High-Performance Liquid Chromatographic Enantioseparation of Cyclic β-Amino Acids on Zwitterionic Chiral Stationary Phases Based on Cinchona Alkaloids

Article abstract of DOI:10.1002/chir.22458

Stereoselective high-performance liquid chromatographic separations of eight sterically constrained cyclic β-amino acid enantiomer pairs were carried out using the newly developed Cinchona alkaloid-based zwitterionic chiral stationary phases Chiralpak ZWIX(+) and ZWIX(-). The effects of the mobile phase composition, the nature and concentrations of the acid and base additives, the counterions and temperature on the separations were investigated. The changes in standard enthalpy, Δ(ΔH°), entropy, Δ(ΔS°), and free energy, Δ(ΔG°), were calculated from the linear van't Hoff plots derived from the ln α vs. 1/T curves in the studied temperature range (10-50°C). The values of the thermodynamic parameters depended on the nature of the selectors and the structures of the analytes. Unusual temperature behavior was observed on the ZWIX(-) column: decreased retention times were accompanied by increased separation factors with increasing temperature. On the ZWIX(+) column only enthalpically, whereas on the ZWIX(-) column both enthalpically and entropically driven separations were observed. The elution sequence was determined in all cases and was observed to be the opposite on ZWIX(+) and on ZWIX(-). Chirality 27:563-570, 2015.

Full text of DOI:10.1002/chir.22458

CHIRALITY 27:563–570 (2015)
Special Issue Article
High-Performance Liquid Chromatographic Enantioseparation of
Cyclic β-Amino Acids on Zwitterionic Chiral Stationary Phases Based
on Cinchona Alkaloids
ISTVÁN ILISZ, ZSANETT GECSE, GYULA LAJKÓ, ENIKÕ FORRÓ, FERENC FÜLÖP, WOLFGANG LINDNER, AND ANTAL PÉTER1*
1
1,2
1,2
2
2
3
1
Department of Inorganic and Analytical Chemistry, University of Szeged, Szeged, Hungary
2
Institute of Pharmaceutical Chemistry, University of Szeged, Szeged, Hungary
3
Department of Analytical Chemistry, University of Vienna, Vienna, Austria
ABSTRACT
Stereoselective high-performance liquid chromatographic separations of eight
sterically constrained cyclic β-amino acid enantiomer pairs were carried out using the newly de-
veloped Cinchona alkaloid-based zwitterionic chiral stationary phases Chiralpak ZWIX(+) and
ZWIX(À). The effects of the mobile phase composition, the nature and concentrations of the acid
and base additives, the counterions and temperature on the separations were investigated. The
changes in standard enthalpy, Δ(ΔH°), entropy, Δ(ΔS°), and free energy, Δ(ΔG°), were calcu-
lated from the linear van’t Hoff plots derived from the ln α vs. 1/T curves in the studied temper-
ature range (10–50°C). The values of the thermodynamic parameters depended on the nature of
the selectors and the structures of the analytes. Unusual temperature behavior was observed on
the ZWIX(À) column: decreased retention times were accompanied by increased separation fac-
tors with increasing temperature. On the ZWIX(+) column only enthalpically, whereas on the
ZWIX(À) column both enthalpically and entropically driven separations were observed. The elu-
tion sequence was determined in all cases and was observed to be the opposite on ZWIX(+) and
on ZWIX(À). Chirality 27:563–570, 2015. © 2015 Wiley Periodicals, Inc.
KEY WORDS: enantiomer separation; HPLC; cyclic β-amino acids; Cinchona alkaloid-based
chiral stationary phases
The syntheses of both racemic and enantiomeric ß-amino
acids have recently gained in importance as a result of their
widespread use in different fields of pharmaceutics and chem-
istry. For example, biologically active carbocyclic ß-amino
acids (e.g., the antibacterial or/and antifungal cispentacin,
anion-exchange mode for the separation of chiral acids, in
the cation-exchange mode for the resolution of chiral amines,
and in the zwitterionic exchange mode for the enantiose-
paration of amphoteric compounds such as amino acids and
small peptides.
1
,2
icofungipen and BAY Y9379) can be widely used in hetero-
The present article describes the separation of eight pairs
of enantiomers (selectands, SAs) of cyclic β-amino acids
(Fig. 1) on the Cinchona alkaloid-based zwitterionic CSP
3
,4
5,6
7,8
cyclic, peptide, or combinatorial chemistry and in drug
9,10
research.
A search is therefore ongoing for new strategies
7,8,11–13
33
for the synthesis of enantiomeric ß-amino acids.
selectors (SOs) in PIM mode. The effects of the mobile
To investigate the stereochemistry and the enantiomeric
excess of the product β-amino acids, highly efficient analytical
methods are needed. One of the most frequently applied tech-
niques is enantioselective (chiral) high-performance liquid
chromatography (HPLC). HPLC enantioseparations of β-
amino acids have been performed by both indirect and direct
methods and in the past decade new types of chiral
derivatizing agents and chiral stationary phases (CSPs) have
been applied for this purpose and reviewed in numerous arti-
phase composition, the nature of the acid and base additives,
the amount of the counterion, temperature, and the structural
features of the SAs on the retention and enantioselectivity are
discussed. The sequence of elution of the enantiomers was
determined in all cases and is used as the basis of a discus-
sion of stereoselective recognition models.
EXPERIMENTAL
Chemicals and Reagents
1
4–26
cles.
Enantioselective retention and separation are influenced by
The enantiomers of racemic cis-aminocyclopentanecarboxylic acids
(cis-ACPC; 1A,1B) were prepared from cyclopentane via chlorosulfonyl
isocyanate addition, followed by aqueous HCl treatment and ion-
temperature.2
7–32
For mechanistic considerations to be
3
7
exchange chromatography. trans-ACPC (1C,1D) was synthesized by
drawn, a plot of R ln α vs. 1/T has been applied with a slope
of –Δ(ΔH°) and an intercept of Δ(ΔS°), as discussed by
Fornstedt et al.30
*Correspondence to: Prof. Antal Péter, Department of Inorganic and Analyti-
cal Chemistry, University of Szeged, H-6720 Szeged, Dóm tér 7, Hungary.
E-mail: apeter@chem.u-szeged.hu
Received for publication 10 March 2015; Accepted 8 April 2015
DOI: 10.1002/chir.22458
Published online 14 May 2015 in Wiley Online Library
(wileyonlinelibrary.com).
Newly developed Cinchona alkaloid-based zwitterionic ion-
exchange-type CSPs characterized by a weak basic and a
strong acidic site operate best under slightly acidic polar-ionic
mobile phase (PIM) conditions.33–36 They can operate in the
©
2015 Wiley Periodicals, Inc.

5
64
ILISZ ET AL.
consisting of a solvent degasser, a pump, an autosampler, a column ther-
mostat, a multiwavelength UV–vis detector. and a corona-charged aerosol
detector from ESA Biosciences (Chelmsford, MA). Data acquisition and
analysis were carried out with ChemStation chromatographic data soft-
ware from Agilent Technologies.
The Chiralpak ZWIX(+) and ZWIX(À) columns (150 ×3.0 mm I.D., 3-μ
m particle size for both columns) were from Chiral Technologies Europe
(Illkirch, France).
Chromatography was performed in isocratic mode at a flow rate of 0.6
À1
ml min and a column temperature of 25°C (if not otherwise stated). De-
tection was accomplished by UV at selected wavelengths of 215 and 230
nm or with a corona detector. The void volume of the columns (t
0
) was
determined by injecting acetone dissolved in MeOH, with detection at
2
0
80 nm. All SAs were dissolved in MeOH in the concentration range
.5–1.0 mg ml and further diluted with mobile phase.
À1
RESULTS AND DISCUSSION
Effects of Mobile Phase Composition and Additives on
Chromatographic Parameters
All the investigated amino acids (Fig. 1) possess a
cycloalkane skeleton. The carboxy and primary amino groups
provide the charged sites for the ionic interactions. SAs A and
B are the enantiomers of the cis, while C and D are the enan-
tiomers of the trans isomers; all other combinations are dia-
stereomers. Besides carboxy and primary amino groups,
analogs 2, 4, and 5 possess a double bond. These differences
result in different physical properties, such as hydrophobic-
ity, polarity, and rigidity of the molecules and may exert a sig-
nificant influence on the interactions with the quinine- and
quinidine-based chiral zwitterionic SOs.
Fig. 1. Chemical structures of Sas.
3
Michael addition of NH to 1-cyclopentenecarboxylic acid in a steel auto-
clave at 170°C for 80 h, followed by purification via ion-exchange chroma-
3
8
tography. (À)-cis-(1R,2S)-ACPC (1B) and (+)-trans-(1S,2S)-ACPC (1C)
were prepared from the corresponding esters by lipase-catalyzed acyla-
tion, followed by hydrolysis.39 The racemic cis- and trans-
aminocyclohexanecarboxylic acid (cis- and trans-ACHC, 3A-3D) and
cis-(1S,2R;1R,2S) and trans-ACHC-ene (1S,2S;1R,2R) were prepared from
the corresponding commercially available cis- and trans-hexahydro- and
In the PIM mode, a mixture of MeOH as protic solvent and
MeCN as aprotic solvent in combination with acid and base
additives ensured the best enantioseparation on the Cinchona
34,36
alkaloid-based zwitterionic CSP ion-exchangers.
To inves-
tetrahydrophthalic anhydrides by ammonolysis, followed by Hoffmann
degradation and ion-exchange purification.4
0,41
In the cases of cis and
tigate the effects of the organic bulk solvent, chromato-
graphic separation was carried out in MeOH/MeCN (75/25,
50/50, and 25/75 v/v) mobile phases containing 25 mM
TEA and 50 mM AcOH for representative SAs, i.e., saturated
and unsaturated analogs of cis isomers (1A,1B and 4A,4B)
and trans analogs (1C,1D and 4C,4D), respectively. In the
presence of different MeOH/MeCN mixtures as mobile
phase bulk solvent on ZWIX(+) and ZWIX(À), the application
of a higher MeCN content in the mobile phase in all cases
significantly increased the retention (Fig. 2). The electrostat-
ically driven interactions involved in the formation of the SO-
SA complexes with zwitterionic CSPs are obviously less
strong in protic MeOH solvents which may be caused 1) by
the weakening of the intermolecular hydrogen bonding
trans-ACHC-ene (4A-4D), Hoffmann degradation was performed with
NaOCl instead of the usual NaOBr. For the preparation of the enantio-
mers of compounds 4A-4D, lipase PS-catalyzed selective N-acylation of
the ethyl esters of racemic cis- or trans-β-amino acids was used.3 To a so-
lution of the ethyl esters of the racemic compounds and 2,2,2-
trifluoroethyl chloroacetate in diethyl ether, a lipase PS preparation was
added. The mixture was stirred at room temperature for 10–40 min, a
few drops of ethanol were then added, and the enzyme was filtered off.
The ethereal solution was cooled to 0°C and gaseous HCl was slowly bub-
bled through the solution for 30 min. The hydrochlorides of the ethyl es-
ters of the (1R,2S) and (1S,2S) enantiomers that precipitated out were
filtered off to give white crystals. The (1R,2S) and (1S,2S) enantiomers
were obtained after small-scale alkaline hydrolysis of the ethyl esters.
Their identities were confirmed by means of melting point determination,
9
1
mass spectrometry, H-NMR spectroscopy, and optical rotation
effect, and 2) by the change of the solvation shells of the elec-
measurement.
33,34,43–45
trostatic interacting ionic sites.
The observed effect
Enantiomeric β-amino acids 2A, 2B, 5A, and 5B were prepared
through CAL-B-catalyzed enantioselective (E > 200) ring opening of race-
mic 6-azabicyclo[3.2.0]hept-3-en-7-one and 7-azabicyclo[4.2.0]oct-4-en-8-
one (prepared by the above-mentioned cycloaddition of chlorosulfonyl
isocyanate to the corresponding cycloalkadienes) with 1 equivalent of
of a higher content of MeCN on the selectivity was not consis-
tent, but it most often led to slightly enhanced
enantioselectivity. In accordance with the former results for
a
β-amino acids,3
6,43–46
an increase in separation performance
H
2
O in 2-propyl ether (iPr
Methanol (MeOH) and acetonitrile (MeCN) of HPLC grade, and NH
ethylamine (EA), triethylamine (TEA), propylamine (PA) tripropylamine
TPA), butylamine (BA), tributylamine (TBA), formic acid (FA), and gla-
2
O) at 70°C.42
was observed at higher MeCN content. However, on ZWIX
(+) for the enantiomer pairs 1A,1B and 4A,4B and on
ZWIX(À) for 4A,4B slight decreases in α were observed,
3
,
47
(
similar to the case of α-amino acids. The resolution (R )
S
cial acetic acid (AcOH) of analytical reagent grade were purchased from
VWR International (Radnor, PA).
was in most cases highest in MeOH/MeCN (50/50 v/v)
containing 25 mM TEA and 50 mM AcOH.
For optimization of the enantioseparation on Cinchona
alkaloid-based CSPs, not only variation of the main compo-
nents of the eluent system, but also variation of the nature
of the acid and base additives in the mobile phase may offer
Apparatus and Chromatography
Chromatographic measurements were performed on a 1100 Series
HPLC system from Agilent Technologies (Waldbronn, Germany),
Chirality DOI 10.1002/chir

HPLC ENANTIOSEPARATION OF CYCLIC β-AMINO ACIDS
565
Fig. 2. Effects of the compositions of the bulk solvents on the chromatographic parameters for SAs 1A,1B, 1C,1D, 4A,4B, and 4C,4D on ZWIX(+) or ZWIX(–).
Chromatographic conditions: column, Chiralpak ZWIX(+) and ZWIX(–); mobile phase, MeOH/MeCN (75/25, 50/50 or 25/75 v/v) containing 25 mM PA and 50
-
1
mM AcOH; flow rate, 0.6 ml min ; detection, 230 nm; temperature 25ºC; symbols, k
α for 1A,1B: α, for 1C,1D: , for 4A,4B: and for 4C,4D:
1
.
for SA 1A,1B:
, for 1C,1D:
, for 4A,4B:
and for 4C,4D:
;
a relatively easy choice. An acid-to-base ratio of 2:1 was
previously found to be most effective for successful
increased (Fig. 3). The increased retention observed is
probably due to the reduced capacity of TBA, TPA, or TEA
in their roles as counterions as compared with BA, PA, or
EA, respectively. As concerns the influence of the nature of
the bases on the separation factor, a slight change in α was
observed, but without any general trend. When the effects
of the base additives on the retention and selectivity are taken
into account, the application of TEA appears favorable.
The nature of the acids exerted a slight effect on the reten-
tion. For SAs 1A,1B, 2A,2B, 3C,3D, 4C,4D, and 5A,5B,
the presence of FA with a few exceptions resulted in slightly
36,43–46
enantioseparation,
with the concentration of the acid
being kept at 50 mM, and that of the base at 25 mM. The acid
excess applied in the mobile phase ensured that the bases
(
amines) were present in their “ammonium ion” forms.
For comparison of the effects of the nature of the base and
the acids as mobile phase additives, separations of saturated
and unsaturated analogs of cis isomers (1A,1B, 2A,2B, and
5
A,5B) and trans analogs (3C,3D and 4C,4D) were carried
out with the same mobile phase composition on both ZWIX
(
+) and ZWIX(À) columns, the MeOH/MeCN content being
smaller k values with the same base additive, as seen previ-
1
ously.4
7–50
kept constant (75/25 v/v), while the nature of the base (25
mM base) and the acid (50 mM) was varied. Seven different
As regards the effect of the acid modifier (FA or AcOH) on
the separation factor and resolution, again no general trend
could be observed. When separation occurred, α differed
slightly in the presence of FA from that obtained with AcOH,
bases (NH , EA, TEA, PA, TPA, BA, and TBA) and two acids
3
(
FA and AcOH) were selected for these experiments.
The experimental results for the pairs 1A,1B and 5A,5B
on ZWIX(+) and ZWIX(À) columns (Figs. 3 and 4) revealed
that the retention factors in several cases slightly increased
in the sequences EA<PA<BA and TEA<TPA<TBA (in sev-
eral cases BA resulted in smaller k values than with PA; data
not shown). Increasingly apolar and bulkier amine additives,
probably having a reduced effect on the competitive ion-ion
interactions lead to an increase in k. With a few exceptions
on both columns, the retention factors increased as the
degree of ethyl, propyl or butyl substitution of the nitrogen
while slightly higher R values were obtained on both CSPs
S
when AcOH was applied.
For both anion-exchange and cation-exchange SOs under
PIM conditions, a predominant ion-exchange-driven retention
51
mechanism has been confirmed and the observed effect
could be explained by a simple displacement model.5 In or-
der to gain deeper insight into the retention mechanism in
the cases of the given zwitterionic CSPs, the effects of varia-
tion of the concentrations of the base additives on the
2
Fig. 3. Effect of the nature of acid and base additives on chromatographic parameters on Chiralpak ZWIX(+) and ZWIX(–) for SAs 1A,1B and 5A,5B. Chromato-
graphic conditions: column, Chiralpak ZWIX(+) and ZWIX(–); mobile phase, MeOH/MeCN (75/25 v/v) containing c, 50.0 mM AcOH and 25.0 mM TEA, d, 50.0
mM AcOH and 25.0 mM NH
5.0 mM BA, i, 50.0 mM AcOH and 25.0 mM TBA, j, 50.0 mM FA and 25.0 mM NH
FA and 25.0 mM PA, n, 50.0 mM FA and 25.0 mM TPA, o, 50.0 mM FA and 25.0 mM BA, p, 50.0 mM FA and 25.0 mM TBA; flow rate, 0.6 ml min ; detection, corona
detector; symbols, k1, , α , R
3
, e, 50.0 mM AcOH and 25.0 mM EA, f, 50.0 mM AcOH and 25.0 mM PA, g, 50.0 mM AcOH and 25.0 mM TPA, h, 50.0 mM AcOH and
2
3
, k, 50.0 mM FA and 25.0 mM EA, l, 50.0 mM FA and 25.0 mM TEA, m, 50.0 mM
-1
S
.
Chirality DOI 10.1002/chir

5
66
ILISZ ET AL.
Fig. 4. Influence of the counter-ion concentration on retention of the first eluting enantiomer (k
1
) of 1A,1B, 2A,2B, 3C,3D, 4C,4D and 5A,5B. Chromato-
graphic conditions: column, ZWIX(+) and ZWIX(–); mobile phase, MeOH/MeCN (25/75 v/v) containing 5, 12.5, 25 and 50 mM TEA and 10, 25, 50, and 100
-
1
mM AcOH (acid-to-base ratio being kept at 2:1); flow rate, 0.6 ml min ; detection, corona detector; symbols, 1A,1B, 2A,2B, 3C,3D, 4C,4D, 5A,5B.
retention were studied. Data displaying the effects of the TEA
on the retention for 1A,1B, 2A,2B, 3C,3D, 4C,4D, and
TABLE 1. Chromatographic data, retention factor (k), selec-
S
tivity factor (α), resolution (R ), and elution sequence of cyclic
ß-amino acids on ZWIX(+) and ZWIX(À) at constant mobile
5
A,5B are depicted in Figure 4. Under the studied condi-
phase composition
tions, linear relationships were found between log k1 and
log cTEA, with slopes varying of around 0.2–0.4. With the
earlier-mentioned simple displacement model, the slopes of
these plots are determined by the ratio of the effective
charges of the solute and the counterions; values of around
Pair of
analytes
Elution
sequence
Eluent
k
1
k
2
α
R
S
ZWIX(+)
1
A,1B
c
c
c
2.35
3.00
2.41
2.75
2.22
3.22
2.86
2.71
2.72
2.66
2.56 1.09 0.88 1R,2S < 1S,2R
3.80 1.27 2.18 1R,2R < 1S,2S
2.41 1.00 0.00
3.41 1.24 1.57 1R,2S < 1S,2R
2.60 1.17 1.60 1R,2S < 1S,2R
3.22 1.00 0.00
3.09 1.08 0.50 1R,2R < 1S,2S
3.16 1.17 1.00 1R,2S < 1S,2R
3.19 1.17 1.14 1R,2R < 1S,2S
3.65 1.38 2.75 1R,2S < 1S,2R
0
.9 have been found for a cation-exchange CSP and different
49
1C,1D
chiral amines. In our case, the significantly lower slopes ob-
2A,2B
—
served reveal a marked difference between a zwitterionic and
*
3
4
d
a “single ionic” CSP, as was found by Hoffmann et al. In the
case of zwitterionic CSPs with enhancement of the counterion
concentration, the retention can be reduced, but in only a
rather limited range. (An order of magnitude enhancement
of the concentration of the counterions resulted in an ~50% re-
duction in the retention factor.) Under the studied conditions
on both CSPs, practically identical slopes were obtained for
each enantiomer, i.e., the enantioselectivity remained almost
constant when the counterion concentration was increased.
However, these findings indicate that the sterically deter-
mined accessibility of the ionizable interaction sites, mainly
of the constraint zwitterionic SO but also of the SAs (Fig. 1),
may cause a deviation from the simplified stoichiometric
displacement model.
3
3
A,3B
C,3D
c
c
d
c
c
c
—
4A,4B
4C,4D
5A,5B
ZWIX(À)
1
A,1B
c
2.81
2.81 1.00 0.00
—
*
*
a
c
c
c
c
c
c
c
11.19 11.86 1.06 0.25 1R,2S < 1S,2R
1C,1D
2A,2B
3A,3B
3.76
3.04
2.90
3.93
3.58
3.33
2.17
4.04 1.07 0.22 1S,2S < 1R,2R
3.04 1.00 0.00
3.72 1.28 1.83 1S,2R < 1R,2S
5.22 1.33 2.58 1S,2S < 1R,2R
4.42 1.23 1.37 1S,2R < 1R,2S
4.65 1.40 2.38 1S,2S < 1R,2R
3.06 1.41 2.82 1S,2R < 1R,2S
—
3C,3D
4A,4B
4C,4D
5A,5B
Structure–Selectivity and Retention Relationships
The steric arrangement of the carboxylic and amino groups
on the constrained SAs (Fig. 1) participating in the SO-SA
associate formation influenced retention and chiral recogni-
Chromatographic conditions: columns, Chiralpak ZWIX(+) and Chiralpak
ZWIX(À); mobile phase. a, MeOH/MeCN (25/75 v/v) containing 25 mM
TEA and 50 mM AcOH, c, MeOH/MeCN (75/25 v/v) containing 25 mM
TEA and 50 mM AcOH, d, MeOH/MeCN (75/25 v/v) containing 25 mM
tion. Table 1 presents the k, α and R values observed with
À1
S
NH
3
and 50 mM AcOH; flow rate, 0.6 ml min ; detection, 230 nm or corona
the most frequently applied mobile phase (eluent c) on the
ZWIX(+) and ZWIX(À) columns.
detector; temperature, 25°C
*10°C
**50°C.
The experimental results obtained with the same mobile
phase under isocratic conditions allow the following
considerations:
On the pseudo-enantiomeric ZWIX(À) column, the ex-
1
. Stereoselectivity
pected reversal of the elution sequence was observed (the
2
A,2B could not be resolved under any conditions applied,
(
i) For all pairs of cis SAs (1A,1B; 2A,2B, 3A,3B,
A,4B, and 5A,5B) on the ZWIX(+) column, the
while 1A,1B could be resolved with mobile phase a at 50°C
[above the Tiso temperature, Tables 1 and 2]).
4
(
1R,2S) SAs were consistently eluted before the
1S,2R) SAs. The SA pair 2A,2B could not be resolved
(
(ii) For all the trans-SAs (1C,1D, 3C,3D, and 4C,4D)
the (1R,2R) SA eluted before the (1S,2S) SA on the
ZWIX(+) column, with a reversal of the elution se-
quence on the ZWIX(À) column. The only exception
under the conditions of eluent c, whereas with mobile
phase d it could be resolved (Tables 1 and 2) and ex-
hibited the same elution sequence.
Chirality DOI 10.1002/chir

HPLC ENANTIOSEPARATION OF CYCLIC β-AMINO ACIDS
567
TABLE 2. Temperature dependence of retention factor of first
2. Retention
1
eluting enantiomer (k ), separation factor (α) and resolution
(
R
S
) of cyclic β amino acids on ZWIX(+) and ZWIX(À)
In general, the trans SAs are better retained than the re-
lated cis SAs on both the ZWIX(+) and the ZWIX(À) columns.
In summary, the clear selectivity and retention trends did
not vary with a change of the MeOH to MeCN ratio or the na-
ture of the acid and the base modifiers. Selected chromato-
grams are depicted in Figure 5.
Temperature (°C)
Mobile
Pair of
analyte
phase
k
1
, α, R
S
10
20
30
40
50
ZWIX(+)
2.39
1.1
1
2
3
4
5
A,1B
A,2B
C,3D
C,4D
A,5B
c
k
α
R
k
α
R
k
α
R
k
α
R
k
α
R
1
2.21
1.09
0.41
2.75
1.24
1.57
2.86
1.08
0.48
2.82
1.17
0.86
1.99
1.42
2.1
1.96
1.09
0.32
2.58
1.22
1.45
2.58
1.07
0.4
2.52
1.15
0.94
1.8
1.76
1.08
0.22
2.41
1.21
1.36
2.34
1.07
0.37
2.26
1.14
0.75
1.62
1.34
1.78
1.59
1.07
0.24
2.26
1.19
1.23
2.12
1.06
0.44
2.05
1.13
0.74
1.47
1.3
Temperature Dependence and Thermodynamic Parameters
S
0.32
2.93
1.25
1.63
3.18
1.09
0.52
3.12
1.18
0.88
2.23
1.46
2.22
In order to investigate the effects of temperature on the
chromatographic parameters, a variable-temperature study
was carried out over the temperature range 10–50°C. Experi-
mental data for all SAs on both columns in the mobile phases
(a) MeOH/MeCN (25/75 v/v) containing 25 mM TEA and
50 mM AcOH, (c) MeOH/MeCN (75/25 v/v) containing 25
mM TEA and 50 mM AcOH, and (d) MeOH/MeCN (75/25
d
d
c
1
S
1
S
1
v/v) containing 25 mM NH and 50 mM AcOH, are listed in
3
S
Table 2. The slightly different mobile phase conditions with
regard to the MeOH/MeCN ratio were selected in order to
achieve separations for all pairs of enantiomers.
c
1
1.38
2.00
S
1.6
The tabulated data indicate that the retention factor k in
most cases decreased with increasing temperature. Transfer
of the SA from the mobile phase to the stationary phase is
generally an exothermic process and consequently k (and α)
decreases with increasing temperature. However, on the
ZWIX(À) column with mobile phase a, the behavior of the
pair 1A,1B was unusual: with increasing temperature, k de-
ZWIX(À)
16.04 15.66 14.81 13.82 11.99
1
1
3
4
5
A,1B
C,1D
C,3D
C,4D
A,5B
a
a
c
c
c
k
α
R
k
α
R
k
α
R
k
α
R
k
1.00
0.00
4.97
1.10
0.36
3.83
1.30
1.5
3.29
1.40
1.93
2.35
1.47
2.17
1.02
0.08
4.84
1.08
0.3
3.67
1.27
1.5
3.15
1.37
1.85
2.26
1.42
2.09
1.04
0.13
4.70
1.06
0.27
3.53
1.24
1.47
3.00
1.34
1.69
2.17
1.38
1.89
1.05
0.17
4.57
1.04
0.22
3.39
1.22
1.81
2.88
1.31
1.67
2.08
1.34
1.8
1.06
0.21
4.41
1.03
0.13
3.25
1.19
1.45
2.76
1.28
1.62
1.98
1.31
1.58
S
S
S
S
S
creased, but α and R increased. Such behavior was recently
S
43–45
observed for other unusual amino acids too.
This unexpected behavior occurred only on the ZWIX(À)
CSP for the pair 1A,1B. In the studied temperature range,
the solvation of the SO of the ZWIX(À) column and the SAs
probably does not change significantly. However, for the pair
1
A,1B an unexpected reversal of the elution sequence was
α
R
observed above the Tiso temperature.
As a general trend, van’t Hoff analysis of the separation fac-
tors (ln α vs. 1/T) gave linear plots, as indicated by the corre-
lation coefficients in Table 3. The differences in Δ(ΔH°) and
Δ(ΔS°), are presented in Table 3. Δ(ΔH°) ranged from À2.2
Chromatographic conditions: column, Chiralpak ZWIX(+) and ZWIX(À); mo-
bile phase. a, MeOH/MeCN (25/75 v/v) containing 25 mM TEA and 50
mM AcOH, c, MeOH/MeCN (75/25 v/v) containing 25 mM TEA and 50
mM AcOH, d, MeOH/MeCN (75/25 v/v) containing 25 mM NH
3
and 50
À1
À1
À1
to À0.6 kJ mol on ZWIX(+), and from À2.2 to +1.1 kJ mol
mM AcOH; flow rate, 0.6 ml min ; detection, corona detector.
on ZWIX(À). The trend in the change in Δ(ΔS°) was similar
to that in Δ(ΔH°). Under the conditions where Δ(ΔH°) was
negative, Δ(ΔS°) was also negative, and positive Δ(ΔH°)
was accompanied by positive Δ(ΔS°). The interactions of
5A,5B with ZWIX(+) and ZWIX(À) were characterized by
the highest negative Δ(ΔH°) and Δ(ΔS°) values at eluent
composition c.
was the pair 3C,3D, which could not be resolved on
ZWIX(+) with mobile phase c, whereas with mobile
phase d it could (Tables 1 and 2).
(
iii) The above elution sequence holds in principle for the
five- and six-membered cycloalkanes, although it
should be mentioned that the pair 2A,2B could not
be separated under the given conditions on the
ZWIX(+) and the ZWIX(À) column, although at com-
position d they could (Tables 1 and 2).
When the selectivity increased with increasing tempera-
ture, Δ(ΔH°) and Δ(ΔS°) were positive. In these cases, the
change in Δ(ΔH°) with increasing temperature had a positive
effect on the enantioselectivity. On the other hand, the posi-
tive Δ(ΔS°) compensated the positive Δ(ΔH°) and resulted
in a negative Δ(ΔG°), the selectivity increasing with increas-
ing temperature. Thermodynamically, this unusual behavior
may be attributed to the positive Δ(ΔS°) values, indicating
the importance of the entropy contribution to the chiral
separation.
There is no clear trend as to whether the cis or the trans en-
antiomer pairs can be better separated. However, it is obvious
that the similar cyclopentene- and cyclohexene-ß-amino acids
(
2A,2B and 5A,5B) have different steric constraints, signifi-
cantly influencing the intermolecular SO-SA interactions.
At this point it should be mentioned that, when the differ-
ences in the retention factors of the SAs are small, the peaks
may not be resolved, as this depends on the efficiency of the
columns.
The thermodynamic parameter –Δ(ΔG°)298 suggests that,
both on ZWIX(+) and on ZWIX(À), mobile phases c and d in-
duced binding to the SO more efficiently, as reflected by the
larger –Δ(ΔG°) (1A,1B on ZWIX(À) could be separated only
under mobile phase condition a).
Chirality DOI 10.1002/chir

5
68
ILISZ ET AL.
Fig. 5. Selected chromatograms for SAs 1-5. Chromatographic conditions: columns, ZWIX(+) for SAs 1A,1B, 1C,1D, 2A,2B, and 3A,3B, ZWIX(–) for SAs
C,3D, 4A,4B, 4C,4D, and 5A,5B; mobile phase: MeOH/MeCN (75/25 v/v) containing 25mM TEA and 50 mM AcOH for SAs 1A,1B, 1C,1D, 3A,3B, and
3
4
3
A,4B, MeOH/MeCN (75/25 v/v) containing 25 mM EA and 50 mM FA for SA 3C,3D, MeOH/MeCN (75/25 v/v) containing 25 mM NH and 50 mM AcOH
-
1
for SA 2A,2B and 4C,4D and MeOH/MeCN (75/25 v/v) containing 25 mM PA and 50 mM AcOH for SA 5A,5B; flow rate, 0.6 ml min ; detection, 230 nm or
corona detector; temperature, 25ºC for all SAs (with exception for SA 2A,2B, 10ºC).
2
TABLE 3. Thermodynamic parameters, Δ(ΔH°), Δ(ΔS°), TxΔ(ΔS°), Δ(ΔG°), correlation coefficients, (R ) and Tiso temperature of
cyclic βÀamino acids on ZWIX(+) and ZWIX(À)
Pair of
analyte
Mobile
phase
-Δ(ΔH°)
(kJ mol
-Δ(ΔS°)
Correlation 2
-TxΔ(ΔS°)298K
À1
-Δ(ΔG°)298K
À1
À1
À1 À1
)
(J mol
K
)
coefficients (R )
(kJ mol
)
(kJ mol
)
T
iso (°C)
ZWIX(+)
0.9936
0.9823
0.9832
0.9955
0.9993
1A,1B
2A,2B
3C,3D
4C,4D
5A,5B
c
d
d
c
0.6
0.9
0.6
0.7
2.2
1.3
0.4
0.4
0.4
0.4
1.4
0.2
0.5
0.2
0.3
0.8
189
431
179
322
201
1.3
1.2
1.2
4.7
c
ZWIX(À)
0.9949
0.9978
0.9982
0.9978
0.9985
1A,1B
1C,1D
3C,3D
4C,4D
5A,5B
a
a
c
c
c
À1.1
1.3
1.7
1.7
2.2
À4.0
3.8
3.7
3.8
4.5
À1.2
1.1
1.1
1.1
1.3
0.1
0.2
0.6
0.6
0.9
9
69
179
254
211
Chromatographic conditions: column, Chiralpak ZWIX(+) and ZWIX(À); mobile phase. a, MeOH/MeCN (25/75 v/v) containing 25 mM TEA and 50 mM AcOH,c,
and 50 mM AcOH; flow rate, 0.6 ml
MeOH/MeCN (75/25 v/v) containing 25 mM TEA and 50 mM AcOH, d, MeOH/MeCN (75/25 v/v) containing 25 mM NH
3
À1
2
min ; detection, corona detector; R , correlation coefficient of van’t Hoff plot, ln α – 1/T curves; Tiso,, temperature of ln k – 1/T curves where enantioselectivity
cancels.
Chirality DOI 10.1002/chir

HPLC ENANTIOSEPARATION OF CYCLIC β-AMINO ACIDS
569
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On ZWIX(À), the positive Δ(ΔS°) for 1A,1B compensated
for the positive Δ(ΔH°) and resulted in a negative Δ(ΔG°)
value (Table 3). For 1A,1B in this temperature range,
enantioresolution is entropically driven, and the selectivity in-
creases with increasing temperature.
The data were used to calculate the temperature Tiso at
which the enantioselectivity cancels out (Table 3). In most
cases, Tiso was considerably higher than room temperature;
while for 1A,1B Tiso was 9°C and the reversed elution se-
quence at higher column temperature was observed
1
1
13. Kiss L, Fülöp F. Synthesis of carbocyclic and heterocyclic
β-aminocarboxylic acids. Chem Rev 2014; 111: 1116–1169.
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1
4. Ilisz I, Berkecz R, Péter A. Application of chiral derivatizing agents in the
high-performance liquid chromatographic separation of amino acid enan-
tiomers: A review. J Pharm Biomed Anal 2008; 47: 1–15.
5. Ilisz I, Pataj Z, Aranyi A, Péter A. High-performance liquid chromatogra-
phy of biologically important, small epimeric peptides and their L,D-
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(
Table 1).
CONCLUSION
HPLC methods were developed for separation of the enan-
16. Ilisz I, Pataj Z, Aranyi A, Péter A. Macrocyclic antibiotic selectors in direct
tiomers of cyclic β-amino acids by using Cinchona-based zwit-
terionic CSPs i.e., Chiralpak ZWIX(+) and ZWIX(À) in PIM
mode. The effects of the composition of the mobile phase,
the nature of the acid and base additives, the structures of
the Sas, and temperature were discussed. The changes in
Δ(ΔH°), Δ(ΔS°), and Δ(ΔG°) were calculated from the linear
van’t Hoff plots derived from the ln α vs. 1/T curves in the
temperature range 10–50°C. The values of the thermody-
namic parameters depended on the nature of the SOs and
the structures of the SAs. On the ZWIX(+) column only
enthalpically, while on the ZWIX(À) column both
enthalpically and entropically driven separations were ob-
served. The latter was registered on the ZWIX(À) column
for analyte 1A,1B. By variation of the chromatographic pa-
rameters, the separations of the stereoisomers were opti-
mized and baseline resolution was achieved for most of the
investigated SAs in at least one chromatographic system.
The elution sequence was determined in all cases and was
found to be opposite on the ZWIX(+) and ZWIX(À) columns.
HPLC enantioseparations. Sep Purif Rev 2012; 41: 207–249.
17. Ilisz I, Aranyi A, Pataj Z, Péter A. Recent advances in the
enantioseparation of amino acids and related compounds: A review. J
Pharm Biomed Anal 2012; 69: 28–41.
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8. Ilisz I, Aranyi A, Pataj Z, Péter A. Enantiomeric separation of
nonproteinogenic amino acids by high-performance liquid chromatogra-
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9. Ilisz I, Aranyi A, Pataj Z, Péter A. Enantioseparations by high-performance
liquid chromatography using macrocyclic glycopeptide-based chiral sta-
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0. Berthod A. Chiral recognition mechanisms in enantiomers separations: a
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ACKNOWLEDGMENTS
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4. Berkecz R, Ilisz I, Misicka A, Tymecka D, Fülöp F, Choi HJ, Hyun MH,
Péter A. HPLC enantioseparation of ß -homoamino acids using crown
ether-based chiral stationary phase. J Sep Sci 2009; 32: 981–987.
25. Gecse Z, Ilisz I, Nonn M, Grecsó N, Fülöp F, Agneeswari R, Hyun MH,
Péter A. High-performance liquid chromatographic enantioseparation of
2
This work was supported by Hungarian National Science
Foundation grants OTKA K 108847 and 108943. A.P. grate-
fully acknowledges the support of Pilar Franco (Chiral Tech-
nologies Europe) for the Chiralpak columns.
isoxazoline-fused 2-aminocyclopentanecarboxylic acids on
a chiral
ligand-exchange stationary phase. J Sep Sci 2013; 36: 1335–1342.
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