Separation of StereoisomersResolution of Racemic Mixtures

1

The separation of a racemic mixture into the individual

pure enantiomers is called resolution.

Since enantiomers have identical physical properties, such

as solubility, boiling point and chromatographic retention

time, they can not be resolved by common physical

techniques such as crystallization, distillation or basic

chromatography.

However, diastereomers have different physical properties

and can be separated by conventional physical techniques,

a difference commonly exploited in resolution of racemic

mixtures by placing the racemate in a chiral environment to

initiate diastereomeric interactions.11:06 AM

Resolution of Racemic MixturesConversion of enantiomers to diastereomers

211:06 AM

Separation of StereoisomersResolution of Racemic Mixtures

3

All methods for separating or characterizing enantiomers

exploit the formation of diastereomeric interactions.

Although the interactions that create diastereomers out of

enantiomers are commonly:

(i) Ionic interactions or

(ii) Covalent interactions,

However, this is not a strict requirement for separations.

Weaker, non-covalent complexes or interactions based on

van Der Waal or hydrogen bonding interactions are often

discriminating enough to allow separation of enantiomers.11:06 AM

Methods of Resolving Racemic MixturesSummary

4

The four general strategies that take advantage of the

formation of diastereomeric interactions to separate a

racemic mixture or enantiomerically enriched mixture are:

a) Formation of diastereomeric salts with an enantiopure

resolving agent.

b) Formation of diastereomeric compounds with an

enantiopure resolving agent.

c) Enzymatic resolution using an appropriate enzyme.

d) Use of chiral stationary phases for chromatographic

resolution of racemic mixtures.

11:06 AM

Resolution of Racemic MixturesDiastereomeric Salt Formation

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In 1849, Louis Pasteur, in his pioneering work, was able to

isolate the enantiomers of tartaric acid because they

crystallize from solution as crystals with differing symmetry

and shape.

But such cases are very rare, but profoundly influenced

the study of stereochemistry: Due to this observation,

concept of enantiomerism was born.11:06 AM

Resolution of Racemic MixturesFormation of Diastereomeric Salts

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(S)-1-Phenylethylamine combines with a racemic mixture

of lactic acid to form diastereomeric salts. The

diastereomers are separated by fractional crystallization.

11:06 AM

Resolution of Racemic MixturesFormation of Diastereomeric Salts

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After the separation process, each of the diastereomers is

subsequently treated with a strong acid such as HCl to

regenerate the corresponding enantiomer of lactic acid.

11:06 AM

Note that since lactic acid would be soluble in the organic

layer, while the ammonium salt of the resolving agent

would be in the water layer, routine solvent extraction with

an organic solvent would allow recovery of R-lactic acid.

Resolution of Racemic MixturesFormation of Diastereomeric Salts

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Since enantiomerically pure compounds are very

expensive, it is usually necessary to recover and reuse the

chiral amine.

This is achieved by treating the (S)-1-phenylethyl

ammonium chloride salt with a strong base such as sodium

hydroxide to regenerate and recover the chiral amine.

11:06 AM

Resolution of Racemic MixturesDiastereomeric Salt of Mandelic Acid

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The natural enantiomer, (S)-(+)-mandelic acid (sweet

almonds), combines with a racemic mixture of 2-

aminobutane to form diastereomeric salts.

11:06 AM

Resolution of Racemic MixturesFormation of Diastereomeric Compounds

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An equally effective strategy of resolving racemic mixtures

is by reacting with an enantiopure resolving agent leading

to formation of covalently bonded diastereomeric

compounds.

After separating the diastereomers through conventional

techniques, such as gas or liquid chromatography, the

resolving agent is recovered by cleaving the covalent bond

formed in the earlier step.

11:06 AM

Resolution of Racemic MixturesFormation of Diastereomeric Esters of Menthol

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(-)-Menthol, a chief component of peppermint oil, can be

readily used as a chiral resolving agent.

11:06 AM

Resolution of Racemic MixturesEnzymatic Resolution

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In enzymatic or kinetic resolution, there is preferential

reaction of just one enantiomer resulting in an

enantioenriched sample of the less reactive enantiomer.

11:06 AM

Resolution of Racemic MixturesChiral Stationary Phases

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A more versatile method for resolving a racemic mixture is

through the use of chromatography on chiral stationary

phases. These are applied in gas chromatographic and

liquid chromatographic techniques.

In the resolution of racemic 2-aminobutane on a

chromatographic system in which an enantiomer of

mandelic acid is attached to a stationary phase, new,

transient, diastereomeric interactions between 2-

aminobutane and the stationary phase lead to separable

diastereomers with different retention times.11:06 AM

Resolution of Racemic MixturesChromatography on Chiral Stationary Phases

1411:06 AM

Resolution of Racemic MixturesChiral Stationary Phases

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Chiral chromatography

exploits the development of

transient, diastereomeric

interactions between the

enantiomers and the chiral

stationary phase.

The S-enantiomer has

stronger interactions with

stationary phase hence tails

in the column.

Chiral stationary phases can

also be used to assess the

efficacy of the separation.11:06 AM

Resolution of Racemic MixturesAssessing the Efficacy of a Resolution

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Using a chromatogram of the resolved compound on a

chiral stationary phase, the percentage composition can be

determined.

11:06 AM

Racemic mixture

Enantiomerically

enriched

Retention time

Resolution of Racemic MixturesAssessing the Efficacy of a Resolution

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The efficacy of the different strategies for separating a

racemic mixture can be determined by assessing the purity

of each of the enantiomers obtained.

The purity of each of the enantiomers is usually expressed

as enantiomeric excess (ee).

Enantiomeric excess (optical purity) is a measure of how

pure an enantiomer is (i.e. the extent to which one

enantiomer is present in excess of the racemic mixture).

It is denoted by the symbol ee and calculated as a %.

ee= % of major enantiomer -% of the minor enantiomer.11:06 AM

Assessing Purity of an EnantiomerCalculating Enantiomeric excess (ee) by Percentage

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If the percentages of each of the two enantiomers in the

mixture is calculated from the area under the peak in a

chromatogram, the enantiomeric excess can be calculated

directly using the formula below.

ee= % of major enantiomer -% of the minor enantiomer.

Consider the case of a mixture containing 95% of one

enantiomer and 5% of the other, the enantiomeric excess

of the mixture is 95% - 5% = 90%.

In essence, there is a 90% excess of one enantiomer over

the racemic mixture, which is 10%.11:06 AM

Assessing Purity of an EnantiomerCalculating Enantiomeric excess (ee) by Mass

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The enantiomeric excess (purity) of a mixture can be

calculated using the masses of each of the enantiomers in

the mixture.

If the masses of each of the pure enantiomers isolated

after separation is known, the enantiomeric excess can be

calculated using the equation below:

11:06 AM

(Where R and S are the amounts of each enantiomer in

the mixture)

Resolution of Racemic MixturesCalculating Enantiomeric Excess (Assignment)

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The enantiomeric excess can also be calculated if the

specific rotation [α] of a mixture and the specific rotation [α]

of a pure enantiomer are known.

ee= ([α] mixture/[α] pure enantiomer) x 100.

A sample of mandelic acid analysed in a polarimeter gave

an observed specific rotation of -75 degrees. If the specific

rotation of (S)-mandelic acid is +154 degrees;

(i) Which enantiomer is in excess? (R or S)

(ii) Calculate the enantiomeric excess of the mixture.

(iii) Calculate the percentage of each enantiomer in the

mixture.

Show your work and explain how each value is obtained.11:06 AM

Resolution of Racemic MixturesCalculating Enantiomeric Excess (Answer)

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If (S)-mandelic acid has a specific rotation of +154 degrees

then its enantiomer has a specific rotation of -154 degrees.

As the specific rotation of the mixture is negative, (R)-

mandelic acid is the dominant one.

ee = [α]obs / [α]max x 100

where ee is the enantiomeric excess and [ ] is the modulus

sign that makes negative values positive.

ee = (75 /154) x 100 = 48.7%

Let the % of R-enantiomer be R, that of the S-enantiomer

be S, then R + S = 100, while R – S = enantiomeric

excess.

R – (100 – R) = enantiomeric excess = 48.7

2R – 100 = 48.7, which implies that 2R = 148.7

The major R-enantiomer = 74.4%

The minor S-enantiomer = (100 – R) = 25.6%11:06 AM

AssignmentSeparation of Stereoisomers

A synthetic chemist investigated the reduction of (S)-2-

amino-3-oxobutanoic acid to L-threonine with sodium

borohydride shown below:

2211:06 AM

After purification of the reaction mixture by column

chromatography, 8.2 g of L-threonine and 1.6 g of L-

allothreonine were isolated:

(i) Determine the diastereomeric excess of the reaction

(ii) What was the diastereoselectivity of the reduction?