Chiral Separation Techniques: A Practical Approach, Second, completely revised and updated edition

Edited by G. Subramanian
Copyright © 2001 Wiley-VCH Verlag GmbH
ISBNs: 3-527-29875-4 (Hardcover); 3-527-60036-1 (Electronic)

1 Techniques in Preparative
Chiral Separations
Pilar Franco and Cristina Minguillón

1.1 Introduction

The recognition of differences in the pharmacological activity of enantiomeric

molecules has created the need to administer them – and therefore to obtain them –
as isolated enantiomers. However, nowadays this problem affects not only the phar-
maceutical industry, but also the agrochemical industry and food additive producers,
both of which are increasingly concerned by this subject.
When chiral, drugs and other molecules obtained from natural sources or by
semisynthesis usually contain one of the possible enantiomeric forms. However,
those obtained by total synthesis often consist of mixtures of both enantiomers. In
order to develop commercially the isolated enantiomers, two alternative approaches
can be considered: (i) enantioselective synthesis of the desired enantiomer; or (ii)
separation of both isomers from a racemic mixture. The separation can be performed
on the target molecule or on one of its chemical precursors obtained from conven-
tional synthetic procedures. Both strategies have their advantages and drawbacks.
The separation of the enantiomers of a racemic mixture, when only one of them
is required, implies an important reduction in yield during the production step of the
target molecule. Techniques to racemize and recycle the unwanted enantiomer are
used to reduce the extent of this problem. However, the same fact becomes an advan-
tage in the development step of a drug, because it is the quickest way to have avail-
able both enantiomers in order to carry out the individual tests needed. In fact, even
if the separation/racemization approach is considered to be “not elegant“ by organic
synthetic chemists, it is nowadays the most often used for the production of single
enantiomers. The enantioselective synthetic approach has the main disadvantage of
the cost and time that could take the development of a synthetic path leading to the
desired enantiomer. Moreover, often the enantiomeric excesses obtained from an
enantioselective procedure are not sufficient to fulfil the requirements of the regula-
tory authorities. In that case, an enrichment step must be added to the enantioselec-
tive process.
All separation techniques which allow the isolation of a certain amount of prod-
uct can be qualified as being “preparative”. In contrast, analytical techniques are
devoted to detect the presence of substances in a sample and/or quantify them. How-
2 1 Techniques in preparative chiral separations

ever, not all preparative techniques are useful at the same scale; some are more eas-
ily adapted to the manipulation of large amounts of material, while others may only
be applicable to the isolation of few milligrams, or even less, though this may be
enough for given purposes.
In this chapter a number of the preparative techniques used in the resolution of
enantiomers is presented. Some of these techniques will be developed more fully in
following chapters.

1.2 Crystallization Techniques

Although crystallization is used routinely to separate solid compounds from impuri-

ties and by-products coming from secondary reactions in their synthesis, it may also
be applied in the isolation of individual enantiomers from a racemic or an enan-
tiomerically enriched sample [1–3]. Indeed, until the development of chiral chro-
matographic techniques, crystallization was one of the few existing ways to resolve
enantiomers. Although crystallization is a very powerful technique for preparative
purposes, few industrial applications have been reported [3] for reasons of confi-
dentiality. Moreover, the technique is far from being generally applicable, and thus
only those compounds which behave as conglomerates (different crystals for both
enantiomers) can be resolved from their equimolecular enantiomeric mixtures, either
by seeding their solutions with crystals of one enantiomer (preferential crystalliza-
tion) [4–6], or by using a chiral environment to carry out the crystallization. The lim-
itation of the preferential crystallization is, therefore, the availability of crystals of
the pure enantiomer.
A chiral environment can be produced by using a chiral solvent in the crystalliza-
tion, but most of these are organic and therefore not useful for highly polar com-
pounds. Therefore, a chiral co-solute is often used [7–9]. Applying this methodol-
ogy, d,l-threonine was resolved into its enantiomers using small amounts of L-serine
or 4-(R)-hydroxy-L-proline [7]. Moreover, an inhibitory effect on the crystallization
of D-glutamic acid from its racemic mixture of some D-amino acids, such as lysine,
histidine or arginine has been described, while their L-counterparts inhibit crystal-
lization of the L-enantiomer [8].
Unfortunately, the occurrence of conglomerates in nature is not common. On
occasion, the probability of obtaining a conglomerate can be increased by trans-
forming the considered compound into a salt, when possible. Racemic compounds
(both enantiomers in the same lattice) are more frequently encountered in nature.
Therefore, it is useful to know which is the behavior of the considered product, tak-
ing into account that it can change depending on the temperature of crystallization.
Several methods exist to determine such a point easily [3]. Racemic compounds may
be enriched by crystallization of a nonracemic mixture, in which case the success
and yield of the enrichment depends (among others) on the composition of the orig-
inal mixture.
 1.3 Chromatographie Techniques 3

On that basis, crystallization is often used in combination with other enantiose-

lective techniques, such as enantioselective synthesis, enzymatic kinetic resolution
or simulated moving bed (SMB) chromatography [10, 11]. In general, when refer-
ring to crystallization techniques, the aim is to obtain an enantiomeric enrichment in
the crystallized solid. However, the possibility of producing an enrichment in the
mother liquors [12, 13], even if this is not a general phenomenon [14], must be taken
into account.
An additional strategy that is frequently used due to the reduced probability of the
preceding situations is the separation of diastereomeric mixtures obtained from the
reaction of the original enantiomeric mixture with chiral derivatizing agents [3,
15–18]. These should be easily cleaved from the target molecule with no racemiza-
tion, and thus be readily available. Low cost and confirmed enantiomeric purity, in
addition to their being recoverable and reusable, are also highly recommended prop-
erties for a chiral derivatizing agent.

1.3 Chromatographic Techniques

1.3.1 Liquid Chromatography

Despite the fact that in former days liquid chromatography was reputed to be a very
expensive and inefficient purification technique for preparative purposes, it is nowa-
days one of the first choices to carry out a large-scale chiral separation. On the one
hand, some technical developments, related to the equipment as well as to the pack-
ing materials have improved the efficiency of the technique. On the other hand,
applications such as the resolution of enantiomers, where the resulting products have
a high added value, can partially balance the classically attributed high costs of li-
quid chromatography. Furthermore, the relative short time necessary to develop a
chromatographic method, and the availability of chromatographic systems, are inter-
esting features that should be taken into account when the enantiomers under con-
sideration need to be separated in the minimum time.
Analogously to crystallization techniques, the chromatographic separation pro-
cess can be applied either to a mixture of enantiomers or to diasteromeric derivatives
obtained by reaction with chiral derivatizing agents. In this case, it is a conventional
chromatographic process which can be performed in achiral conditions, and the
same drawbacks as with any other indirect method might be encountered. Thus, such
indirect resolutions are strongly dependent on the enantiomeric purity of the deriva-
tizing agent which must be cleaved without affecting the configuration of the stereo-
genic elements in the target molecule.
Several chromatographic modes will be reviewed in this respect, and most will
make use of a chiral support in order to bring about a separation, differing only in
the technology employed. Only countercurrent chromatography is based on a li-
quid–liquid separation.
4 1 Techniques in preparative chiral separations

1.3.1.1 High-Pressure/Medium-Pressure Liquid Chromatography

(HPLC/MPLC)

HPLC separations are one of the most important fields in the preparative resolution
of enantiomers. The instrumentation improvements and the increasing choice of
commercially available chiral stationary phases (CSPs) are some of the main reasons
for the present significance of chromatographic resolutions at large-scale by HPLC.
Proof of this interest can be seen in several reviews, and many chapters have in the
past few years dealt with preparative applications of HPLC in the resolution of chi-
ral compounds [19–23]. However, liquid chromatography has the attribute of being
a batch technique and therefore is not totally convenient for production-scale, where
continuous techniques are preferred by far.
In order to carry out a direct preparative chromatographic resolution of enan-
tiomers in batch elution mode, the same methods as are used in other nonchiral
large-scale chromatographic separations are applied [24], such as multiple close
injections, recycling or peak shaving. All of these are addressed to reduce cost and
increase yield, while saving solvent and making full use of the stationary phase.
Thus, the injections are sometimes performed repeatedly (multiple close injection),
in such a way that most of the chromatographic support is involved in the separation
at any moment. When the resolution is not sufficient, it can be improved by recycling
of the partially overlapped peaks [23]. This has almost the same effect as is obtained
when using a longer column, but clearly a broadening of peaks occurs. However,
after several cycles partially overlapped peaks can be completely resolved. This
method is often combined with the so-called peak shaving, which allows the recov-
ery of the part of the peaks corresponding to pure enantiomers while the overlapped
region is recycled. In fact, to date this method is the most often used.
The chiral environment needed for enantiomeric separations is furnished by the
chiral support into the column. Scale-up of a chiral separation can be made having
as a reference an analytical resolution, but optimization of the preparative process is
critically dependent upon the nature of the CSP. In order to increase the throughput,
the column is usually used in overloading conditions. The loading capacity of a chi-
ral stationary phase depends not only on the chiral selector density, but also on the
type of selector. Therefore, some types of CSPs are more suitable than others for
preparative purposes [21, 25]. Not all the commercially available CSPs used for ana-
lytical purposes are appropiated for large-scale resolutions (Table 1-1). CSPs with a
large application domain, such as those derived from proteins with a vast applica-
bility for analytical purposes, have a very low loadability and, therefore, they are not
well adapted to preparative separations [26, 27]. This is also the case of molecular
imprinted polymers (MIPs) [28–30]. The limited number of recognition sites
restricts their loading capacity and thus also their use in large-scale chromatography.
Some ligand-exchange CSPs have been used at preparative level [31, 32]. In this
case it must be taken into account that an extraction process, to remove the copper
salts added to the mobile phase, must be performed following the chromatographic
process [33]. Teicoplanin, in contrast, resolves all ordinary α and β-amino acids with
mobile phases consisting of alcohol/water mixtures. No buffer is needed in the
 1.3 Chromatographie Techniques 5

mobile phase [34]; hence evaporation of the solution derived from the preparative
separation leaves a pure product. Cyclodextrin-based CSPs [35, 36], antibiotics [34,
37–39] such as the above-mentioned teicoplanin, and certain types of Pirkle phases
have been utilized with preparative purposes. In those cases, although the loading
capacity is not high, other advantages, such as a broad application domain for antibi-
otics, or the ease of preparation and the chemical stability, for multiple interaction
supports, can balance this limitation. Polyacrylamides [40, 41] have also been used
extensively, often in nonreported separations. Nevertheless, polysaccharide-derived
CSPs are the most commonly applied for preparative chromatography. This is due
both to their substantial loading capacity and broad enantiodiscrimination scope.
They are the best adapted supports for this purpose either in HPLC, or in medium-
pressure liquid chromatography (MPLC) [19].
Several types of polysaccharide-derived CSPs can be considered (Table 1-1). The
support with the highest number of applications described, either at high or medium
pressure, is microcrystalline cellulose triacetate (CTA). Other polysaccharide deriva-
tives, mainly some benzoates, are used in their pure form, as beads which are
directly packed [42, 43]. Coated CSPs, consisting of a polysaccharide derivative,
benzoate or aryl carbamate of cellulose or amylose, on a matrix of aminopropylsi-
lanized silica gel, are also among the most extensively utilized CSPs [44–49]. How-
ever, these supports have the limitation of the choice in the mobile phase composi-
tion. CSPs whose chiral selectors are bonded to the chromatographic matrix (usually
silica gel) can perform in a number of different conditions and compositions in the
mobile phase. This is the case of the already mentioned Pirkle phases, cyclodextrin,
antibiotic or polyacrylamide-derived CSPs. Unfortunately, polysaccharide deriva-
tives in coated CSPs often swell, or even dissolve, in a number of solvents. Thus, the
compatible mobile phases with these supports are mixtures of a hydrocarbon (hex-
ane or heptane) with an alcohol (ethanol or isopropanol), though many compounds
have a reduced solubility in these mixtures. This feature, which is not a problem
when these CSPs are used for analytical purposes, can be a major disadvantage at
preparative scale. The low solubility limits the amount of product that can be
injected in a single run and, therefore, the maximum loadability of the column can-
not be attained [45]. This limitation can be overcome when the chiral selector is
bonded to the chromatographic matrix [50–57]. In this case, a broader choice of sol-
vents can be considered as mobile phase or simply to dissolve the racemate to be
separated [58, 59]. It must be taken into account that, changes in selectivity as well
as in the loading capacity of the CSP occur when solvents are changed [58].
In this context, the enantiomeric pair containing the eutomer of cyclothiazide can
be resolved by HPLC on cellulose-derived coated CSPs. Nevertheless, the poor solu-
bility of this compound in solvents compatible with this type of support makes this
separation difficult at preparative scale. This operation was achieved with a cellulose
carbamate fixed on allylsilica gel using a mixture of toluene/acetone as a mobile
phase [59].
On occasion, the broad choice of existing phases is not enough to resolve a par-
ticular problem successfully. Derivatization with achiral reagents can be useful to
introduce additional interacting groups in a poorly functionalized substract, or to
6 1 Techniques in preparative chiral separations

adapt it to be resolved in a particular CSP. On these occasions, derivatization can

increase the chances of success in a given resolution [60].

Table 1-1. Preparative chiral separations.

Examples of Supplier or
Packing name Chiral selector (semi)preparative reference of the
applications CSP

CTA Crystalline cellulose triacetate [19,42,61–62] Daicel, Merck

TBC Cellulose tribenzoate beads [19,42] [42]
MMBC Cellulose tris(3-methylbenzoate) beads [19,42] [43]
PMBC Cellulose tris(4-methylbenzoate) beads [19,42] [43]

Chiralcel OD Cellulose tris(3,5-dimethylphenylcarbamate) [11,19,67,68] Daicel, [67,68]

coated on silica gel
Chiralcel OC Cellulose tris(phenylcarbamate) coated on silica gel [19] Daicel
Chiralcel OJ Cellulose tris(4-methylbenzoate) coated on silica gel [19] Daicel
Polysaccharides Chiralcel OB Cellulose tribenzoate coated on silica gel [19] Daicel
Chiralpak AD Amylose tris(3,5-dimethylphenylcarbamate) coated on [19,69] Daicel
silica gel
Chiralpak AS Amylose tris[1-(S)-phenylethylcarbamate] coated on [70] Daicel
silica gel

– Mixed cellulose 10-undecenoate/tris(3,5-dimethyl- [59,71] [51]

phenylcarbamate) bonded on silica gel
– Mixed amylose 10-undecenoate/tris(3,5-dimethyl- [71] [56]
phenylcarbamate) bonded on silica gel

Cyclodextrins Cyclobond I Cyclodextrin immobilized on silica gel [19,22,72] Astec

Hyd-β-CD Hydroxypropyl-β-cyclodextrin [23] Merck,[23]

DNBPG-co 3,5-Dinitrobenzoylphenylglycine covalently bonded [19,21,73–78] Regis

on silica gel
ChyRoSine-A 3,5-Dinitrobenzoyltyrosine butylamide [79] Sedere
Pirkle-1J 3,5-Dinitrobenzoyl-β-lactam derivative [80] Regis
Pirkle type α-Burke 2 Dimethyl N-3,5-dinitrobenzoyl-α-amino-2,2-dimethyl- [80] Regis
4-pentenyl phosphonate bonded to silica
ULMO N-dinitrobenzoyl-N’-undecenoyl-diphenylethanediamine [81] Regis
– Cis-3-(1,1-dimethylethyl)-4-phenyl-2-azetidinone [82] [82]
Quaternary ammonium derivative of 3,5-Dinitro-
benzoyl-L-leucine on α-zirconium phosphate [83] [83]
– (S)-N-undecenoylproline 3,5-dimethoxyanilide [80] [80]
bonded on silica gel

Poly-PEA Poly[(S)-N-acryloylphenylethylamine ethyl ester] [21,84,85] [84]

Polyacrylamides PolyCHMA Poly[(S)-N-methacryloyl-2-cyclohexylethylamine] [84] [84]
D-ChiraSpher Poly[(S)-N-acryloylphenylalanine ethyl ester] [11,23,86] Merck

Polystyrene-Prol L or D-proline bonded to polystyrene [87] [87]

LEC Chirosolve-pro L or D-proline bonded to polyacrylamide [88] UPS Chimie
NucleosilChiral-1 L-hydroxyproline Cu2+ complexes bonded on silica gel [33] Macherey-Nagel

Antibiotics Teicoplanin Chirobiotic T [89] Astec

Vancomicin Chirobiotic V [89] Astec
 1.3 Chromatographie Techniques 7

1.3.1.2 Flash Chromatography

Flash chromatography is widely employed for the purification of crude products

obtained by synthesis at a research laboratory scale (several grams) or isolated as
extracts from natural products or fermentations. The solid support is based on silica
gel, and the mobile phase is usually a mixture of a hydrocarbon, such as hexane or
heptane, with an organic modifier, e.g. ethyl acetate, driven by low pressure air.
(Recently the comparison of flash chromatography with countercurrent chromato-
graphy (CCC), a technique particularly adapted to preparative purposes, has been
studied for the separation of nonchiral compounds [90].)
With regard to the resolution of enantiomers, some applications can be found with
modified silica gel supports. Thus, a Pirkle-type CSP was used for the separation of
200 mg of a racemic benzodiazepinone [75]. Also tris-(3,5-dimethylphenyl)carba-
mate of cellulose coated on silica C18 [91, 92] was applied successfully to the reso-
lution of the enantiomers of 2-phenoxypropionic acid and to oxprenolol, alprenolol,
propranolol among other basic drugs. However, the low efficiency of this technique
and the relative high price of the CSPs limits its use to the resolution of milligram
range of sample.

1.3.1.3 Simulated Moving Bed (SMB)

The simulated moving bed (SMB) technology was patented in the early 1960s as a
binary continuous separation technique. It consists of a series of several columns
connected to each other head-to-tail, simulating an annular column. The eluent
source, the feed of mixture to process and the two collecting positions move along
this circle in such a way that mimics a relative countercurrent movement between the
mobile and the stationary phases. This makes compatible the continuous injection of
mixture to be purified, and the recovery of two different fractions with the chro-
matographic process [93]. The feature of being continuous was considered an advan-
tage in order to be included in a production chain, when related to other existing sep-
aration techniques that act mainly in a batch basis. Although the ability to obtain two
fractions from a mixture might be seen as a limitation, SMB found very important
applications in the petro-chemical and sugar industries [94]. However, it was not
until some decades later that such a binary technique was realized to be advanta-
geous and especially suited to the separation of enantiomeric mixtures.
Since the first separation of enantiomers by SMB chromatography, described in
1992 [95], the technique has been shown to be a perfect alternative for preparative
chiral resolutions [10, 21, 96, 97]. Although the initial investment in the instrumen-
tation is quite high – and often prohibitive for small companies – the savings in
solvent consumption and human power, as well as the increase in productivity,
result in reduced production costs [21, 94, 98]. Therefore, the technique would be
specially suitable when large-scale productions (≥100 g) of pure enantiomers are
needed. Despite the fact that SMB can produce enantiomers at very high enan-
tiomeric excesses, it is sometimes convenient to couple it with another separation
8 1 Techniques in preparative chiral separations

technique, often crystallization [11, 94], in order to increase the global productivity
[10].
The type of CSPs used have to fulfil the same requirements (resistance, loadabil-
ity) as do classical chiral HPLC separations at preparative level [99], although dif-
ferent particle size silica supports are sometimes needed [10]. Again, to date the
polysaccharide-derived CSPs have been the most studied in SMB systems, and a
large number of racemic compounds have been successfully resolved in this way
[95–98, 100–108]. Nevertheless, some applications can also be found with CSPs
derived from polyacrylamides [11], Pirkle-type chiral selectors [10] and cyclodex-
trin derivatives [109]. A system to evaporate the collected fractions and to recover
and recycle solvent is sometimes coupled to the SMB. In this context the application
of the technique to gas can be advantageous in some cases because this part of the
process can be omitted [109].
Enantiomeric drugs or intermediates in their synthesis are the compounds most
often purified with this technology and reported in the literature, although many res-
olutions performed in the industry have not been published for reasons of confiden-
tiality. Some of the most recent examples in the field are summarized in Fig. 1-1.

1.3.1.4 Closed-loop Recycling with Periodic Intra-profile Injection (CLRPIPI)

An intermediate approach between HPLC and SMB chromatography, called

“closed-loop recycling with periodic intra-profile injection” (CLRPIPI) has been
described recently [110]. This is a new binary preparative separation technique
whose concept implies the combination of recycling with peak shaving and SMB.
Thus, once the pure fractions of the peaks are collected, the partially resolved frac-
tion is recycled into the column. A new injection of fresh sample is then produced
just between the two partially resolved peaks. The new mixture passes through the
column, at the end of which pure fractions are collected while the partially resolved
fraction is recycled again, and the process is repeated. This is similar to SMB as it is
a binary technique, but it is not continuous. The capital cost of this system is sub-
stantially lower than that of SMB devices but a high productivity is maintained. It
can be a good alternative when the amount of enantiomers to purify is not high
enough to justify the investment of a SMB instrument. Some examples of the use of
this technique in the purification of enantiomers, either by derivatization and sepa-
ration, on a nonchiral column [111], or by direct resolution on a CSP (Chiralpak AS)
[112] can be found in the literature.

1.3.1.5 Countercurrent Chromatography (CCC/CPC)

Countercurrent chromatography (CCC) refers to a chromatographic technique which

allows the separation of solutes in a two-phase solvent system subjected to a gravi-
tational field. Two immiscible liquid phases, constituted by one or more solvents or
solutions, are submitted to successive equilibria, where the solutes to be separated
 1.3 Chromatographie Techniques

Fig. 1-1. Some of the structures of the racemates resolved recently by SMB.
9
10 1 Techniques in preparative chiral separations

are partitioned on the basis of their different affinity for one or the other phase. The
chromatographic process occurs between them without any solid support. The CCC
instruments maintain one of the liquid phases as stationary by means of the cen-
trifugal force, while the other is pumped through as mobile phase [113–115]. Inter-
est in the technique has favored the development of improved devices based on the
same principle, namely the retention of the liquid phases by means of a centrifugal
field, but with slight technical modifications. Thus, classical CCC devices use a vari-
able-gravity field produced by a two-axis gyration mechanism, while centrifugal
partition chromatography (CPC) devices are based on the use of a constant-gravity
field produced by a single-axis rotation mechanism [113–115]. Both CCC and CPC
preparative-scale instruments are available commercially [116].
The technique has some advantages relating to the traditional liquid–solid sepa-
ration methods. The most important of these is that all the stationary phase takes part
in the separation process, whereas the activity of a solid phase is mainly concen-
trated in the surface of the support, an important part of this being completely inert.
This fact increases the loading capacity of the phase, and this is the reason why CCC
is especially suited for preparative purposes. Therefore, modern CCC overcomes the
disadvantages of direct preparative chromatography by HPLC with regard to the
high cost of the chiral solid stationary phase and its relatively limited loadability.
From the pioneering studies of Ito et al. [117], CCC has been mainly used for the
separation and purification of natural products, where it has found a large number of
applications [114, 116, 118, 119]. Moreover, the potential of this technique for
preparative purposes can be also applied to chiral separations. The resolution of
enantiomers can be simply envisaged by addition of a chiral selector to the station-
ary liquid phase. The mixture of enantiomers would come into contact with this li-
quid CSP, and enantiodiscrimination might be achieved. However, as yet few exam-
ples have been described in the literature.
The first partial chiral resolution reported in CCC dates from 1982 [120]. The sep-
aration of the two enantiomers of norephedrine was partially achieved, in almost 4
days, using (R,R)-di-5-nonyltartrate as a chiral selector in the organic stationary
phase. In 1984, the complete resolution of d,l-isoleucine was described, with N-
dodecyl-L-proline as a selector in a two-phase buffered n-butanol/water system con-
taining a copper (II) salt, in approximately 2 days [121]. A few partial resolutions of
amino acids and drug enantiomers with proteic selectors were also published [122,
123].
However, it was not until the beginning of 1994 that a rapid (<1.5 h) total resolu-
tion of two pairs of racemic amino acid derivatives with a CPC device was published
[124]. The chiral selector was N-dodecanoyl-L-proline-3,5-dimethylanilide (1) and
the system of solvents used was constituted by a mixture of heptane/ethyl
acetate/methanol/water (3:1:3:1). Although the amounts of sample resolved were
small (2 ml of a 10 mM solution of the amino acid derivatives), this separation
demonstrated the feasibility and the potential of the technique for chiral separations.
Thus, a number of publications appeared subsequently. Firstly, the same chiral selec-
tor was utilized for the resolution of 1 g of (±)-N-(3,5-dinitrobenzoyl)leucine with a
modified system of solvents, where the substitution of water by an acidified solution
 1.3 Chromatographie Techniques 11

ensured the total retention of the chiral selector in the stationary phase [125]. The
separation of 2 g of the same leucine derivative employing the pH-zone refining
technique with the same instrument was later described [127]. (The elution pattern
of pH-zone-refining CCC bears a remarkable resemblance to that observed in dis-
placement chromatography and allows the displacement of ionizable molecules
through the CCC column by means of a pH gradient [116, 126].)
Recently, two examples of the separation of enantiomers using CCC have been
published (Fig. 1-2). The complete enantiomeric separation of commercial d,l-
kynurenine (2) with bovine serum albumin (BSA) as a chiral selector in an aque-
ous–aqueous polymer phase system was achieved within 3.5 h [128]. Moreover, the
chiral resolution of 100 mg of an estrogen receptor partial agonist (7-DMO, 3) was
performed using a sulfated β-cyclodextrin [129, 130], while previous attempts with
unsubstituted cyclodextrin were not successful [124]. The same authors described
the partial resolution of a glucose-6-phosphatase inhibitor (4) with a Whelk-O
derivative as chiral selector (5) [129].

Fig. 1-2. Several racemates resolved by CCC (2, 3, 4) and some of the chiral selectors used (1, 5)
(see text).

The CCC instruments have even been used as enzymatic reactors to carry out
enantioselective processes. Thus, the hydrolysis of 2-cyanocyclopropyl-1,1-dicar-
boxylic acid dimethylester including a bacterial esterase in the stationary phase was
reported [131]. After 8 h, the procedure yielded the desired product automatically,
without any extraction and with an 80 % e.e.
12 1 Techniques in preparative chiral separations

1.3.2 Subcritical and Supercritical Fluid Chromatography

Supercritical fluid chromatography (SFC) refers to the use of mobile phases at tem-
peratures and pressures above the critical point (supercritical) or just below (sub-
critical). SFC shows several features that can be advantageous for its application to
large-scale separations [132–135]. One of the most interesting properties of this
technique is the low viscosity of the solvents used that, combined with high diffu-
sion coefficients for solutes, leads to a higher efficiency and a shorter analysis time
than in HPLC.
As a matter of fact, the main advantage in comparison with HPLC is the reduc-
tion of solvent consumption, which is limited to the organic modifiers, and that will
be nonexistent when no modifier is used. Usually, one of the drawbacks of HPLC
applied at large scale is that the product must be recovered from dilute solution and
the solvent recycled in order to make the process less expensive. In that sense, SFC
can be advantageous because it requires fewer manipulations of the sample after the
chromatographic process. This facilitates recovery of the products after the separa-
tion. Although SFC is usually superior to HPLC with respect to enantioselectivity,
efficiency and time of analysis [136], its use is limited to compounds which are
soluble in nonpolar solvents (carbon dioxide, CO2). This represents a major draw-
back, as many of the chemical and pharmaceutical products of interest are relatively
polar.
Although some applications for preparative-scale separations have already been
reported [132] and the first commercial systems are being developed [137, 138],
examples in the field of the resolution of enantiomers are still rare. The first prepar-
ative chiral separation published was performed with a CSP derived from (S)-N-(3,5-
dinitrobenzoyl)tyrosine covalently bonded to γ-mercaptopropyl silica gel [21]. A
productivity of 510 mg/h with an enantiomeric excess higher than 95 % was
achieved for 6 (Fig. 1-3).
Examples with other Pirkle-type CSPs have also been described [139, 140]. In
relation to polysaccharides coated onto silica gel, they have shown long-term stabil-
ity in this operation mode [141, 142], and thus are also potentially good chiral selec-
tors for preparative SFC [21]. In that context, the separation of racemic gliben-
clamide analogues (7, Fig. 1-3) on cellulose- and amylose-derived CSPs was
described [143].

Fig. 1.3. Chemical structures of racemic compounds resolved by SFC.

 1.4 Enantioselective Membranes 13

1.3.3 Gas Chromatography

Gas chromatography (GC) has also been used for preparative purposes, but is
restricted to relatively volatile racemates such as anesthetics, pheromones or
monoterpenes and, therefore, very few applications are reported. Nevertheless, in the
cases to which GC may be applied, it could be considered as an economical alterna-
tive to HPLC. Most of the resolutions of enantiomers were performed on cyclodex-
trin-derived CSPs [109, 144–153], and only on very few occasions were other chiral
selectors used [153].
One of the latest resolutions of the anesthetic enflurane (8) has been performed by
preparative GC on a γ-cyclodextrin CSP, the process later being scaled-up via SMB
[109] (Fig. 1-4). This is the first GC-SMB separation described.

Fig. 1-4. Resolution of enflurane by GC.

1.4 Enantioselective Membranes

Membrane-based separation techniques constitute nowadays well-established pro-

cess methods for industrial treatments of fluids. Like SMB, membrane-based sepa-
rations can be performed in continuous mode. In the field of preparative-scale enan-
tiodiscrimination, much effort has been invested in this subject due its high potential
[154, 155]. (Chapter 5 of this book is devoted to the subject, and further discusses
the advantages and applications of membrane technologies.)
The first successful chiral resolutions through enantioselective membranes have
been published recently, but few cases are applicable to the preparative scale, mainly
due to mechanical and technical limitations. Low flow rates, saturation of the chiral
selectors and loss of enantioselectivity with time are some of the common problems
encountered and that should be solved in the near future.
Enantioselective transport processes can be achieved either with solid or liquid
membranes (Fig. 1-5). In this latter case, the liquid membrane can be supported by
a porous rigid structure, or it can simply be an immiscible liquid phase between two
solutions with the same character (aqueous or nonaqueous), origin and destination
14 1 Techniques in preparative chiral separations

Fig. 1-5. Enantioselective transport processes.

of the compound to be transported [154]. The membrane is then simply a technical

tool which permits a type of liquid–liquid extraction to be performed. In all cases the
membrane should contain the chiral selector to carry out the separation of enan-
tiomers.
The nature of enantioselective solid membranes can be very diverse. Chiral syn-
thetic and semisynthetic polymers have been applied directly for this purpose, but
other chiral molecules have also proved to be useful after immobilization on a
nonchiral porous membrane. Polysaccharide derivatives, especially cellulose carba-
mates [156–159], acrylic polymers, poly(α-amino acids) [160–162] and polyacety-
lene-derived polymers are some of the polymeric selectors that have been successful
in the resolution of racemic mixtures by this method. The high loadability of these
compounds, already demonstrated in HPLC and other classical applications, makes
them very attractive in continuous processes. Moreover, the filmogenic properties of
some of them, such as the polysaccharide derivatives, are interesting characteristics
when the formation of a membrane is envisaged. More recently, the introduction of
molecular imprinted polymers (MIPs) to membrane technologies has been described
as a promising alternative [163–166]. Among the chiral molecules immobilized on a
nonchiral rigid support membrane to perform an enantioselective separation are
amino acids and proteins, such as BSA [167–169]. The main limitation in the case
of solid membranes is the silting that occurs when all recognition sites have been
occupied and there is no real transport through the membrane. An ingenious system
has been described [159] to take advantage of this phenomenon for the separation of
enantiomers.
Liquid membranes can be constituted by liquid chiral selectors used directly [170]
or by solutions of the chiral molecules in polar or apolar solvents. This later possi-
bility can also be an advantage since it allows the modulation of the separation con-
 1.5 Other Methods 15

ditions. Chiral crown ethers [171–173], cyclodextrins [174] and amino acid deriva-
tives [19–22] have been successfully used in the resolution of free amino acids
[175–178], amino acid derivatives [175], cyclic and heterocyclic compounds [174]
and also racemic drugs, such as the β-blockers propranolol and bupranolol [177].
Another possibility of constructing a chiral membrane system is to prepare a solu-
tion of the chiral selector which is retained between two porous membranes, acting
as an enantioselective liquid carrier for the transport of one of the enantiomers from
the feed solution of the racemate to the receiving side (Fig. 1-5). This system is often
referred to as membrane-assisted separation. The selector should not be soluble in
the solvent used for the elution of the enantiomers, whose transport is driven by a
gradient in concentration or pH between the feed and receiving phases. As a draw-
back common to all these systems, it should be mentioned that the transport of one
enantiomer usually decreases when the enantiomer ratio in the permeate diminishes.
Nevertheless, this can be overcome by designing a system where two opposite selec-
tors are used to transport the two enantiomers of a racemic solution simultaneously,
as it was already applied in W-tube experiments [171].
Most of the chiral membrane-assisted applications can be considered as a modal-
ity of liquid–liquid extraction, and will be discussed in the next section. However, it
is worth mentioning here a device developed by Keurentjes et al., in which two mis-
cible chiral liquids with opposing enantiomers of the chiral selector flow counter-
currently through a column, separated by a nonmiscible liquid membrane [179]. In
this case the selector molecules are located out of the liquid membrane and both
enantiomers are needed. The system allows recovery of the two enantiomers of the
racemic mixture to be separated. Thus, using dihexyltartrate and poly(lactic acid),
the authors described the resolution of different drugs, such as norephedrine, salbu-
tamol, terbutaline, ibuprofen or propranolol.

1.5 Other Methods

1.5.1 Chiral Extractions

Liquid-liquid extraction is a basic process already applied as a large-scale method.

Usually, it does not require highly sophisticated devices, being very attractive for the
preparative-scale separation of enantiomers. In this case, a chiral selector must be
added to one of the liquid phases. This principle is common to some of the separa-
tion techniques described previously, such as CCC, CPC or supported-liquid mem-
branes. In all of these, partition of the enantiomers of a mixture takes place thanks
to their different affinity for the chiral additive in a given system of solvents.
The instrumentation which until now has been used in chiral extraction experi-
ments is very diverse, ranging from the simple extraction funnel [123, 180], the U-
or W-tubes [171, 181], to more sophisticated devices, such as hollow-fiber extraction
apparatus [175] or other membrane-assisted systems. Most of these experiments
16 1 Techniques in preparative chiral separations

have been brought about at a reduced scale, though the potential of the extraction
techniques is very promising. In principle, the type of chiral additives used can be
the same as the selectors applied to supported-liquid membranes or CCC. Neverthe-
less, as all the chiral recognition process occurs in solution, and an aqueous phase is
often involved, the solvation of selector and racemate molecules competes with the
chiral interactions selector-enantiomers, especially those implying hydrogen bonds.
Therefore, it is very often the case that chiral selectors with very high chiral recog-
nition abilities are needed [171, 182, 183]. The main disadvantage of liquid–liquid
extraction in the separation of enantiomers is the need for an additional treatment to
separate the chiral selector from the phase containing one of the enantiomers of the
resolved racemate.
Early examples of enantioselective extractions are the resolution of α-aminoalco-
hol salts, such as norephedrine, with lipophilic anions (hexafluorophosphate ion)
[184–186] by partition between aqueous and lipophilic phases containing esters of
tartaric acid [184–188]. Alkyl derivatives of proline and hydroxyproline with cupric
ions showed chiral discrimination abilities for the resolution of neutral amino acid
enantiomers in n-butanol/water systems [121, 178, 189–192]. On the other hand,
chiral crown ethers are classical selectors utilized for enantioseparations, due to their
interesting recognition abilities [171, 178]. However, the large number of steps often
required for their synthesis [182] and, consequently, their cost as well as their lim-
ited loadability makes them not very suitable for preparative purposes. Examples of
ligand-exchange [193] or anion-exchange selectors [183] able to discriminate amino
acid derivatives have also been described.
Proteins (BSA or ovomucoid, OVM) have also been successful in the preparative
resolution of enantiomers by liquid–liquid extraction, either between aqueous and
lipophilic phases [181] or in aqueous two-phase systems (ATPS) [123, 180]. The res-
olution of d,l-kynurenine [180] and ofloxacin and carvediol [123] were performed
using a countercurrent extraction process with eight separatory funnels. The signi-
ficant number of stages needed for these complete resolutions in the mentioned ref-
erences and others [123, 180, 189], can be overcome with more efficient techniques.
Thus, the resolution of d,l-kynurenine performed by Sellergren et al. in 1988 by
extraction experiments was improved with CCC technologies 10 years later [128].
It is worth noting that the extractive process can be performed continuously. Thus,
the separation of (±)-mandelic acid into its enantiomers was achieved with a liquid
particle extractor described by Abe et al. [190–192] using N-docecyl-L-proline as
chiral selector.

1.5.2 Preparative Gel Electrophoresis and Thin-Layer

Chromatography

Recently, the separation of some milligram quantities of terbutaline by classical gel

electrophoresis has been reported [194]. A sulfated cyclodextrin impregnated on the
agarose gel was used as a chiral selector and the complete resolution was achieved
in 5 h. Analogously, small amounts of enantiomers can be isolated using thin-layer
 1.5 Other Methods 17

chromatographic supports impregnated with chiral selectors [195–197]. However,

these techniques seem, at present, far from being applicable to the resolution of
important amounts of racemates.

1.5.3 Enantioselective Distillation and Foam Flotation

Among the existing separation techniques, some – due to their intrinsic characteris-
tics – are more adapted than others to processing large amounts of material. Such pro-
cesses, which already exist at industrial level, can be considered in order to perform
an enantioselective separation. This is the case for techniques such as distillation and
foam flotation, both of which constitute well-known techniques that can be adapted
to the separation of enantiomers. The involvement of a chiral selector can be the clue
which changes a nonstereoselective process into an enantioselective one. Clearly, this
selector must be adapted to the characteristics and limitations of the process itself.
Several chiral selectors have been used in the separation of enantiomers by distil-
lation [198]. Among them, the bisalcohol 8 (Fig. 1-6) has permitted obtainment of
the ketone (+)-9 with an enantiomeric excess of 95 %. This example shows the fea-
sibility of the process even though, in this particular case, the price of the chiral
selector might prohibit scale-up of the separation.

Fig. 1-6. Chemical structures

of the chiral selector (8) used
in the resolution of 9 by
distillation.

In another example of enantioselective distillation, it was the enantiomeric mix-

ture to resolve itself which contributed to create a chiral environment. Thus, non-
racemic mixtures of α-phenylethylamine were enantiomerically enriched by sub-
mitting to distillation different salts of this amine with achiral acids [199].
The main advantage of distillation over other separation techniques is the absence
of solvent involved. This feature can contribute to a reduction in the price of an enan-
tioselective separation. In the search of other economical process-scale enantiomeric
separations, foam flotation or froth flotation can be considered. To our knowledge,
only one example has been described [200] regarding the application of foam flota-
tion to the separation of enantiomers that shows the method to be feasible. Several
derivatives of L-hydroxyproline, β-cyclodextrin derivatives, such as permethylated
β-cyclodextrin, vancomicin and digitonin were used as chiral foaming agents for the
separation of racemic amino acid derivatives and drugs such as warfarin, and with
different results. The best separation described is the obtainment of N-tert-butoxy-
carbonyl-D-phenylalanine (10) with a 76 % e.e. when using permethyl-β-cyclodex-
trin in a foaming column of 40 cm length (Fig. 1-7).
18 1 Techniques in preparative chiral separations

Fig. 1-7. Racemic aminoacid derivatives

resolved and chiral selector used as foam-
ing agent.

1.6 Global Considerations

All enantioselective separation techniques are based on submitting the enantiomeric

mixture to be resolved to a chiral environment. This environment is usually created
by the presence of a chiral selector able to interact with both enantiomers of the mix-
ture, albeit with different affinities. These differences in the enantiomer–selector
association will finally result in the separation that is sought.
Ideal chiral selectors to be used in preparative separations should fulfil certain
properties. In general, high loadability is one of the most interesting features for
large-scale purposes, but high enantioselectivity, high chemical stability, low cost
and broad applicability are also very important issues. None of these properties can
be considered independently.
Very high values of enantioselectivity can be attained with specific selectors for
particular enantiomers [182, 201–204]. Nevertheless, the application domain is
reduced consequently. Values of chromatographic enantioselectivity over 40 have
been reported either in normal phase conditions [201, 202] or in aqueous mobile
phases [204]. These high enantioselectivities can represent an increase in the load-
ing capacity that could cause a reduction of the global cost of the separation. How-
ever, the long elution time of the second enantiomer would not be convenient for
practical purposes when using chromatography. In contrast, other separation tech-
niques would take profit of this characteristic. Thus, liquid–liquid extraction of aro-
matic amino acids was successfully achieved with a highly enantioselective syn-
thetic receptor by Mendoza et al. [182]. In this case, however, the scale-up of this
separation would be hardly feasible due to the large number of steps needed for the
synthesis of the receptor.
A compromise among all the properties mentioned herein should be established,
depending on the technique used and on the particular application. Preparative sep-
aration of enantiomers is still an open subject which requires further investigation in
the search of new chiral selectors and techniques well adapted to large scale pro-
cesses.
 References 19

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