How to Separate Enantiomers

Core Concepts

This article will cover the different techniques that could be used to separate enantiomers. After understanding the different resolution techniques, you will be able to utilize these techniques to ensure that the each compound is able to play its designated role without unfavorable interference from its enantiomer.

Topics Covered in Other Articles

Significance of the Process

A racemic mixture is a solution containing both the S and R enantiomers of a particular compound.

Enantiomers have the same physical and chemical properties since they have identical mass numbers and identical electron configurations, respectively. However, the arrangement of the bonds around the central atom confers different reactivities in biological systems. This is since enzymes are stereospecific, so their active sites are tailored to recognize a specific spatial order of bonds, thus, while an enzyme could bind a compound, it is unable to bind its enantiomer.

One important example would be our protease enzymes. We are only able to digest L-amino acids since our bodies have enzymes that recognize L-amino acids, we do not have enzymes to recognize D-amino acids, and hence we can’t digest them.

how to separate enantiomers. l and d amino acids which need advanced techniques to separate.

Due to different reactions in the human body, separating enantiomers becomes especially important in drug design. While one enantiomer might lead to therapeutic and desirable effects, the other enzyme could be inactive or could lead to detrimental effects. To illustrate, consider the Thalidomide Crisis of 1960. The R isomer of Thalidomide was a good remedy for pregnant women with morning sickness. However, doctors did not know that the S enantiomer was a teratogen, a mutagen that crosses the placental barrier and affects fetal development. Since the doctors were unaware of that, the drug was given as a racemic mixture, leading to a significant number of fetal malformations and birth defects. Feel free to check out this article to read more about the magnitude of the defects this drug caused.

Separation Techniques

There are a number of ways to separate enantiomers, the most important of which will be discussed below:

Resolving Agents

We have mentioned that enantiomers have identical physical and chemical properties. Therefore, we are unable to utilize any chemical or physical separation techniques to segregate the R and S stereoisomers. However, we also mentioned that diastereomers have different properties. 

So what we will do here is transform the racemic mixture into a mixture of diastereomers. We do this by combining the mixture with an enantiomerically pure compound (so ONE enantiomer of a different compound) that is able to react with the racemic mixture. The enantiomerically pure compound is called a ‘resolving agent’. The result would be a mixture of diastereomers. These can easily be separated. 

Let’s illustrate with an example. If we know that compounds 1 and 2 are able to react to produce a new product (which retains both chiral centers) then we would have a reaction like this :

(R) Compound 1 + (R) Compound 2 = (R,R) product
(S) Compound 1 + (R) Compound 2 = (S,R) product

After we obtain the products, which are diastereomers, we take advantage of their different physical and chemical properties to separate them. We can then perform a reaction to regenerate the enantiomers.

Chiral Column Chromatography

When we usually use column chromatography, the beads in the column are able to preferentially bind one compound and not the other. You can check out this article if you want a recap on chromatography. Separation by chromatography is done by utilizing property differences such as size, polarity, affinity, or charge. If we want to separate enantiomers, then the beads must be able to preferentially bind one enantiomer and not the other. This is infact possible, because as you might remember, it is through binding affinity that our proteases are able to bind L-amino acids and not D-amino acids.

So, the column (stationary phase) contain a chiral compound that is has a significantly higher affinity for one enantiomer and not the other. And the racemic mixture would be the mobile phase. The enantiomers in the racemic mixture exit the chromatography columns at different rates, which is how we are able to isolate them.

Reactions with Enzymes

Similar to the technique above, we utilize the affinity of enantiomers to separate them. Knowing that an enzyme is selective to one enantiomer and not the other, we the enzyme to our mixture and wait for the reaction to occur. The enantiomer that reacts with the enzyme will be transformed into a different compound. We are able to differentiate this compound from the other enantiomer.

However, keep in mind that by doing this, you lose one enantiomer and retain the other, so make sure to react the non-desired enantiomer so you are left with the one you want.

Kinetic Separation

Similar to both techniques above, we utilize affinity differences. So we react the racemic mixture with an enantiomerically pure compound, such that one enantiomer reacts faster than the other. So, while the enantiomer of higher affinity undergoes the reaction, the enantiomer with lower affinity is consumed slower and disproportionally left behind, allowing us to collect it.


When we crystallize a racemic mixture, we are able to visually differentiate the R and S enantiomers – with the aid of a microscope. Therefore, we can manually separate the enantiomers.

Further Reading