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Home , Enantiomer, Enantiomeric excess, Racemic mixture

Separation of Stereoisomers Resolution of Racemic Mixtures The separation of a into the individual pure 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 , distillation or basic . However, 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 11:06 AM initiate diastereomeric interactions. 1 Resolution of Racemic Mixtures Conversion of enantiomers to diastereomers

11:06 AM 2 Separation of Stereoisomers Resolution of Racemic Mixtures 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

11:06 AM discriminating enough to allow separation of enantiomers. 3 Methods of Resolving Racemic Mixtures Summary 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 . d) Use of chiral stationary phases for chromatographic resolution of racemic mixtures.

11:06 AM 4 Resolution of Racemic Mixtures Diastereomeric Salt Formation In 1849, , in his pioneering work, was able to isolate the enantiomers of because they crystallize from solution as crystals with differing symmetry and shape.

But such cases are very rare, but profoundly influenced the study of : Due to this observation, 11:06 AM concept of enantiomerism was born. 5 Resolution of Racemic Mixtures Formation of Diastereomeric Salts (S)-1-Phenylethylamine combines with a racemic mixture of to form diastereomeric salts. The diastereomers are separated by fractional crystallization.

11:06 AM 6 Resolution of Racemic Mixtures Formation of Diastereomeric Salts After the separation process, each of the diastereomers is subsequently treated with a strong acid such as HCl to regenerate the corresponding of lactic acid.

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 extraction with

11:06 AM an organic solvent would allow recovery of R-lactic acid. 7 Resolution of Racemic Mixtures Formation of Diastereomeric Salts 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 8 Resolution of Racemic Mixtures Diastereomeric Salt of The natural enantiomer, (S)-(+)-mandelic acid (sweet almonds), combines with a racemic mixture of 2- aminobutane to form diastereomeric salts.

11:06 AM 9 Resolution of Racemic Mixtures Formation of Diastereomeric Compounds

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 10 Resolution of Racemic Mixtures Formation of Diastereomeric Esters of (-)-Menthol, a chief component of peppermint oil, can be readily used as a chiral resolving agent.

11:06 AM 11 Resolution of Racemic Mixtures Enzymatic Resolution In enzymatic or , there is preferential reaction of just one enantiomer resulting in an enantioenriched sample of the less reactive enantiomer.

11:06 AM 12 Resolution of Racemic Mixtures Chiral Stationary Phases 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

11:06 AM diastereomers with different retention times. 13 Resolution of Racemic Mixtures Chromatography on Chiral Stationary Phases

11:06 AM 14 Resolution of Racemic Mixtures Chiral Stationary Phases 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

11:06 AM efficacy of the separation. 15 Resolution of Racemic Mixtures Assessing the Efficacy of a Resolution Using a chromatogram of the resolved compound on a chiral stationary phase, the percentage composition can be determined.

Racemic mixture

Retention time

Enantiomerically enriched

11:06 AM 16 Resolution of Racemic Mixtures Assessing the Efficacy of a Resolution 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 (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 17 Assessing Purity of an Enantiomer Calculating Enantiomeric excess (ee) by Percentage

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 11:06 AM 18 the racemic mixture, which is 10%. Assessing Purity of an Enantiomer Calculating Enantiomeric excess (ee) by Mass 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:

(Where R and S are the amounts of each enantiomer in 11:06 AM the mixture) 19 Resolution of Racemic Mixtures Calculating Enantiomeric Excess (Assignment) The enantiomeric excess can also be calculated if the [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 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. 11:06 AM Show your work and explain how each value is obtained. 20 Resolution of Racemic Mixtures Calculating Enantiomeric Excess (Answer) 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%

11:06 AM The minor S-enantiomer = (100 – R) = 25.6% 21 Assignment Separation of Stereoisomers A synthetic chemist investigated the reduction of (S)-2- amino-3-oxobutanoic acid to L-threonine with sodium borohydride shown below:

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? 11:06 AM 22