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Gas Chromatographic Separation of Enantiomers on Cyclodextrin Derivatives.

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Volume 29 . Number 9
September 1990
Pages 939 - 1076
International Edition in English
Gas Chromatographic Separation of Enantiomers
on Cyclodextrin Derivatives **
New Analytical
Methods (42)
By Volker Schurig * and Hans-Peter Nowotny
In investigations concerned with the phenomenon of molecular chirality, the use of gas chromatography for the enantiomeric analysis of stable, volatile compounds is a technique of
steadily growing importance.['] In the last three years an important breakthrough in gas-chromatographic separation of enantiomers has been achieved by using alkylated cyclodextrins (a,
p, and y) as chiral stationary phases in high-resolution capillary columns. In academic and
commercial practice two different and complementary strategies have been adopted up to now.
In the first, alkylated cyclodextrins are diluted with polysiloxanes and coated on glass or fused
silica capillary columns. In the second, lipophilic per-n-pentylcyclodextrins and hydrophilic
di-n-pentyl- and hydroxyalkylpermethylcyclodextrinsare coated directly in the form of liquid
phases onto suitably pretreated glass or fused silica surfaces. These techniques permit enantiomer separations not only for polar diols and alcohols, derivatized hydroxycarboxylic acids,
amino acids, sugars, and alkyl halides, but also for nonpolar alkenes, cyclic saturated hydrocarbons, and metal 7~ complexes. An important aspect for practical applications is that in many
cases the enantiomers can be separated without previous derivatization. Whereas the resolution of racemates of unfunctionalized hydrocarbons is attributed to an enantioselective hostguest inclusion complex, some observations indicate that for polar guest molecules additional
enantioselective interactions are also involved. The new chiral stationary phases can be used
over a wide range of temperatures (25 to 250 "C). The technique described is likely to become
widely adopted as a simple, accurate and highly sensitive method for the enantiomeric analysis
of chirdl compounds that can be vaporized without decomposition. It will also stimulate future
research aimed at finding universal cyclodextrin phases and elucidating the mechanisms of
1. Introduction
The problem of determining enantiomeric compositions
(enantiomeric excess, ee) is central to all contemporary re[*] Prof. Dr. v. Schurig, Dr. H.-P. Nowotny [']
Institut fur Organische Chemie der Universitat
Auf der Morgenstelle 18, D-7400 Tiibingen (FRG)
Present address: F.Hoffmann-La Roche
CH-4002 Basel (Switzerland)
Gas Chromatographic Separation of Enantiomers on Optically Active
Metal-Complex-Free Stationary Phases, Part 2: Part 1. 171.
Angen. Chem. I n [ . Ed. Engl. 29 (1990) 939-957
search concerned with the synthesis, characterization, and
use of chiral compounds.['] Gas chromatography offers an
accurate and reliable modern analytical method for separating enantiomers of compounds that can be vaporized without decomposition. Its inherent advantages include simplicity, speed, reproducibility, and sensitivity.['The availability of high-resolution capillary columnsthat is, wall-coated open-tubular (WCOT) columns-and
the use of highly sensitive chromatographic detectors allow
analyses to be carried out on nanogram quantities of sample,
often without the need for tedious sample pretreatment.
Verlagsgesellschaft mbH, D-6940 Weinheim,1990
0570-0833190/0909-0939 3 3.50f ,2510
Mixtures of different enantiomers can be separated simultaneously (e.g., all proteinogenic amino acids). By combining
the method with ancillary techniques such as multidimensional chromatography and/or coupling to spectrometric
methods, one can analyze enantiomers even in complex biological matrices. The use of an inert gas as the mobile phase
avoids complications due to solvent effects and other variables, such as gradient elution and the use of buffers, modifiers, and additives, which influence liquid-chromatographic
enantiomer separation.
The chiral stationary phases that have become particularly
important in gas-chromatographic enantiomer separation
belong to three classes:
1 . amino acid derivatives which form hydrogen bonds13]
(e.g., the commercially available phases Chirasil-Val [41
and XE-60-L-Val(R)-~~-pea)[~l
2. carbohydrate derivatives, notably cyclodextrins with a
propensity to form inclusion complexes
3. metal complexes (“complexation gas chromatography”)
Stationary phases derived from amino acids have already
been reviewed;”] the present article deals with the use of
cyclodextrin derivatives for gas-chromatographic separation
of enantiomers.
In contrast to the stationary phases of classes 1 and 3,
cyclodextrins have only been applied for routine enantiomer
separation by high-resolution capillary gas chromatography
during the last three years.
2. Cyclodextrins
Cyclodextrins-cycloamyloses, cycloglucans, or cyclomaltooligoses-were isolated as degradation products of
starch by Villiers as long ago as 1891,[s1but it was not until
1904 that they were characterized by Schardinger as cyclic
oligosaccharides (the “Schardinger dextrin~”).[~]
two decades before Pedersen’s synthesis of crown ethers,“
Freudenberg and Cramer in 1948 recognized the ability of
cyclodextrins to form molecular inclusion complexes.“ Cyclodextrins are a homologous series of nonreducing cyclic
oligosaccharides made up of six or more (a)-D-glucopyra-
y- cyclodextrin
Fig. 1. a-Cyclodextrin (cyclohexaamylose), P-cyclodextrin (cycloheptaamylose), and y-cyclodextrin (cyclooctylamylose) [14].
nose units linked together by a-l,4-glycoside bonds[”. 13]
(Fig. 1[I4]).
Cyclodextrins are obtained biotechnologically via enzymatic degradation of the glucose units of the polysaccharide
starch by cyclodextrin glycosyltransferases of Klebsiella
pneumonia, Bacillus macerans, or other types of bacillus. This
reaction results in detachment of a turn from the starch helix
accompanied by cyclization. The relative quantities of the
individual cyclodextrins depend on the type of enzyme employed and can be influenced by the addition of organic
So far, CL-,b-, y-, and 6-cyclodextrins with six to nine
glucopyranose units have been isolated and characterized.
Cyclodextrins with up to twelve glucopyranose units have
been observed,[”. 1 6 ] but only the three lowest members of
Volker Schurig was born in 1940 in Dresden and began studies in chemistry in f959 at the
Universitat Tiibingen, where he received his doctorate under Ernst Bayer in 1968. He then spent
two years in Israel as a postdoc at the Weizmann Institute of Science, where he began his work
on the gas-chromatographic separation of enantiomers with Gil-Av. After pursuing this work at
the University of Houston, he completed his “Habilitation” in Tiibingen with work on complexation gas chromatography. He has been a guest professor at the Universitt Paris-Sud and the
Weizmann Institute. His areas of interest include homogeneous catalysis with soluble polymeric
metal compounds, the formation of highly ordered solid-state structures by chiral control, enzymatic epoxidations, chiral N M R shift reagents, chromatographic enantiomer separations, and the
stereochemical analysis and synthesis of flavors and pheromones.
Hans-Peter Nowotny was born in Herrenberg in 1959 and began studies in chemistry in 1979 at
the Universitat Tiibingen, where he received his doctorate under Volker Schurig in 1989 on
enantiomer separation by inclusion gas chromatography on peralkylated cyclodextrins. He is
presently employed by Hoffmann-La Roche in Basel.
Angew. Chem. Int. Ed. Engl. 29 (1990) 939-9S7
the homologous series are at present commercially available
(a, p, y ) . Strained cyclodextrins with less than six glucopyranose units have not been observed up to now.
Each of the chiral glucose units of the cyclodextrins possesses a rigid 4C, chair conformation. The macrocyclic conformation of the cyclodextrins corresponds to a torus in both
the solid state and in solution; the wider opening is occupied
exclusively by secondary hydroxyl groups (C2-OH and C3OH), while the opposite, narrower opening is occupied exclusively by primary hydroxyl groups (C6-OH). Table 1 lists
some molecular dimensions and physical properties of the
Table 1. Molecular dimensions and physical data for cyclodextrins
Number of glucose units
Number of chiral centers
Molecular mass
External diameter [pm]
Internal diameter [pm]
Volume of cavity [nm3]
pK, of hydroxyl groups
Solubility in water
[g per 100 mL. 25 "C]
Molarity of saturated solution [MI
Melting and decomposition point [K]
0 114
1530- 1540 1690-1750
12.1 - 12.6 (all)
1 85
It can be seen that the height of the molecular cavity is
constant, whereas its diameter varies. The inside face of the
torus comprises two sets of C-H groups (C3 and C5) and,
between them, sets of glycoside ether linkages (C1 and C4).
The cavity has a high electron density due to the lone pairs
of the oxygen atoms. Because of the absence of hydroxyl
groups directed inward, the cavity is both hydrophobic and
nonpolar.[171The outer hydroxyl groups at the cavity openings make the underivatized cyclodextrins hydrophilic. The
macrocycle derives additional rigidity from the formation of
stable intramolecular hydrogen bonds between secondary
hydroxyl groups, 0 3 - H . . - 0 2 and 0 3 . . . H - 0 2 .
The complexing properties of cyclodextrins, which can be
regarded as empty molecular cavities of different sizes, have
been thoroughly studied.[I2- 201 Hydrophobic interactions
favor the formation of inclusion complexes. The propensity
of cyclodextrins to form complexes with guest molecules
is employed in many different technological applications." 5 , "I The observation that, by formation of inclusion
complexes, the stability, solubility, bioavailability, residence
time, toxicity, and odor properties of guest molecules are
beneficially altered commands particular interest for the
pharmaceutical and food industries.['4- 16, z21
Numerous X-ray structural studies have been carried out
on a variety of inclusion complexes of P-cycl~dextrin.[~~.
In addition to 1 :1 inclusion complexes, 2: 1 host-guest complexes, in which the cyclodextrin molecules are linked by
hydrogen bonds between the secondary hydroxyl groups,
can also be formed. Studies on heptakis-(2,6-U-dimethyl)-Pcyclodextrin showed that protection of the free hydroxyl
groups prevents the formation of cyclodextrin dimers and
also that the presence of the methoxy groups can slightly
increase the space available to a hydrophobic guest molecule
Angew. Chrm. Inr. Ed Engl. 29 ( ( 9 9 0 ) 939-957
compared with that of the cyclodextrin cavity itself.1241This
example illustrates that the conformation and complexing
ability of cyclodextrins can be tailored by chemical modification.
In addition to host-guest selectivity, cyclodextrins exhibit
some catalytic properties related to those of enzymes (competitive inhibition and Michaelis-Menten kinetic^)."^. 16]
Cyclodextrins have therefore been studied as enzyme models
for a-chymotrypsin, carbonic anhydrase, and ribonuclease.I2'1
A total synthesis of a-cyclodextrin has been achieved.r261
Derivatizations of cyclodextrins, such as alkylations and
acylations, can be carried out regioselectively at the hydroxyl
groups.[271Monofunctionalizations,[Z81 a specific bifuncti~nalization,'~~]
and bridging across the cavity (yielding
"capped cyclodextrins") have also been reported.'301
The inherent molecular asymmetry of cyclodextrins,
which arises from the fact that the cyclooligomers contain
only D-glucose units, widens considerably the scope of potential applications. As a consequence of their biotechnological origin from natural starch, only the dextrorotatory
enantiomers of cyclodextrins are known; the racemic forms
and the levorotatory antipodes are not available. In many
cases, the inclusion of chiral guest molecules into cyclodextrins represents an enantioselective process. Thus, different
complex formation constants were found for the two fenchone enantiomers in ESR studies on the formation of inclusion complexes of P-cyclodextrin with (+)- and (-)-fenchone via the competitive displacement of a paramagnetic
species from the cyclodextrin cavity.[31Cyclodextrins have
also been used as chiral auxiliary reagents in NMR spectroscopy. In the cyclodextrin complex of 2,2,2,-trifluoro-lphenylethanol, for example, the CF, resonance of the (8(+) enantiomer appears at higher field than that of
the (R)-(
-) enanti~mer.[~''
A direct proof of the enantioseZective complexation of (R)-and (S)-2-(3-phenoxyphenyl)propanoic acids (fenoprofen) with P-cyclodextrin has recently been accomplished by crystal structure analysis.[331The
inclusion of optically inactive compounds in cyclodextrins
can lead to a measurable circular dichroism, caused by the
induction of chiral conformations.[' 8 , 351 Furthermore,
diastereomers formed in P-cyclodextrin by the inclusion of a
prochiral compound were distinguished by ENDOR meas u r e m e n t ~ . [Numerous
asymmetric catalytic reactions using cyclodextrins have also been described." 6, 3 7 1 Kinetic
studies on the cyclodextrin-catalyzed hydrolysis of racemic
oxazolones provided evidence for enantioselectivities that
were of opposite senses for a-and ~ - c y c l ~ d e x t r i n s . [ ~ ~ ~
An obvious method for separating racemic mixtures using
enantioselective complexation in cyclodextrins is to carry
out a fractional crystallization on the diastereomeric inclusion complexes. Indeed, racemic carboxylic acid esters, menthol and several carboxylic acids,[39,401 0-alkyl alkylphosphinates,14'] s u l f ~ x i d e s , [0-alkyl
~ ~ ~ alkyls~Ifinates,[~~~
2-bromo-2-chloro-I ,1 ,I-trifluoroethane
have been separated into their enantiomers in this way.
However, the enantiomeric purity (ee) of the products was
low in most cases. The highest reported value was ee = 84 YO
for U-isopropyl methylph~sphinate,[~']
but this was only
achieved after repeating three times the cycle of complexation, fractional crystallization, and release of the enriched
product. This cumbersome procedure is avoided when the
reversible enantioselective inclusion reaction is coupled to
a chromatographic (i.e., a multistage) separation process,[45-471
3. Chromatographic Separation of Enantiomers
on Cyclodextrins
Cyclodextrins are used in many chromatographic procedures for selective separations of compounds, including
e n a n t i ~ m e r s . [481
~ ~Table
2 gives a summary of representative applications.
Table 2. Applications of cyclodextrins in chromatography [47,48] S = in the
stationary phase, M = in the mobile phase. Electrokinetic methods: electroosmosis, electrophoresis, isotachophoresis.
Type of chromatography
GasGel in- HighAffinity
solid and clusion pressure
subcritical carbon dioxide (subFC) as the mobile phase.[631
Enantiomer separations by liquid chromatography on acetyl
and carbamoyl derivatives of P-cyclodextrin have also been
recently reported.[64’651
In HPLC, as in thin layer chromatography, cyclodextrins
were initially used not as immobilized stationary phases but
as additives in the mobile phase in conjunction with conventional reversed phases (RP-I 8).145* An important advantage of this approach is that the formation of the inclusion
complex enhances the
giving an appreciable
reduction in the lower limit of detection when a fluorescence
detector is used. This principle has also been employed in a
method whereby P-cyclodextrin as a chiral stationary phase
is combined with f+-cyclodextrinas an additive in the mobile
phase.[68]Partially and fully methylated a- and P-cyclodextrins have also been applied as additives in the mobile
The use of cyclodextrins for gas-chromatographic separation of enantiomers will be described in Section 4.2.
4. Cyclodextrins in Gas Chromatography
Soluble cyclodextrin polymers
Insoluble cyclodextrin polymers
In thin layer chromatography (TLC) cyclodextrins can be
used as either the stationary or the mobile phase.[491For
example, aqueous solutions of a-cyclodextrin, dimethy1-Pcyclodextrin, y-cyclodextrin, and soluble polymers of f+-cyclodextrin have been used as chiral mobile phases.r471p-Cyclodextrin itself is not suitable, since it is only sparingly
soluble in water. However, its hydrophilicity can be improved by adding urea 1501 or by partial derivatization to
hydroxyethyl- or hydroxypropyl-~-cyclodextrin.~sll
P-Cyclodextrins chemically bound to silica gel have also been
used for separating enantiomer~.[~’.
5 3 J Furthermore, cyclodextrins have been employed for separating enantiomers
in electrokinetic chromatographic techniques such as capillary isotachophoresis (CITP)[541and high-performance capillary zone electrophoresis (HPCZE).[’ 51 Especially noteworthy is the use of cyclodextrins with singly charged substituents in inverse micellar electrokinetic chromatography,
as described by Terabe et al.[571In gel inclusion chromatography (GIC)[58]mandelic acid derivatives and indole alkaloids have been separated into their enantiomers both analytically and on a preparative scale on insoluble polymers of
P - c y ~ l o d e x t r i n 47,
. ~ ~591
The use of cyclodextrins for separating enantiomers in
high-pressure liquid chromatography (HPLC) has proved
especially successfu1.[60-621Unlike many of the well-known
chiral stationary phases, the cyclodextrin stationary phases
described so far are compatible with aqueous and strongly
polar mobile phases in the reversed phase mode of operation.
The cyclodextrin stationary phases can also be used with
4.1. Gas Chromatographic Separation of Achiral
As long ago as the early sixties, peracylated a- and f+-cyclodextrins were used above their melting point (i.e., above
220 “C) as stationary phases in packed columns for the selective separation of fatty acid esters.[701The analysis of the
retention data did not provide conclusive evidence for the
formation of inclusion complexes in this case. However, permethylated a- and f+-cyclodextrins,when used above their
melting point or as solutions in polysiloxane (DC-710) in
packed columns, gave retention times that were greater for
iso-alkanes than for n-alkanes, and this was attributed to the
formation of inclusion complexes.[71] In gas-solid chromatography (GSC) a- and P-cyclodextrin polyurethane
resins,[721a-, p-, and y-cycl~dextrins,[~~*
741 hexakis(2,6-di0-methyl)-a-cyclodextrin, and heptakis(2,6-di-O-methyl)-f+cyclodextrin[751were applied for the selective separation of
positionally isomeric arenes (e.g., xylene and lutidine isomers); P-cyclodextrin polymers were found to be unsuitable
for this purpose.[761The packed columns used have low efficiencies, and consequently it was not possible to observe
separation of enantiomer~.[~~I
For applications in gas-liquid
chromatography (GLC) the hydrophilic nature of underivatized cyclodextrins proved to be a disadvantage, since these
require highly polar solvents as achiral stationary phases.
This difficulty can be avoided by using peralkylated cyclodextrins, which are lipophilic and low-melting. The first
breakthrough was obtained with permethyl-0-cyclodextrin
and perpropanoyl-f+-cyclodextrin,
the latter being covalently
bonded onto a poIy~iloxane.[~~’
Underivatized a- and P-cyclodextrins, dissolved in formamide or ethylene glycol allowed the separation of positional isomers o f xylene, diethyland dimethylcyclohexane, as well as cis- and
tvans-decalin.[801Even more remarkable is that with these
packed columns the first gas-chromatographic separations
of enantiomers on cyclodextrin phases were observed for
pinenes and pinanes.r8‘.821
Angew. Chem. Int. Ed. Engl. 29 (1990) 939-957
4.2. Gas Chromatographic Separation of Enantiomers
of Chiral Compounds
Although numerous gas-chromatographic studies had already been carried out with cyclodextrin stationary phases,
notably the pioneering experiments of Smolkova-Keulemansovu et al.,[73*7 9 , 8 3 1 the important breakthrough in enantiomer separation was only achieved quite recently through
the use of high-resolution capillary columns. Thus, in a 1987
monograph on the practice of enantiomer separation by capillary gas chromatography, the great potential of cyclodextrins for separating enantiomers of nonpolar molecules is
mentioned only in the preface by GiI-Av.r841
The attempts by
Kim in 1981 to separate enantiomers of saturated aliphatic
hydrocarbons with the general formula CR'R2R3R4 (R =
alkyl), such as 2,2,3-trimethylheptane, on stainless steel capillaries coated with a solution of permethyl-o-cyclodextrin in
polysiloxane (ZOO/, in OV-101) were unsuccessful, although
the retention data led to the conclusion that inclusion complexes were formed.[85a1Finally, KoScielski et al., from 1983
onwards, achieved enantiomer separations of some chiral
bicyclic alkenes (a- and 0-pinene and carene) and bicyclic
alkanes (cis- and trans-pinane) on packed columns in which
the supporting material was coated with a solution of a-cyclodextrin in formamide (Fig. 2).[*', "I
t [rninl
Fig. 2. Separation of the enantiomers of rr-pinene on rr-cyclodextrin in formamide solution supported on Celite at 44°C. Packed column, 2 m x 4 mm.
The chromaiogrdm shown as a broken line was obtained on pure formamide
(without cyclodextrin) supported on Celite. Carrier gas: helium, 2.75 bar [82].
As mentioned earlier, although these columns give large
separation factors IX for the enantiomers, they have very low
efficiencies. Thus, the peak shapes are unusually broad, even
for packed columns, as is evident from the low theoretical
plate numbers of about 950-1250 for columns two meters
long.[791These columns therefore have lower efficiencies
than cyclodextrin columns in HPLC, which give about 8000
theoretical plates for a length of 25 cm.[611The method is
also found to have other disadvantages, namely, the limited
temperature range (maximum 70 "C), the short lifetime of
Angew. Chem lnt. Ed. Enungl. 29 (1990) 939-957
the columns owing to the continual loss of solvent and water
molecules, which are necessary for separation,@5b1
by bleeding, and the fact that the method cannot be adapted for use
with capillary columns. This has led to a considerable
growth of interest in the development of thermally stable
cyclodextrin phases for use in high-resolution capillary
4.3. The Choice of Cyclodextrin Derivatives as Chiral
Stationary Phases for High-Resolution
Gas Chromatographic Separation of Enantiomers
Because of the physical properties of underivatized cyclodextrins, especially their decomposition on melting and
their insolubility in polysiloxanes, they appear to be unsuitable for coating capillary columns. On the other hand, many
cyclodextrin derivatives with more favorable melting points
and/or solubilities are known.[271Owing to the lability of
cyclic oligosaccharides in acid media, only derivatizations
carried out in alkaline media can be considered. Peracylated
and partially or fully methylated cyclodextrins have already
been used as stationary phases in gas chromatography on
packed columns.[70.''I It is essential that the C-0 bonds at
the chiral centers C2 and C3 remain intact during the acylation or alkylation of the hydroxyl groups in order to avoid
epimerizations. In planning the derivatization strategy one
must take into account the fact that the newly introduced
groups at the wider end of the cyclodextrin torus may participate in the chiral recognition process.1623
Alkylation offers a number of advantages over acylation:
1. Since the ether group is more stable than the ester
group, alkylated derivatives are chemically and thermally
more stable and less susceptible to hydrolysis. It can be assumed that the alkoxy groups at the periphery of the
cyclodextrin molecule have thermal stabilities similar to
those of the glycoside ether linkages in the cyclodextrin
skeleton, and the maximum temperature for use as a stationary phase is therefore limited solely by the stability of the
cyclodextrin skeleton.
2. Per-0-alkyl derivatives are less polar than per-0-acyl
derivatives. Consequently, the former have lower melting
points and are more soluble in nonpolar polysiloxanes.
The C2 hydroxyl group is more acidic and the C6 hydroxyl
group is less sterically hindered than the C3 hydroxyl group.
Consequently peralkylations do not always proceed to completion. However, this fact can be used advantageously for
regioselective derivati~ation.['~]
In the first enantiomer separations performed by high-resolution capillary gas chromatography, molten permethylated 0-cyclodextrin was used, initially by Juvancz, Alexander,
and Szefjtli[88,891and later by Venema and Tol~rna.[~~~
the other hand, Schurig and N o ~ o t n y [ ~ ' employed
a solution of permethylated b-cyclodextrin in a moderately polar
polysiloxane (e.g., OV-I 701) as enantioselective stationary
phase. The following cyclodextrin derivatives have successfully been used in solution as stationary phases for gas-chromatographic separation of enantiomers (see Section
5):[91 - 9 8 1
hexakis(2,3,6-tri-O-methyl)-a-cyclodextrin (2)
heptakis(2,6-di-O-methyl-3-O-trifluoroacetyl)-~cyclodextrin (4)
octakis(2,6-di-O-methyl-3-O-trifluoroacetyl)-y-cyclodextrin (5)
heptakis(3-O-heptafluorobutanoyl-2,6-di-O-methyl)P-cyclodextrin (6).
The following derivatives have also been investigated,
and, although they can be used, they exhibit lower separation factors cl:[921
heptakis(2,3,6-tris-O-(5')-2'-methylbutyl)P-cyclodextrin (11)
The high melting points of undiluted permethylated cyclodextrins are found to be a disadvantage. Only one of
these, 1, is liquid over a relatively broad range of temperatures and is suitable in the molten state as a stationary phase.
Values for the melting point given in the literature vary from
83 to 175 OC.['*,
These discrepancies may be explained by the presence of inhomogeneous, incompletely
methylated products. Venema and T u l ~ m aon, ~the
~ basis
~ ~ of
calorimetric measurements, arrived at a melting point of
150 "C for 1. However, on cooling, 1 did not crystallize but
instead gave a supercooled melt, which afforded very good
chromatographic results at temperatures above 76 "C. Commercially available 1 can contain 2,6-di-O-methyl-P-cyclode~trin.~~~~
Konig and Wenzflool found that per-n-pentylated cyclodextrins are liquids even below room temperature. They
employed the following cyclodextrin derivatives very successfully for separating enantiomers of many different
racemic mixtures on deactivated glass capillary columns (see
Section 6):""- ' ' ' I
heptakis(3-O-acetyl-2,6di-O-n-pentyl)-~-cyclodextrin (16)
octakis(3-O-butanoyl-2,6-di-O-n-pentyl)-y-cyclodextrin (18)
Armstronget al." 1 8 - 1 2 3 1 used the following partially alkylated, liquid cyclodextrin derivatives as stationary phases on
untreated fused silica capillary columns for gas-chromatographic separation of enantiomers:
hexakis(O-(S)-2-hydroxypropyl-per-O-methyl)a-cyclodextrin (PMHP-a-CD, 19)
heptakis(O-(S)-2-hydroxypropyl-per-O-methyl)P-cyclodextrin (PMHP-P-CD, 20)
(dipentylE-CD, 21)
heptakis(2,6-di-O-n-pentyl)-P-cyclodextrin(dipentylP-CD, 22)
heptakis(2,6-di-O-n-pentyl-3-O-trifluoroacetyl)-Pcyclodextrin (DPTFA-P-CD, 23)
Compounds 19-22 all have free hydroxyl groups which
make these moderately polar derivatives hydrophilic. As has
been shown by 252Cfmass spectrometry, the derivatives are
present as partially alkylated mixtures, which give rise to the
desired liquid consistency. For many racemic mixtures, the
order in which the enantiomers are eluted is reversed if the
stationary phase is changed from 19 to 20 or from 21 to
22." 181
5. Gas Chromatographic Separation of
Enantiomers on Diluted Cyclodextrin Derivatives
Solutions of peralkylated cyclodextrins in moderately polar polysiloxanes, either in packed columns as described by
Casu et aI.["] or in capillary columns as described by Schurig
and Nowotny,[".
are useful for the gas-chromatographic
separation of enantiomers for the following reasons:
1. The diluted phase can be used over a wide range of temperatures. The essential requirement is that the cyclodextrin derivatives must be soluble in polysiloxanes; their
melting points and phase transitions are unimportant. In
principle, it is also possible to employ mixtures of different cyclodextrins (a, fl, and y) simultaneously and to incorporate suitable additives for competitive inclusion.
2. Coating the solutions onto glass or fused silica capillary
columns, whose surfaces can be deactivated by high-temperature silylation to give very inert columns,[*241 results
in separations with high efficiency, even for strongly polar
3. The choice of appropriate solvents allows the polarity of
the stationary phase to be varied over a wide range, independently of the nature of the derivatization of the cyclodextrins.
4. Only small quantities of the substances in solution are
needed, allowing the economical use of cyclodextrins that
are difficult to obtain (y and 6 cyclodextrins).
5. The low concentrations of cyclodextrins give short analysis times, and experience so far indicates that this does not
reduce the separation factors a.
6. Thermodynamic parameters involved in enantiomer discrimination (AR,JAG0), AR,JAH0), and AR.JAS0)) can
readily be determined from relative retention data using
the concept of the retention increase R' derived for complexation gas chromatography.[63"'l
Peralkylated cyclodextrins of low molecular mass are preferred because, for example in n-pentylated cyclodextrins,
the conformationally flexible n-pentyl groups probably do
not contribute to chiral recognition, and they also shield the
cavity to some extent.
The polydimethylsiloxane OV-101 (100 % methyl groups)
has proved to be a versatile solvent for optically active metal
chelates in complexation gas chromatography.'61 On suitably prepared glass or fused silica capillary columns it forms
stable films. Glass capillary columns coated with a solution
of 1 in OV-101 can separate enantiomers of many chiral
compounds above 80 0C;f921
below this temperature, peak
broadening occurs. It appears that at low temperatures the
limit of the solubility of cyclodextrins in the highly nonpolar
polysiloxane is reached. Compound 1 is soluble in the polyAngew. Chem. Int. Ed. Engi. 29 (1990) 939-957
methylphenylsiloxanes DC-550 (75 YOmethyl, 25 % phenyl)
and OV-17 (50% methyl, 50% phenyl) even at room temperature, thus allowing enantiomer separations to be performed
also at low temperatures; however, these phases do not form
stable films on glass surfaces.
5.1. Heptakis(2,3,6-tri-O-rnethyl)-fl-cyclodextrin (1)
in OV-1701
OV-1701 is a polysiloxane of higher viscosity (a "gum"
phase, with 5 % cyanopropyl groups, 7 % phenyl groups,
and 88% methyl groups). It has a polarity comparable to
that of OV-17 and forms very Stable films on suitably pretreated glass or fused silica capillary columns. Solutions of 1
in OV-1701 are ideal for gas-chromatographic separation of
selected enantiomers between 25 and 200 0C.191- 9 3 1 Enantiomer separations can be performed for the following classes of compounds: cyclic ethers such as 2,5-dimethyltetrahydrofuran, underivatized cyclic and acyclic ketones, y-lactones, terpene ketones (e.g., pulegone), underivatized secondary aliphatic alcohols, aromatic alcohols, terpene alcohols (e.g., 3-menthanols), underivatized aliphatic diols, esters of 2-bromopropanoic acid, 1,3-dioxolanes, aliphatic and
Fig. 4. Separation of the enantiomers of the allene ethylhexa-3,4-dienoate (left)
and of the propellane modhephene (right) on 1 in OV-1701 (0.07 M) at 105°C
(left) and 65 "C (right). Deactivated fused silica capillary columns. 40 m x
0.25 mm I.D. (left) and 25 m x 0.25 mm I.D. (right). Carrier gas' H,, 0.7 bar
(left) and 1 bar (right).
t Iminl
t [minl
aromatic oxiranes such as phenyloxirane, bicycloalkenes (CLand P-pinene), and bicycloalkanes (cis- and trans-pinane).
Selected examples of the first enantiomer separations
achieved are shown in Figure 3.
Enantiomer separations of particular interest are those for
an allene and for an unsaturated propellane (see Fig. 4), as
well as the enantiomer separation of unfunctionalized saturated monocyclic hydrocarbons (Fig. 5).[941
The high degree of deactivation of the fused silica capillary
columns used can be seen from the enantiomer separation of
underivatized polar d i ~ l s ; [ ~no
* ] adsorption effects are pres-
t Iminl
t iminl-
t Iminl
Fig. 3. Separation of the enantiomers of a) 2-ethyl-6-methyl-5,6c) 4,7,11 -trioxapentacyclodihydro-y-pyrone. b) 2,2,4-trimethyl-l,3-dioxane,
['.03'0.05-9]undecane[126], and d) 2,2-dimethyl-3-phenyloxiraneon
1 in OV-1701 (0.07 M) at 100°C (a), 60 "C (b and e). 125 "C (c). and 50 "C (d).
Glass capillary column, 40 m xO.25 mm I D. Carrier gas: 1 bar N2(a), 0.7 bar
N,(b), 1 bar He (c and d) [93j.
Angew. Chem. 1121. Ed. Engl. 29 (1990) 939-957
20 30
t Imtnl
Fig. 5. Separation of the enantiomers of cis/truns-l-ethyl-2-methylcyclohexane
and of cis/truns-l -methyl-2-n-propylcyclohexane
on 1 in OV-1701 (0.07 M) at
50°C. Deactivated fused silica capillary column, 30 m x 0.25 mm I.D. Carrier
gas: H,, 1.5 bar [94].
served enantioselectivity. This assumption was only partly
verified in practice,[92]since the enantioselectivities of 1 and
2 are found to overlap. However, 2 shows a distinct selectivity for phenyloxirane~.[~*I
The molecular cavity in 3 is large enough to accommodate
compounds such as the spiroketals rvans-2,3-dimethyl-l,4dioxaspiro[4.4]nonane (Fig. 8) and 2-ethyl-l,6-dioxaspiroOH
f Iminl
f Iminl
Fig. 6. Separation of the enantiomers of n-hexan-1,2-diol and 2-methyl-3buten-1,2-diol on 1 in OV-1701 (0.07 M) at 75°C. Deactivated fused silica capillary column, 25 m x 0.25 mm I.D. Carrler gas: H,, 1 bar [Y7].
t Lrninl
Fig. 8. Separation of the enantiomers of 1rans-2,3-dimethyl-l.4-dioxaspiro[4.4]nonane on 3 in OV-1701 (0.07 M) at 60°C. Glass capillary column,
40 m x 0.25 mm (I.D.). Carrier gas. 1 bar H, [Y2].
[4.4]nonane and to separate them into their e n a n t i o m e r ~ . [ ~ ~ ]
For many of the enantiomers that can be separated on 3,
overlapping was observed with 1 and 2.[921Surprisingly,
however, 3 showed no enantioselectivity for chiral saturated
cyclic hydrocarbons.
5.3. Higher ALkylated Cyclodextrin Derivatives
in OV-1701
t [minl
Fig. 7. Separation of the enantiomers of neomenthol, menthol, and isomenthol
on 1 in OV-1701 (0.07 M) at 85°C. Deactivated fused silica capillary column,
25 m x 0.25 mm I.D. Carrier gas: H,, 1 bar [Y7].
ent, as is evident from the symmetrical shape of the peaks
and the high efficiency of separation (Fig. 6).
Besides enantioselectivity, 1 in OV-1701 shows a pronounced diastereoselectivity, which can be exploited for analytical purposes, as illustrated by the separation of the
diastereomeric 3-menthanols (Fig. 7).19']
5.2. Hexakis(2,3,6-tri-O-methyl)-a-cyclodextrin(2)
and Octakis(2,3,6-tri-O-methyl)-y-cyclodextrin (3)
in OV-1701
It was thought that altering the dimensions of the cyclodextrin cavity would offer a way of influencing the ob946
Peralkylated cyclodextrins with longer n-alkyl groups are
also soluble in OV-1701 and are suitable for gas-chromatographic separation of enantiomers, giving enantioselectivities that are in some cases different.t921Since these cyclodextrin derivatives have greater molar masses (with 21 alkyl
groups per p-cyclodextrin molecule), their solutions must
contain a larger mass fraction of the cyclodextrin to give the
same molar concentration. Thus, a 0.07 M solution of permethyl-P-cyclodextrin (1) corresponds to 10 wt %, whereas
0.07 M solutions of perethyl-p-cyclodextrin (8), per-n-propylP-cyclodextrin (9), and per-n-butyl-p-cyclodextrin (10) correspond to 12, 14, and 16 wt%, respectively.
Enantiomer separations using diluted per-n-pentyl-p-cyclodextrin (15) have also been described recently.['271Surprisingly, blocking of the wider opening of the cyclodextrin
torus in 11 by (9-configurated sec-pentyl groups did not
result in a change in enantioselecti~ity.[~~~
5.4. Heptakis(2,6-di-O-methyl-3-O-trifluoroacetyl)$-cyclodextrin (4), Octakis(2,6-di-O-methyl-3-0trifluoroacety1)-y-cyclodextrin (5), and Heptakis(2,QdiO-methyl-3-O-heptafluorobutanoyl)-$-cyclodextrin (6)
in OV-1701
The P-cyclodextrin derivatives 4-6,[93*951 derived from
heptakis(2,6-di-O-methyl)-~-cyclodextrinby selective perAngeu. Chem. Int. Ed. Enxl. 29 (1990) 939-957
fluoroacylation at the 3-position, give different enantioselectivities in some cases. This is attributed to the fact that the
perfluoroacyl groups are able to form additional hydrogen
bonds and to induce dipole-dipole interactions. The separation factors for y-lactones are considerably greater on 4
(Fig. 9) than on 1, and the increase in size of the cyclodextrin
cavity in 5 gives favorable enantioselectivities for 6-lactones.
On the other hand, these perfluoroacylated cyclodextrin
derivatives cannot be employed for enantiomer separations
of chiral monocyclic alkenes and alkanes. Compound 6 is
particularly suitable for separating enantiomers of cyclic and
acyclic ketones.195,961
tive reaction of the cyclodextrin with n-pentyl bromide in the
presence of a threefold excess of NaOH in dimethyl sulfoxide
yields the 2,6-di-n-pentylated derivatives." 301 The 3-hydroxyl groups are then further alkylated (in this example, with
n-pentyl bromide/NaOH) or are acylated using an acyl
halide. Per-n-pentylcyclodextrins are thermally stable, viscous liquids, which are colorless at room temperature and
can be coated onto appropriately deactivated glass surfaces.
The results of Konig et al.['OsI more than confirmed the
prediction made in 1987 by S ~ e j t l i , [one
~ ~ 'of the pioneers of
cyclodextrin research, who wrote: "It is expected that good
complex forming low-melting derivatives of cyclodextrins in
capillary columns will result in excellent chiral recognition
and resolution of many racemates."
Indeed, the regioselective alkylation and acylation open
up the way to a wide variety of possible cyclodextrin stationary phases for gas-chromatographic separation of enantiomers. The range of enantiomer-separation applications
I _I
t [rninl
Fig. 9. Separation of the enantiomers ofy-lactones on 4 in OV-I 701 (0.07 M ) at
160°C (4'C min-'). Deactivated fused silica capillary column, 25 m x 0.25 mm
I.D. Carrier gas: H,, 1.3 bar [97].
A dimeric cyclodextrin derivative, bis(permethy1-P-cyclodextrin)glutarate, showed no enantiomer separating proper tie^,"^^ despite the fact that this compound was expected
to exhibit cooperative inclusion behavior.['2s, 1 2 9 ]
6. Gas Chromatographic Separation
of Enantiomers on Undiluted Liquid Cyclodextrin
By introducing n-alkyl groups with longer chains (pentyl,
hexyl, lauryl), Konig et al. were able to obtain liquid cyclodextrin derivatives preferentially suited for gas-chromatographic separation of many types of compounds on
deactivated glass capillary
" ' 1 These are prepared from a-, p-, or y-cyclodextrin: for example, regioselecAngen. Chem. Inr. Ed. Engl. 29 (1990) 939-957
I [rnin]
Fig. 10. Separation of the enantiomers of pertrifluoroacetylated carbohydrates
and anhydroalditols on 13 at I 1 5 "C and 120 "C. Glass capillary column, 40 m.
Carrier gas: H,, 1 bar. Top: a- and P-glucopyranose. Bottom: 1.5-anhydroglucitol, -galactitol, and -mannitol (R=CF,CO) [lOS].
extends from trifluoroacetylated amino and hydroxyl compounds, including carbohydrates, derivatized amino acids,
olefins, alkyl halides, and spiro- and bicyclic acetals to diolefin n: complexes of metal carbonyls.
6.1. Hexakis(2,3,6-tri-O-n-pentyl)-a-cyclodextrin (13)
This lipophilic cyclodextrin (Lipodex A) stationary phase,
which is soluble in dichloromethane, can be used on glass
capillary columns to separate racemates of trifluoroacetyiated alcohols,[1021epoxy alcohols,"
diols, triols, cdrbohydrates (Fig. 10),r1081 underivatized 4-methyl-3-heptanone,11061alkyl halides," lo] spiroacetals,"
and glycerol
derivatives. This phase, or its per-n-hexylated analogue, has
also been used to resolve enantiomers of N-alkylated barbiturates.[' l61
6.2. Hexakis(3-O-acetyl-2,6-di-O-n-pentyl)-a-cyclodextrin (14)
This lipophilic cyclodextrin (Lipodex B) stationary phase
is particularly effective for separating racemic five-membered heterocycles such as y-lactones, cyclic carbonates of
1,2-diols, and s u c ~ i n i m i d e s . High
~ ~ ~ ~separation
factors are
also achieved with this phase for racemic trifluoroacetylated
carbohydrates and hydroxy compounds such as aldols,
cyanohydrins, and amino alcohols.r1061
6.3. Heptakis(2,3,6-tri-O-n-pentyl)-fl-cyclodextrin (15)
This lipophilic cyclodextrin (Lipodex C) stationary phase
allows trifluoroacetylated alcohols, trifluoroacetylated carbohydrates, lower homologues of hydroxycarboxylic acid
esters, and cyanohydrins to be separated into their enant i ~ m e r s . ~Especially
noteworthy is the separation of the
enantiomers of the pheromone grandisol, which succeeded
despite the fact that the trifluoroacetylated hydroxyl group
is separated from the chiral center by two carbon atoms
(Fig. 11).
This phase can also be used to resolve the enantiomers of
acyclic, monocyclic, and bicyclic olefins and d i e n e ~ . ~ " ~1 3.1
Here, the enantioselective inclusion process was found to be
sensitive to small changes in the structure of the substrate.
Thus, 4-methyl-I -hexene and 4-methylcyclohexene could
not be separated, in contrast to the 3-methyl isomers. This
phase also showed enantioselectivity for cyclic and acyclic
alkyl halides with up to eight carbon
For example, racemic mixtures of the 2-chloroalkanes from 2chlorobutane up to 2-chlorooctane have been resolved
(Fig. 11). In contrast to the higher homologues 1,2-dibromoheptane and 1,2-dibromooctane, 1,2-dibromohexane could
also be separated into its enantiomers.
Fig. 11. Separation of the enantiomers of grandisol (top) and alkyl halides
(bottom) on 15 at 90°C and 50°C. respectively. Glass capillary column, 42 m.
Carrier gas. H,, 1 bar [113].
6.4. Heptakis(3-O-acetyl-2,6-di-O-n-pentyl)-flcyclodextrin (16)
This lipophilic cyclodextrin (Lipodex D) stationary phase,
whose acetyl groups are capable of forming hydrogen
bonds,['061 allows enantiomer separations to be carried out
Fig. 12. Separation of the enantiomers of trifluoroacetylated amines and amino alcohols on 16 at 140 "C (2 "C min- '). Glass capillary colum, 45 m. Carrier
gas: H,, 1 bar 11031.
Angew. Chem. In! Ed. Engl 29 (1990) 939-957
on polar compounds such as trifluoroacetylated amines with
2- and 0-chirality, amino alcohols, esters of p-amino acids,
and cyclic tran~-diols['~~1
(Figs. 12 and 13).
The presence of the acetyl group gives particularly high
separation factors CL for trifluoroacetylated amines and
amines containing additional functional groups. Many of
these derivatized amines could not previously be separated
into their enantiomers on amino acid derivatives as station-
t Irntnl
Fig. 14. Separation of the enantiomers of 2,4-dimethyl-1-heptene (top left),
rrans-3,3,8,8-tetramethylcyclooctene(top right), and tricarbonyl(q4-2-methylene-l,3-butanediyl)iron(o) and tricarbonyl(q4-2-methyl-l
[I 171(bottom) on 17 at 60"C, 95 "C, and 8 0 T , respectively. Deactivated glass
capillary column, 50m. Carrier gas: H,, 1 bar (1151
R g . 13. Separation of the enantiomers of trifluoroacetylated amphetamine,
mexiietine, pholedrin. and tranylcypromin (from right to left) on 16 at 175 "C.
Glass capillary column, 45 m. Carrier gas: H,, 1 bar [103].
ary phases. Separation of the enantiomers of the methyl ester
of a-lipoic acid can also be achieved using 16." 151
0-trifluoroacetylated esters of a- and (3-hydroxycarboxylic acids can also be resolved, including the esters of
tartaric, malic, and mandelic acid.
Racemic alcohols and diols have been resolved in derivatized form as trifluoroacetyl esters, and a number of terpene
ketones such as fenchone, menthone, isomenthone, and camphor have been resolved without derivatization (Fig. 15).
6.5. Octakis(2,3,6-tri-O-n-pentyl)-y-cyclodextrin (17)
and Octakis(3-O-butanoyl-2,6-di-O-n-pentyl)-y-cyclodextrin (18)
For y-cyclodextrins the greater diameter of the cavity, the
increased conformational flexibility of the macrocycle, and
the fact that these compounds have been found to form
unstable inclusion complexes[491lead one to expect a reduction in enantioselectivity. Instead, Konig et a1.[114* found
that 17 and 18 were suitable for separating enantiomers of
various types of compounds, giving large separation factors
in some cases. Especially noteworthy is the separation of
enantiomers of nonpolar substrates such as alkenes with (3chirality : for example, 2,4-dimethyl-I -heptene, the planarchiral alkene trans-3,3,8,8-tetramethylcyclooctene,and the
planar-chiral metal carbonylx complexes tricarbonyl (q4-2methylene-I ,3-butanediyl)iron(o), and tricarbonyl(q4-2-methyl-I ,3-butadiene)iron(o) have been separated on the hydrophobic liquid 17 (Fig.
. - 141.I' 15]
Racemic a-alkyl and N-methyl a- and p-amino acids in the
Of N-trifluoroacetyl-o-methy'
derivatives have
been resolved into their enantiomers on 18.11141
Except in the
case of proline, the D enantiomers of a-amino acids are eluted before the L antipodes, thus facilitating determination of
the enantiomeric purity of naturally occurring L-amino
acids. However, histidine, tyrosine, and arginine have so far
turned out to be unsuitable for analysis.
f lminl
Angew. Chcm. In!. Ed. Engl. 29 (1990) 939-957
Fig. 15. Separation of the enantiomers of underivatized fenchone, menthone,
isomenthone, and camphor on 18 at 100°C (2"Cmin-'). Deactivated glass
capillary column, 45 m x 0.25 mm I.D. Carrier gas: H,, 1 bar (1141.
Bicyclic and tricyclic acetals (bark beetle pheromones such
as frontalin, exo- and endo-brevicomin, and lineatin), which
were previously separated by complexation gas chromatog949
6.8. Heptakis(2,6-di-O-n-pentyl-3-O-trifluoroacetyl)p-cyclodextrin (DPTFA-p-CD, 23)
This phase is especially suitable for separating racemic
trifluoroacetylated carbohydrates into their enantiomers." 231 For a-m-galactose (pyranose) the separation factor CL at 320°C is 2.14.
7. Applications
t lrnini
Fig. 16. Separation of the enantiomers of a-hexachlorocyclohexane on 17 at
190 "C. Deactivated glass capillary column, 50 m x 0.25 mm I.D. Carrier gas:
H,, 1 bar 11141.
raphy,['] give large separation factors CL with these stationary
phases, as also do y-, 6-, and e-lactones. Of particular interest
is the separation of the enantiomers of a-hexachlorocyclohexane at 190°C (Fig. 16).11141
6.6. Heptakis(O-(S)-2-hydroxypropyl-per-O-methyl)B-cyciodextrin (PMHP-B-CD, 20)
This viscous liquid, which is hydrophilic, relatively polar,
and water-soluble, is a suitable enantioselective stationary
phase for coating onto fused silica capillary columns.[1201
Mass-spectrometric analysis has indicated that the product
is a mixture of partially alkylated derivatives. Various types
of compounds have been separated into their enantiomers on
this phase, including trifluoroacetylated secondary amines,
trifluoroacetylated amino alcohols, epoxides, j3- and y-lactones, derivatives of norbornene and norbornane, and
monocyclic acetals with five- and six-membered rings. Surprisingly, introducing an additional chiral center via the 2hydroxypropyl substituent has no effect on the observed
enantioselectivity for a wide range of guest molecules, as was
shown by comparative experiments using derivatives with
substituents having the (S) and ( R ) configurations.['201
Apart from some isolated cases, no advantages resulted from
using the corresponding a- (19) or y-cyclodextrin derivatives.'12'1
The main area of application of chiral cyclodextrin phases
at present is in enantiomer analysis by gas chromatography
of chiral compounds that can be vaporized without decomposition. Such applications include the determination of the
enantiomeric purity of products obtained by enantioselective
reactions and elucidation of the enantiomeric composition
of natural substances (e.g., pheromones and flavors) and of
pharmaceutical products.['311 An advantage of the method
is that many compounds can be analyzed directly without
derivatization or sample pretreatment. A few representative
examples are briefly mentioned here.
The enantioselective telomerization of butadiene and
formaldehyde to cis- and trans-2,s-divinyltetrahydropyran
and the enantiomeric analysis of the products has been described." 2 7 , 1321 The stereochemistry of the rhodium(1)-catalyzed hydrogenation of 1-ethyl-2-methylcyclohexeneto 1ethyl-2-methylcyclohexane has been investigated; no enantioselectivity was observed in this case.[961An analysis of the
flavor constituent of hazel nuts, (E)-5-methylhept-2-en-4one (filbertone), revealed a mixture with a preponderance of
the S antipode.11331
6.7. Hexakis(2,6-di-O-n-pntyl)-u-cyclodextrin
(Dipentyl-u-CD,21) and Heptakis(2,6-di-O-n-pentyI)B-cyclodextrin (Dipentyl-B-CD, 22)
These stationary phases, which are more polar than the
per-n-pentyl cyclodextrin derivatives, can easily be coated
onto fused silica capillary columns.['221For these phases the
liquid consistency is again attributed to the presence of a
mixture of partially alkylated derivatives. In experiments
with 65 racemic compounds, striking differences were found
between the enantioselectivities obtained with the a-, @-, and
The range of compound
types that can be separated on 21 and 22 is similar to that for
per-n-pentylated cyclodextrins.
t tminl
t lminl
Fig 17. Separation of the enantiomers of a-pinene and dipentene (Iimonene)
on 1 in OV-1701 at 50°C. Fused silica capillary column, 25 m x0.25 mm I.D.
Carrier gas: H, 0.9 bar. Left: racemic mixtures. Right: determination of the
enantiomeric purity of the aikenes in a pharmaceutical formulation by headspace analysis [ 1341
Angew. Chem. Int. Ed. Engl. 29 (1990) 939-9S7
Enantiomeric excesses ee differing considerably from
100 YOwere found for optically active a-pinene and limonene
in commercial samples and in a pharmaceutical preparation
(Fig. 1 ,).I1341 The enantiomeric composition of y-lactones in
foodstuffs has been determined by multidimensional gas
chromatography.[’ 351
The absolute configuration of a-damascone and a-ionone
in black tea has been investigated (Fig. 18).[1361A number of
The results obtained so far on the gas-chromatographic
separation of enantiomers on cyclodextrin derivatives show
a remarkably wide range in the types of racemates which can
be resolved. They include strongly polar chiral diols, free
carboxylic acids, derivatized amino acids and carbohydrates,
metal coordination compounds, allenes, propellanes, planarchiral cycloalkenes, and unfunctionalized monocyclic hydrocarbons. In all cases, molecules that are identical in constitution but differ in their configurations (incongruent mirrorimage structures) are discriminated by means of an aracemic
chiral stationary phase, thereby allowing chromatographic
enantiomer separation. The interaction necessary for this to
occur can be strong or weak[61and can consist of an inclusion process and/or other chemical interactions.
With regard to the nature of the host-guest interaction in
cyclodextrins, a number of different contributions have been
considered!”. 13,
(1) steric fit by conformational
change of the guest molecule and/or of the cyclodextrin molecule (induced fit) during the molecular inclusion proc e ~ s , [ ~1401
~ * - (2) hydrogen bonding,[24*14’] (3) van der
Waals interactions (London dispersion forces and dipoleinduced-dipole interactions), (4) hydrophobic interactions,
( 5 ) dipole-dipole interactions, (6) charge-transfer interactions, (7) electrostatic interactions, (8) release of “highenthalpy” water molecules from the cyclodextrin cavity,
(9) release of solvent molecules from the cyclodextrin cavity
with a gain in entropy, (10) relief of the ring strain of the
To achieve enantiomer separation by the inclusion process, the molecular geometry should play an important role
(shape selectivity and size selectivity). For polar guest
molecules chemical interactions can also be important
(chemical functionality selectivity). The results obtained so
far surprisingly show that the molecular shape of the guest
can be varied over a wide range. Thus, in addition to cyclic
compounds, branched acyclic molecules such as underivatized ketones, haloalkanes, olefins, and allenes can be separated into enantiomers. Moreover, the molecular size does
not appear to be a crucial factor. Armstrong et al. have even
questioned the importance of the inclusion process, since
certain guest molecules do not exhibit selectivity with respect
to the diameter of the cyclodextrin cavity (a, p, or y).[lzol
Furthermore, peak broadening, which often occurs with inclusion mechanisms due to delayed mass transfer, is not observed here.
Whereas the enantiomer separation of saturated cyclic hydrocarbons such as trans-l,2-dimethylcyclohexanecan be
unambiguously attributed to a molecular inclusion proc e s ~ , [for
~ ~polar
substrates one must also take into account
additional enantioselective interactions (e.g., hydrogen
bonding with cyclodextrins having acyl groups at the 3-position). In this connection it is significant that Schurig et al.
have also observed enantiomer separations (e.g., of racemic
cyclic ethers and aromatic oxiranes) using open-chain per-npentylated a m y l o ~ e . [ ’Thus,
~ ~ ~ enantiomer discrimination
can also presumably occur at the outer surface of the cyclodextrin molecule. Surprisingly, modifying the cyclodextrin cavity opening by incorporating chiral anchoring groups
has no effect on the enantioselectivity of the host molecule.
For example, heptakis(2,3,6-tri-0-(S)-2’-methylbutyl)-P-cyclodextrin (11) does not allow enantiomer separation,[921
1 5 3
t [rninl
Fig. 18. a ) Separation of the enantiomers of a-ionone on octakis(3-0-methyl2,6-di-O-n-pentyl)-y-cyclodextrin.
b) Gas chromatogram of a fraction from an
extract of black tea. c) Coinjection of a) and b). Deactivated glass capillary
column, 60m, 115°C. Carrier gas: H,, 1 bar [136].
olefinic algal pheromones (multifidene, aucantene) were separated into their enantiomers using cyclodextrin derivatives,
thus laying the basis for clarifying their stereochemical interrelationships.[’371 The determination of the enantiomeric
composition of chiral cis- and trans-2,3-epoxy alcohols,
which can be prepared by asymmetric epoxidation and have
numerous applications, was achieved using per-n-pentylated
a-cyclodextrin!’ 21 A particularly interesting application is
the gas-chromatographic determination of the enantiomeric
purity of tricarbonyl(q4-2-methylene-1
and tricarbonyl(q4-2-methyl-I ,3-butadiene)iron(o).[’ ’1
8. Remarks on the Separation Mechanism
As early as 1952, Cramer, in a pioneering study of the
resolution of racemates on c y c l o d e ~ t r i n s ,wrote:
[ ~ ~ ~ “From
this we have discovered a relationship between molecular
shape and biochemical activity. Here the cyclodextrin molecule exerts its effect solely as a result of its geometrical shape,
not by means of functional groups. However, only those
reagents that can enter the molecular cavity can be considered. Thus, striking parallels with the lock and key relationships of biochemical processes are revealed here.” On the
basis of thermodynamic parameters obtained for cyclodextrin inclusion complexes, Saenger“ concluded that “the
inclusion process does not depend primarily on the (chemical) nature of the guest molecule”.
Angew. Chem. I n r . Ed. EngI. 29 (1990) 939-957
and the configuration of the 2-hydroxypropyl group in
derivatives of 19 and 20[1181does not affect the observed
enantioselectivity.l’201On the other hand, the separation of
the enantiomers of acylated alcohols and amines on polar
cyclodextrin derivatives is dependent on the nature of the
Since the acetyl and trifluoroacetyl
acyl group (Fig. 19).r1191
groups are similar in size and shape, the difference in enantioselectivity is attributed to the different electronic properties of these two acyl groups (polarity and propensity to form
hydrogen bonds).“ 191
smaller opening containing the primary methoxy groups is
Huruta et al. therefore predicted that permethylated cyclodextrins should have a greater propensity for
enantiomer differentiation than the corresponding underivatized compounds.[’38- I4O1 Contrary to this prediction it is
found that, compared with underivatized cyclodextrins in
formamide solution (cf. Fig. 2),‘*’, 8 2 1 the separation factors
c( for pinenes (Fig. 17) and pinanes, for example, on the
alkylated and acylated cyclodextrins investigated so far,
both in their undiluted and diluted forms, are very small. It
is only the very high efficiency of the high-resolution capillary columns used that allows quantitative enantiomer separation in these cases with CL values of 1.02 and above. This
corresponds to a value of only about 20 cal mol- for the
difference in the free energies for enantiomer discrimination
AR,s(AGo).[71Enantiomer separations based on such small
free energy differences are thus attributed to a rather unpredictable “random effect”,[93]which is unlikely to lead to
mechanistic conclusions. Accordingly, the observation that
minor changes in the host structure can result in reversals of
the elution order of enantiomers (Fig. 20)f”81does not seem
It is known from X-ray crystal structure studies that hydrogen bonding can occur between the host molecule and
suitable guest molecules.[24,86, 1 4 ‘ ] Crystal structures of inclusion complexes formed by the enantiomer of mandelic
acid indicate that such hydrogen bonds can make a cruciaI
contribution to enantiomer differentiation even for permethylated cyclodextrins. Thus, the complex formed between
permethyl-a-cyclodextrin and D-mandelic acid contains a
hydrogen bond between host and guest which is not present
in the complex with L-mandelic
It is not clear to
what extent the structure in the solid state can be extrapolated to that in solution (packing effects). That some free alcohols undergo enantiomer separation, whereas the corresponding acetates do notJg1- 9 3 1 may be due to the
contribution of hydrogen bonding to chiral recognition. Permethylated cyclodextrins, in contrast to the underivatized
compounds, can only act as acceptors for hydrogen bonding
to compounds with protic groups.
Fig. 19. Effect of derivatlzation on the separation of the enantiomers of acyldton 22. Bottom left: 2-amied amines. Top: 1,2,3,4-tetrahydro-l-naphthylamine
no-1-methoxy-propane on 22. Right: 2-amino-1-methoxy-propaneon 20 [119]
In many cases, minor structural differences in the guest
molecules can have dramatic effects on chiral recognition r921
For example, a comparison of different alkyl-substituted ybutyrolactones showed that, whereas the chain lengths of
alkyl substituents at the y-position (Fig 9) and additional
substituents at the P-position have only slight effects on the
separation factor u, introducing a methyl group at the cl-POsition relative to the carbonyl group leads to inferior separation ]’91 Again, whereas 3,3,5-trimethylcyclohexanonewas
successfully separated quantitatively into its enantiomers on
1, no separation was obtained for the 2,2,6 isomer Ig2]
Methylation of cyclodextnns increases the depth of the
molecular cavity (to about 1000- 1100 pm) and enhances its
conical shape, since the larger opening containing the secondary methoxy groups is increased while the opposite
t lminl
Fig. 20. Reversal of the order of elution of enantiomers on polar cyclodextrin
derivatives. Separation of the enantiomers of pertrifluoroacetyl-P-D,r-arabinose (left) and mandelic acid methyl ester (right) on A) 20 and B) 22 at 80°C
and 120°C. respectively Fused silica capillary column, 10 m. Carrier gas: N,
Angew. Chem Int Ed. Engl. 29 (1990) 939-9S7
The so-called classical hydrophobic interactions are mainly caused by entropy effects. Although the formation of the
host-guest complex is accompanied by a decrease in entropy,
the removal of the hydration shell from the guest molecule
and the release of solvent molecules from the cyclodextrin
cavity can result in an overall increase in entropy. It will be
shown later that the latter type of hydrophobic interaction
could also be important for cyclodextrin derivatives diluted
in polysiloxane, whereas the effect of any dehydration can be
neglected, since water molecules should be completely removed during the conditioning of the gas-chromatographic
columns and the injection of the substrate at high temperatures.
Since the formation of the inclusion complex is largely
independent of the chemical properties of the guest molecule, it has been suggested that one of the driving forces for
the inclusion process is the removal of high-enthalpy water
molecules from the molecular cavity, where they reside in an
unfavorable, hydrophobic environment.[
According to
the argument above, this contribution should be negligible
under the conditions of the gas-chromatographic experiment.
Enantioselective interactions between chiral and prochiral
guest molecules and cyclodextrins have been detected by a
number of methods.[’8.31332.
341 Since the use of cyclodextrins for enantiomer separation in gas chromatography only
began quite recently, no theoretical models for the mechanism of chiral recognition have yet been put forward. On the
other hand, for the separation of racemates by fractional
crystallization with cy~lodextrins,[~~1
for cyclodextrin-catalyzed reactions,[’431and for liquid chromatography (HPLC)
on cyclodextrin stationary phases, a three-point interaction
model analogous to the models of Eusson and Stedmanf1441
and of D ~ Z g l i e s hhas
~ ’ ~been
~ ~ proposed.[61.621 According to
this model, a lipophilic, and preferably aromatic, part of the
molecule is included in the cyclodextrin cavity, and two polar
substituents interact with the secondary hydroxyl groups at
the wider opening of the cyclodextrin cavity (e.g., by hydrogen bonding). This model is an appropriate one to account
for enantiomer differentiation of arenes with a chiral center
in the side-chain and several polar groups at the chiral center
(e.g., mandelic acid derivatives and propanolol). However, it
is not suitable for alicyclic compounds with chiral centers in
the ring, nor for compounds that have no functional groups
capable of forming hydrogen bonds. The necessity for an
“aromatic presence”, as observed in liquid-chromatographic
enantiomer separations on carbohydrate stationary phases,
is not found in the gas-chromatographic counterpart.
Although van der Waals forces have only a short range
(being proportional to F 6 ) , the close contact between host
and guest molecules can cause these forces to contribute
appreciably to the selective inclusion process. The quantitative enantiomer separation observed for chiral alkanes on 1
(cf. Fig. 5),IE2, 941 where other interactions can be excluded,
shows that on cyclodextrin phases van der Waals interactions alone are sufficient for differentiating between enantiomers. In a molecular modeling study,[146]force field calculations and molecular dynamics simulations were applied
to the inclusion complexes formed by the enantiomers of cisand trans-1-ethyl-2-methylcyclohexane with 1. From Figure
21 (left) it can be seen qualitatively that there is indeed a close
A n p u , . Chem. f n t . Ed. Engl. 29 (1990) 939-957
contact between the van der Waals surfaces in the complex of
trans-1-ethyl-2-methylcyclohexane (blue) with 1 (red). The
complex is viewed from the side of the secondary methoxy
groups, that is, from the wider end of the cavity. Although
the guest molecule is completely enclosed by the host, it was
not possible on the basis of such a small free energy difference to make predictions about the diastereo- and enantioselectivities, and the calculated values for the stabilities of the
complexes were not in agreement with the experimentally
observed gas-chromatographic elution order of the cis and
trans isomers (cf. Fig. 5).f921As already mentioned, carrying
out molecular calculations on the basis of crystal structure
data and of thermodynamic parameters (AR,JAG0),
AR,JAH0), AR.JAS0)), determined by gas chromatography
for highly enantioselective host-guest complexes, is a worthwhile task for the future.
Fig. 21. Molecular modeling study of the inclusion complex of rrans-l-ethyl-2methylcyclohexane (one enantiomer) with 1 11461. Left: with van der Wdals
radii. Right: without van der Waals radii.
Gas-chromatographic separation of enantiomers is a thermodynamically controlled process. The enantiomer discrimination involving a racemic guest and an aracemic (enantiomerically pure) host i s described by the Gibbs-Helmholtz
equation (a) and corresponds to the sum of the enthalpy
and entropy differences in the complexation of the enantiomers with the chiral stationary phase. The process is temperature-dependent, and it has recently been observed that
the order of elution of the enantiomers on a given chiral
phase (amino acid derivatives or metal complexes) is reversed above the “isoenantioselective temperature” Ti,, .[14’]
For a thermodynamic characterization of the mechanism
of enantiomer discrimination it is useful to distinguish between the total interaction, as described by -AGO, and the
difference in free energy between the complexes of the two
enantiomers, -AR,s(AG)o.[61 One can assume that for a given
interaction between host and guest, which can be strong or
weak, small additional interactions are sufficient to produce
enantiomer discrimination. Accordingly, for the cyclodextrin inclusion process the crucial factor is the competitive
behavior of the enantiomers, as determined by the enthalpy
and entropy contributions to chiral recognition at a given
temperature. Thermodynamic data on enantiomer discrimination have been determined for cis-I -ethyl-2-methylcyclo953
hexane and cis-pinane on 1 in OV-1701 (0.07 M) (cf. Table
961 The thermodynamic parameters were obtained
from relative retention data, using the concept of the retention increase R adopted from complexation gas chromatography,[6. 1251
Table 3. Thermodynamic parameters for the separation of the enantiomers of
and cis-pinane on 1 in OV-1701 (0.07 M) obtained from temperature-dependent measurements between 30 ‘C and 70 ‘C
using n-octane and n-decane as standards 195, 961.
[kJ mol-’1
[J K - ’ mol-’1
Surprisingly, AR,JAH0)and A&AS0) have opposite signs
for the cycloalkanes investigated. It follows from this that no
(positive) isoenantioselective temperature exists, and therefore no peak inversions are expected in this system (see also
Ref. [I 181). The increase in entropy, which is unusual for an
association process, may be explained by the fact that the
solvent (polysiloxane) molecules released from 1 on inclusion of the hydrocarbon molecules gain individual degrees of
freedom. Both - A,,,(AH”) and AR.JAS0) thus contribute
to the enantiomer discrimination, as described by
-AR,S(AGo), and a quantitative enantiomer separation occurs even for small entropy and enthalpy differences. A low
value of A,,,(AH”) implies that the enantiomer separation
shows only a slight temperature dependence, and quantitative separation is obtained even at high temperatures (e.g.,
250 “C), as is generally found using cyclodextrin stationary
A systematic search for enantioselective host-guest systems with high separation factors (a > 2) is not only a prerequisite for the preparative applications of racemate separation, but is also warranted in order to optimize model
systems that can be used for mechanistic studies, thermodynamic measurements of enantioselectivity, crystal structure
studies, and computer-aided calculations on the different
diastereomeric association complexes.
9. Practical Aspects and Outlook
High-resolution gas-chromatographic separations of
enantiomers using cyclodextrin derivatives are achieved at
present only with analytical capillary columns. Separations
on a semipreparative scale can therefore only be expected in
rare cases with the separation factors a that are currently
obtained. Enantiomer separations can be performed in the
usual temperature range of 25 to 250 “C. For special applications it is also possible to use lower temperatures (-25 “C).
The enantioselective phases have a long lifetime if oxygenfree carrier gases are employed (e.g., nitrogen, helium, hy954
drogen). For samples diluted in solvents the quantities injected should be minimized by using the split injection technique
(1 :100). The successful coating of glass or fused silica capillary columns with undiluted or diluted cyclodextrin derivatives requires some expertise and can only be carried out
satisfactorily on pretreated, deactivated surfaces.“ 241 Established methods of treatment are leaching, persilylation, or
Carbowax deactivation.[’24]Capillary columns coated with
cyclodextrins are commercially available from well-known
chromatography suppliers, including Chrompack (Middelburg, Netherlands) (diluted cyclodextrin 1 on fused silica
capillary columns, cf. Section 5),[981 Macherey-Nagel
(Diiren, FRG) (undiluted n-pentylated/acylated cyclodextrin phases 13-16 (Lipodex) on glass capillary columns, cf.
Sections 6.1-6.5;[’051 see also Ref. [I271 for the same on
fused silica capillary columns), and Astec, Advanced Separation Technologies (Whippany, NJ, USA), and ICT (Frankfurt/Main, FRG) (undiluted polar cyclodextrin phases 1923 on fused silica capillary columns, cf. Sections 6.66.8).[1201
For the enantioselective cyclodextrin phases described up
to now the user must distinguish between the following factors: availability, performance, reproducibility, long-term
stability. The inspection of chromatograms, insofar as they
are available, often provides the expert with decisive information. The use of cyclodextrins as solutions in polysiloxanes has resulted in columns of high efficiency, even for
strongly polar substrates such as diols (Fig. 6)197,981and
Thus, for the first eluted peak of trans-2,3dimethyl-1,4-dioxaspiro[4.4]nonane (Fig. 8), the number of
effective plates was measured as N = 79 400. For a column
length of 40 m this corresponds to a height equivalent theoretical plate (HETP) of 0.50 mm. Accordingly, despite the
small separation factor (a = 1.02), an almost complete resolution of the peaks (R= 1.33) is achieved. For capillary
columns with 1 in the molten state at 14O-15O0C, HETP
values of only 1.6 and 1.4 mm were obtained, and a comparable efficiency (0.3 mm) was only reached at 200 0C.[891
On undiluted polar cyclodextrin derivatives, peak resolution
is not always ideal either (Fig. 19).[1191
Poorer HETP values
were measured for packed columns with P-cyclodextrin in
formamide solution (1.8 mm;1791
cf. also Fig. 2) and for gassolid packed columns with p-cyclodextrin on Chromosorb
W (10.8 mm).[741
Chemically binding the cyclodextrin derivatives to polysiloxanes, in a manner analogous to “Chirasil-Val”[51 or
“Chirasil-Metal”,[12s1is expected to further increase the efficiency and lifetime of the columns.[*1The immobilization of
such “Chirasil-Dex” stationary phases on the capillary surface is required for using supercritical gases for enantiomer
separation (supercritical fluid chromatography, SFC) or for
employing electrically driven chromatographic systems. As
mentioned before, the low separation factors CI observed up
to now render it unlikely that the cyclodextrin phases available at present can generally be used for preparative-scale
gas-chromatographic enantiomer separations. Also, there is
at present no universal enantioselective cyclodextrin station-
[*] Note added in proof Successful experiments have been reported recently
[148]: e.g.. “Chirasil-Dex” [149].
Angew. Chem. h t . Ed. Engl. 29 (1990) 939-957
ary phase available, and unsuccessful separation attempts
are, regrettably, hardly ever reported in the literature. In our
experience, the success rate for quantitative enantiomer separations with a given cyclodextrin phase such as 1 lies well
below 50%. Minor changes in the structure of the guest
molecules often have a great effect on the enantioselectivity.
Even for compounds that are structurally related, often only
certain isomers or members of a homologous series can be
separated into their enantiomers on cyclodextrin phases. It is
therefore difficult to make predictions.
The objective for the future is to discover enantioselective
stationary phases based on cyclodextrins that will have as
wide a range of applications as possible. A further development still awaited is the preparation of phases with inverse
configuration by total synthesis;1261they are required for
carrying out enantiomeric analyses on highly enriched substrates in cases where the trace component is eluted after the
major component, making integration difficult. Owing to
the lack of such inversely configurated phases, in no case has
a formal proof of successful enantiomer separation on
cyclodextrins, in contrast to phases consisting of amino
acids[31or metal
been possible. Such proof is
normally provided by using the racemic form, which gives
rise to peak coalescence, or the mirror-image stationary
phase, which causes peak inversion.
In the next few years a steady growth of interest in chiral
stationary phases based on cyclodextrins for capillary gas
chromatography can be expected. Selective achiral separations (of geometrical isomers, diastereomers, positional isomers, etc.), as well as methods for modifying complex elution
profiles, will also play an important role. However, cyclodextrin stationary phases are not expected to entirely replace or supersede the amino acid or metal complex stationary phases mentioned in the introduction. Instead, they
represent a considerable extension to the range of techniques
available to the enantiomer analyst, in accordance with the
comment by Gil-Av, the pioneer of gas-chromatographic
enantiomer separation, that “every phase, whether low or
high molecular, will be recognized as suiting best a certain
niche of applications”.r841
The authors thank the Deutsche Forschungsgemeinschaft
and the Fonds der Chemischen Industrie for supporting this
work. We are indebted to Professor Berson (New Haven) for
providing a sample of ethyl hexa-3,l-dienoate and to Professor
Fitjer (Gottingen) for providing a sample of modhephene. We
thank Dip1.-Chem. Dieter Schmalzing, Michael Schleimer,
and Martin Jung for their valuable contributions to the gaschromatographic enantiomer separation on cyclodextrin stationary phases. I/: S . thanks Professor Konig (Hamburg),
Professor Armstrong (Rolla, Missouri), and Professor
Smolkova-Keulernansova (Prague) for reprints and preprints
of recent work, and especially Professor Gil-Av (Rehovoth)
and Professor Bayer (Tiibingen) for fruitful scientqic exchange over the years.
Received: March 1, 1990 [A 780 IE]
German version: Angew. Chem. 102 (1990) 969
Translated by Dr. Jack Beeconsall, Pwllheli (UK)
Angew. Chem. I n t . Ed. Engl. 29 (1990) 939-957
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