1. METHODS OF RESOLUTION OF RACEMIC MIXTURE
Dr. A.G. Nikalje

2. (+-)α- Chloropropanoic acid
RACEMIC MIXTURE: “An equimolar mixture of a pair of enantiomers is called as racemate or racemic mixture.”
Denoted by (+-) +
(+-)α- Chloropropanoic acid

3. Resolution is necessary to prepare optically pure chiral auxiliaries, to purify products of low enantiomeric excess and it is valid strategy for chiral synthesis.
RESOLUTION: “Separation of enantiomers from either racemic mixture of enantiomerically enriched mixture.”
Mefloquine (Antimalarial)
Marketed in the form of its racemic mixture where the erythro form is more active than threo form.

4. Resolution of enantiomers

5. Racemic mixture is optically inactive(Zero rotation of plane polarized light). The difficult part is to produce exclusively one enantiomer.
METHODS OF RESOLUTION
1)Mechanical separation (crystallisation method)
3 types of crystalline forms are as follows:
1)Conglomerate with homochiral assemblies of enantiomers in a single crystal.
2)Racemic compounds containing equal no. of (+) & (-) isomers in the unit cell of crystals.
3) Pseudoracemates with no preference for enantiomers in the crystal structure.

7. RACEMIC CONGLOMERATE :
If the molecules of the substance have greater affinity for same enantiomer than for the opposite one, mechanical mixture of enantiomerically pure crystals will result. Melting point of racemic conglomerate is always lower then that of pure enantiomer.
Addition of small amt of one enantiomer to conglomerate increases the melting point.
Preferential crystallisation depends on solubility of enantiomer which is less then solubility of racemic form.

8. Mechanical separation
Crystals are nonsuperimposable their appearance is not identical and trained crystallographer can separate them with pair of tweezers.
Seeding of racemic mixture with something will cause only one enantiomer to crystallize.
Spontaneous resolution is achieved by sublimation.
The best method for inducing crystallization is to inoculate or seed a supersaturated solution with small particles of the material to be crystallized.

9. Fractional crystallization : A process to separate different solutes from a solution.
Method for the separation of diastereomers.
Techniques such as Gas chromatography and preparative liquid chromatography prove useful from separation point of view where the quantities to be resolved are small.

10. 2) RESOLUTION THROUGH FORMATION OF DIASTEREOMERS. (Chemical method)
Pasteur developed method for formation of diastereomeric salts & their fractional crystallisation is best method of resolution.
Example
Resolution of racemic acid (+-) A with optically pure base (+)-which combines with racemic acid gives 2 diastereomeric salts.
(+-)A+(+-)B
(+)-A.(+)-B+(-)-A.(+).B
Two salts differ in properties such as solubility, boiling point and adsorption coefficient .
Disposition of the salt with mineral acid would furnish (+-)A in
enantiomerically pure form.
Separated either by fractional distillation or crystallisation.

11. Eg; A- terpinol, isocarvomenthal & phenolic compounds give
3) RESOLUTION THROUGH THE FORMATION OF MOLECULAR COMPLEXES
Instead of forming stable salts or covalent compounds with the substrates and the resolving agents, have molecular complexes which form and decompose easily for the suitable for resolution.
Eg : Digitonin steroidal saponin forms addition complexes with various alcohols.
Eg; A- terpinol, isocarvomenthal & phenolic compounds give
(+)2 Napthylcamphylamine.

12. 4) Resolution by chromatography
Objective: Chromatographic methods are developed for direct seperation of enantiomer.
Trifluoroacetyl derivative of optically active amino acid used for
gas chromatographic resolution of racemic alcohols with esters.
Paper chromatography effects partial resolution of camphorsulfonic acid.
Gas chromatography is used for analysis and separation of enantiomers.
Optically active substances are selectively adsorbed by optically active adsorbent. Wool and casein selectively adsorb (+)-Mandelic acid from aqueous solution of (+-_)- Mandelic acid.
Resolution is by Troger’s base.
Amines resolved via acylation by optically active acid chlorides.

13. Chromatographic resolution of enantiomers:
Prepare column containing stationary phase coated with a chiral compound
Enantiomers form diastereomeric complexes with the chiral stationary phase.
Separate the diastereomeric complexes based on differences in affinity for stationary phase
Strongly complexed: elutes slowly.
Weakly complexed: elutes more quickly.

14. Stereoselective adsorbents prepared in the presence of a suitable reference compound of known configuration.eg: silica gel in the presence of quinine. Silica gel adsorbs quinine more readily then its stereoisomer quinidine.
Cinchonidine (configurationally related to quinine ) is adsorbed more readily then its stereoisomer cinchonine (configurationally related to quinidine)

15. 5) Channel complex formation
Means of carrying out resolution WITHOUT chiral reagents.
TRI-O- THYMOTIDE

16. Tri- o- thymotide forms clathrates with ethanol, n-hexane.
It crystallize as racemate but that resolution takes place when it forms clathrates with
n.- hexane
Chloroform
Benzene
By means of seeding and slow growth of a single crystal, one can obtain (+) and (-) form depending on the nature of the seed.
Tri- o- thymotide is itself a racemic modification.

17. 6) Chiral recognition
Chiral hosts are used to form diastereomeric inclusion compounds.
Host which form an inclusion compound with one enantiomer of a racemic guest is known as chiral recognition.
Only one enantiomer fits into the chiral host. Chiral crown ethers partially resolve the racemic amine salt.
Enzymes act as host for chiral recognition.
Clathrates: During crystallisation, certain substances form a cage like lattice in which the coordinating compound is entrapped.eg: hydroquinone molecules crystallize in the cage like structure with Hydrogen bonding.
Cyclodextrins:
Cyclic oligosaccharides with minimum 6 D-(+)- glucopyranose units attached by α-1,4 linkage.

18. Cyclodextrins(CD) act as host molecules.
Cyclic oligosaccharides with minimum 6 D-(+)- glucopyranose units attached by α-1,4 linkage.
Cyclodextrins(CD) act as host molecules.
Structure assume a truncated cone and accommodate a wide variety of compounds. Interior cavity is hydrophobic and the entrance of the cavity is hydrophilic.
Α Cyclodextrins
β Cyclodextrins
γ Cyclodextrins
Smallest cavity 6A* diameter
Larger internal diameter 6-7A*
Larger internal diameter 6-8A*
Resolve chronic diarrhoea
And constipation .
Lowers blood triglyceride levels.
Use in pharmaceutical technology.
Enhance solubility
Dissolution: Famotidine & Tolbutamide.
Stability
Sustained release of Diltiazem, isosorbide dinitrate.
Used in food industry.
As they have the ability of complexing fragrances.

19. HYDROPHILIC ENTRANCE HOST MOLECULE (GAMMA- CYCLODEXTRIN)
CAVITY FOR GUEST MOLECULE
HYDROPHOBIC INTERIOR

20. Structures of alpha, Beta and gamma Cyclodextrins

21. 7) Resolution through equilibrium asymmetric transformation
This resolution based on 2 steps
Epimerization of diastereomeric species.
Precipitation of predominant epimers.
Epimerization of diastereomeric species
Eg: Treat 2 (p carboxybenzyl) 1 hydrindanone with brucine (Bitter alkaloid related to strychnine) in acetone solution,
diastereomers precipitate in 90% yield.
Precipitation of predominant epimers
Eg: (-)-menthylester of racemic phenylchloroacetic acid treat with
Base, it epimerizes and give equilibrium mixture of esters with
57%(-)- acid and 43% (+)- acid.

22. Brucine is used as standard for bitterness.
The ability of brucine, and to a lesser extent strychnine, to function as resolving agents for amino acids was reported by Fisher in The formation of diastereomeric brucine salts has been reported for thousands of organic compounds.
Brucine is used as standard for bitterness.

23. 8) Resolution by biochemical transformation
Biochemical method have important application in the resolution of (+-)- amino acids.
Acetylated (+-)- amino acids treated with enzyme acylase 1 till the half of the acetyl groups are hydrolysed. i.e. L amino acids and unhydrolysed group i.e. D amino acid.
When certain bacteria or moulds are added to dilute solution of racemic mixture, destroy one enantiomer more rapidly than the other.
Penicillin glaucum,(a mould), when grown in solution of racemic ammonium tartarate, attacks the (+) form & leaves the (-) form.

24. Limitations of Biochemical method:
Use dilute solutions to obtain small amounts.
Need is to find a microorganism which will attack only one of the enantiomer.
One form is always destroyed & the other is not always obtained In 50%yield since some forms may also be destroyed.

25. 9) KINETIC RESOLUTION
Enantiomer react with chiral compounds at different rates, so it possibly affects a partial separation by stopping the reaction before completion.
Application:
Resolution of racemic alkenes: Alkenes are used because they don’t easily convert to diastereomers if other functional groups are absent.
Resolution of racemic alkenes is done by treatment with diisopinocamphenylborane.
Only one of the enantiomer of the original racemic mixture is obtained, but 2 ways of getting the other are
Use of other enantiomer of the chiral reagent.
Convert pdt to starting compound by reaction that preserves the stereochemistry.
Reactions catalysed by enzymes use for kinetic resolution.

26. Resolution by means of formation of diastereomers
Resolution by acids
Resolution by Bases
Resolution by
Alcohols
Aldehydes and
ketones
Optically active acids: Brucine,
Morphine, Quinine,
Strychnine
Optically active acids: Tartaric acid.
Converted to acid ester derivative by succinic anhydride.
Resolved by optically active hydrazines (-)-menthylhydrazines
Optically active bases: Benzimidazoles,
menthylamine.
Resolved by diastereomer formation with optically active Acyl chlorides
(to from ester)
With optically active isocyanates (urethanes)

27. 10)Preferential crystallisation by inoculation
Supersaturated solution of racemic modification is treated with crystal of one enantiomer, whereupon this form is precipitated.
Resolution of glutamic acid is done by inoculation.
It is a major excitatory neurotransmitter in the brain and spinal cord. It is converted into either glutamine or Gamma-Aminobutyric Acid (GABA), two other amino acids that help pass messages to the brain.
Preferential crystallisation depends on solubility of enantiomer which is less then solubility of racemic form.
Copper complex of DL-Aspartic acid resolved by inoculation.
Resolution was effected by seeding the Supersaturated aqueous solution with pure crystals of D or L isomer of the amino acids.
10)Preferential crystallisation by inoculation

28. 11)ATROPISOMERISM
DFEFINE: “Optical rotation due to restricted rotation in bulky group (orto-ortho position) substituted biphenyl derivative compound.
In the crystal, both benzene rings of biphenyls lie in the same plane.
In solution & vapor phase the 2 rings are twisted w.r.t. each other by angle of 45 degrees due to steric interaction between 2,2’ and 6,6’ pair of hydrogen's.
Isolable stereoisomers resulting from restricted rotation about single bonds.
45degrees

29. 12)Deracemization
One enantiomer is converted to the other so that a racemic mixture is converted to a pure enantiomer or to mixture enriched in enantiomer.
Eg: Racemic thioester was placed in contact with certain optically active amide. After 28 days the solution contained 89% of one enantiomer and 11% of the other.
To effect deracemization 2 conditions are necessary:
Enantiomer must complex differently with the optically active substance
They must interconvert under experimental conditions.
Presence of base(Et3N) was necessary for the interconversion to take place.

30. 13) Optical purity : Optical purity . = observed rotation x 100%
Ratio of the rotation of a mixture to the rotation of a pure enantiomer
Optical purity . = observed rotation x 100%
rotation of pure enantiomer
Another method to express (or determine) the relative amounts of enantiomers present in a mixture is enantiomeric excess.
Numerically identical to optical purity
e.e. = o.p. = excess of one over the other x 100%
entire mixture
Optical purity = percent enantiomeric excess is given as:

31. Enantiomeric excess (e
Enantiomeric excess (e.e) is a measurement of purity used for chiral substances. It reflects the degree to which a sample contains one enantiomer in greater amounts than the other. A racemic mixture has an e.e of 0%, while a single completely pure enantiomer has an e.e of 100%. A sample with 70% of one enantiomer and 30% of the other has an e.e of 40%.
A mixture of enantiomer whose purity is to be determined converted using optically active reagent into mixture of 2 diastereomers.
2 diastereomers are separated by gas chromatography and the ratios determined from the peak areas. The ratios of diastereomers is the same as that of the original enantiomers.
Direct seperation of enantiomer by gas or liquid chromatography on chiral column used to determine o.p.

32. Methods of optical purity
Isotopic dilution method,
Use of chiral lanthanide shift reagent,
High pressure liquid chromatography &
Gas chromatography.
Miscellaneous methods:
Kinetic resolution,
C13 NMR relaxation rates of diastereomeric complexes,
Circular polarization of luminescence.
Isotope dilution analysis is a method of determining the quantity of chemical substances.

33. ISOTOPIC DILUTION METHOD
Steps involved are as follows:
Find the weight of an enantiomer (A)as well as its racemic modification(B).
Take that enantiomer & mix in solution with known weight of its racemic modification.(A+B)
Label(A+B) with an isotope & Perform recrystallisation.
After recrystallisation, Isotope content of the racemic modification is determined.
If the enantiomer under consideration is (+)-form.
The recovered racemic modification contain unlabelled(+)-and labelled(-)- form.
Calculate the dilution factor.

34. 8. If the (+)-enantiomer is not optically pure some unlabelled (-)- form will also be present in the recovered racemic modification.
In this case, the isotopic dilution factor will be less then the predicted one.

35. Basic principle of isotope dilution
Adding of an isotopically altered standard to the sample changes the natural isotopic composition of the analyte. By measuring the resulting isotopic composition, it is possible to calculate the amount of the analyte present in the sample.