. (2016, August 1) Nuclear magnetic resonance spectroscopy of stereoisomers. Wikipedia. Wikimedia Foundation. 
12. Pastuer, L. (1860) The Asymmetry of Naturally Occurring Organic Compounds. Pastuer Brewing. lecture. 
13. Scott, R. P. W. Cyclodextrin Chiral Stationary Phases from Chiral Gas Chromatography. Chromatography Online (Scott, K., Ed.).