Single isomer cyclodextrins as chiral selectors in capillary electrophoresis

Journal of Chromatography A 1627 (2020) 461375

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Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Single isomer cyclodextrins as chiral selectors in capillary
electrophoresis
˝ a, Eszter Kalydi a,b, Milo Malanga b, Gábor Benkovics b, Szabolcs Béni a,∗
Ida Fejos
a
b

˝ út 26, Hungary
Department of Pharmacognosy, Semmelweis University, Budapest, H-1085 Ülloi
CycloLab, Cyclodextrin R&D Ltd, Budapest, H-1097 Illatos út 7, Hungary

a r t i c l e

i n f o

Article history:
Received 11 May 2020
Revised 24 June 2020
Accepted 28 June 2020
Available online 5 July 2020
Keywords:
Capillary electrophoresis
Synthesis of single isomer cyclodextrin
derivatives

Chiral separation
Enantiomer migration order
Enantiorecognition
Supramolecular interactions

a b s t r a c t
Since decades, cyclodextrins are one of the most powerful selectors in chiral capillary electrophoresis
for the enantioseparation of diverse organic compounds. This review concerns papers published over the
last decade (from 2009 until nowadays), dealing with the capillary electrophoretic application of single
isomer cyclodextrin derivatives in chiral separations. Following a brief overview of their synthetic approaches, the inventory of the neutral, negatively and positively charged (including both permanently
ionic and pH-tunable ionizable substituents) and zwitterionic CD derivatives is presented, with insights
to underlying structural aspects by NMR spectroscopy and molecular modeling. CE represents an ideal
tool to study the weak, non-covalent supramolecular interactions. The published methods are reviewed
in the light of enantioselectivity, enantiomer migration order and the fine-tuning of enantiodiscrimination by the substitution pattern of the single entity selector molecules, which is hardly possible for their
randomly substituted counterparts. All the reviewed publications herein support that cyclodextrin-based
chiral capillary electrophoresis seems to remain a popular choice in pharmaceutical and biomedical analysis.
© 2020 The Author(s). Published by Elsevier B.V.
This is an open access article under the CC BY-NC-ND license.
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1. Introduction
The understanding of chiral recognition is not only essential in
biology and supramolecular chemistry, but also instructive in stereoselective synthesis and chiral separation. The analytical scale separation of chiral analytes is one of the most popular applications
in capillary electrophoresis (CE), commonly achieved by adding
chiral selectors, most frequently cyclodextrins (CDs) to the background electrolyte (BGE). These popular selectors are composed
of (1,4)-linked α -d-glucopyranose units forming a truncated cone
shape and contain a hydrophilic outer surface surrounding a rather
lipophilic cavity [1]. The latter enables inclusion complex formation with a wide variety of small molecules ranging from endogenous bioactive compounds, through agrochemicals, fragrances
to oligopeptides, pharmaceuticals to food components. The chiral
recognition by CDs is mostly based on the different interaction
affinities between the selector and the enantiomers of the analyte

where diastereomeric host-guest type complexes with subtle structural differences are formed. Complexation of the guest molecules
∗

Corresponding author.
E-mail address: (S. Béni).

often occurs via their inclusion into the CD cavity either from the
narrower or the wider side of the truncated cone, displacing solvent molecules from the cavity, however outer sphere interactions
may also contribute to the differential recognition of the enantiomers. Van der Waals, hydrophobic and electrostatic interactions
as well as hydrogen bonding are considered as driving forces of
complex formation besides the steric factors. Contrary to chiral
separations by liquid or gas chromatography, a successful enantioseparation in CE can still be accomplished in the case of equal
binding constants due to different mobilities of the diastereomeric
complexes [2].
CE represents one of the most powerful analytical techniques
not only for the physical separation of enantiomers but also for
a better understanding of the molecular basis of (enantioselective) intermolecular interactions [3]. A conceptual benefit of CE
for the investigation of peculiar stereochemical mechanisms of
non-covalent interactions is the cumulative character of this technique. CE offers significant advantages compared to other separation methods such as the high efficiency (the high plate numbers) allowing even the observation of weak enantioselective intermolecular interactions, that are invisible to other techniques,
the fast separation time, the miniaturization and the amenabil-

/>0021-9673/© 2020 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license.
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I. Fejos,

ity to mathematical modeling and “dry-chemistry” optimization

[4,5]. Capillary electrophoresis also offers an easy, fast and costeffective way to study the enantioselectivity of new chiral selector
candidates, as the selector has to be simply added to the background electrolyte. A wide range of chiral test compounds could
be screened in a short time and due to the high flexibility of chiral capillary electrophoresis (CCE) and the possibility of automation, several parameters influencing the enantioseparation (such
as the selector concentration in the BGE or the pH of the running buffer, co-solvents, application of external pressure/vacuum)
could be studied. Furthermore, no need of selector immobilization and the low consumption of selectors, selectands and running buffers enables the use of rather expensive chiral selectors as
well as dual or multiple selector systems. Moreover, this technique
also carries the possibility of enantiomer migration order (EMO)
reversal without affecting the affinity pattern between the selector
and the analyte [6]. On the other hand, CE suffers from the disadvantage of being incapable to provide any direct structural information about the intermolecular diastereomeric associates involved in chiral recognition/separation. The combination of CE and
NMR spectroscopy is a powerful tandem for a better understanding
of chiral recognition mechanisms, while extending these investigations by state-of-the-art molecular modeling studies, a deeper insight into the pertinent noncovalent host-guest interactions could
be achieved [7]. The orthogonal techniques of spectroscopy and
equilibrium chemistry may help to explain and predict beneficial
CCE characteristics, such as opposite migration order of the enantiomers when different CDs are used, or they may assist in development of the optimal conditions for a CCE enantioseparation.
2. Single isomer CD derivatives (SIDs)
Although cyclodextrins have been known since the end of 19th
century [8], their first application in electromigration methods
awaited nearly 100 years. CDs were originally applied for the separation of achiral compounds [8]. Later, specifically in 1988, the
first papers on the chiral separation utilizing CDs in CE format
were published [9–12]. Since that time, a plethora of CD derivatives
has been synthesized and employed as chiral selectors. Native CDs
were historically among the first selectors to be evaluated. Their
applications may be limited by inadequate aqueous solubility, especially for the most commonly used β -CD, since the optimal CD
concentration necessary for the enantiomer separation could not
be reached. The lack of charged functionalities are also restricting
regarding intermolecular interactions, therefore, tailor made synthetic modifications of the native CDs have been extensively performed to provide a large variety of selectors decorated with various substituents, improving water solubility and sometimes also
the enantiorecognition towards a specific class of chiral analytes
[13,14]. The complexity of delicate factors influencing enantiomerselector interactions makes the ab initio prediction or design of
a successful enantioseparation particularly difficult. There is currently a consensus that no generally applicable model has resulted
from the large body of structural studies to predict enantiomeric

discrimination, i.e. how to design cyclodextrin substitution in order to improve the resolution of enantiomers.
In order to satisfy the urgent need of structurally diverse selectors in the pharmaceutical, cosmetic and food industries, a wide
range of various CD derivatives (such as methylated, sulfated, carboxymethylated, sulfobutylated ones) became commercially available as randomly substituted derivatives [15]. Despite the ease of
their preparation and their extensive application in CCE, a randomly substituted CD product is in fact a complex mixture of cyclodextrin isomer species, variable both in degrees and position(s)
of substitution. The manufacturer usually declares only their degree of substitution (DS): the average number of derivatized OH

groups. While the DS became nowadays synthetically reproducible,
the same substitution pattern could not be guaranteed for every
lot, resulting potentially in poor reproducibility of CE separation
and the failure in method validation by practitioners. With a dual
aim of gaining a better control on the structural diversity of CD
derivatives and a more systematic way to investigate molecular
recognition processes, potentially leading to more predictable separations, single isomer CD derivatives have been synthesized for
ca. 20 years now and characterized both structurally and from the
viewpoint of CE separation efficiency [13]. Based on several earlier studies of Vigh and co-workers [16–18], the importance of using chemically uniform, better-defined pure isomers as chiral resolving agents from both fundamental and practical points of view
was reviewed in 2009 [13]. To understand the influence of the substituent moiety, the site and extent of substitution on enantiodiscrimination, CD derivatives with systematically varied substitution
patterns are still highly demanded. With the help of SIDs both
NMR spectroscopic characterization and molecular modeling at the
atomic level are feasible contrary to the randomly substituted selectors, where neither theoretical predictions nor accurate explanations of the experimental results are possible.
The use of single isomer selectors in the studies reviewed
herein has been emphasized for two reasons. One is to eliminate the drawback of dozens of isomers in commercially available randomly substituted CDs, resulting in selector mixtures illdefined both structurally and in enantiodifferentiation; the other
is to provide a solid and comprehensive theoretical framework on
the enantiomer migration in electrophoresis and identify the variables in order to predict successful separations [19–21]. Typical
SIDs are the mono-substituted CDs (having only one substituent
per CD molecule in a defined position) and the persubstituted CDs
(having all hydroxyl groups substituted in the same positions). Besides the open-chain derivatives, less common SIDs are the capped
CD derivatives, bonding a chain to two different points of the rim,
forming a bridge (hemispherodextrins, HSDs).
2.1. Synthetic challenges and strategies to obtain single isomer CDs
SIDs are generally prepared via multistep synthesis and, when

commercially available, are rather expensive products. In the following sections we provide a brief overview of the synthetic strategies for the preparation of SIDs.
2.1.1. General chemistry of CDs
CDs have 18, 21 and 24 free hydroxyl- (OH) groups in case of
the α -, β - and γ -CD respectively, one primary and two secondary
hydroxyls on each glucopyranose units, where the chemical modifications can take place [22] (see Fig. 1).
The primary hydroxyl groups (OH-6) are the most basic, most
nucleophilic and less hindered. By careful selecting a weak base,
the primary rim of the CD can be promptly modified with a large
variety of electrophiles, even the bulky ones. The hydrogen bond
between the OH-2 and OH-3 of the adjacent glucopyranose units

Fig. 1. Cartoon representation of cyclodextrins along with the sites of modification.


˝ E. Kalydi and M. Malanga et al. / Journal of Chromatography A 1627 (2020) 461375
I. Fejos,

and the neighboring electron-withdrawing anomeric acetal function are responsible for the highest acidity of OH-2. These hydroxyl moieties can be selectively deprotonated, for example, in
anhydrous basic conditions. The hydroxyl groups at the 3-positions
(OH-3) are the less reactive and most hindered. Usually, modification of the 3-positions requires extensive use of protective groups,
nevertheless, the use of reagents able to strongly interact with the
CD cavity can result in direct regioselective substitution of these
positions.
2.1.2. Persubstitution
In persubstituted CDs, all the glucopyranose units are equally
modified, either on selected positions or on all the OH groups.
Per-2,3,6-tri-O-substituted CDs can be prepared in a straightforward manner by utilizing native CDs as starting material. In particular, a large variety of per-2,3,6-tri-O-alkyl and -O-aryl substitution has been achieved in anhydrous conditions with polar, aprotic
solvents (dimethyl sulfoxide (DMSO) and N,N-dimethylformamide
(DMF), mostly) and strong base (mainly hydride). Most recently, efficient and exhaustive alkylation procedures, based on phase transfer catalysis (PTC) have been reported by our group [23]. The
application of PTC conditions allows the industrial scale-up of a

plethora of per-alkylated CDs by utilizing as starting material commercially available partially alkylated CDs.
Per-2,3,6-tri-O-acylation is also easily achieved from native CDs.
In this case, organic acid anhydride (for example, acetic anhydride)
or mixture of anhydride and the corresponding carboxylic acid
(for example, acetic anhydride and acetic acid) are utilized as solvents and the addition of a catalyst (Lewis acid, mostly) allows the
exhaustive substitution in reasonable time [24]. The use of acyl
halide to produce per-2,3,6-tri-O-acylated CDs in inert solvent is
also a widely explored strategy.
Persubstitutions on selected positions are carried out by exploiting the different reactivity of the OH groups. The primary hydroxyls, for example, are better nucleophiles than the secondary
ones, so they can be selectively modified using a weak base.
The most important representative of the per-6-substituted family are the per-6-halogenated and the per-6-silylated CDs. Per-6halogenated CDs can be prepared by reacting a halogen source,
triphenylphosphine (TPP) and DMF (or N-methyl-2-pyrrolidone)
in Vilsmeier-Haack-type reaction [25]. If a strong halogenating
reagent is applied (thionyl chloride/bromide, phosphorous pentachloride/tribromide), then TPP can be omitted. According to this
classical procedure per-6-chloro-, -bromo- and -iodo-CDs have
been prepared in industrial scale. The per-6-halo-CDs are fundamental key-intermediates as they can be readily displaced by most
nucleophiles, such as azides, amines or thiols.
The tert-butyldimethylsilyl chloride (TBDMSCl) in DMF with imidazole as a base, or in pyridine, gives access to per-6-O-silylated
CDs, the most common primary-side protected CDs [26]. These
compounds can easily be prepared in kg scale according to the
aforementioned synthetic approaches.
Per-2,3-substitution is generally carried out by using per6-silylated CDs as starting material. The exhaustive per-2,3-Oalkylation for example have been achieved by utilizing harsh conditions (hydrides, aprotic solvent, alkylating agent and heating) or,
more recently, by applying soft and up-scalable PTC conditions
(catalyst, aprotic solvent, alkylating agent, room temperature). Per2-O-alkylation of per-6-O-silylated CDs has been also reported by
using aprotic solvent and by carefully selecting a strong base, however, in this case, chromatographic purification is mandatory for
the isolation of the corresponding compounds.
Per-2,6-O-difunctionalization of CDs is also possible. Native
CDs can be selectively alkylated if barium salts are applied to
the mixture. By using mixtures of Ba(OH)2 /BaO in polar, aprotic solvents (usually DMSO/DMF in different ratios), several per-

3

2,6-O-alkylated CDs have been prepared. Among these derivatives,
per-2,6-O-dimethyl-β -CD (DIMEB) has been prepared in industrial
scale. Particularly important for this class of compounds are the
per-2,6-O-silyl-CDs as they are versatile synthons towards per-3-Osubstitued and per-2-O-substitued CDs. Per-2,6-O-silyl-CDs can be
effectively prepared in pyridine, with an excess of silylating agent
at reflux with a catalytic amount of 4-dimethylaminopyridine
(DMAP).
Direct per-3,6-O-disubstitution and/or per-3-O-substitution of
CDs have not achieved effectively, yet. The preparation of these
compounds is usually been achieved by applying multistep synthesis based on different protecting groups.

2.1.3. Monosubstitution
In monosubstituted derivatives, only one glucopyranose unit is
modified in a selected position. Due to the similar reactivity of the
hydroxyl groups, even when a limiting amount of reagent is used,
oversubstitution and/or isomer formation is inevitable. Mono-6substituted CDs are easily prepared by applying a weak base to the
media.
The most important CDs in this class are the mono-6-tosyl-CDs
[27]. The β -analogue is classically prepared by reacting β -CD with
tosyl-chloride in pyridine and isolated in kg scale without the necessity of chromatography. A lot of variations/improvements of this
fundamental reaction have been introduced by time to time. The
α - and the γ -analogues can be prepared in a similar fashion, but
chromatography is necessary to achieve a suitable purity.
Nucleophilic substitution of the tosyl moiety generates the
widest variety of CDs. Mono-6-halo, azido-, thio-, hydroxylamino,
alkylamino CDs have been prepared in this way. Treatment of
mono-6-tosyl-CDs with aqueous alkaline conditions leads to the
formation of peculiar epoxide, the 3,6-anhydro-CD.

Selective secondary side modification is more challenging due
to the higher number of OH groups present, which are forming a
H-bond belt making the molecule more rigid and the OH-groups
less reactive. However, benefiting from the pronounced acidic character of the 2-OH function, by using a controlled amount of a
strong base (usually sodium hydride), selective tosylation at the
2-position has been reported. In this manner, mono-2-O-alkyl-CDs
have been prepared. Recently, mono-2-O-propargyl-β -CDs have
been prepared in high yield by using lithium hydride in DMSO and
propargyl bromide with a catalytic amount of lithium iodide [28].
In the CD field, lithium-based hydrides seem more selective compared to the sodium counterparts.
A more convenient method to obtain selectively 2-OH or 3-OH
substituted derivatives is by the exploitation of the inclusion complex forming ability of the CDs. When a reagent is used, which
forms a stable complex with the CD, then the orientation of the
guest will dictate the site of the substitution. In this manner, the
use of m-nitrophenyl tosylate affords 2-O-tosyl-β -CD in good purity, while the application of 3-nitrobenzenesulfonyl chlorides allows the preparation of 3-O-tosyl-β -CD in good yield.
The regiospecific 3-O-cinnamylation of CD derivatives has also
been reported recently.
A recent and versatile approach to obtain selectively substituted CDs have been widely applied and it is based on the selective deprotection of persubstituted CDs using diisobutylaluminium
hydride (DIBAL). When a permethylated α - or β -CD is treated
with DIBAL, the reaction affords permethylated 2A ,3B -dihydroxy α or β -CD and permethylated 6A -hydroxy α - or β -CD in 55% and
20% yields, respectively. When a perbenzylated α -, β -, or γ -CD is
treated with DIBAL, the reaction leads to perbenzyl-mono-6-OHα /β /γ -CD. The deprotected 6-OH position can be further modified and after the removal of the benzyl protection, mono-6-Osubstituted CDs can be prepared in good overall yield.


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I. Fejos,

Similarly, selective desilylation by DIBAL has also been reported

on various per-6-O-tert-butyldimethylsilylated compounds, to afford the corresponding mono-6A -OH or di-6A ,6D -OH derivatives.
2.1.4. Disubstitution
The selective DIBAL deprotection can be used as starting point
for the preparation of new disubstituted CDs. This approach is
time-consuming and based on extensive chromatography for the
purification of the products, however, the achieved regioselectivity
is highly remarkable. An alternative strategy to get single isomer
disubstituted CDs is based on the use of suitable disulfonyl capping agent followed by the subsequent substitution by the desired
nucleophile. This method can be applied to synthetize 6A , 6B -, 6A ,
6C - and 6A , 6D -disubstituted CDs by using 4,6-dimethoxybenzene1,3-disulfonyl chloride, benzophenone-3,3 -disulfonyl chloride or
trans-stilbene-4,4 -disulfonyl chloride, respectively.
The preparation and unambiguous characterization of heterodisubstituted CDs is a challenging task. Heterodisubstituted
derivatives are usually obtained by the further modification of a
mono-substituted SID. In case of the β -CD, this approach leads to
the formation of three possible pairs of pseudoenantiomers [29].
A higher order single isomer multisubstitution is only rarely reported, however, A, C, E-6-O-trisubstitution of α -CD is easily accessible using trityl chloride as a protecting group. Trityl chloride
is a bulky molecule, therefore has a steric hindering effect, allowing only the substitution of the more accessible primary side, and
only every second glucopyranose unit can be accessed. The remaining free OH functions can be subsequently modified and the
removal of the protecting group results in A, C, E-trisubstituted α CD derivatives.
3. SIDs in chiral capillary electrophoresis
3.1. Application of neutral cyclodextrins in CCE
Neutral CD derivatives are still frequently used in order to
broaden the chiral recognition abilities and fine-tune the physicochemical properties (such as solubility) of the native CDs. The
methylated CDs are the most common members of this group,
composed of selectively mono-, di-, and trimethylated single isomers. Even a slight modification in their structure (degree and/or
the position of methylation) largely influences their properties
such as solubility, complexation ability and enantiorecognition,
thus an in-depth characterization of the β -CD derivatives was
performed recently by our group [23]. In this study, the detailed synthetic procedures clearly indicated that the production of heptakis(2,3,6-tri-O-methyl)-β -CD (TRIMEB or TM-β -CD)
was found to be the most straightforward and easily scalable

to kilogram scale [23]. The syntheses of the per-dimethylated
and the per-monomethylated derivatives require multiple steps
and extensive use of protecting groups, therefore the industrial scale-up was found to be challenging. The introduction of
PTC conditions to each alkylating step allowed the production
of the methylated derivatives in multi-gram (10–100 g) scale
[23]. The synthesis of the primary-side homogeneously substituted, heptakis(6-O-methyl)-β -CD (6-MEB or 6-Me-β -CD) has been
only described in the pioneering work of Takeo et al. (based
on extensive chromatographic purification, resulted in low yields)
[30] and in the work of Uccello-Barretta et al. (using of hazardous reagents) [31]. Our proposed method for the efficient synthesis for per-monomethylated heptakis(6-O-methyl)-β -CD (and
in general for 6-O-alkylated CDs) is better suited for industrial scale up and completely chromatography-free at each step
[32].
The enantiorecognition ability of various selectively methylated
CDs on several amino acid derivatives has already been inves-

tigated previously by Tanaka and his research group [33–38]. A
study dealing with the influence of the extent of the modification of the CD with a specific substituent on chiral separation has
been carried out by Maruszak et al. in 2001 [39]. They studied
three different methylated β -CDs: the randomly methylated β -CD
(DS 1.6–2.0), heptakis(2,6-di-O-methyl)-β -CD (DIMEB or DM-β -CD)
and heptakis(2,3,6-tri-O-methyl)-β -CD, but no significant differences were found in their separation ability of four neurotransmitters. Chankvetadze et al. conducted several studies on the EMO reversal in the presence of native β -CD and the fully methylated TMβ -CD [40–42]. The effect of degree of methylation on the enantiomer migration order has been shown for peptide enantiomers
reviewed by Scriba [43]. The separation of the enantiomers depending on the degree of methylation of CDs was reported for
medetomidine enantiomers by Krait et al. [44]. Randomly substituted M-β -CD as well as the trisubstituted TM-β -CD partially resolved the medetomidine enantiomers, while no enantioseparation
was observed using the 2,6-disubstituted DM-β -CD. The observed
enantioseparation in the case of randomly substituted M-β -CD was
accomplished by constituent CD species with a substitution pattern
differing from that of the 2,6-disubstitution. Schmitt et al. studied
the enantioseparation of several mixture of the single isomer fully
methylated TM-β -CD and the native β -CD, comparing with randomly substituted M-β -CD. For a robust enantioseparation method
development, the application of mixtures of defined single isomer
CDs has priority [45].

It has been shown recently, that the degree of methylation can
also affect the enantiomer migration order as well, independently
of the isomeric purity [46]. In the case of methylated derivatives,
the enantiomer migration order also depended on the substitution pattern of the CD. Thus, opposite migration sequence was observed for the enantiomers of certain cyclic β -amino acids when
randomly methylated M-β -CD or fully methylated TM-β -CD was
used compared to DM-β -CD, regardless of its isomeric purity (50,
75 or 95%).
The comparison of chiral recognition abilities of single isomer mono-, di-, and trimethylated CD derivatives with the
structurally-related randomly substituted CDs (RAMEB, CRYSMEB,
and DIMEB50) was briefly studied by Varga et al. [23]. In this case
the isomeric mixtures were found to be more versatile chiral selectors over the single isomer derivatives. As an alternative, the single
isomer 2,6-DIMEB showed exceptional enantiorecognition abilities,
however, TRIMEB, or 2-MEB could also provide similar advantages.
2D ROESY NMR experiments were performed with terbutaline and
it was confirmed that 2-O- and 6-O-methylation extends the cavity to accommodate terbutaline in an enantiospecific manner. Our
study also showed that single isomer 3,6-DIMEB could not be included in the chiral screening study due its low aqueous solubility.
Several works dealing with the enantioselectivities of permethylated CDs, the applied mono-, di- and trimethylated SIDs
are listed in Table 1. Among the per-dimethylated SIDs, the
heptakis(2,3-di-O-methyl-6-hydroxy)-β -cyclodextrin (HDM-β -CD)
was found to be the most widely studied derivative (see Table 1.).
The recent applications of per-trimethylated CDs, hexakis(2,3,6tri-O-methyl)-α -CD (TM-α -CD, TRIMEA), TRIMEB and octakis(2,3,6tri-O-methyl)-γ -CD (TM-γ -CD, TRIMEG) are also summarized in
Table 1.
Besides the methylated derivatives the less hydrophobic acetyl
functionalized CDs were also studied in CCE recently (see Table 1).
Salgado studied the impact of acetylation and noticed the enantiomer migration order reversal in the case of clenpenterol
with native β -CD and the heptakis(2,3-di-O-acetyl-6-hydroxy)-β cyclodextrin (HDA-β -CD) [47]. NMR experiments augmented with
molecular modeling and molecular dynamics (MD) simulations
provided insight into the structural and energetic determinants of
the distinct binding of clenpenterol enantiomers to the two CDs

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I. Fejos,

5

Table 1
Abbreviated names, structures and recent application of neutral SIDs in CCE. Substituents are numbered according to Fig. 1.
Abbreviation

Name

2-MEB
(HM-β -CD)

Heptakis(2-O-methyl)β -CD

25 basic and zwitterionic analytes [56], 27 basic analytes [63]

Heptakis(3-O-methyl)-

27 basic analytes [63]

3-MEB

Substituents

β -CD

Analytes separated

6-MEB
(6-Me-β -CD)

β -CD

Heptakis(6-O-methyl)-

methylene-dioxypyrovalerone [32], 27 basic analytes [63]

2,6-DIMEB
(DM-β -CD)

Heptakis(2,6-di-Omethyl)-β -CD

27 basic analytes [63], bicyclic β amino acids [46], medetomidine [44], 6
Tröger’s base derivatives [64], aspartate and glutamate [65], tryptophan
methyl ester [66], ephedrine [67], enilconazole [68], terbutaline [69],
propranolol [70], norephedrine [71], 6,6 -dibromo-1,1 -binaphthyl-2,2 -diol
[72], repaglinide [73], imidazole enantiomers [74], vinpocetine [75],
dapoxetine and its impurities [76], warfarin [77], aspartame [78], pregabalin
[79], warfarin and its metabolic enantiomers [80], 13 amphetamine-like
designer drugs [81], 6 stimulants [82], fluoxetine [83], ofloxacin and its five
impurities [84], hydrobenzoin [85], 3 glitazone compounds [86], peptides
[87], phenethylamines [88], talinolol [59], fluoxetine and norfluoxetine [89],
hirsutine and hirsuteine [90], clemastine and its related substances [91]

2,3-DIMEB

Heptakis(2,6-di-Omethyl)-β -CD

25 basic and zwitterionic analytes [56], 27 basic analytes [63]

3,6-DIMEB

Heptakis(3,6-di-Omethyl)-β -CD

27 basic analytes [63]

TRIMEB
(TM-β -CD)

Heptakis(2,3,6-tri-Omethyl)-β -CD

27 basic analytes [63], 4 neutral compounds [60], profens [61,92], seven
2-arylpropionic acid nonsteroidal anti-inflammatory drugs [62], ketoprofen
[93,94], lipoic acid [95], fluoxetine [83], ketoconazole [65], lercanidipine [96],
propranolol [70], carprofen, pentobarbital [97], norephedrine [71],
6,6 -dibromo-1,1 -binaphthyl-2,2 -diol [72], talinolol [59], amphetamine [98],
sibutramine [99], ephedrine [67], enilconazole [68], terbutaline [69], three
β -blockers [100], cis-β -lactam [83], five antimalarial drugs [101], aspartame
[78], pregabalin [79], warfarin [102], asenapine [103], vinpocetine [75],
dapoxetine and its impurities [76], six phenoxy acid herbicides [104],
fluoxetine and norfluoxetine [89], meptazinol and its three intermediate
enantiomers [105], elaidic and vaccenic trans fatty acid isomers [106],
coumarin derivatives [107]

TRIMEA(
TM-α -CD)

Hexakis(2,3,6-tri-Omethyl)-α -CD

Ketoprofen [93], aspartame [78], pregabalin [79]

TRIMEG(
TM-γ -CD)

Octakis(2,3,6-tri-Omethyl)-γ -CD

amine derivaties [108], pregabalin [79], ketoprofen [93], enilconazole [68],
terbutaline [69], aspartame [78],

HDA-β -CD

Heptakis(2,3-di-Oacetyl)-β -CD

25 basic and zwitterionic analytes [56], clenpenterol [47], ephedrine [67],
enilconazole [68], norephedrine [71], terbutaline [109]

HMA-β -CD

Heptakis(2-O-methyl3-O-acetyl)-β -CD

25 basic and zwitterionic analytes [56]

and the migration order reversal of their respective inclusion complexes in CCE.
The research groups of Holzgrabe and Chankvetadze conducted
several studies evaluating the application of both HDM-β -CD and
HDA-β -CD as a chiral selector in aqueous CE systems [48–55].
Our group has investigated the neutral, synthetic precursors

of the frequently applied single isomer sulfated CDs in order

to determine whether chiral selectivity is associated only with
the sulfate group [56]. Four neutral single isomer CDs substituted on the secondary side with acetyl and/or methyl functional
groups, heptakis(2-O-methyl-3,6-dihydroxy)-β -cyclodextrin (HMβ -CD), HDA-β -CD, HDM-β -CD, heptakis(2-O-methyl-3-O-acetyl6-hydroxy)-β -cyclodextrin (HMA-β -CD), and their sulfated analogues the negatively charged heptakis(2,3-di-O-methyl-6-sulfo)-β -


6

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I. Fejos,

cyclodextrin (HDMS-β -CD) and heptakis(2,3-di-O-acetyl-6-sulfo)β -cyclodextrin (HDAS-β -CD) were investigated by non-aqueous
capillary electrophoresis (NACE) for the enantiodiscrimination of
various drugs and related pharmaceutical compounds. The possibility of extending the applicability of CCE to non-aqueous conditions increases the versatility of method development in CE, especially in the case of sparingly soluble analytes, also opening the
floor to chiral selectors with poor aqueous solubility. The advantages of non-aqueous BGEs, such as low conductivity, improved
compatibility with mass spectrometric detectors, feasibility of ionpair formation in non-aqueous BGEs, reduced adsorption onto the
capillary wall, reduced generated electric current and Joule heating
etc. render this technique a viable extension to aqueous CCE. Moreover, NACE gives an opportunity to alternative separation mechanisms: changing the type of the BGE could result in different CDanalyte complex structures, which may manifest as the reversal of
the EMO [57–59]. This work focused on the chiral selectivity of the
neutral derivatives, which are the synthesis intermediates of the
sulfated products. The chiral recognition experiments proved that
among the neutral compounds the HMA-β -CD shows remarkable
enantioselectivity towards chiral guests in NACE, while HM-β -CD,
HDA-β -CD and HDM-β -CD failed to resolve any of the 25 studied racemates under the applied experimental conditions. In order
to get deeper insight into the molecular interactions between the
studied SIDs and racemic fluoroquinolones (ofloxacin, gatifloxacin
and lomefloxacin) and β -blockers (propranolol), 1 H and ROESY
NMR experiments were performed. The 2-O-methylation in combination with the 3-O-acetylation of the host was evidenced to exclusively carry the essential spatial arrangement necessary for chiral recognition. Thus, it was shown that the non-sulfated synthetic
precursor HMA-β -CD bears/is responsible to the chiral selectivity

prior to the final sulfation step.
The main drawback of neutral chiral selectors in CE is the lack
of self-mobility, therefore enabling the separation of only charged
enantiomers. Liu et al. demonstrated that ionic liquids (ILs) surfactants in conjunction with neutral CDs (TRIMEB) can resolve also
neutral enantiomers [60]. Ionic liquids are known as organic salts,
possessing low melting points close to room temperature. These
ionic components exhibit beneficial characteristics, such as varying the viscosity, conductivity or miscibility with different solvents. Wang et al. [61] reported the combined use of the chiral
IL-type surfactant N-undecenoxy-carbonyl-L-leucinol bromide (LUCLB) and TM-β -CD as a dual chiral selector system for the simultaneous enantioseparation of 5 profens. This MEKC method was
optimized regarding the chain length and concentration of the IL
surfactant, and could be applied for the quantitative determination
of ibuprofen in pharmaceutical tablets.
A binary system of trimethyl-β -CD and a chiral amino acid
ester-based ionic liquid (L-alanine tert-butyl ester lactate, lAlaC4Lac), was developed for the chiral separation of seven 2arylpropionic acid nonsteroidal anti-inflammatory drugs (NSAIDs)
by Mavroudi et al. [62] Comparing to the system with TM-β -CD as
the sole chiral selector, addition of l-lactate as an anion a synergistic effect (improvement in resolution values and in peak efficiency)
could be demonstrated.

3.2. Negatively charged CDs
Semisynthetic CD derivatives bearing ionizable functional
groups possess self-electrophoretic mobility. The self-mobility of
charged CDs makes the enantioseparation of uncharged enantiomers possible and it is also advantageous for charged analytes
due to strong ionic interaction between the oppositely charged
species [110]. All these advantages led to the development of a
wide range of various ionic CD derivatives.

3.2.1. Persubstituted derivatives
3.2.1.1. Permanently charged derivatives. The negatively charged
CDs are favorable for the enantioseparation of neutral and cationic
compounds. Considering the wide application of basic drug
molecules, negatively charged CDs have become the most frequently used chiral selectors, especially the sulfate-substituted β CDs. This may result from the introduction of sulfate groups carrying the negative charge(s) over the entire pH range studied in CCE.

This anionic site of the selectors offers an electrostatic-supported
interaction with cationic guests, enhancing the enantiorecognition
capacity at any pHs. This simplifies the CCE method development
and helps to predict and understand the enantioseparation process,
since the separation selectivity could be determined as a function of the selector concentration and the pH of the BGE. This is
the basis of the CHARM-model (charged resolving agent migration model) [111]. The largest possible enantioselectivities can be
achieved by simulations based on measurements in a single lowpH and high-pH BGE as well as varying only the concentration of
the chiral selector.
The synthesis and the application of persubstituted anionic
SIDs in CCE was introduced by Vigh et al. [16] with the most
widely used single isomer resolving agents in CCE, the sulfated CD
derivatives. Vigh and his group prepared and characterized various families of structurally well-defined sulfated CDs in high isomeric purity. In the first generation of these sulfated SIDs, position C-6 was persubstituted with sulfo groups, while the remaining C-2 and C-3 positions of the glucopyranose units were
unmodified (as heptakis(6-O-sulfo)-β -CD [HS-β -CD]), or bearing
identical acetyl or alkyl substituents (as heptakis(2,3-diacetyl-6-Osulfo)-β -CD [HDAS-β -CD] and heptakis(2,3-dimethyl-6-O-sulfo)-β CD [HDMS-β -CD], respectively) [16–18].
Cucinotta et al. [13] has already reviewed the first applications
of these SIDs, in Table 2 the recent applications are summarized
from the last 10 years.
Several studies compared the enantiorecognition and complexation behavior of the randomly sulfated CD derivatives (with DS
of 7–11 or highly sulfated analogs with DS of 12–15) and the 6O-sulfated SID, the HS-β -CD. Wang presented examples for both,
the superiority of the SIDs (in the case of chlorpheniramine) and
the advantage of randomly substituted analogs (in the case of atropine) in enantioseparation [112]. Comparative studies on enantioseparation of thioridazine in a citrate buffer at pH 3.0 were
performed by dynamic CE, using sulfated-β -CDs with different DS
and positions of sulfate substituent, such as randomly sulfated β CD, 18-sulfate-substituted β -CD and heptakis(2,3-dihydroxy-6-Osulfo)-β -CD. These studies revealed the role of interactions between chiral selectors and thioridazine in the enantioseparation
and enantiomerization of thioridazine [113]. Moreover, a substitution pattern-dependent migration order reversal of medetomidine enantiomers was observed in the study of Krait et al. in the
presence randomly sulfated β -CD (DS~12–15) and heptakis(6-Osulfo)-β -CD [44]. Levomedetomidine interacted stronger with the
SID, while dexmedetomidine formed more stable complexes with
randomly sulfated β -CD. They investigated the complexation of
the enantiomers of medetomidine with various CDs by CE, NMR
and molecular modeling. However, a complete picture could not
be obtained unfortunately by NMR and molecular modeling in the

case of the randomly substituted CD since the mixture of positional and substitutional isomers present, highlighting the disadvantage of the random analogs in structural follow-up studies. Applying the multiple chiral selector (multi-CS) model, Dubsky demonstrated the difference between the enantiorecognition of
single isomer HS-β -CD and randomly substituted, commercial mixture of sulfated CDs [114]. He supported the enhanced enantioselectivity of multi-CS systems with an additional, electrophoretic,
enantioselective mechanism resulting from different limiting mo-


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Table 2
Abbreviated names, structures and recent application of negatively charged SIDs in CCE. Substituents are numbered according to Fig. 1.
Abbreviation

Name

Substituents

Analytes separated

Permanently charged SIDs
per-sulfated β -SIDs
HS-β -CD

Heptakis(6-O-sulfo)-β -CD

Thioridazine [113], propranolol [70], phenolic acids,
flavones [167], lorazepam [114], tryptophan methyl ester
[66], β -blockers, phenethylamines, anticholinergic agents
[112], phenylalanine, 1-phenylethanol, chlorpheniramine,
promethazin [168], medetomidine [44],
3-chiral-1,4-benzodiazepines [115], oxazolidinones
(tedizolid +precursors) [122], oxazolidinones (radezolid)

[123], oxazolidinones (sutezolid+ precursor) [124],
ephedrine [67], α -diimine Ru(II) and Fe(II) complexes
[139], pindolol [141]

HDMS-β -CD

Heptakis-(2,3-di-O-methyl-6O-sulfo)-β -CD

10 β -blockers [116], fenbendazole (prochiral),
oxfendazole, nonchiral fenbendazole sulfone [120],
synthetic intermediate of
3,4-dihydro-2,2-dimethyl-2H-1-benzopyrans [119], 10
basic drugs [143], propranolol [125], carvedilol [57],
propranolol [70], bupivacaine and propranolol [126],
norephedrine [71], talinolol [59], acebutolol [128],
enilconazole [68], terbutaline [69], brombuterol [147],
ephedrine [67], 3-chiral-1,4-benzodiazepines [115],
oxazolidinones (tedizolid +precursors) [122],
oxazolidinones (radezolid) [123], oxazolidinones
(sutezolid+ linesolid) [124], 8 β -blocker drugs [127], 25
basic and zwitterionic analytes [56],

HDAS-β -CD

Heptakis(2,3-di-O-acetyl-6-Osulfo)-β -CD

dexamphetamine, 1R,2S(-)norephedrine,
1S,2S(+)norpseudoephedrine [117], 10 β -blockers [116],
fenbendazole (prochiral), oxfendazole, nonchiral
fenbendazole sulfone [120], synthetic intermediate of

3,4-dihydro-2,2-dimethyl-2H-1-benzopyrans [119], 10
basic drugs [143], propranolol [125], carvedilol [57],
propranolol [70], bupivacaine and propranolol [126],
alprenolol, bupranolol, terbutaline, tiaprofenic acid,
suprofen, flurbiprofen [169], propranolol [58],
norephedrine [71], talinolol [59], basic analytes [118],
amphetamine [98], acebutolol [128], enilconazole [68],
terbutaline [69], brombuterol [147], ephedrine [67],
3-chiral-1,4-benzodiazepines [115], oxazolidinones
(tedizolid +precursors) [122], oxazolidinones (radezolid)
[123], oxazolidinones (sutezolid+ linesolid) [124], 8
β -blocker drugs [127], 25 basic and zwitterionic analytes
[56], α -diimine Ru(II) and Fe(II) complexes [139],
phenethylamines [88], Ru(II)- and Fe(II)-polypyridyl
associates [170]

per-sulfated α - and γ -SIDs
HxS-α -CD

Hexakis(6-O-sulfo)-α -CD

α -diimine Ru(II) and Fe(II) complexes [139]

HxDMS-α -CD

Hexakis(2,3-di-O-methyl-6-Osulfo)-α -CD

–

HxDAS-α -CD

Hexakis(2,3-di-O-acetyl-6-Osulfo)-α -CD

α -diimine Ru(II) and Fe(II) complexes [139], Ru(II)- and

OS-γ -CD

Octakis(6-O-sulfo)-γ -CD

l- and d-amino acids [140], pindolol [141], α -diimine
Ru(II) and Fe(II) complexes [139]
(continued on next page)

Fe(II)-polypyridyl associates [170]

7


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I. Fejos,

8
Table 2
(continued)
Abbreviation

Name

ODMS-γ -CD

Octakis(2,3-di-O-methyl-6-Osulfo)-γ -CD

α -diimine Ru(II) and Fe(II) complexes [139]

ODAS-γ -CD

Octakis(2,3-di-O-acetyl-6sulfo)-γ -CD

oxazolidinones (tedizolid+precursors) [122], α -diimine
Ru(II) and Fe(II) complexes [139], Ru(II)- and
Fe(II)-polypyridyl associates [170]

II. generation sulfated β -SIDs

Substituents

Analytes separated

HMS-β -CD

Heptakis(2-O-methyl-6-Osulfo)-β -CD

–

HMAS-β -CD

Heptakis(2-O-methyl-3-Oacetyl-6-O-sulfo)-β -CD

10 basic drugs [143], non-ionic and weak base analytes
[148], carvedilol [57]

HMdiSu-β -CD
(HMDS)
IV. generation

Heptakis(2-O-methyl-3,6-di-Osulfo)-β -CD

Enilconazole [68], terbutaline [69], brombuterol [147],
non-ionic and weak base analytes [148]

HAMS

Heptakis(2-O-sulfo-3-Omethyl-6-O-acetyl)-β -CD

non-ionic and weak base analytes [148]

DBSB-β -CD

Heptakis(2,3-di-O-benzyl-6-Osulfobutyl)-β -CD

fluorescent cyanobenzylindole derivatives of d/l–serine
[151]

DBSB-α -CD

Hexakis(2,3-di-O-benzyl-6-Osulfobutyl)-α -CD

fluorescent cyanobenzylindole derivatives of d/l–serine
[151]

6-(SB)7 -β -CD

Heptakis(6-O-sulfobutyl)-β -CD

basic and noncharged analytes [152]

HDHSA-β -CD

Heptakis(2,6-di-O-[2-hydroxy3(sulfoamino)propoxy])-β -CD

3 β -adrenoreceptor agonists [153]

III. generation

sulfoalkylated SIDs

(continued on next page)


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9

Table 2
(continued)
Abbreviation

Name

Substituents

Analytes separated

Adjustable anionic charge
Per substituted derivatives

HDMCM

Heptakis(2,3-di-O-methyl-6-Ocarboxymethyl)-β -CD

ODMCM

25 noncharged, basic, and zwitterionic analytes [156]

Octakis(2,3-di-O-methyl-6-Ocarboxymethyl)-γ -CD
Mono substituted derivatives

basic analytes [157]

2CMα CD

2A -O-carboxymethyl-α -CD

Tryptophan, baclofen, primaquine and Tröger’s bases
[159,163]

3CMα CD

3A -O-carboxymethyl-α -CD

Tryptophan, baclofen, primaquine and Tröger’s bases
[159,163]

6CMα CD

6A -O-carboxymethyl-α -CD

Tryptophan, baclofen, primaquine and Tröger’s bases
[159,163]

2CMβ CD

2A -O-carboxymethyl-β -CD

Tröger’s bases, baclofen, mefloquine, and tryptophan
methyl ester [160,163]

3CMβ CD

3A -O-carboxymethyl-β -CD

Tröger’s bases, baclofen, mefloquine, and tryptophan
methyl ester [160,163]

6CMβ CD

6A -O-carboxymethyl-β -CD

Tröger’s bases, baclofen, mefloquine, and tryptophan

methyl ester [160]

2CMγ CD

2A -O-carboxymethyl-γ -CD

Tröger’s bases, mefloquine, primaquine and tryptophan
methyl ester [162,163]

3CMγ CD

3A -O-carboxymethyl-γ -CD

Tröger’s bases, mefloquine, primaquine and tryptophan
methyl ester [162,163]

6CMγ CD

6A -O-carboxymethyl-γ -CD

Tröger’s bases, mefloquine, primaquine and tryptophan
methyl ester [162,163]

mono-Suc-β CD
di-Suc-β -CD
tri-Suc-β -CD

mono-6A -O-succinyl-β -CD
di-6-O-succinyl-β -CD
tri-6-O-succinyl-β -CD

Catechin [165]

SET-β -CD
SMHT-β -CD

6A -sulfoethylthio-β -CD
6A -(6-sulfooxy-5,5-bissulfooxymethyl)hexylthio-β CD

Basic analytes [166]
Basic analytes [166]

bilities in the case of chiral selector mixtures. It is verified that the
two enantiomers of lorazepam under interaction with a mixture
of CSs are very likely to differ in their limit mobilities, which is
opposite to single CS systems where the two limit mobilities are
likely to be the same. This additional mechanism generally makes

the multi-selector systems more selective than the single selector
systems.
The effect of the sulfation of the primary hydroxy groups of β CD is highlighted: enantiomer migration order reversal could be
observed in the case of propranolol enantiomers applying native


10

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I. Fejos,

β -CD (and the partially resolving TM-β -CD) or the 6-O-sulfated

analogs, the HS-β -CD (and also in the case of the further substituted HDAS-β -CD and HDMS-β -CD) in aqueous buffer [70]. Significant structural differences were also confirmed by 1D ROESY measurements, observed between the complexes of propranolol with
native β -CD and its single isomer sulfated analogue, HS-β -CD. Inclusion type complexes were formed in both cases, however, the
naphthyl moiety entered the cavity from opposite directions and
the extent of the penetration into the cavity was also different for
the two CDs. These significant structural differences between the
complexes can be responsible for the opposite migration order of
the propranolol enantiomers observed in CE using these two CDs
as chiral selectors.
The molecular mechanisms of enantiorecognition and the opposite enantiomer migration order of ephedrine enantiomers was
investigated in the presence of native α - and β -CD, as well as with
HDAS-β -CD by CE, NMR and high-resolution MS [67]. 1D ROESY
experiments prevailed a striking difference between the structures
of ephedrine complexes with the native and HDAS-β -CDs. Enhanced chiral recognition ability could be observed in the case of
HDAS-β -CD towards the enantiomers of ephedrine. The EMO were
followed for norephedrine enantiomers with native α - and β -CD,
as well as with HDAS-β -CD and HDMS-β -CD by CE and NMR [71].
The complexes between the enantiomers of norephedrine and the
sulfated CDs, HDMS-β -CD, and HDAS-β -CD, were substantially different, however, EMO of norephedrine was identical in the presence of these CDs. HDAS-β -CD proved to be the most suitable chiral selector for the enantioseparation of norephedrine. Based on 1D
ROESY NMR experiments, the interactions with the sulfated CDs
occurred through at the primary rim: in the case of HDAS-β -CD,
an inclusion complex is formed, whereas only a superficial binding
of the analyte is observed for HDMS-β -CD. A clear enantiomeric
bias could be seen between norephedrine and HDAS-β -CD, as the
1S,2R-isomer seemed to form a stronger complex compared to its
1R,2S counterpart.
The three 6-O-sulfated CD derivatives, differing in the substituent at C2 and C3 positions: hydroxyl, acetyl and methyl derivatives as HS-β -CD, HDAS-β -CD and HDMS-β -CD respectively, were
examined by experimental design for enantioseparation of four
chiral benzodiazepines by CE [115]. The highest resolution values
were obtained with the addition of 5% HS-β -CD and 15% methanol
as an organic modifier to 20 mM borate buffer, pH 9.0.

The two most widely applied sulfated SIDs, HDAS-β -CD and
HDMS-β -CD are both completely sulfated at the primary rim, and
additionally substituted at their secondary rims with moderately
hydrophobic (acetyl) or hydrophobic (methyl) functional groups.
Several studies compared these two SIDs in CCE (see Table 2.).
Rousseau et al. investigated the NACE separation of ten β -blockers
with d-optimal design [116] to estimate the effects of the nature of the CD and the BGE anion as well as their concentrations on the enantioseparation. A generic NACE system (10 mM
ammonium acetate and 40 mM HDAS-β -CD in methanol acidified with 0.75 M formic acid) was able to completely resolve the
enantiomers of all β -blockers, with a minimal Rs value of 4. The
optimal conditions were compared to the optimal conditions obtained by modeling resolution, mobility difference and selectivity. Kokiashvili also found HDAS-β -CD advantageous for the simultaneous determination of the enantiomeric purity of dexamphetamine as well as the analysis of 1R,2S-(−)-norephedrine and
1S,2S-(+)-norpseudoephedrine as potential impurities [117]. The
validated method was successfully used for the analysis of commercial dexamphetamine sulfate samples where 3–4% of levoamphetamine were detected, indicating the preparation method of
amphetamine. In comparison with the polarimetric measurements
0.06% of levoamphetamine can be detected by capillary electrophoresis. Yao and his group demonstrated the utility of HDAS-

β -CD for chiral separation of 12 pairs of basic analyte enantiomers
under the optimized conditions (50 mM Tris-H3 PO4 and 6 mM
HDAS-β -CD at pH 2.5) [118]. Furthermore, a molecular modeling
strategy was established with 4 model compounds (clenbuterol,
oxybutynin, salbutamol and penehyclidine) to confirm and explain
the possible chiral recognition mechanism: the binding energy difference between a pair of enantiomers towards the chiral selector
was a significant factor contributing to enantioselectivity.
Rousseau found the more hydrophobic dimethylated SID,
HDMS-β -CD preferable for the analysis of synthetic intermediate
of new 3,4-dihydro-2,2-dimethyl-2H-1-benzopyrans in NACE [119].
However, high resolution and efficiency values could be achieved
only by the addition of a chiral ionic liquid (IL), i.e. ethylcholine
bis(trifluoromethylsulfonyl)imide (EtChol NTf2 ), to the BGE containing HDMS-β -CD, indicating the synergistic effect of the anionic CD and the chiral IL. The validated method permitted the
determination of 0.1% of each enantiomer in the presence of its
stereoisomer. In a further study of this group, a NACE method

has been established using the combination of 10 mM HDAS-β CD and 10 mM HDMS-β -CD for the simultaneous determination
of a prochiral drug, fenbendazole, and its chiral (oxfendazole) and
nonchiral (fenbendazole sulfone) metabolites [120].
After the determination of enantiomeric impurity of linezolid
by capillary electrophoresis using heptakis-(2,3-diacetyl-6-sulfo)β -cyclodextrin [121], Michalska and her research group investigated the enantioseparation of further oxazolidinones using SIDs
in CCE [122–124]. During method development for the simultaneous separation of the non-charged tedizolid enantiomers and the
weak base linezolid enantiomers, hydrophilic negatively charged
single isomer and moderately hydrophobic and hydrophobic CDs
were also tested including HS-β -CD, HDAS-β -CD and HDMS-β -CD,
respectively [122]. Only CDs with acetyl moieties at the C2 and
C3 positions (HDAS-β -CD or its gamma analog octakis(2,3-di-Oacetyl-6-sulfo)-γ -CD (ODAS-γ -CD)) provided baseline separation.
The best enantioseparation of tedizolid (Rs = 4.1) was obtained
with HDAS-β -CD in 50 mM formate buffer (pH 4.0) with the addition of acetonitrile. The separation mechanism of the more hydrophilic, dibasic radezolid, together with its precursor linezolid
was investigated to reveal the relationship between the oxazolidinone structure and the complexation process applying HS-β CD, HDAS-β -CD and HDMS-β -CD [123]. The CDs having an acetyl
or methyl group at the C2 and C3 positions (HDAS-β -CD and
HDMS-β -CD), exhibited partial and baseline separation of enantiomers in a low pH buffer, respectively. However, higher temperatures were required for the separation with HDAS-β -CD and
acetonitrile addition was required for HDMS-β -CD. Some further
structure-enantioselectivity relations were also deduced in their
study. As the further step of the mechanistic investigation of the
enantioseparation of oxazolidinones, a NACE method has been developed for the less water-soluble sutezolid enantiomers [124]. HSβ -CD, the most hydrophilic selector tested was incompatible with
NACE buffers. HDAS-β -CD and HDMS-β -CD provided the baseline
separation of sutezolid enantiomers, however, with different EMO:
a substitution dependent enantiomer migration order reversal occurred. Instead, enantiomers of linezolid were separated only by
HDMS-β -CD.
CCE separations in non-aqueous BGEs are as well established
as the separations in aqueous buffers. While achiral separation
mechanisms in NACE are most likely similar to those in aqueous
buffers, remarkable differences can exist between the molecular
mechanisms of the separation of enantiomers in aqueous and nonaqueous buffers. Servais and co-authorss resolved the enantiomers
of propranolol using CE in aqueous and non-aqueous methanolic

BGEs with the two single isomer sulfated derivatives HDMS-β -CD
and HDAS-β -CD [125]. The enantiomer migration order of propranolol was reverted when an aqueous BGE was replaced with non-


˝ E. Kalydi and M. Malanga et al. / Journal of Chromatography A 1627 (2020) 461375
I. Fejos,

aqueous BGE in the presence of HDMS-β -CD but remained the
same in the case of HDAS-β -CD. The possible molecular mechanisms leading to this reversal of EMO were as follows: propranolol
formed inclusion type complexes with HDMS-β -CD in an aqueous
buffer but external type complexes in a methanolic BGE. Contrary
to that, for HDAS-β -CD external type complexes were formed in
the case of aqueous solutions but inclusion type complexes resulted in a methanolic BGE. In addition, the inclusion type complex with HDMS-β -CD in the aqueous solution was formed by insertion of the naphthyl moiety of (R)-propranolol via the narrower
primary rim of the CD. In the case of HDAS-β -CD, the inclusion
complex in the methanolic BGE was due to the penetration of the
alkyl chain of (R)-propranolol into the HDAS-β -CD cavity through
the wider opening. Despite the different type complexes in aqueous and non-aqueous BGEs for both studied CDs, the EMO reversal
between aqueous and non-aqueous BGEs was observed only in the
case of HDMS-β -CD. In order to understand the fine mechanisms
of EMO of propranolol with HDAS-β -CD, further binding studies
were performed by NMR and high-resolution mass spectrometry
(HRMS) by the same research group [58]. Enantioselective nuclear
Overhauser effect was observed for the first time in this study:
enantioselective ROESY experiments indicated that (S)-propranolol
formed a tighter complex with HDAS-β -CD than (R)-propranolol.
Thus, the ROESY experiment may provide valuable qualitative information regarding the strength of selector–analyte binding in the
cases when the binding constants cannot be measured due to the
formation of nonuniform complexes or any other reason.
Besides NMR, molecular modeling constitutes an essential tool
for exploring mechanisms of chiral recognition at the atomic level.

In a further study of the same group [126], bupivacaine and propranolol were investigated by the single isomer highly sulfated CD
derivatives HDMS-β -CD and HDAS-β -CD. Quantum chemistry calculations were compared to the EMO observed in NACE as well as
to the structures of complexes provided by NMR experiments. Interaction energies calculated for bupivacaine and propranolol correlated with the EMO observed in the NACE experiments using
both SIDs. The interaction between propranolol and HDMS-β -CD
or HDAS-β -CD should be considered as a dynamic process giving
rise to a set of external and inclusion complexes with different statistical populations.
The EMO and the selector-selectand interactions of the β blocker talinolol was also studied with HDMS-β -CD and HDAS-β CD in aqueous and non-aqueous CE and NMR spectroscopy [59].
The enantiomer affinity pattern of talinolol toward these two SIDs
was opposite in both aqueous and non-aqueous CE, however, the
EMO did not change for a given CD derivative when the aqueous buffer was replaced with non-aqueous BGE (for the electropherograms see Fig. 2A.). Inclusion complex formation between
talinolol and HDAS-β -CD was confirmed in aqueous buffer, while
with HDMS-β -CD it was of the external type (for the structure of
the complexes see Fig. 2B.). The complex with HDAS-β -CD in nonaqueous electrolyte was also of the external type. In spite of external complex formation, excellent separation of the enantiomers
was observed in NACE. The changes in the chemistry of CDs (the
nature of the substituents on the wider secondary rim) can lead
to a reversal of enantiomer affinity pattern of this type of chiral
selectors both in aqueous and non-aqueous electrolytes, however
the changes in the geometry of analyte–CD complexes do not always result in a substantial change in the chiral separation ability
or chiral recognition pattern.
Beyond propranolol and talinolol, six further chiral β -blocker
drugs, acebutolol, atenolol, carazolol, carteolol, carvedilol and sotalol have been enantioseparated and studied systematically using
the same SIDs in aqueous CE and NACE by Feng et al. [127] regarding the influence of the substituents on the wider rim of the
CD and that of the electrophoretic medium type on the enantios-

11

electivity and enantiomer affinity pattern. In general, higher affinity and enantioselectivity were obtained in the presence of HDASβ -CD in both BGEs. Substituent dependent reversals of EMO were
observed toward these two CDs for almost all compounds in NACE
and aqueous CE. It is particularly noteworthy that opposite EMO
was also found using the same SID when the aqueous BGE was

replaced with a methanolic one. Similar observations were also
found during the study of enantiomer affinity pattern of acebutolol with the sulfated SIDs [128]. The EMO was found to be opposite when an aqueous background electrolyte was replaced with
non-aqueous BGE in the presence of HDAS-β -CD, but remained the
same in the presence of HDMS-β -CD.
Müllerová et al. developed a method allowing the comparison of EMOs when applying two different selectors without the
need of the pure enantiomeric form(s) of the analytes but applying a specific mixture of the two selectors [98]. They demonstrated the method on a racemic sample of amphetamine with
three CDs, (2-hydroxypropyl)-β -cyclodextrin, heptakis(2,3,6-tri-Omethyl)-β -cyclodextrin and heptakis(2,3-di-O-acetyl-6-O-sulfo)-β cyclodextrin. The developed method can be applied when the absolute EMO in a new selector is unknown, while the EMO in a
reference selector is determined in previous experiments or is obtained from the literature data.
Regarding the sulfated α -SIDs [20,129–131] and the sulfated γ SID analogues [132–138], only limited new results can be found
from the last decade (see Table 2.). Sálezová et al. studied both
α -derivatives, as hexakis(6-O-sulfo)-α -CD (HxS-α -CD), hexakis(2,3di-O-acetyl-6-O-sulfo)-α -CD (HxDAS-α -CD), and γ -derivatives, as
octakis(6-O-sulfo)-γ -CD (OS-γ -CD), ODAS-γ -CD and octakis(2,3di-O-methyl-6-O-sulfo)-γ -CD (ODMS-γ -CD), besides the beta SID
HS-β -CD and HDAS-β -CD for the chiral analysis of α -diimine Ru(II)
and Fe(II) complexes [139]. Fradi et al. simultaneously separated 10
pairs of in-capillary derivatized amino acids using β -CD and OS-γ CD as chiral selectors, SDS as surfactant and IPA as organic modifier [140]. Also applying OS-γ -CD as chiral selector, a validated
separation method for pindolol enantiomers was published [141].
Michalska et al. demonstrated, that the acetyl groups at the C2
and C3 positions for beta (HDAS-β -CD) and gamma (ODAS-γ -CD)
derivatives are essential to provide baseline separation of tedizolid
enantiomers.
The first generation of sulfated SIDs was followed by their
second generation where the C-6 positions were bearing the
sulfo groups, but the C-2 and C-3 positions were substituted
differently: the heptakis(2-O-methyl-6-O-sulfo)-β -CD (HMS-β -CD)
and the heptakis(2-O-methyl-3-O-acetyl-6-O-sulfo)-CD (HMAS-β CD) [19,142]. These SIDs did not appear in any further application
in CCE, until now no results from HMS-β -CD, and only few results
for HMAS-β -CD are available among which the work of Rousseau
et al. [143] showed the enantioseparation of ten basic drugs in
NACE.
Servais et al. studied the impact of the BGE composition on the

ionization performance of carvedilol in achiral and chiral conditions by alternative infusion method in CE-MS applying HDMS-β CD, HDAS-β -CD and HMAS-β -CD [57]. It has been concluded that
in order to carry out a sensitive chiral NACE-MS analysis, the chiral
selector must be selected not only according to its enantioselectivity but also according to its effect on the ionization efficiency in
mass spectrometry.
The only single isomer heptakis(2-O-methyl-3,6-di-O-sulfo)-β CD (HMdiSu-β -CD, also abbreviated as HMDS-β -CD) carrying 14
sulfate groups has been synthesized and introduced to CCE by
Maynard and Vigh [144,145]. The high negative charge of HMdiSuβ -CD over the entire pH range is responsible for strong ionic interaction. However, an unexpected migration behavior was experienced for the weakly binding cationic analyte when applying
HMdiSu-β -CD due to the drastically different ionic strength de-


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I. Fejos,

Fig. 2. (A) CE separation of talinolol enantiomers in aqueous electrolyte (100 mM phosphoric acid adjusted to pH 3 with triethanolamine, R:S = 1:2, 48.5/40 cm, 50 μm
i.d. capillary, 25 kV; 15 °C; 230 nm) in the presence of 50 mg/ml HDMS-β -CD (a), and 60 mg/ml HDAS-β -CD (reversed polarity mode) (b), and in non-aqueous electrolyte
(10 mM ammonium formate in methanol acidified with 0.75 M formic acid) in the presence of 50 mg/ml HDMS-β -CD (c) and 50 mg/ml HDAS-β -CD (d) (B) Tentative
structure of talinolol/HDMS-β -CD (a) and talinolol/HDAS-β -CD (b) complexes in aqueous BGE (From Ref. [59]).

pendencies of the noncomplexed monocationic enantiomer and its
complexed, polyanionic counterpart. The unexpected effective mobility extrema were rationalized by extending the CHARM model
[111] to include ionic strength effects.
After the early fundamental experiments on the chiral selectivity and the complex formation by Chankvetadze et al. [146],
Gogolashvili et al. studied briefly the enantioselectivity and enantiorecognition pattern of HMdiSu-β -CD in the recent years. Opposite EMO of enilconazole were observed with HMDiSu-β -CD and

β -CD [68]. Inclusion complex formation of both enilconazole enantiomers with β -CD was confirmed by NMR, while no interaction of
enilconazole with HMdiSu-β -CD could be detected. Most likely the
interaction between enilconazole and HMdiSu-β -CD leads to the
formation of a shallow external complex that is sufficient for separation of enantiomers in CE, but cannot be evidenced based on

NMR ROESY experiment. In this way the high sensitivity of CE for
the detection of weak intermolecular interactions was also demonstrated experimentally.


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13

Fig. 3. (A) CE separation of terbutaline enantiomers (R:S = 2:1, 40/31 cm, 50 μm i.d. capillary, 50 mM KH2 PO4 –100 mM H3 PO4 pH 2.0 buffer, 150 μA; 20 °C; 200 and
220 nm) in the presence of 18 mg/ml β -CD (a), 21 mg/ml HMDiSu-β -CD (b), and 0.6 mg/ml HDAS-β -CD (c) (B) Structures of terbutaline/β -CD (a), terbutaline/HMDiSu-β -CD
(b), and terbutaline/HDAS-β -CD complexes (c) as deduced from ROESY experiments. (From Ref. [69]).

In the next study of Gogolashvili similar trends were observed:
the affinity pattern of terbutaline enantiomers was the same towards all studied CDs (higher affinity towards (S)-terbutalin) except for HMDiSu-β -CD, where the (R)-terbutalin showed higher
affinity [69]. For representative electropherograms see Fig. 3A. Further studies using NMR spectroscopy and molecular modeling calculations were performed on terbutaline complexes with β -CD,
HMdiSu-β -CD and the best enantiodiscriminating HDAS-β -CD. Significant structural differences were observed between the complexes, as depicted in Fig. 3B. In the case of native β -CD hydrophobic interactions can play a primary role in complex formation, leading to the inclusion of the aromatic moiety of terbutaline
into the cavity of β -CD. In the case of anionic CDs electrostatic
interactions could be predominant, causing inclusion of aliphatic

alkylamino moiety of the analyte into the cavity of HMDS-β -CD
and HDAS-β -CD, although from different sides of the CD truncated
cone.
In the third study of this group, the opposite affinity pattern of
brombuterol enantiomers toward β -CD and its two sulfated derivatives, HDAS-β -CD and HMDiSu-β -CD was again associated with
major differences in the structure of the complexes [147].
Recently, Vigh’s group reported the synthesis and first CE application of a completely original class of sulfated-CDs [148]. The
sulfo groups were located exclusively on the C-2 positions, while
the remaining C-3 and C-6 positions were non-identically persubstituted with methyl and acetyl groups, respectively. The C-2
sulfation provides an anionic site at the wider rim (contrary to

the above-mentioned C-6 sulfated SIDs), where the cationic part


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of chiral guests could reside. This electrostatic-supported interaction may play role in separation and complex formation behaviors. Heptakis(2-O-sulfo-3-O-methyl-6-O-acetyl)-β -CD (HAMS) was
studied in CCE applying 14 non-charged and 33 weak base analytes. In order to investigate the influence of the sulfo group’s position on enantiorecognition, HMAS, which carries the sulfo group
at the C6 position, and HMdiSu, bearing the sulfo group at both
the C3 and C6 positions, was compared with HAMS for the separation of the enantiomers of chlophedianol, homatropine, and metoprolol. All three of these SIDs carry non-identical substituents at
the C2 and C3 positions. The binding strength for chlophedianol
decreases in the order of HAMS>HMdiSu>HMAS, while for homatropine the order was HMdiSu>HAMS>HMAS. HAMS offers the
highest separation selectivity for both chlophedianol and homatropine at low SID concentrations, where the effective mobilities of
the enantiomers remain cationic. These observations underline the
fact that different SIDs offer different separation selectivities and
emphasize the need for NMR experiments to better understand the
underlying enantiorecognition mechanism.
Besides the so far discussed sulfated CDs, where the functional
group is directly attached to the CD moiety, the sulfoalkylated CD
derivatives contain an alkyl linker between the CD core and the
permanently negatively charged group, allowing the extension of
the cavity and widening the chiral selectivity spectrum with additive hydrophobic interactions outside of the CD cavity. The randomly substituted sulfobutyl ether of β -cyclodextrin (Betadex Sulfobutyl Ether Sodium, SBE-β -CD) is widely applied in the pharmaceutical field due to its outstanding in vivo parenteral safety (authorized as excipient in the United States Pharmacopeia (USP)), as
well as in analytical separation techniques, in particular as chiral
selector in CCE for its effective complexation ability.
The preparation of single isomer sulfoalkylated CDs requires
multi-step synthetic strategies with extensive use of protecting groups, making the production time-consuming and expensive. As the first example of a single isomer sulfoalkyated
CD, heptakis-(2,3-di-O-methyl-6-O-sulfopropyl)-β -cyclodextrin was
synthesized and characterized by Kirshner and Green [149] as inverse phase transfer catalyst in biphasic Tsuji–Trost and hydroformylation reactions. They prepared a set of per-2,3-O-alkyl-6O-sulfoalkylated-CDs in order to optimize and improve the properties of the compounds as mass-transfer promoters in an aqueous biphasic hydroformylation reaction [150]. The first application

of single isomer per-2,3-O-substituted-6-sulfobutylated CDs in CE
was reported by McKee and Green [151]. Hexakis(2,3-O-dibenzyl6-O-sulfobutyl)-α -cyclodextrin and heptakis(2,3-O-dibenzyl-6-Osulfobutyl)-β -cyclodextrin were synthesized and studied, as new
chiral surfactants in CE by pyrene fluorescence studies. These CD
derivatives present 12 and 14 aromatic moieties, respectively, on
one side of the CD while 6 and 7 anionic sulfobutyl moieties,
respectively that protrude outside from the other side of the
molecule. Both CD derivatives form micelles and exhibit critical
micelle concentrations (CMCs) of about 90 μM. The beta analog
baseline resolved fluorescent cyanobenzylindole derivatives of D/L–
serine at concentrations of 50–200 μM. The authors demonstrated
that these micelle-forming amphiphilic CDs can be used as chiral
surfactants in capillary electrophoresis.
Our group published the five-step synthesis of the novel single
isomer per-6-O-sulfobutylated-β -CD, the heptakis-(6-O-sulfobutylether)-β -cyclodextrin (6-(SB)7 -β -CD) carrying the negative functionalities exclusively on its primary side and it is unmodified
on the lower rim [152]. The negatively charged single isomer
chiral selector was able to separate 9 out of the 10 test racemates of various positively charged primary, secondary and tertiary
amines, moreover, all the four stereoisomers of the non-charged
tadalafil. These results, along with the enantiomer migration order
and the selector concentration-dependent enantioselectivity, high-

lighted the broad applicability of these SIDs offering an alternative
to the widely applied sulfated analogs. 1 H and ROESY NMR experiments confirmed the enantioselective interactions between dapoxetine and 6-(SB)7 -β -CD, and the 2D ROESY results showed the inclusion complexation of the aromatic moiety of dapoxetine in the
CD cavity [152].
In the work of Li et al. [153] a rational, systematic strategy firstly combined de novo design and molecular modeling to
expedite the design, manipulation and selection of the effective
CD derivative for a given analyte enantioseparation in CE. According to characteristics of the analyte, the manipulation of the
chiral discrimination site of the selector host at molecular level
could improve the chiral recognition ability. The novel single isomer sulfamic acid modified CD derivative, heptakis {2,6-di-O-[2–
hydroxy-3-(sulfoamino)propoxy]}-β -cyclodextrin (HDHSA-β -CD) has
been designed, synthesized and tested as a tailor-made chiral CE

selector for the enantiomeric separations of β -adrenoreceptor agonists.
3.2.1.2. pH-adjustable negatively charged SID derivatives. In spite of
the great advantages of permanently ionic SIDs, Cucinotta et al.
[13] proposed an alternative approach for the development of an
ideal chiral selector. They demonstrated that carefully selected substituents extend the range of intermolecular interactions offered by
the CDs and provide effective tools for tuning separation selectivity. Their argument was based on the theory of Wren and Rowe
[154] stating that the experimental conditions maximizing the difference in the formation degree of diastereoisomeric complexes are
preferred over those maximizing the degree of formation of both
diastereomeric complexes. This approach justifies the development
of other types of single isomer selectors besides the strong anionic persubstituted SIDs. In this aspect, the development of welldefined structured single isomer chiral selector with an additional
tunable ionization state as well as the mono-derivatives could be
promising tools in CCE. Carboxy, carboxymethyl (CM), succinyl and
phosphate SIDs could become ideal candidates for this purpose,
since the presence and magnitude of their negative charge is pHdependent. The comparison of the enantioselectivity of the proposed weak anionic SIDs at various pH values would provide experimental evidence for the necessity of strong ionic interaction
between the selector and the analyte. If high selectivities were
found even in the case of an uncharged selector, the role of ionic
interaction in the enantioselectivity has to be reconsidered.
The synthesis of α - and β -CD derivatives permethylated on
their C-2 and C-3 OHs and per-carboxymethylated on their C-6 position has been introduced by Kraus et al. in 1997 [155] as the first
SIDs, however the CCE application of heptakis(2,3-di-O-methyl-6O-carboxymethyl)-β -CD (HDMCM) was conducted by our group
[156] focusing on the adjustable, pH-dependent ionic character of
the SID. The selector concentration- and pH-dependent enantioselectivity has been investigated applying three distinct pHs as well
as methanolic NACE conditions. The increase in the selector concentration and in the gross negative charge of HDMCM improved
the enantioseparation that could be observed in the majority of
the cases. At pH 2.5, where HDMCM is uncharged, and the hydrogen bonds are responsible for the enantioselective complex formation, only seven out of the 26 chiral test molecules could be
resolved. Increasing the pH to 4.5, twelve additional enantiomer
pairs were separated, and at pH 7.0, where HDMCM is fully ionic
(deprotonated), almost all chiral analytes were resolved. These results along with the pH-dependent enantioselectivity for zwitterionic compounds highlighted the broad applicability and the advantages of HDMCM over other chiral hosts bearing permanent
charges.
The four-step synthesis, characterization and CCE application

of the larger cavity size persubstituted CM-CD, octakis(2,3-di-O-


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15

Fig. 4. (A) pH-dependent 1 H NMR spectra of ODMCM (600 MHz Varian DDR, H2 O:D2 O = 9:1 by volume, 0.05 M NaCl and 0.05 M Na2 HPO4, 1 mM ODMCM, 0.05 mM
methanol, pH range 1.31–9.64 with 0.5 M HCl and NaOH), (B) Representative CE electropherograms applying ODMCM demonstrating the effect of the cyclodextrin concentration effect of the BGE pH on the enantioseparation of dapoxetine enantiomers (58.5/50 cm, 50 μm i.d. capillary, 20 mM H3 PO4 -NaOH pH 2.5, and 20 mM NaH2 PO4 -NaOH
pH 7.0 buffer, 1–10 mM ODMCM, 30 kV; 20 °C; 200 nm).

methyl-6-O-carboxymethyl)-γ -CD (ODMCM), has been described
by our group as well [157]. The acid-base profile of the SID was determined by 1 H NMR-pH titration (see Fig. 4A.) indicating the overlapping protonation of the eight carboxylates occuring in the pH
range of 7.7 to 2.0. The impact of the ionization state of ODMCM
and the selector concentration was investigated on the enantiorecognition properties by CCE: increasing the pH of the BGE and
the selector concentration usually led to higher enantioseparations
(for the representative electropherograms in the case of dapoxetine
enantiomers see Fig. 4B.). The pH-dependent enantioselectivity of
ODMCM was proved by both CCE and 1 H NMR spectroscopy. These
results clearly show that the per-carboxymethylated selectors offer
a wide variability in their ionization state; therefore, these selectors could be considered as primary choice candidates to study the
influence of the selectors ionization state on the enantioseparation.

Besides all the listed CD derivatives, no CCE studies
can be found using Sugammadex (octakis-(6-deoxy-6-Smercaptopropionyl-γ -CD), one of the well characterized pHadjustable negatively charged SID derivative, therefore our research
group has started to elaborate its enantiorecognition potential as
well.
3.2.2. Mono-derivatives
Besides the extensively investigated per-substituted SIDs, a few

studies deal with the mono-substituted analogs as single isomer
chiral selectors in CCE (see Table 2.). Depending on the pH of the
BGE these compounds can carry a single negative charge or exist
in neutral form, and perfectly suited for the investigation of the
role of substituent position on chiral separation. It was the goal
of Chankvetadze et al. when investigating single isomer mono-


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substituted anionic CD derivatives, 6-deoxy-6-monocarboxy-β -CD
and 6-monophosphate-β -CD [158]. Since in the case of monosubstituted CDs three regioisomers exist, Rezanka and his research
group focused on the impact of the position of the CM group on
the enantioselectivity of the chiral selector. They prepared a complete set of regioisomers of monosubstituted CM-CDs and compared the enantioselectivities of the regioisomers. The enantioselectivity of all the three individual regioisomers of the monosubstituted CM-α -CDs was studied and compared with the mixture of the three monosubstituted CM-α -CDs and with native α cyclodextrin [159]. Their experiments revealed a significant influence of the location of the carboxymethyl group on the α cyclodextrin skeleton on the enantioselectivity for all the studied analytes. Interestingly, the least common 3-O regioisomer provided significantly better resolution than the native α -CD and
its monosubstituted carboxymethyl derivatives. Comparison of the
three monosubstituted carboxymethyl-β -CD regioisomers individually, their 1:1:1 mixture along with the native β -CD, and the
commercially available random CM-β -CD (DS~3) [160] indicated
that substituent position has a significant influence on the enantioseparation. For most of the investigated analytes the commercially available derivative of CD provided better resolutions than
the monosubstituted carboxymethyl CD derivatives.
A complete set of mono-carboxymethylated γ –CDs were also
synthesized by the same research group [161] besides the
full sets of peracetylated 2-O-, 3-O-, and 6-O-allyl, -propargyl,
and -formylmethyl, derivatives of γ -CD. 2-O-, 3-O-, and 6-Ocarboxymethyl-γ -CD, as well as the native γ -CD and the random
CM-γ -CD analog were studied and the results confirmed that the
position of carboxymethyl group influences the enantioseparation
efficiency toward all the studied analytes [162]. The 2-O-and 3-Oregioisomers provide a significantly better resolution than native
γ -CD, while the 6-O-regioisomer gives only a slightly better enantioseparation. In general, higher number of carboxymethyl groups

led to better resolution.
A comprehensive study on the position of the carboxymethyl
group as well as the cavity size of the individual CD was deeply
investigated applying all nine regioisomers of mono- substituted
2-O-, 3-O-, and 6-O-carboxymethyl-α -, β -, and γ -CDs and native
α -, β -, and γ -CDs at pH 2.5 [163]. Based on CCE data, the apparent
stability constants of all CD–Tröger’s base complexes were deduced
and a significant influence of the substituent location in the monosubstituted CD as well as the size of the CD cavity on both the
chiral separation and the apparent stability constants were found.
Regarding only the native CDs, the most stable complexes were
formed with β -CD, however from all of the studied CD derivatives, the highest apparent stability constants were obtained for
3-O-carboxymethyl-γ -CD and the highest selectivity was achieved
for 2-O-CM-β -CD. This comparative analysis could serve a basis for
development of models aiming to describe chiral separation processes in real case systems.
The mono-6-O-succinyl-β -CD (CDsuc6) was introduced by Cucinotta [164], and they demonstrated promising chiral recognition
ability towards catecholamines in CCE. Kim et al. synthesized and
applied in CCE three kinds of negatively charged Suc-β -CDs with 1,
2, and 3 succinyl moieties at the primary hydroxyl groups of β -CD,
the mono-Suc-β -CD, di-Suc-β -CD, and the tri-Suc-β -CD, respectively [165]. The effects of nature and concentration of Suc-β -CDs
and BGE pH on the migration time and resolution of (±)-catechin
are discussed. They have concluded that the optimal separation
conditions have been reached utilizing monosubstituted CD. On the
other hand, the chiral selectors with the higher degree of substitution had a broader pH range of (±)-catechin separation when compared with mono-succinyl-β -CD.
To improve the resolution power of the chiral selector and
enantiomeric peak efficiency in CE, single isomer perma-

nently negatively charged β -CD derivatives, mono(6-deoxy-6sulfoethylthio)-β -CD (SET-β -CD) bearing one negative charge and
mono[6-deoxy-6-(6-sulfooxy-5,5-bis-sulfooxymethyl)hexylthio]-β CD (SMHT-β -CD) carrying three negative charges, were synthesized [166]. The apparent binding constants and mobilities of the
complexed analytes were determined in order to gain an improved
understanding on the effect of the number of negative charges

on a given enantioseparation. SMHT-β -CD exhibited significantly
greater enantioseparation over SET-β -CD at lower concentrations
due to its higher number of negative charges providing a wider
separation window resulting from an increased countercurrent
mobility of the selector and higher binding affinity to the analytes.
3.3. Positively charged CDs
Positively charged CD-derivatives are less widely used in (chiral) CE applications. While they potentially shorten the analyte migration times towards the cathode via complexation, allowing realization of shorter analysis times, their tendency to adsorb to the
negatively charged inner surface of the silica capillary may lead to
decreased resolution power and reproducibility problems. Cationic
CDs may hold permanently charged functional groups (strong electrolytes) or nitrogen bases (weak electrolytes), gaining positive
charge only upon protonation at suitable pH values of the BGE.
The first cationic CD synthesized in 1978 was mono-(6A trimethylammonium)-β -cyclodextrin hydrogencarbonate (tma-β CD) [171]. This permanently charged derivative was prepared as a
simple enzyme model and its ligand-binding and catalytic properties were examined.
The application of positively charged CD derivatives in CE was
first described by Terabe in 1989 [11]. He introduced the concept
of electrokinetic chromatography (EKC), where a charged (macrocyclic) selector is dissolved in the BGE and functions as a carrier
of the analytes. The separation is based on the same principle as
ordinary chromatography, but the carrier is not immobilized nor
form a distinct phase, rather it is homogeneously distributed in the
BGE solution forming a quasi-stationary phase. When an analyte
is added, a certain population of analyte molecules gets incorporated in the carrier through a partition mechanism and transported
via the migration of the carrier. The unbound fraction of analyte
molecules migrates according to its own electrophoretic mobility
and the electroosmotic flow. As a result, different net migration velocities arise for each analyte, dictated by their affinity towards the
carrier host and its molar concentration.
The application of charged CD as a resolving agent in CE offers
special advantages compared to the neutral derivatives. Insertion
of ionogenic groups may enhance the solubility of the CD itself,
but more importantly, neutral molecules lacking an intrinsic electrophoretic mobility also may become resolvable by charged CDs.
In this pioneering report of EKC [172], Terabe used the negatively charged carboxymethyl-β -CD and the positively charged

mono-6A -(2-aminoethylamino)-β -cyclodextrin
(AEA-β -CD)
to
demonstrate the realization of EKC. Six enantiomeric dansyl amino
acids (DNS-AAs) were resolved by EKC with the cationic AEA-β -CD
derivative.
3.3.1. pH-adjustable positively charged SID derivatives
Weak base CD derivatives can be differentiated according to
their substitution degree (mono-, di-, multi- or persubstituted
derivatives, where also non-ionizable substituents may be present)
or the number of protonable groups (mono-, di- or multivalent
[173] derivatives). There are also examples for monosubstituted
derivatives holding multiple charges. The presence of several basic sites gives the opportunity of more precise tailoring of the pHdependent degree of protonation and overall charge, thus modulating the strength of analyte-selector interaction. This dry chemistry-


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type design of the CE separation would require determination of
precise pKa dissociation constants of the CD derivatives, which remain usually unknown.
Besides mono- [11,174,175] and diamino [176] compounds,
their alkylated derivatives [177–183], histamine-modified derivatives [184–187], hemispherodextrins [188–190], a family of
hydroxyalkylamino-β -CDs [191–193] and amino-alkylamino-β -CDs
[176] were reported between the late 1990s to ca. 2010. Comparative screening studies of these selectors provided deeper insights
into the intrinsic mechanism of chiral recognition. The majority
of works in the past decade focused on the extension of previous
studies by either introducing a new representative of a previously
synthesized family of structurally homologue cationic selectors or
explored the applicability of previous successful CD derivatives for
enantioresolution of novel families of compounds.

The simplest form of cationic CDs, mono- and peramino derivatives were studied by several groups for the separation of various
Dns-AAs, anionic and ampholytic analytes. [11,174–176] In these
applications, α -CD and β -CD derivatives were usually functionalized on their primary or secondary side. Among the γ -CD derivatives, only mono-6-amino-γ -CD (6NH2 -γ -CD) [174,194] was explored for the chiral separation of Dns-AAs. Cucinotta et al. used
mono-3A -amino-γ -CD (3NH2 -γ -CD) as chiral selector for the separation of Dns-AAs [195] (see Table 3.). In a subsequent study, the
same SID was able to separate only eight of the thirteen investigated amino acids as fluorescein isothiocyanate (FITC) derivatives
[196], while the mono-3A -amino-β -CD (3NH2 -β -CD) analogue exhibited a better selectivity. The cavity size was found to play a key
role in the chiral recognition and (compared to Dns-AAs) it was
hypothesized that the FITC-moiety of the labelled AAs does not get
included into the CD cavity, it solely improves the detection limit.
Permethyl-mono-6A-amino-β -CD (PMMAB-CD) was proved to
be an effective selector to separate pyrethroic acid and profen
enantiomers [197], Németh et al. used it in 2014 also for the separation of Dns-AAs [101].
In the early 20 0 0s, Cucinotta’s group implemented ligand exchange capillary electrophoresis (LECE) to CE. The method relies on
the principle that a suitable metal ion is added to the BGE, which
can coordinate both the CD derivative and the analyte molecules
to form mixed metal-ligand complexes. This phenomenon can be
exploited to separate enantiomers (chiral LECE, CLECE). Cucinotta
et al. conducted several studies to explore the exact mechanism of
complex formation with copper(II) ion. Besides CE, they combined
several techniques such as potentiometric titrations, UV/VIS, circular dichroism and NMR spectroscopies to understand the structural
influence on enantioseparation on an array of mono- or multivalent cationic CDs, such as amino- or histamine-derivatized CDs,
mono-6A -(2-aminoethylamino)-β -CD (AEA-β -CD), and mono-6A [N-(2-methylamino)pyridine)]-β -CD (CDampy), including the different impact of their primary or secondary side modification
[190,198–200]
The potential of LECE was extended by its coupling to LIF
and TOF-MS detection, both yielding similarly low detection limits
[201]. Moreover, CE-MS proved to be capable of detecting non-UVabsorbing analytes or permitted to use strongly UV-absorbing CD
selectors, thus widening the space for chiral LECE method development [202]. The researchers demonstrated the suitability of CE-MS
by analyzing real-word samples such as transgenic and wild soy
and vinegar [202].
Although two histamine-modified CDs, mono-6A -N-histaminoβ -CD (CD-hm) and mono-6A -[4-(2-aminoethyl)imidazolyl]-β -CD

(CD-mh) were among the first published members of cationic CD
selectors [184–187], a new histamine derivative of β -CD, functionalized at the secondary rim (CDhm3) was applied in chiral LECE
towards the enantiomeric pairs of certain AAs only more than
twenty years later [176]. Comparing the structures of the success-

17

fully applied CD derivatives revealed that the factor to determine
the EMO of AAs is the presence of histamine and not the position
of derivatization on the CD.
Recently, the AEA-β -CD derivative was also used successfully
for the achiral separation of hirsutine and hirsutein, two pharmacologically active ingredients of Uncaria rhynchophylla, applied in
the therapy of mental and cardiovascular diseases [90].
Mono-6-hydroxyalkylamino-β -CD derivatives are a family of
well-characterized chiral CE selectors, reported by Iványi et al.
[192], tested for the resolution of mandelic acids, pyrethroic acids
and profens as model compounds.
Jakó et al. used the mono-6A -(3-hydroxy)propylamino-β -CD
(HPA-β -CD) to develop a method for quantitative determination
of amino acid neurotransmitters and neuromodulators using CELIF hyphenation [203]. The analytes determined were aspartate
and glutamate enantiomers, derivatized with the fluorophore 4fluoro-7-nitro-2,1,3-benzoxadiazole. The method was validated on
animal brain samples. The enantioselectivity could further be increased by addition of DIMEB to the BGE. The potential of HPA-β CD was further demonstrated in the validated CE-LIF method targeting also D–serine in mice brain samples [204]. These examples
illustrate that in spite of the underutilization of positively charged
CD derivatives as chiral selectors in CE, they can be successfully
applied for AA enantiomer analysis even in complex biological matrices.
In 2014, Yu et al. investigated the applicability of a previously
unreported derivative, mono-6A -piperidine-β -CD (Pip-β -CD) both
alone and together with neutral CDs (β -CD, TRIMEB and HP-β CD) for the enantioseparation of meptazinol and its three enantiomeric intermediates [105]. It turned out that Pip-β -CD alone
performed well for intermediate II, while the dual system with
β -CD provided the best enantioseparation for intermediate III. In

addition, the Pip-β -CD/HP-β -CD dual system excelled at the chiral resolution of meptazinol and intermediates III and IV in a single run. Pip-β -CD was successfully applied to separate folinic acid
diastereomers, where computational techniques were also used to
model the separation mechanism [205]. The results identified electrostatic interactions as the decisive factor for the enantioseparation. Three years later the same SID was compared by Zhu et al.
to five other CD derivatives: cyclohexylamine-β -CD (CHA-β -CD),
dimethyl- and hydroxypropyl-β -CD, carboxymethyl- and sulfatedβ -CD for the enantiomeric separation of ofloxacin and its five related substances [84]. No enantioresolution could be achieved with
the cationic CDs, since in the investigated pH range of 2.5–4.5, both
the analyte carboxylic acids and the selectors were protonated, disabling the attractive electrostatic forces to aid enantiorecognition.
Pip-β -CD augmented with DIMEB and sodium cholate as micellar
modifier in the BGE lead to a twofold increase in resolution.
Besides the further utilization of previously reported SID
cationic selectors, a few attempts have been made to synthesize
novel derivatives to further broaden the portfolio of adjustable
cationic SIDs.
A unique multicationic CD, mono-6A -((2S,3S)-(1)−2,3-Oisopropylidene-1,4-tetramethylenediamine)-β -CD (MIPTACD) has
been synthesized in 2010 by Liu et al. [206]. Ten Dns-AAs racemates and N-acetylphenylalanine served as model compounds
for this aminoalkylamino derivative, holding a sidechain with
two additional chiral centers. MIPTACD showed excellent chiral
recognition towards the 11 mentioned amino acid derivatives.
Although this pioneering application was promising, no additional
chiral CE study appeared using MIPTACD, presumably due to its
7-step synthesis compared to the less laborious preparation of
simpler cationic derivatives.
Hemispherodextrins, synthesized by Cucinotta et al., represent a special class of disubstituted CDs: a saccharidic unit is
attached to the primary side of the CD, connecting the oppo-


18

Table 3
Abbreviated names, structures and recent application of positively charged SIDs in CCE. Substituents are numbered according to Fig. 1.

Abbreviation

Name

Substituents

Analytes separated

Adjustable cationic charge
mono-3A -amino-β -CD

FITC-AAs [196], AAs [202]

3NH2 -γ -CD

mono-3A -amino-γ -CD

FITC-AAs [196,195]

PMMAB-CD

Permethyl-mono-6A -amino-β -CD

Dns-AAs [230]

CDhm3
(C3-histamine-substitutedβ -CD)

mono-3A -deoxy-3A -[2-(4imidazolyl)ethylamino]-β -CD

AAs [176]

HPA-β -C

mono-6A -(3-hydroxypropylamino)-β -CD

Amino acid neurotransmitters
[204,203]

Pip-β -CD

mono-6A -piperidine-β -CD

Meptadizol and synthetic
intermediates [105]
Folinic acid diastereomers [205]
Ofloxacine and its related compounds
[84]

CHA-β -CD

mono-6A -cyclohexylamine-β -CD

Ofloxacine and its related compounds
[84]

AEA-β -CD
(CDen)

mono-6A -(2-aminoethylamino)-β CD

AAs [202]
Hirsutine and hirsuteine [90]

MIPTACD

mono-6A -[(2S,3S)-(1)−2,3-OIsopropylidene-1,4tetramethylenediamine]-β -CD

Dns-amino acids,
N-acetylphenylalanine [206]

THLYSH
(Lysine bridged HSD)

Di-6A ,6D -[6,6 -dideoxy-6,6 diLysamino-α ,α ’-trehalose]-β -CD

Terbutaline and non-steroidal
anti-inflammatory drugs [207]

HEtAMCD

mono-6A -(2-hydroxyethyl-1ammonium)-β -CD
chloride

Acidic and ampholytic racemates
[191,212]
(continued on next page)

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I. Fejos,

3NH2 -β -CD


Table 3
(continued)
Name

Substituents

Analytes separated

MPrAMCD

mono-6A -(3methoxypropylammonium)-β -CD
chloride

Dns-amino acids, α -hydroxyl and
carboxylic acids [210,212]

MEtAMCD

mono-6A -(2methoxyethylammonium)-β -CD
chloride

ampholytic and acidic racemates
[211,212]

MBuAMCD

mono-6A -(4methoxybutylammonium)-β -CD
chloride

16 acidic racemates including three
Dns-amino acids [231]

HPrAMCD

mono-6A -(3-hydroxypropyl-1ammonium)-β -CD
chloride

AA neurotransmitters and
neuromodulators [203,204,212]

HBuAMCD

mono-6A -(4-hydroxybutyl-1ammonium)-β -CD
chloride

Carboxylic acids [212]

BHEtAMCD

mono-6A -[bis(2-hydroxyethyl)−1ammonium]-β -CD
chloride

Carboxylic acids [212]

THEtAMCD

mono-6A -[tris(2-hydroxyethyl)−1ammonium]-β -CD
chloride

Carboxylic acids [212]

BMEtAMCD

mono-6A -[bis(2-methoxyethyl)−1ammonium]-β -CD
chloride

Carboxylic acids [212]

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I. Fejos,

Abbreviation

(continued on next page)
19


20

Table 3
(continued)
Name

Substituents

Analytes separated

dhypy-CDCl

mono-6A -(3R,4Rdihydroxypyrrolidinium)-β -CD

Dns-AAs, anionic and ampholytic
acids [213]

pyCDCl

mono-6A -pyrrolidinium-β -CD
chloride

Dns-amino acids, α -hydroxyl and
carboxylic acids [214]

N-CH3 -pyCDCl

mono-6A -(N-methylpyrrolidinium)-β -CD
chloride

Dns-amino acids, α -hydroxyl and
carboxylic acids [214]

N-EtOH- pyCDCl

mono-6A -(N-(2-hydroxyethyl)pyrrolidinium)-β -CD
chloride

Dns-amino acids, α -hydroxyl and

carboxylic acids [214]

mono-6A -(2-hydroxymethylpyrrolidinium)-β -CD
chloride
Permanently cationic derivatives

Dns-amino acids, α -hydroxyl and
carboxylic acids [214]

PEMEDA-β -CD

mono-6A -(N,N,N’,N’,N’pentamethylethylenediammonium)-β -CD
dichloride

Anionic, weak-acids and neutral
analytes [216]

PEMPDA-β -CD

mono-6A -(N,N,N’,N’,N’pentamethyl-propylene-1,3diammonium)-β -CD
dichloride

Anionic, weak-acids and neutral
analytes [216]

mono-6A -(3-methylimidazolium)-

Tetracyclines [220]

2-MeOH-pyCDCl

MIMCDOTs

β -CD

tosylate
(continued on next page)

˝ E. Kalydi and M. Malanga et al. / Journal of Chromatography A 1627 (2020) 461375
I. Fejos,

Abbreviation


Table 3
(continued)
Substituents

Analytes separated

PrIMCD

mono-6A -(propylimidazolium)-β CD
chloride

Dns-AAs [221]

MPrIMCD

mono-6A -(3methoxypropylimidazolium)-β -CD

chloride

Dns-AAs [221]

AllIm-β -CD

mono-6A -(1-allylimidazolium)-β CD
chloride

Kynurenine [222]

4-ATMCDCl

mono-6A -(4-amino-1,2,4triazolium)-β -CD
chloride

Dansyl-AAs, Naproxen [223]

AMBuIMCD

mono-6A -[3-(4-ammoniumbutyl)imidazol-1-ium]-β -CD chloride

Dns-AAs, acidic racemates [232]

AMBIMCD

6A -Ammonium-6C butylimidazolium-β -CD
chloride

Dns-amino acids, α -hydroxyl and

carboxylic acids [226,227]

HEtTrMEtIm-β -CD

6A -(4-Hydroxyethyl-1,2,3triazole)−6C -(2methoxyethylimidazolium)-β -CD
chloride

Dns-AAs [228]

HEtTrMPrIm-β -CD

6A -(4-Hydroxyethyl-1,2,3triazole)−6C -(3methoxypropylimidazolium)-β -CD
chloride

Dns-AAs [228]

HEtTrMPrAm-β -CD

6A -(4-Hydroxyethyl-1,2,3triazole)−6C -(3methoxypropylammonium)-β -CD
chloride

Acidic racemates [229]

21

Name

˝ E. Kalydi and M. Malanga et al. / Journal of Chromatography A 1627 (2020) 461375
I. Fejos,

Abbreviation


22

˝ E. Kalydi and M. Malanga et al. / Journal of Chromatography A 1627 (2020) 461375
I. Fejos,

site sides of the rim as a bridge, forming a hemispherical saccharidic system. The first representatives, di-6A ,6D -(6,6 -diaminoα ,α ’-trehalose)-β -CD (THAMH) [188,190], Di-6A ,6D -[6,6 -dideoxy6,6 -di(S-cysteamine)-α ,α ’-trehalose]-β -CD (THCMH) [189] and di6A ,6D -N-[6,6 -di-(β -alanylamido)−6,6 -dideoxy- α ,α ’-trehalose]-β CD (THALAH) [188,190] were successfully applied for the separation of phenoxy acids, profens and DNS-AAs. They were followed
in 2017 by di-6A ,6D -[6,6 -dideoxy-6,6 diLys-amino-α ,α ’-trehalose]β -CD (THLYSH or lysine-bridged HSD), which was used to separate terbutaline and non-steroidal anti-inflammatory drugs, in both
chiral and achiral separations [207]. Chiral separations could be
achieved by the lysine-bridged HSD without addition of another
CD derivative, which was not the case for the earlier HSDs.
In contrary to their negative counterparts, the family of weak
base-type CD derivatives contains relatively few persubstituted
members, reported around 20 0 0 or earlier. The first representatives was the per-6-hydroxyalkylamino-derivative [208], followed
by per-6-amino-β -CD in 2004 [173]. The resolving power of persubstituted weak bases has a pronounced pH-dependence, as their
degree of protonation can be varied sensitively in a larger pH interval. The charge repulsion of the substituents can alter the shape
of the CD cavity, influencing complexation, selectivity and affecting
enantiomeric resolution as well [209]. Moreover, at acidic pH values, the multiple positive charges present on the selector molecule
promote its adsorption propensity to the capillary wall, leading
to limited reproducibility. These factors may be the reason of the
scarcity of studies with persubstituted cationic CDs.
Although primary, secondary and tertiary amine functionalized CDs (with n-alkyl or cycloalkyl moieties on the nitrogen) do
not possess a pH-independent positive charge over the entire pH
range, at pH << pKa values they can be considered and usually referred to in the literature as permanently cationic derivatives, since
they are rarely applied at pH values where the amine groups are
deprotonated.
Alkylammonium-β -CD derivatives have been studied by various research groups. Tang et al. investigated the effect of chainlength for enantiomer resolution by the comparison of monoalkylammonium CD derivatives, while Mikus and Kaniansky compared N-methylated mono-6A -amino-β -CDs with different degrees
of methylation to separate DNP-AAs [183]. The results revealed that

the charge of the selector plays a significant role in the separation selectivity. The permanently cationic quaternary ammoniumβ -CD trimethylammonium-β -CD showed significantly better resolution power at high pH compared to its mono- and dimethylated
counterparts.
In 2013, Zhou et al. used mono-6A -(2-hydroxyethyl-1ammonium)-β -CD chloride (HEtAMCD), prepared by the addition
of diluted hydrochloric acid to the EA-β -CD [191] (see Table 3.).
Using the protonated form of this formerly reported derivative,
a new recognition site was implemented. By introducing the
ammonium cation into the selector, three different driving forces
are in action: inclusion complex formation, hydrogen bonding
and electrostatic interaction, the latter one induced by the presence of the ammonium group. Although a detailed comparison
was described with three previously reported ammonium CDs,
mono-6A -(3-propylammonium)-β -CD chloride (PrAMCD), mono6A -(3-methoxypropylammonium)-β -CD chloride (MPrAMCD) and
CD-NH3 Cl. The comparison focused on the effect the hydroxyalkyl chain (compared to CD-NH3 –Cl), the role of the OH group
(compared to PrAMCD, lacking the OH group and to MPrAMCD,
where the OH is etherified). The study indicated that the hydroxyethylammonium group of HEtAMCD significantly increased
the enantioselectivity. 1D and 2D NMR study of complexes with
selected analytes has revealed a hydrogen bond between the
hydroxy groups of CD and that of the analytes as an additional
driving force for enantiomer recognition. The same hydrogen

Fig. 5. Spatial differences in MEtAMCD/ 3-Phenillactic acid (3-PLA) systems. Left:
MEtAMCD/R-3-PLA complex: The additional H-bond, responsible for the chiral selectivity is highlighted with the dotted circle. Right: MEtAMCD/S-3-PLA complex
lack of the additional H-bond revealed. The inclusion complexation behavior was
obtained by 1 H NMR experiments Figure redrawn from Ref. [211].

bond-enhanced chiral resolution was observed by Dai et al.
when compared the chiral selector capabilities of MPrAMCD to
PrAMCD using hydroxyl acids and ampholytic racemates. The
chiral recognition was further enhanced with the presence of an
extra methoxy group, potentially forming an extra hydrogen-bond
between MPrAMCD and the analytes [210].

Subsequently, two additional homologues with different chainlength were synthesized, mono-6A -(2-methoxyethylamino)-β -CD
(MEtAMCD) [211] and mono-6A -(4-methoxybutylamino)-β -CD
(MBuAMCD) [210]. In comparison with the counterpart aminesubstituted CDs, MEtAMCD yielded better enantioselectivities for
Dns-AAs, hydroxy acids and phenoxyalkanoic acids, presumably
again due to the hydrogen bonds. MBuAMCD yielded baseline
separation for most racemates even at 1.0 mM concentration,
while Rs > 6 was achieved in certain cases at 3.0 mM CD. The
hydrogen-bond-enhanced enantioseparation was supported by
auxiliary NMR spectroscopic studies (see Fig. 5).
Both the number and the length of the alkyl substituents on the
nitrogen turned out to influence significantly the chiral separation
in a later study [212]. Mono-6A -bis(2-hydroxyethyl)-1-ammoniumβ -CD chloride (BHEtAMCD), Mono-6A -tri(2-hydroxyethyl)-1ammonium-β -CD chloride (THEtAMCD), and mono-6A -bis(2methoxyethyl)-1-ammonium-β -CD chloride (BMEtAMCD) were
first described as chiral selectors in CE. Six carboxylic acid racemates were chosen as test analytes at different concentrations.
The results demonstrated, that with the increasing number of
hydroxylalkyl groups at the N-atom, the chiral resolving power
significantly declined, as the aqueous solubility of the derivatives
deteriorated. The best enantioseparation was achieved by mono6A -(3-hydroxypropyl)-1-ammonium-β -CD chloride (HPrAMCD)
and MPrAMCD. These CD derivatives were further investigated by
NMR experiments using mandelic acid as a guest, proving that
besides the inclusion complex formation, electrostatic attraction
and hydrogen bonded interactions are also present as additional
chiral driving forces.
A series of mono-6-pyrrolidinium-β -CD derivatives have been
characterized as chiral selectors by Xiao et al. [213,214]. Originally, they have been synthesized to overcome analyte detection limit problems caused by the UV absorption of imidazoliumderivatized CDs. A non-planar pyrrolidine ring or its derivative was
attached to the primary side of β -CD [213]. The first synthesized
SID was mono-6A -(3R,4R-dihydroxypyrrolidinium)-β -CD chloride
(dhypy-CDCl) for the enantioseparation of anionic and ampholytic
acids. A baseline resolution could be achieved for a mixture of five
enantiomer pairs. Although its analogue mono-6A -pyrrolidine-β CD chloride (pyCDCl) holds achiral center sidechain, it surprisingly

˝ E. Kalydi and M. Malanga et al. / Journal of Chromatography A 1627 (2020) 461375
I. Fejos,

showed a higher selectivity and resolution. The authors proposed
the explanation that the two hydroxyl groups on the side-chain
might produce a steric hindrance during chiral recognition, thus
lowering the degree of complexation and the binding constant.
In another study, mono-6A -pyrrolidinium-β -CD chloride (pyCDCl), mono-6A -(N-methyl-pyrrolidinium)-β -CD chloride (N-CH3 pyCDCl), mono-6A -(N-(2-hydroxyethyl)-pyrrolidinium)-β -CD chloride (N-EtOH-pyCDCl), mono-6A -(2-hydroxymethyl-pyrrolidinium)β -CD chloride (2-MeOH-pyCDCl) were synthesized and explored
for the enantioresolution of carboxylic acids, hydroxycarboxylic
acids and Dns-AAs [214]. Interestingly, the unsubstituted pyCDCl
performed best. The other three cationic CDs with N-alkyl substituents afforded higher anionic effective mobilities but much
lower selectivity and resolution for most of the analytes. Exceptions were Dns-Asp- and Dns-Glu, due the role of the carboxylic
group in chiral recognition.
3.3.2. Permanently positively charged SID derivatives
In certain chiral CE separations, the appropriate protonation
state of the analyte dictates the pH of the BGE. In these cases,
application of a permanently positively charged CD irrespective of
pH may be beneficial during method development. The permanently cationic derivatives can be categorized according to their
charge numbers or the number of substituents. The simplest and
most widely employed members are the monosubstituted, monocationic CDs. Additional charge can be either introduced on the
same substituent, constituting the group of monosubstituted, dually cationic (or even multiple charged) derivatives or onto a different substituent forming the disubstituted, dicationic members.
In the recent literature, there is a trend for the synthesis of the
latter group of SID selectors.
The ever-growing variety of cationic CD selectors is mainly built
up of alkylimidazolium-derivatives, various types of alkyltriazolium
derivatives and quaternary ammonium-CD derivatives. Their counterion is generally chloride, coming synthetically from either a
strong cation-exchanger resin or HCl salt formation, and this anion does not interfere with the detection.
In the literature of disubstituted cationic SIDs, a permanently
cationic substituent is typically combined either with a protonable

side-chain or with a non-basic substituent.
A series of permanently positively charged, novel SI α /β /γ CDs were synthesized by Popr et al., introducing one, two
or three tetraalkylammonium groups on the primary side of
the rim [215]. However, only two of the prepared compounds
were reported, the mono-6A -(N,N,N,N,N-pentamethyl-ethylene1,2-diammonium)-β -CD dichloride (PEMEDA-BCD) [216], which
was previously prepared by Nzeadibe et al. [217] and the
newly synthesized mono-6A− (N,N,N,N,N-pentamethylpropylene-1,3diammonium)-β -CD (PEMPDA-BCD) [216]. Both derivatives are dicationic compounds, bearing two quaternary ammonium groups in
their sidechain. They were tested as additives in BGE systems, including also an organic modifier. Fourteen analytes including native amino acids, N-protected amino acids and profens were tested.
Both chiral selectors enabled the enantioseparation of N-Boc-D,Ltryptophan due to an favorable ionic interaction.
In the past few years, ionic-liquid functionalized CDs raised
more attention in the field of enantioseparation. Tang and Ong
et al. introduced a new family of mono-6-substituted-CDs as permanently positively charged chiral selectors in CE. They synthesized and characterized the series of IL-functionalized SIDs, mono6A -(3-alkylimidazolium)-β -CDs with different chain-length. CDs
with a shorter alkyl chain (R = Cn H2 n +1, n ≤ 4) demonstrated a better resolution during the enantioseparation of Dns-AAs [218,219].
In a recent study, Zhou et al. used mono-6A -(3methylimidazolium)-β -CD tosylate (MIMCDOTs) for the separation
and quantification of tetracyclines at the same time [220] (see

23

Table 3.). The cationic CD served simultaneously as electroosmotic
flow modifier by coating the capillary wall and also as a resolving
agent via inclusion complex formation with tetracyclines
After the successful enhancement of chiral resolution of
amino acids and hydroxyl acids by MPrAMCD compared to
the PrAMCD exploiting the extra interaction provided by the
methoxy group [210], mono-6A -methoxypropylimidazolium-β -CD
chloride (MPrIMCD) and mono-6A -propylimidazolium-β -CD chloride (PrIMCD) were compared for the separation of eight Dns-AAs
[221]. The novel single isomer CD enhanced the interactions between the CD and the amino acids to afford better chiral resolutions in case of all the investigated AAs.
As a novel chiral selector, mono-6A -(1-allylimidazolium)-β -CD
chloride (AllIm-β -CD) was introduced by Rizvi et al. [222]. Along
with the native α -CD, β -CD and the randomly substituted HPBCD the derivative was investigated for the chiral separation of

kynurenin from biological samples like urine and serum. Both
enantiomers have a role in the development of neurological diseases. The authors revealed the synergetic effect of allIm-β -CD and
α -CD leading to baseline separation, while these two CDs alone afforded no or only partial separation at lower concentration. The cooperative effect was confirmed by molecular modeling (see Fig 6).
The benefit of the dual system was outstanding at 7.4, when the
dual system provided resolution of the two constituents.
In the study of Li et al., a novel IL amino triazolium functionalized SID, mono-6A -(4-amino-1,2,4-triazolium)-β -CD chloride (4ATMCDCl) was synthesized to separate dansyl amino acids and
naproxen [223]. Besides its permanent positive charge on the triazole ring, the amino substituent adds an adjustable positive charge
to the selector. A molecular modeling method was additionally applied, which demonstrated that amino triazolium could provide
more additional interactions, such as π -π stacking, π -cation and
hydrogen bonding, increasing the enantioselectivity towards the
analytes.
A similar class of cationic CDs were synthesized by Boffa et al.
[224]. They combined the advantages of CDs and ILs using the
copper—catalyzed azide-alkyne cycloaddition applicability in CE
was not explored, only one of the prepared compounds was investigated as a stationary phase in a later chiral GC study.
Similarly to 4-ATMCDCl, mono-6A -[3-(4-ammoniumbutyl)imidazol-1-ium]-β -CD chloride (AMBuIMCD) [225], reported
by Zhou et al. bears an additional adjustable charge besides
its permanently positive imidazolium moiety, providing high
aqueous solubility irrespectively to pH. This SID provided good
enantiomeric recognition towards Dns-AAs and acidic analytes,
deprotonated at pH 6 of the BGE. Synthesis of two different sets
of structurally analogous dicationic derivatives have also been
reported mono-6A -[3-(4-ammoniumalkyl)-imidazol-1-ium]-β -CD
chlorides and mono-6A -[3-(3-imidazolalkyl)-ammonium]-β -CD
chlorides, with the chain lengths between 2 and 6).
The last decade witnesses a trend to synthesize multisubstituted SIDs for chiral CE separations. Dai et al. prepared a series
of SID dicationic AC regioisomer CDs: mono-6A -ammonium-6C alkylimidazolium-β -CD chlorides [226]. Although four derivatives
with chain-lengths of 1–4 have been synthesized, only mono-6A ammonium-6C -butylimidazolium-β -CD chloride (AMBIMCD) was
evaluated as a chiral selector towards Dns-AAs and acidic racemates [226,227]. Seven out of eighteen studied analytes were baseline separated at 0.5 mM CD concentration. At the same separation conditions, these dicationic CDs displayed better enantioseparations than their mono-imidazolium and mono-ammonium counterpart CDs.
In 2014 Tang et al. synthesized two AC-regioisomer

SIDs of β -CD, functionalized with methoxyalkylimidazolium
and 4-(2-hydroxyethyl)-1,2,3-triazole to form mono-6A -4(2-hydroxyethyl)-1,2,3-triazole-6C -methoxyethylimidazolium-β -CD


˝ E. Kalydi and M. Malanga et al. / Journal of Chromatography A 1627 (2020) 461375
I. Fejos,

24

Fig. 6. (A) Separation of D, l-kynurenin (KYN) in α -CD/AllIm-β -CD dual selector system. BGE: 50 mM borax borate buffer, pH 9.0. CE conditions: 15 kV, 50 mbar × 5 s,
25 °C, λ= 226 nm, d-KYN, l-KYN 10 0 0 nM each (B) Synergetic effect of α -CD/AllIm-β -CD on the chiral separation of D,L-kynurenin. Right: l-KYN/ α -CD/AllIm-β -CD Left:
l-KYN/ α -CD/AllIm-β -CD. The details of the separation mechanism were revealed by molecular docking and molecular mechanics. Figure redrawn from Ref. [222].

chloride (HEtTrMEtIm-β -CD) and mono-6A -4-(2-hydroxyethyl)1,2,3-triazole-6C -methoxypropylimidazolium-β -CD
chloride
(HEtTrMPrIm-β -CD)
[228].
mono-6A -4-(2-hydroxyethyl)-1,2,3triazole-6C -3-methoxypropylimidazolium β -CD chloride was
shown to separate Dns-amino acids and acidic enantiomers. Compared to the aforementioned AC-regioisomers, the latter SID could
achieve satisfactory enantioseparation in shorter time.
The third type of SID AC-regioisomer positively charged CD
was reported by Zhou et al. [229] As a new member of the
AC disubstituted SID, mono-6A -4-(2-hydroxylethyl)-1,2,3-triazole6A -3-methoxypropylamino-β -CD (HETz-MPrAMCD) was compared
to its monosubstituted counterpart, MPrAMCD, showing better
enantioseparations for dansyl AAs at low concentrations.
3.4. Zwitterionic CDs
A new, inner-salt type 6-O-(2-hydroxyl-3-betainyl-propyl)-

β -cyclodextrin (6-HBP-β -CD) was prepared by a ‘‘synthesis-

deprotection one pot’’ method and this zwitterionic CD was found
to be an efficient chiral selector in CCE comparing with native β CD and 2-HP-β -CD for drug racemates including chlorphenamine
[233].
An amino acid modified single isomer CD derivative,
heptakis{2,6-di-O-[3-(1,3-dicarboxyl
propylamino)−2hydroxypropyl]}-β -cyclodextrin (glutamic acid-β -cyclodextrin,
glu-β -CD) was synthesized and used as a chiral selector in CE for
the enantioseparation of 12 basic drugs [234]. Glu-β -CD switches
its charge state at the isoelectric point of the glutamic acid (pH
3.2), thus the effect of the BGE pH and the selector concentration
was studied during the method optimization. To confirm and

explain the possible chiral recognition mechanism computational
modeling strategy was used with three antihistamines.

4. Concluding remarks
CCE has become an established and powerful analytical scale
separation for enantiomers in pharmaceutical, agrochemical, cosmetic and food industries. The multitude of experimental variables along with the possibility of solid physicochemical modeling of electromigration phenomena with predictive outcome makes
this technique particularly attractive for easy and fit-for-purpose
method development. The most versatile and commonly used
supramolecular selectors for the discrimination of enantiomers remain to be cyclodextrins, available now in a plethora of derivatives varying in positions, degrees and types of functionalization.
The structurally uniform single isomer cyclodextrins offer unique
opportunities for in-depth, systematic studies of the structural aspects of the selector-analyte interactions in particular with NMR
spectroscopy and molecular modeling, contributing to better understanding and experimental design of chiral CE separations. Reviewing the past decade of this field emphasized the importance of
CD structure-enantioresolution relationships to achieve previously
unattained separations, including also specific combinations of CDs
(dual systems), ligand-exchange metal-ion complexation, ionic liquids or micelles. Capillary electrophoresis as the youngest member of the separation science family is flourishing for chiral separations, which is clearly attributed to the synthetic efforts leading
to the huge variety of CD-based selectors reviewed herein.

˝ E. Kalydi and M. Malanga et al. / Journal of Chromatography A 1627 (2020) 461375
I. Fejos,

Declaration of Competing Interest
None.
CRediT authorship contribution statement
Ida Fejos:
˝ Writing - original draft, Supervision, Writing - review
& editing. Eszter Kalydi: Writing - review & editing, Visualization.
Milo Malanga: Writing - review & editing. Gábor Benkovics: Writing - review & editing. Szabolcs Béni: Conceptualization, Funding
acquisition, Writing - review & editing.
Acknowledgements
This work was partially supported by the János Bolyai Research
Scholarship of the Hungarian Academy of Sciences and by the
Bolyai+ New National Excellence Program (grant number: ÚNKP19-4-SE-53) of the Ministry of Human Capacities.
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