**2. Separation of enantiomeric mixtures without chiral reagent**

## **2.1. Formation of macroscopically helical crystals**

The enantiomeric mixtures form crystals of a given helicity corresponding to the major configuration (**Scheme 1**).

In case of purification of enantiomeric mixtures of threonine was observed, that the majority of crystals have a convolution corresponding to the helical structure of the excess, while the minor enantiomer, crystallized near the excess, have the opposite convolution. The ratio between the major and minor helical crystals is in good correlation with the eutectic composition

**Scheme 1.** Purification of enantiomeric mixtures of threonine from water (ee<sup>0</sup> ≠ 0).

of the enantiomeric mixture of threonine. So the eutectic composition (eeEu) precipitates during evaporation, dominated by the helicity of the excess, along with the crystallization of the minor enantiomer as well.

Mirror-image crystals are formed from the supramolecular helical structures, which contain one of the enantiomer in excess. The helicity of the crystals is determined by the optical rotation of the enantiomer in excess [13, 14].

#### **2.2. Particle-size-controlled crystallization**

followed by the purification of the mixture, is applied [1, 2, 4]. In most cases, mixtures of diastereomers received with appropriate resolving agents, or mixtures of enantiomers isolated thereof, have to be separated. It is common in the two separation methods, that the distribution of the mixtures between two phases, and the phase separation can be applied [4–6]. However, the phase distribution of the mixtures of chiral compounds is not linear, but the distributions follow the binary melting phase diagrams of the mixtures, or the ternary phase

Besides the effect of the applied solvents, the phase distribution of the mixtures is also determined by kinetic or thermodynamic control [9]. The phase distribution is also determined by the eutectic composition of the chiral molecules in the mixtures [10, 11]. The equilibrium of the supramolecular helical structures, which participate in the phase distribution, determines the formation of the phase equilibriums [12]. A remarkable consequence of the effect of the helical structures is that the mirror-image macroscopic enantiomers form not only mirror-image crystals, but by attaching together, mirror-image helical crystals are formed [13, 14]. At the same time, mainly one of the helicities can be attributed to a given enantiomer, most probably this is the reason behind the results of separations. In the followings, the most characteristic

The enantiomeric mixtures form crystals of a given helicity corresponding to the major con-

In case of purification of enantiomeric mixtures of threonine was observed, that the majority of crystals have a convolution corresponding to the helical structure of the excess, while the minor enantiomer, crystallized near the excess, have the opposite convolution. The ratio between the major and minor helical crystals is in good correlation with the eutectic composition

diagrams characteristic also for the applied solvent [7, 8].

100 Laboratory Unit Operations and Experimental Methods in Chemical Engineering

examples of the above-mentioned methods will be discussed.

**Scheme 1.** Purification of enantiomeric mixtures of threonine from water (ee<sup>0</sup> ≠ 0).

**2.1. Formation of macroscopically helical crystals**

figuration (**Scheme 1**).

**2. Separation of enantiomeric mixtures without chiral reagent**

The ethanol solution of the conglomerate racemic *trans*-hydrobenzoin [15] (**THB**) was seeded with different amounts of (*S*,*S*)-**THB** and (*R*,*R*)-**THB** seeds of different particle size during a specified cooling program. After crystallization, the received crystals were separated to different ranges of particle size by sieving. Thus, enantiomeric mixtures of (*S,S*)-**THB** and (*R,R*)- **THB** of 83% and 87% enantiomeric excess were gained, respectively (**Scheme 2**) [16].

#### **2.3. Gravity-based enantiomer separation**

According to Soloshonok et al., the SDE (self disproportionation of enantiomers) appears in three main areas: gravitational field, phase transition, and the achiral chromatography [17]. Basically, the gravity-based SDE applies the differences in crystal density. The racemate enantiomeric mixture can be considered as the mechanical mixture of the racemic and enantiopure crystals, which can have different crystal densities. This difference can be applied for the separation of the racemic and enantiopure fraction. For example, from a enantiomeric mixture of phenylalanine (**Phe**) having 50% enantiomeric purity, two phases of 90 and 13% enantiomeric purity, respectively, could be separated after stirring in an inert solvent of appropriate density, set between the densities of the racemic and enantiopure crystals (**Scheme 3**) [18, 19].

Based on these results, separation of amino acid enantiomeric mixtures was carried out via density gradient ultracentrifugation, applying an iodinated gradient (*Nycodenz*) used in the isolation of nucleic acids and proteins. Recently, the density difference between the racemic and enantiopure Ibuprofen was utilized in an apparatus based on principle of magnetic levitation [20].

**Scheme 2.** Application of particle-size-controlled crystallization for resolution.

**Scheme 3.** Application of density difference for the purification of enantiomeric mixtures.

#### **2.4. Distribution between phases, enantiomeric separation**

In the case of phase transitions, the SDE phenomenon is not uniform, it highly depends on the type of the phase transition [17].

#### *2.4.1. Fractionated crystallization*

In the case of the recrystallization of enantiomeric mixtures, by plotting the enantiomeric purity of the solid phase in function of the starting enantiomeric purity, a curve similar to binary and ternary phase diagrams can be obtained (*ee*<sup>0</sup> -*ee* curve) (**Scheme 4**). Regarding a racemate enantiomer mixture, by recrystallizing a mixture having lower purity than the eutectic composition, in any case increased purity will be gained in the solution/melt phase, while above the eutectic composition, the enantiomeric enrichment is expected in the solid phase [2]. The recrystallization is not successful in all the cases to reach enantiomeric enrichment, for example the recrystallization experiments of the enantiomer mixtures of *N*-formyl-phenylalanin (*N*-formyl-**Phe**) and *N*-acetyl-phenylalanin (*N*-Ac-**Phe**), were unsuccessful [21].

A possible mechanism of the recrystallization of racemate-type enantiomeric mixtures is described by Tamura [24–28].

**Scheme 4.** Typical curve received from the recrystallization of a conglomerate-type enantiomer mixture (*ee*<sup>0</sup>

from the recrystallization of a racemate-type enantiomer mixture (*ee*<sup>0</sup>

**Scheme 5.** Sublimation of enantiomer mixtures of mandelic acid (MA).

crystallization from solution (lower diagrams) [23].



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and an example of *ee*<sup>0</sup>



#### *2.4.2. Distribution between solid and gas phases, enantiomer separation*

In the case of mandelic acid, the vapor phase has a eutectic composition, which is independent from the composition of the starting mixture and this composition will sublimate [29]. Independently from the preparation of the starting mixture, enantiomeric mixtures of mandelic acid of 30–54% enantiomeric purity were received as sublimates (**Scheme 5**), which approximates well the eutectic composition determined from the binary and ternary phase diagrams of mandelic acid (*ee*eu: 32% [30, 31]). In the case of the sublimation of several racemate-type amino acids, the purities received in the sublimates [32–34] were identical to the eutectic compositions determined from the ternary phase diagrams [35].

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**Scheme 4.** Typical curve received from the recrystallization of a conglomerate-type enantiomer mixture (*ee*<sup>0</sup> -*ee* diagram) and an example of *ee*<sup>0</sup> -*ee* diagram for crystallization from melt [22] (upper diagrams); and a typical curve received from the recrystallization of a racemate-type enantiomer mixture (*ee*<sup>0</sup> -*ee* diagram) and an example of *ee*<sup>0</sup> -*ee* diagram for crystallization from solution (lower diagrams) [23].

**Scheme 5.** Sublimation of enantiomer mixtures of mandelic acid (MA).

**2.4. Distribution between phases, enantiomeric separation**

102 Laboratory Unit Operations and Experimental Methods in Chemical Engineering

**Scheme 3.** Application of density difference for the purification of enantiomeric mixtures.

binary and ternary phase diagrams can be obtained (*ee*<sup>0</sup>

*2.4.2. Distribution between solid and gas phases, enantiomer separation*

eutectic compositions determined from the ternary phase diagrams [35].

type of the phase transition [17].

*2.4.1. Fractionated crystallization*

unsuccessful [21].

described by Tamura [24–28].

In the case of phase transitions, the SDE phenomenon is not uniform, it highly depends on the

In the case of the recrystallization of enantiomeric mixtures, by plotting the enantiomeric purity of the solid phase in function of the starting enantiomeric purity, a curve similar to

a racemate enantiomer mixture, by recrystallizing a mixture having lower purity than the eutectic composition, in any case increased purity will be gained in the solution/melt phase, while above the eutectic composition, the enantiomeric enrichment is expected in the solid phase [2]. The recrystallization is not successful in all the cases to reach enantiomeric enrichment, for example the recrystallization experiments of the enantiomer mixtures of *N*-formyl-phenylalanin (*N*-formyl-**Phe**) and *N*-acetyl-phenylalanin (*N*-Ac-**Phe**), were

A possible mechanism of the recrystallization of racemate-type enantiomeric mixtures is

In the case of mandelic acid, the vapor phase has a eutectic composition, which is independent from the composition of the starting mixture and this composition will sublimate [29]. Independently from the preparation of the starting mixture, enantiomeric mixtures of mandelic acid of 30–54% enantiomeric purity were received as sublimates (**Scheme 5**), which approximates well the eutectic composition determined from the binary and ternary phase diagrams of mandelic acid (*ee*eu: 32% [30, 31]). In the case of the sublimation of several racemate-type amino acids, the purities received in the sublimates [32–34] were identical to the


#### *2.4.3. Distribution between liquid and gas phases, enantiomer separation*

During the distillation of enantiomer mixtures of isopropyl-(*S*)-trifluorlactate (isopropyl- (*S*)-**TLAK**), the purity of the enantiomer mixtures gained in the distillate and in the residue was different from the starting composition (**Scheme 6**) [36, 37]. Another example for the enantiomer enrichment received by fractionated distillation is that an enantiomeric mixture of 91% enantiomer purity of *N*-trifluoracetyl-(*S*)-valine-methyl-ester (*N*-trifluoracetyl-**Val-Me**) could be further separated to two parts of 88.0 and 97.6% enantiomeric excess, respectively [38].

#### *2.4.4. Separation of enantiomeric mixtures by achiral chromatography*

The SDE phenomenon prevails in the case of enantiomeric enrichment by achiral chromatography. Applying achiral stationary phase and an appropriate eluent, the enantiomeric mixtures can be separated to a polar and a less polar phase, which have different enantiomer purity from the staring composition due to the formation of homo- and heterochiral associations. For example, an enantiomeric mixture of *N*-acetyl-1-phenylethylamin (*N*-Ac-**PhEA**) having 71% enantiomeric excess could be further separated on silica gel stationary phase to two fractions of 99 and a 28% *ee* values, respectively (**Scheme 7**) [39].

Such a separation was first described by Cundy and Crooks [40], but this method is applied by others as well, for the purification of enantiomeric mixtures [17, 41].

*2.4.6. Kinetic control at the fractionated precipitation*

**Scheme 8.** Fractionated precipitation of enantiomer mixture of Tisercin.

second fraction will be enriched in the other one (**Scheme 10**) [21].

mer will stay in the organic phase, while the salt in the water [45].

function of the starting enantiomer purity, a diagram similar to the *ee*<sup>0</sup>

composition around *ee*<sup>0</sup>

*2.4.7. Precipitation and extraction*

*2.4.8. Precipitation and distillation*

In the case of the fractionated precipitation of the enantiomer mixtures of *N*-propionylphenylalanine (*N*-propionyl-**PhA**), the curve expected from the binary phase diagram is significantly different from the received one. The crystals of the enantiomeric excess catalyze (instead of the separation of a low enantiomeric excess, expected under thermodynamic control) the separation of much higher enantiomer purity. For example, in the case of a starting

**Scheme 7.** Purification of enantiomeric mixture of *N*-acetyl-phenylethylamine applying achiral chromatography [45].

With the combination of precipitation and extraction, for example by liberating a part of the enantiomer mixture in the mixture of water and a water-immiscible solvent, the free enantio-

The purification of enantiomer mixtures can also be carried out by the transformation of the racemic percentage of the enantiomer mixture into solid phase as salt, followed by the distillation of the free enantiomeric excess [46, 47]. This method was applied in the case of enantiomer mixtures of salts of 1-phenylethyl-amine (**PhEA**) composed with nonequivalent amounts of dicarboxylic acids. By plotting enantiomer purity of the distillate and the residue in the

: 20%, in the first fraction one of the enantiomers is enriched, while the

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#### *2.4.5. Separation of enantiomers by fractionated precipitation*

After partial liberation of the achiral salt of the enantiomeric mixtures, the purity of the received enantiomeric mixture may be different from the starting composition. By the addition of base equivalent to the enantiomeric excess to the hydrochloric salt of the conglomerate *Tisercin* (Levomepromazine) (**TIS**) in every case the liberating enantiomeric mixture is purer than the starting composition (**Scheme 8**) [42, 43].

By the resolution of the racemic *cis*-permethric acid (**CPA**), a mixture enriched in (*S,S*) enantiomer was received. Further purification of the **CPA** was carried out by precipitation from its *Na*-salt with hydrochloric acid (**Scheme 9**) [44].

**Scheme 6.** Separation of enantiomer mixtures by distillation.

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**Scheme 7.** Purification of enantiomeric mixture of *N*-acetyl-phenylethylamine applying achiral chromatography [45].

**Scheme 8.** Fractionated precipitation of enantiomer mixture of Tisercin.

#### *2.4.6. Kinetic control at the fractionated precipitation*

In the case of the fractionated precipitation of the enantiomer mixtures of *N*-propionylphenylalanine (*N*-propionyl-**PhA**), the curve expected from the binary phase diagram is significantly different from the received one. The crystals of the enantiomeric excess catalyze (instead of the separation of a low enantiomeric excess, expected under thermodynamic control) the separation of much higher enantiomer purity. For example, in the case of a starting composition around *ee*<sup>0</sup> : 20%, in the first fraction one of the enantiomers is enriched, while the second fraction will be enriched in the other one (**Scheme 10**) [21].

#### *2.4.7. Precipitation and extraction*

*2.4.3. Distribution between liquid and gas phases, enantiomer separation*

104 Laboratory Unit Operations and Experimental Methods in Chemical Engineering

*2.4.4. Separation of enantiomeric mixtures by achiral chromatography*

two fractions of 99 and a 28% *ee* values, respectively (**Scheme 7**) [39].

by others as well, for the purification of enantiomeric mixtures [17, 41].

*2.4.5. Separation of enantiomers by fractionated precipitation*

than the starting composition (**Scheme 8**) [42, 43].

**Scheme 6.** Separation of enantiomer mixtures by distillation.

from its *Na*-salt with hydrochloric acid (**Scheme 9**) [44].

respectively [38].

During the distillation of enantiomer mixtures of isopropyl-(*S*)-trifluorlactate (isopropyl- (*S*)-**TLAK**), the purity of the enantiomer mixtures gained in the distillate and in the residue was different from the starting composition (**Scheme 6**) [36, 37]. Another example for the enantiomer enrichment received by fractionated distillation is that an enantiomeric mixture of 91% enantiomer purity of *N*-trifluoracetyl-(*S*)-valine-methyl-ester (*N*-trifluoracetyl-**Val-Me**) could be further separated to two parts of 88.0 and 97.6% enantiomeric excess,

The SDE phenomenon prevails in the case of enantiomeric enrichment by achiral chromatography. Applying achiral stationary phase and an appropriate eluent, the enantiomeric mixtures can be separated to a polar and a less polar phase, which have different enantiomer purity from the staring composition due to the formation of homo- and heterochiral associations. For example, an enantiomeric mixture of *N*-acetyl-1-phenylethylamin (*N*-Ac-**PhEA**) having 71% enantiomeric excess could be further separated on silica gel stationary phase to

Such a separation was first described by Cundy and Crooks [40], but this method is applied

After partial liberation of the achiral salt of the enantiomeric mixtures, the purity of the received enantiomeric mixture may be different from the starting composition. By the addition of base equivalent to the enantiomeric excess to the hydrochloric salt of the conglomerate *Tisercin* (Levomepromazine) (**TIS**) in every case the liberating enantiomeric mixture is purer

By the resolution of the racemic *cis*-permethric acid (**CPA**), a mixture enriched in (*S,S*) enantiomer was received. Further purification of the **CPA** was carried out by precipitation

With the combination of precipitation and extraction, for example by liberating a part of the enantiomer mixture in the mixture of water and a water-immiscible solvent, the free enantiomer will stay in the organic phase, while the salt in the water [45].

#### *2.4.8. Precipitation and distillation*

The purification of enantiomer mixtures can also be carried out by the transformation of the racemic percentage of the enantiomer mixture into solid phase as salt, followed by the distillation of the free enantiomeric excess [46, 47]. This method was applied in the case of enantiomer mixtures of salts of 1-phenylethyl-amine (**PhEA**) composed with nonequivalent amounts of dicarboxylic acids. By plotting enantiomer purity of the distillate and the residue in the function of the starting enantiomer purity, a diagram similar to the *ee*<sup>0</sup> -*ee* curve, received in

To the enantiomeric mixture of AML in solution (in acetone), achiral fumaric acid (**FUM**) was given in equal amount to the racemic percentage. The mixture was dissolved by heating. After cooling, the fumaric acid salt of the racemic percentage was filtered out, while the residue was evaporated, resulting in enantiopure (*S*)-**AML** and (*R*)-**AML** base, respectively. From a starting **AML** enantiomeric mixture of *ee*: 68%, reacted with 0.16 equivalent fumaric acid (equivalent to the racemic percentage), after the filtration of the precipitated crystalline neutral fumaric acid salt, (*S*)-**AML** of *ee*: 99.9% enantiomeric excess can be separated from the

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**3. Separation of diastereomeric mixtures (recent results)**

ences the chiral recognition and thus the result of the separation [51, 52].

upgraded in order to be safely applicable in the search of resolving agents.

**3.1. Chiral salt of helical supramolecular structure as resolving agent (separation of** 

The salt of a chiral amine of supramolecular helical (double helix) structure and an achiral acid precipitates from the solvent (methanol) containing racemic alcohol as well, in the form of supramolecular helical crystals, which are composed of chiral amine, acid and one enantio-

According to Kinbara, the most suitable resolving agent of a racemic molecule can be selected by the design of a stable hydrogen bond system [50]. Saigo et al. concluded after the analysis of several single crystals of pairs of diastereomeric salts, that the formed CH/π interactions play a significant role in the solubility difference of the diastereomers, which clearly influ-

Others estimated well by quantum chemical computations the difference between the lattice energies of the pairs of diastereomeric salts, without preliminary knowledge on the crystal structure [53, 54]. However, it is confessed by the authors that these calculations need to be

The conclusions drawn from the preparative results can facilitate the choice of the resolving agent. For example, it is already trivial, that very good separations can be reached with the application of a resolving agent of similar molecular structure (structurally related) to the

mother liquor (**Scheme 11**).

**diastereomeric molecular complex)**

racemic compound [10, 21, 55–58].

mer of the racemic alcohol (**Scheme 12**) [49].

**Scheme 11.** Purification of enantiomeric mixture of amlodipine.

**Scheme 9.** Fractionated precipitation of *cis*-permethric acid.

**Scheme 10.** Fractionated precipitation of N-propionyl-phenylalanine.

course of recrystallizations, can be obtained, and also, the joins are in accordance with the eutectic composition of the ternary phase diagram [48].

#### *2.4.9. Precipitation of neutral salts of dicarboxylic acid*

The racemic amlodipine with the chiral dicarboxylic tartaric acid crystallizes as the neutral salt of the racemic compound from solvents, without the presence of solvates or solvate-like molecules. Consequently, in the case of enantiomeric mixtures with achiral dicarboxylic acids, the crystallization of the neutral salt of the racemic percentage seemed to be logical.

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**Scheme 11.** Purification of enantiomeric mixture of amlodipine.

course of recrystallizations, can be obtained, and also, the joins are in accordance with the

The racemic amlodipine with the chiral dicarboxylic tartaric acid crystallizes as the neutral salt of the racemic compound from solvents, without the presence of solvates or solvate-like molecules. Consequently, in the case of enantiomeric mixtures with achiral dicarboxylic acids,

the crystallization of the neutral salt of the racemic percentage seemed to be logical.

eutectic composition of the ternary phase diagram [48].

**Scheme 10.** Fractionated precipitation of N-propionyl-phenylalanine.

*2.4.9. Precipitation of neutral salts of dicarboxylic acid*

**Scheme 9.** Fractionated precipitation of *cis*-permethric acid.

106 Laboratory Unit Operations and Experimental Methods in Chemical Engineering

To the enantiomeric mixture of AML in solution (in acetone), achiral fumaric acid (**FUM**) was given in equal amount to the racemic percentage. The mixture was dissolved by heating. After cooling, the fumaric acid salt of the racemic percentage was filtered out, while the residue was evaporated, resulting in enantiopure (*S*)-**AML** and (*R*)-**AML** base, respectively. From a starting **AML** enantiomeric mixture of *ee*: 68%, reacted with 0.16 equivalent fumaric acid (equivalent to the racemic percentage), after the filtration of the precipitated crystalline neutral fumaric acid salt, (*S*)-**AML** of *ee*: 99.9% enantiomeric excess can be separated from the mother liquor (**Scheme 11**).
