Chiral Resolution Techniques

Global Chiral Compounds Market

Many chemicals used in pharmaceutical, agricultural, flavors, fragrances and other specialty products are chiral in nature. Chiral chemicals exist in two different forms, or enantiomers, which are mirror images of each other. These compounds can have very different properties, such as smell, taste and efficacy. In the most extreme case, one enantiomer can be a potent drug and the other, a poisonous substance. A well-known example of this is the notorious drug thalidomide, which was prescribed worldwide in the late 1950s to combat symptoms associated with morning sickness in pregnant women. While R-enantiomer has safe sleep inducing effects; the co-existing S-enantiomer is believed to be responsible for thousands of cases of birth defects.

With such a dramatic difference in biological activities, it is not surprising that the demand for single enantiomers has been increasing. In fact, in recent years, US-FDA has mandated that every isomer of a chiral drug molecule must be studied for possible side-effects, and where possible, companies must manufacture enantiomerically pure products. Today, active ingredients in nine out of ten world’s top-selling drugs are chiral; six of the compounds in this select group are in the form of single enantiomers, while the rest three are racemates or racemic compounds.

There are two types of stereoisomers:

  • Enantiomers contain chiral centers that are non-superimposable & mirror images. They only come in pairs!

  • Diastereomers contain chiral centers that are non-superimposable but are NOT mirror images. There can be many more than 2 centers depending on the number of stereocenters.

Separation of racemates into their component enantiomers is a process called chiral resolution. Since enantiomers have identical physical properties, such as solubility and melting point, resolution is extremely difficult. Diastereomers, on the other hand, have different physical properties, and this fact is used to achieve resolution of racemates. Reaction of a racemate with an enantiomerically pure chiral reagent gives a mixture of diastereomers, which can be separated.

Diastereomeric Crystallization

Diastereomeric crystallization is a predominant technique employed in the resolution of chiral acids & bases. The principle of crystallization is based on the formation by two enantiomers of the diastereomeric salts of optically pure compound which can be separated easily (because of difference solubility). In most cases, after the desired enantiomer has been separated from the diastereomeric salt, the resolving agent is recovered and becomes available for reuse. If the remaining undesired enantiomer is re-racemized, more desired material can be recovered from the mixture. If this procedure is repeated, the yield of the desired product can approach 100%. Such is the case of a-amino-E-caprolactam, an intermediate for a cholecystokinin antagonist (Merck) named dextropropoxyphene (Darvon). This is one of the reasons, Diastereomeric Crystallization is considered a GREEN METHOD. And because of its easy adoption for manufacturing purposes that does not require costly investment in initial setup, most companies first try this method and use the other methods only if this method does not work. Today 65% of chiral products are made using this technique.

Diastereomeric crystallization chemically separates enantiomers from racemic mixture by producing a salt.

This is done by adding an enantiopure acid or base to the mixture so that the resulting salts are not mirror images of each other. Instead, they are diastereomers with different chemical and physical properties that allow their separation.

Sometimes companies use diastereomeric crystallization as a rapid method before a permanent solution is found. For example, in the case of Pfizer’s nerve pain treatment Lyrica, the initial route for Lyrica used an equimolar quantity of S-Mandelic acid to resolve the desired enantiomer to obtain multi-kilo quantities of Lyrica; before the enzymatic route was developed [that took a few years].

In other cases, diastereomeric crystallization is used to obtain higher purity. In one case, using enzymatic resolution of alcohol, we were unable to increase enantiomeric purity beyond 90%. Finally, desirable “EE” was obtained when we used diastereomeric crystallization of corresponding acid phthalate. We feel this combination of methods has great utility, substantially reducing the quantities of material that needs to be handled in the conventional chiral salt fractionation.

But in most cases, diastereomeric crystallization is the prefered method of choice for manufacturing that can scale up easily for manufacturing purposes as shown below:

  • 2-Amino-5-methoxytetralin is an intermediate to assemble N-0923 (Nagase & Co., Ltd., Japan), a potent dopamine D-2, effective against Parkinson’s disease (currently in clinical trial).This intermediate is resolved by diastereomeric salt formation with (S)-Mandelic acid. The undesired isomer is racemized to increase efficiency of the process.

  • In vivo study on Sulphostin, a diapeptidyl peptidase IV inhibitor, its large-scale method was investigated. During the synthesis the most difficult part was simplified by optical resolution of the end product by basic resolving agent (achieved 2-fold process improvement compared to previous method.)

  • Citalopram, a selective serotonin reuptake inhibitor introduced in 1989 for treatment of depression, is a racemic mixture whose entire inhibitory activity resides in the S (+)-enantiomer, also known as escitalopram. The key intermediate was difficult to purify due to resolution problems, which was addressed nicely by using (+)-di-p-toluoyl tartaric acid and recrystallizing salt twice in medium polar solvent.

  • AG-7404 (Pfizer), a key intermediate acid was resolved by (-)norephedrine. The acid was enriched with undesired isomer, which was then racemized completely. The final compound was obtained by enzymatic resolution. Thus the method was a combination of diastereomeric crystallization as well as enzymatic resolution.


High performance liquid chromatography (HPLC) is a popular method used by most pharmaceutical companies to get quick analytical quantities of pure enantiomers during the early drug-discovery stage. Using the chiral columns offered by many companies like Chiral Technologies, Regis, Millipore Sigma, Phenomenex, scientists can obtain small quantities of pure enantiomer in the matter of days. However, this technology is not feasible for scale-up manufacturing and requires large amount of solvents.

The technique is based on the following technology: A pair of enantiomers is considered to be resolvable if alpha > 1.1. Here one enantiomer is retarded in its passage through the column because of its preferential binding to the chiral stationary phase. Because of this, the two enantiomers of a racemate emerge from the column at different times and with different volume fractions of eluent. Unfortunately, most chiral resolutions involve only small differences in eluent fractions.


Although most preparative chiral separations have been performed in the conventional batch-mode process, interest in simulated moving-bed (SMB) chromatography is growing.

Simulated Moving Bed or SMB chromatography is used to separate one chemical compound or one class of chemical compounds from a mixture to provide significant quantities of the purified or enriched material at a lower cost than could be obtained using simple (batch) chromatography. It combines continuous countercurrent chromatographic separation with chemical reaction. By using multiple columns in series and a complex valve arrangement, which provides for flow of the feed mixture and solvent ("eluent" or "desorbent") at any column, its performance can be increased dramatically.

SMB technology is being used for the separation of the enantiomers of chiral molecules at production scale. For example, it was used during the separation of the enantiomers of the antiasthmatic agent formoterol and of the antitussive agent guaifenesin. Compared to the batch elution chromatography, a reduction of the mobile phase consumption of respectively 81% and 84% was achieved using SMB. The results show that the influence of the feed rate and extract rate on the separation affect the high purity and productivity and enable the production of both the enantiomers.

SMB offers lower production cost by requiring less column volume, less chromatographic separation media ("packing" or "stationary phase"), and less solvent. Although because of its complex assembly, its adoption requires higher initial cost compared to single column operations, and also leads to higher maintenance cost.


The supercritical fluid chromatography technique or SFC is an innovative technology used in the analysis as well as the separation of various racemates. The various enantiomers have been separated using SFC due to its several merits over conventional HPLC methods such as low cost, wide polarity compatibility, higher column efficiency, and fast analyzing capability, which are desirable features for the pharmaceutical industry.

SFC has been found to be a better technique than HPLC for the chiral separations on a preparative scale because of its requirement of a low volume of solvents and easier recovery of the pure enantiomers. However, it is not cost-effective or scalable in a production environment beyond kilo scale levels.

One of the compounds developed using SFC is Diniconazole. It is a broad-spectrum triazole fungicide that is used to prevent plant diseases such as powdery mildew, bunt, rust, smut, and Septoria leaf spot. Diniconazole is a chiral chemical with one chiral center and is sold commercially as a mixture of R- and S-enantiomers. It has been reported that R-(-)-diniconazole has higher bactericidal activity, while S-(+)-diniconazole shows higher plant growth regulator activity. It is important to know if environmental factors have altered the ratio of R- and S-diniconazole.

Chiral Supercritical Fluid Chromatography (SFC) and mass spectrometry detection were used to study the different ratios of diniconazole in various plant materials. A ChromegaChiral™ CCA column was used to provide baseline resolution of the two diniconazole enantiomers. However, they are closely separated. In this study 14 different chiral stationary phases were evaluated for the chiral SFC separation.

ChiroSolve offers Chiral Resolution Kits

Separation of chiral molecules (enantiomers) from racemic mixture using Diastereomeric Crystallization Technique can be achieved and enhanced by the addition of chiral acids or bases. The resulting formation of salts can be separated on the basis of their differing solubility.

Using this principle, ChiroSolve Inc. has developed Chiral Resolution kits that allow scientists to quickly identify most commercially feasible chiral separation conditions for a given racemate within a matter of days. It does this by offering 96-vial high-throughput Chiral Screen Kits that:

  • Are made from chemically inert polypropylene material that can withstand temperatures of -200C to 1200C, so that full resolution can be done inside the vials

  • Contain chirally pure resolving agents that are easily cheaply available in large quantities and that have historically offered chiral separation

  • Contain 384 to 576 resolution conditions that racemate is checked against in parallel

  • Offer results within 24 hours after simple heating and cooling efforts

Typical Resolution procedure

  1. Choose the right type of kit (A1, A2, A3, A4 or B1, B2, B3, B4) depending on whether the unknown racemate is a base or acid respectively

  2. If the racemate is of type alcohol, amino acid, aldehyde or ketone, do the pre-processing to bring the mixture to acidic or basic moity

  3. Remove the aluminum seal and dissolve 12 mmol of racemate (if solid) in solvent of your choice

  4. Add 0.03 mmol of racemate to each of 96 vials of each kit. Use the additional septum-mat provided to reseal the vials after adding the racemate. Note that the seal/caps are pierceable to accommodate direct injection of racemate by an autostation

  5. Heat the rack along with its vials to up to 80º C (1-2 min. few times) or until the mixture becomes homogeneous

  6. Allow the kits to cool to ambient temperature. Then, if required, further cool it to 4º C and finally to 0º C and observe any crystallization. Vials with crystals are considered positive tests and need further investigation

  7. Use crystal initiation techniques to see if you can get more vials with crystals. Vials with no crystals even after this effort are considered negative tests

  8. Separate out the vials with crystals (positive tests) note down their barcode identification

  9. Analyze each of the crystals separately after liberating enantiomers from its diastereomeric salts for specific rotation

Example Chiral Screening done and their results


Atenolol belongs to a class of drugs known as beta blockers, which are prescribed for high blood pressure, angina and to prevent repeat heart attacks. It is the most widely prescribed beta-blockers in the world and is sold under the brand name Tenormin by AstraZeneca Inc. since 1976. The drug is a basic compound with a molecular weight of 266.3 gm, a chemical formula of C14H22N2O3, and a chemical structure of this form:

This is a β 1- adrenergic receptor antagonist. This activity is due to the (-) isomer.

In order to identify the optimal resolution condition for this basic compound and to separate out the enantiomers, acidic ChiroSolve kits A-1, A-2, A-3, and A-4 were used.

Actual work

  1. 9 mmol of atenolol or 2.397 gm was dissolved into 60 mL of methanol. 200 uL was dispensed through the pierceable septums into each of the 288 vials of the kits A-1, A-2 and A-3 using robotic liquid dispenser. This gave each vial approximately 0.03 mmol of atenolol

  2. Kits were placed in an 80°C water bath for a few minutes until the racemate was completely dissolved and then cooled to ambient temperature

  3. To encourage maximum crystallization, kits were placed in a 4°C refrigerator for 15 minutes. In case of some of the vials, some basic initiation was needed for crystal formation

  4. A basic workup of 500 uL 1 N NaOH and 400 uL of ethyl acetate was performed on each of the kits to release the potentially diastereomer drug bound to the chiral resolving acid

  5. The basic workup was mixed with the crystals and then the top layer of ethyl acetate containing the released compound was taken out and added to a pre-weighed tube

  6. The ethyl acetate was then evaporated and the potentially diastereomeric compound was left and weighed

  7. A Rudolph Research Analytical Autopol® III – Automatic Polarimeter was used to measure the optical rotation, α, of the crystals dissolved in methanol

  8. Based on the α rotation and the crystal yield, specific rotation was calculated [α] and the best results that provided highest yield and rotation were identified (shown below)


- Maximum positive specific rotation of +0.133 in the A-1 kit’s D-6 vial which contains (-) malic acid and 90% isopropanol.

- Maximum negative specific rotation of -0.114 in the A-2 kit’s D-7 vial which contains (-) dibenzoyl-l-tartaric acid and 80% methanol.

Exact enantiomeric purity can be further measured using Chiral HPLC that tells the extent of chiral enhancement achieved. We recommend that this procedure should be repeated twice to ensure consistent results. We also recommend that further scale-up of the 2 or 3 best results should be performed to identify the best resolution process during manufacturing.

2. Flurbiprofen

Flurbiprofen is a member of the phenyl alkanoic acid derivative family of non-steroidal anti-inflammatory drugs (NSAIDs) used to treat the inflammation and pain of arthritis and can be used for the treatment of metastatic prostate cancer, and Alzheimer's disease. It is also known by the trade name ANSAID and marketed by Pfizer. The drug is an acidic compound with a molecular weight of 244.27 gm, the chemical formula C15H13FO2, and a chemical structure:

In order to identify the optimal resolution condition for this acidic compound and to separate out the enantiomers, base ChiroSolve® kits B-1, B-2, B-3, and B-4 were used.


It was found that Quinine in methanol would offer the ideal condition (more data available when paper is published).


This is an off the market analgesic and anti-inflammatory drug that may be a starting point for finding a new drug to treat spinal muscular atrophy (SMA), a devastating childhood neurological disorder. The drug is an acidic compound with a molecular weight of 281.31 gm, the chemical formula C17H15NO3, and a chemical structure:

In order to identify the optimal resolution condition for this acidic compound and to separate out the enantiomers, base ChiroSolve® kits B-1, B-2, B-3 and B-4 were used.


It was found that L-prolinol in 90% IPA or 95% Ethanol would offer the ideal condition (more data available when paper is published).