Amino acid ionic liquids as chiral ligands in ligand-exchange chiral separations

Article abstract of DOI:10.1002/chem.200900982

Recently, amino acid ionic liquids (AAILs) have attracted much research interest. In this paper, we present the first application of AAILs in chiral separation based on the chiral ligand exchange principle. By using 1alkyl-3-methylimidazolium L-proline (L-Pro) as a chiral ligand coordinated with copper(II), four pairs of underivatized amino acid enantiomers - DLphenylalanine (DL-Phe), DL-histidine (DL-His), DL-tryptophane (DL-Trp), and DL-tyrosine (DL-Tyr) - were successfully separated in two major chiral separation techniques, HPLC and capillary electrophoresis (CE), with higher enantioselectivity than conventionally used amino acid ligands (resolution (R_s) = 3.26-10.81 for HPLC; R_s = 1.34-4.27 for CE). Interestingly, increasing the alkyl chain length of the AAIL cation remarkably enhanced the enantioselectivity. It was inferred that the alkylmethylimidazolium cations and L-Pro form ion pairs on the surface of the sta-tionary phase or on the inner surface of the capillary. The ternary copper complexes with L-Pro are consequently attached to the support surface, thus inducing an ion-exchange type of retention for the DL-enantiomers. Therefore, the AAIL cation plays an essential role in the separation. This work demonstrates that AAILs are good alternatives to conventional amino acid ligands for ligand-exchange-based chiral separation. It also reveals the tremendous application potential of this new type of task-specific ILs.

Full text of DOI:10.1002/chem.200900982

FULL PAPER
DOI: 10.1002/chem.200900982
Amino Acid Ionic Liquids as Chiral Ligands in Ligand-Exchange Chiral
Separations
Qian Liu, Kangkang Wu, Fei Tang, Lihua Yao, Fei Yang, Zhou Nie, and
Shouzhuo Yao*[a]
Abstract: Recently, amino acid ionic
liquids (AAILs) have attracted much
research interest. In this paper, we
present the first application of AAILs
in chiral separation based on the chiral
ligand exchange principle. By using 1-
electrophoresis (CE), with higher enan-
tioselectivity than conventionally used
amino acid ligands (resolution (Rs)=
3.26–10.81 for HPLC; Rs =1.34–4.27
for CE). Interestingly, increasing the
alkyl chain length of the AAIL cation
remarkably enhanced the enantioselec-
tivity. It was inferred that the alkylme-
thylimidazolium cations and l-Pro
form ion pairs on the surface of the sta-
tionary phase or on the inner surface
of the capillary. The ternary copper
complexes with l-Pro are consequently
attached to the support surface, thus
inducing an ion-exchange type of re-
tention for the dl-enantiomers. There-
fore, the AAIL cation plays an essen-
tial role in the separation. This work
demonstrates that AAILs are good al-
ternatives to conventional amino acid
alkyl-3-methylimidazolium
l-proline
(l-Pro) as a chiral ligand coordinated
with copper(II), four pairs of underiv-
atized amino acid enantiomers—dl-
phenylalanine (dl-Phe), dl-histidine
(dl-His), dl-tryptophane (dl-Trp), and
dl-tyrosine (dl-Tyr)—were successfully
separated in two major chiral separa-
tion techniques, HPLC and capillary
ligands
for
ligand-exchange-based
chiral separation. It also reveals the
tremendous application potential of
this new type of task-specific ILs.
Keywords: amino acids · chirality ·
electrophoresis
ligand design
· ionic liquids ·
Introduction
have summarized the recent application of ILs in separation
and chromatography, spectroscopy, and electrochemistry.[5–13]
In high-performance liquid chromatography (HPLC), ILs
have played a variety of roles in facilitating the chromato-
graphic separation either as mobile-phase additives or as
stationary phases. The IL mobile-phase additives can dy-
namically coat the stationary phase and more effectively
mask the surface silanols than conventionally used amines.
The reason is thought to be the combination of the electro-
static interaction between IL cations and surface silanols
and the hydrophobic interaction between IL cations and
alkyl chains of the stationary support.[14,15] This feature
makes ILs useful for reducing peak tailing and enhancing
the resolution of polar analytes.[16,17] Furthermore, ILs can
serve as ion-pairing agents for ionized analytes[18] or as ion-
exchange-based stationary phases when being covalently
linked to the stationary support.[19] These applications have
also been expanded to capillary electrophoresis (CE). ILs
have manifested some fascinating strengths when being used
as background electrolyte (BGE) additives or capillary-wall
modifiers in a variety of CE applications, such as improving
resolution, reducing wall adsorption, and controlling electro-
osmotic flow (EOF).[20–26] ILs have also been used to en-
New discoveries in chemistry, physics, biology, and material
science may provide new tools for chemical measurements.
A good example is the increasing use of ionic liquids (ILs)
in modern analytical chemistry.[1] ILs are a class of organic
salts with a melting point close to or below room tempera-
ture. They have unique chemical and physical properties, in-
cluding being air- and moisture-stable, exceedingly low
vapor pressure, tunable miscibility with water, a wide elec-
trochemical window, high conductivity, and high heat capaci-
ty.[2–4] Because of these properties, ILs have shown tremen-
dous potential in analytical applications. Several reviews
[a] Q. Liu,+ K. Wu,+ F. Tang,+ L. Yao, F. Yang, Z. Nie, Prof. S. Yao
State Key Laboratory of Chemo/Biosensing and Chemometrics
College of Chemistry and Chemical Engineering
Hunan University, Changsha 410082 (China)
Fax : (+86)731-88821848
E-mail: szyao@hnu.cn
[+] Q. Liu, K. K. Wu, and F. Tang contributed equally to this work.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200900982.
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hance chiral separation with b-cyclodextrin (CD).[27] Conclu-
sively, ILs provide a new approach for the optimization of
HPLC and CE separations.
Results
Current research interest in ILs includes the design of ILs
with controllable chemical and physical properties or even
specific functions (i.e., so-called task-specific ILs).[28,29] One
interesting example is the design and synthesis of chiral ILs
and their use as chiral solvents for optical resolution and
asymmetric synthesis.[30–34] Chiral ILs have also been synthe-
sized and used as chiral selectors in CE.[35–37] Unfortunately,
we have found no reports on the application of chiral ILs in
HPLC. On the other hand, despite their potential, chiral ILs
are not commercially available and their synthesis often re-
quires expensive reagents and complicated synthetic
schemes.[30–33] These limitations severely hinder their applica-
tions.
Enantioseparations by HPLC with AAILs as chiral ligands:
In our HPLC study, the AAILs coupled with CuII were used
as chiral mobile-phase additives. The influencing factors
were studied with dl-Phe as a model analyte. Herein,
AAILs are used instead of conventionally used amino acid
ligands. Our first concern was whether or not the amino
acids can still serve as chiral ligands when being made into
AAILs and what the function is of the AAIL cation in the
separation. Thus, we first performed the separation using
AAILs with different alkyl chain lengths in the cation. As
shown in Figure 1, good baseline separations of dl-Phe en-
Another interesting possibility with task-specific ILs is to
introduce natural amino acids into the cations or anions of
ILs.[38–43] Amino acids have many advantages including low
cost, biocompatibility, and ease of chemically modification.
In most cases, amino acid based ILs are also chiral ILs.
Amino acids provide a valuable approach for functional
design of ILs. Specifically, the term “amino acid ionic
liquid”, or “AAIL”, was coined for the ILs with amino acids
as anions; they were first synthesized by the Ohno group
using an anion-exchange method.[44,45] Because the amino
acids have various functional groups and keep their nature
in AAILs,[46] it is possible to design AAILs for a wide range
of tasks. For instance, Chen et al. have used AAILs as a sup-
port for metal scavenging and heterogeneous catalysis.[47]
However, the study of AAILs is still in its infancy, and the
current research mainly focuses on their synthesis and char-
acterization.[48–51] To our knowledge, there have been no re-
ports of using AAILs for chromatographic separation or
chiral separation.
In this paper, we demonstrate for the first time the appli-
cation of AAILs in chiral separation. Four underivatized
amino acids were used as model analytes. Because the
amino acids maintain their nature in AAILs,[46] we per-
formed the separations based on the ligand-exchange princi-
ple.[52–54] The l-proline ILs coupled with CuII were used as
chiral selectors because l-proline has been proven to be an
outstanding ligand in organocatalysis and organometallic
catalysis.[55] Chiral recognition is achieved based on the for-
mation of ternary mixed-metal complexes between the
AAIL ligand and the analyte ligand. The different complex
stability constants of the mixed complexes with d and l en-
antiomers result in the enantioseparation of racemic amino
acids. The method was validated by means of two major
chiral separation techniques, HPLC and CE. In both tech-
niques, AAILs show a significant superiority over the con-
ventionally used amino acid ligands. Specifically, we have
made an elaborate comparison between the HPLC and CE
results. On the basis of the comparison, the detailed separa-
tion mechanisms, including the function of AAIL cation, are
discussed in this paper.
Figure 1. Separation factor (a; shown in white) and resolution (Rs; shown
in gray) of dl-Phe for different chiral selectors in HPLC. Pure l-Pro, l-
Pro+ACTHNUGTRENUNG[C4mim][Br], and four Pro ILs with different alkyl chain lengths
coupled with CuII were used as chiral mobile-phase additives. Mobile
phase: 1 mm chiral mobile-phase additive, 0.5 mm Cu(Ac)2, and MeOH
(15% v/v) in water; column: Ultimate XB C18 (150ꢁ4.6 mm i.d.); flow
rate: 1 mLminÀ1; temperature: 258C; fluorescence detection: lex/lem
=
215 nm/295 nm.
antiomers are obtained with all the four l-Pro ILs (separa-
tion factor (a) and resolution (Rs)>2.0), thereby indicating
that the AAILs are indeed effective chiral ligands. For com-
parison, we also performed the separation with l-Pro as a
chiral ligand under the same conditions. Although the base-
line separation is also obtained with l-Pro (a=1.82 and
Rs =2.70), its enantioselectivity is evidently poorer than that
of l-Pro ILs. Another interesting control experiment is the
separation using l-Pro and a common IL, 1-butyl-3-methyli-
midazolium bromide, [C4mim][Br], as a binary mobile-phase
additive. Surprisingly, the obtained resolution (Rs =1.46) is
even lower than that of pure l-Pro. Therefore, just the pres-
ence of the [Cnmim] cation in the mobile phase does not
cause the better separation, and it is essential to combine
the [Cnmim] cation and l-Pro into AAILs. Notably, by in-
creasing the alkyl chain length of [Cnmim] from C2 to C8,
the enantioselectivity is significantly enhanced (i.e., Rs in-
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Amino Acid Ionic Liquids
FULL PAPER
creases from 2.80 to 4.73). The above phenomena indicate
that the AAIL cation plays an important role in the separa-
tion mechanism. Furthermore, the retention time of analytes
also increases with the alkyl chain length. Considering that
gets worse at higher pH. Taking the high resolution, short
analysis time, and good peak shape into account, we used
pH 5.8 to perform the separation.
We have also investigated other influencing factors includ-
ing the concentration of CuII and organic solvent in the
mobile phase. The best molar ratio of AAIL to CuII was
found to be 2:1, which is consistent with the expected value
based on the ligand-exchange principle that the tetracoordi-
nated CuII ions combine with bifunctional solute molecules
and bidentate chiral matrix fixed ligands.[52–54] The addition
of MeOH does not exert a significant impact on the enantio-
selectivity. However, it was added into the mobile phase to
adjust the retention of analytes on the column. In this study,
15% v/v MeOH was used to tailor conditions for a suitable
retention time. Incidentally, we also found that adding 7.5%
v/v acetonitrile in the mobile phase can play a similar role.
Figure 3 shows the chromatograms for the enantiosepara-
tions of four pairs of amino acid enantiomers in HPLC. The
corresponding separation data are listed in Table 1. Un-
doubtedly, this AAIL-based method shows excellent enan-
tioselectivity for the tested racemic amino acids: a ranges
from 1.27 to 2.16, and Rs ranges from 3.26 to 10.81. It should
the enantioselectivity for [C6mim]
that of [C8mim][Pro] and that it is more difficult to synthe-
size AAILs with longer alkyl chain lengths because of the
ACHTUNGTREN[NUNG Pro] is comparable to
ACHTUNGTRENNUNG
larger viscosity, we used [C6mim]
the following studies.
ACHTUNGTREN[NUNG Pro] as a model AAIL in
We then investigated the effects of AAIL concentration
and eluent pH on the separation. As shown in Figure 2A,
both a and Rs increase with an increase of the AAIL con-
Figure 2. A) Effects of AAIL concentration and B) mobile phase pH on
*
*
the enantioseparation of dl-Phe in HPLC ( =a, =Rs). For A), the
concentration of Cu(Ac)2 in the mobile phase was kept at half of AAIL
concentration. For B), the mobile phase was 1 mm [C6mim]ACTHNUTRGENU[GN Pro], 0.5 mm
Cu(Ac)2, and MeOH (15% v/v) in water. The pH was adjusted by an
aqueous solution of HCl or NaOH. Other conditions were the same as
indicated in Figure 1.
centration. It should be noted that the baseline separation
can be achieved even at a very low concentration of AAIL
(e.g., 0.25 mm), thus indicating the strong enantioseparation
capability of AAILs. Generally, it is desirable to use a low
concentration of mobile-phase additive to control the back-
ground noise and to minimize the cost. Since the enantiose-
lectivity with 1 mm AAIL is sufficiently high (a>2 and Rs >
4), we used 1 mm AAIL in the following studies. Figure 2B
depicts the dependence of enantioselectivity on the eluent
pH. By increasing the pH from 3.9 to 7.9, both a and Rs in-
crease. For Rs especially, it increases significantly from 0.76
at pH 3.9 to 8.11 at pH 7.9. This phenomenon is similar to
that in a previous report.[56] When increasing the pH over
7.9, however, both a and Rs begin to decline. A further,
higher pH will yield precipitates in the mobile phase. On
the other hand, increasing the pH greatly prolongs the re-
tention time of analytes; for example, at pH 7.9, the reten-
tion time (tR) of l-Phe exceeds 30 min. The peak shape also
Figure 3. Enantioseparations of dl-His, dl-Phe, dl-Trp, and dl-Tyr by
HPLC. Mobile phase: 1 mm [C6mim]ACHTNUGRTENUNG[Pro], 0.5 mm Cu(Ac)2, and MeOH
(15% v/v) in water (pH 5.8); fluorescence detection: lex/lem =280 nm/
348 nm for dl-His, 215 nm/295 nm for dl-Phe, 270 nm/304 nm for dl-Trp,
and 216 nm/295 nm for dl-Tyr. Other conditions were the same as indi-
cated in Figure 1.
Table 1. Separation data of HPLC and CE for racemic amino acids.
HPLC[a]
k’D
CE[b]
tmD [min]
[c]
a
Rs
4.61
8.84
10.81
3.26
a[d]
Rs
dl-Phe
dl-Trp
dl-Tyr
dl-His
3.72
9.11
2.44
3.97
2.16
1.74
1.97
1.27
15.95
16.06
19.52
12.39
1.037
1.080
1.092
1.023
1.48
3.52
4.27
1.34
[a] Experimental conditions for HPLC are the same as indicated in
Figure 3. [b] Experimental conditions for CE are the same as indicated in
Figure 7. [c] tmD =migration time of the d enantiomer. [d] The separation
factor in CE separation is defined as the ratio of migration time of l and
d enantiomers (i.e., a=tmL/tmD).
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S. Yao et al.
also be noted that the chromatographic conditions are opti-
mized based on using dl-Phe as the model enantiomers. As
the aim of this work is to explore the mechanism of AAIL-
based enantioseparation, we did not perform further optimi-
zation with respect to each individual racemic amino acid.
In spite of that, the obtained enantioselectivities for the
other tested racemic amino acids are also satisfactory. To
our knowledge, this should be one of the best achievements
for enantioseparation of underivatized amino acids by chiral
ligand exchange chromatography.[57,58]
venient comparison with HPLC, [C6mim]ACTHUNGTERNNU[G Pro] was also se-
lected as a suitable AAIL in the following studies.
Figure 5 depicts the effects of the concentration of AAIL
and organic solvent on the separation. It can be seen from
Figure 5A that a much higher concentration of AAILs is re-
Enantioseparations by CE with AAILs as chiral ligands: As
another important chiral separation technique, CE is always
regarded as complementary to HPLC due to its distinct sep-
aration mechanism from HPLC. Thus, we think, it should be
interesting to study the same chiral selective system,
AAILs–CuII, in both HPLC and CE. In CE, the AAILs cou-
pled with CuII were used as both chiral selectors and back-
ground electrolytes (BGEs), and thus needed no additional
BGEs. This is the first advantage brought by these bifunc-
tional ILs in CE. We first investigated the effect of alkyl
chain length of the AAIL cation on the separation. As
shown in Figure 4, the Rs is significantly enhanced by the
Figure 5. A) Effects of AAIL and B) MeOH concentration on resolution
of dl-Phe in CE. For A), the concentration of Cu(Ac)2 in buffer was
kept at half of the AAIL concentration. For B), the buffer was 30 mm
[C6mim]ACTHNUGTRNE[UNG Pro], 15 mm Cu(Ac)2 with different percentages of MeOH in
water. Other conditions were the same as indicated in Figure 4.
quired to obtain effective enantioseparation in CE than in
HPLC. The Rs increases with AAIL concentration until the
AAIL concentration reaches 30 mm. At an AAIL concentra-
tion over 30 mm, obvious band broadening is observed that
compromises the selectivity, thus the Rs begins to decline.
For instance, by increasing AAIL concentration from 30 to
50 mm, the efficiency of d- and l-Phe decreases from 83600
and 56000 platesmÀ1 to 75100 and 51200 platesmÀ1, respec-
tively. As a result, 30 mm was used as the optimal AAIL
concentration. Furthermore, it is usually essential to add
buffer additives such as sodium dodecylsulfate (SDS) or or-
ganic solvents to enhance the enantioselectivity in CE.[60–62]
Here, considering that SDS may cause greater background
noise, we chose MeOH to adjust the separation selectivity.
As shown in Figure 5B, the enantioselectivity for dl-Phe is
poor without MeOH in the buffer. Adding MeOH in a con-
centration no higher than 30% v/v into the buffer remarka-
bly improves the selectivity. At the same time, addition of
MeOH greatly prolongs the migration time. At an MeOH
concentration over 30% v/v, the rather long migration time
aggravates the band broadening and thereby compromises
the selectivity. Thus 30% v/v was used as a proper additive
concentration. It is worth noting that the organic solvent has
exerted a larger effect on the enantioselectivity in CE than
in HPLC.
Figure 4. Effect of alkyl chain length of AAIL cation on resolution of dl-
Phe in CE. Buffer: 30 mm AAIL, 15 mm Cu(Ac)2, and MeOH (30% v/v)
in water; capillary: fused silica of 50 cm total length (40 cm to detector)ꢁ
50 mm i.d.; injection: 3.4 kPa for 5 s; detection: 200 nm; applied voltage:
30 kV; temperature: 258C.
long alkyl chain in the AAIL cation. This phenomenon is
consistent with the result obtained in HPLC, thereby dem-
onstrating again that the AAIL cation plays an important
role in the separation mechanism. However, it should be
noted that the resolution in CE is lower than that in HPLC.
For comparison, l-Pro was also tested as a chiral ligand, and
its performance was approximately the same as that of
[C4mim]
ACHTUNGTRENNUNG[Pro] and inferior to [C6mim]ACHTUNGTERN[NUGN Pro] and [C8mim]-
ACHTUNGTRENNUNG
the electroosmotic flow (EOF) was suppressed thoroughly
(ca. 4.3ꢁ10À6 cmÀ2 VÀ1 sÀ1 estimated by using the Williams
and Vigh method[59]) and no analytes were detected within
30 min. This distinct phenomenon of l-Pro from l-Pro ILs
indicates again that the use of AAILs is not simply equal to
the combined use of amino acids and IL cations. As a con-
Figure 6 shows the enantioseparation of racemic Phe over
a range of buffer pH. The effect of pH in CE is, surprisingly,
distinct from that in HPLC: the Rs dramatically decreases as
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Figure 7. Enantioseparations of dl-His, dl-Phe, dl-Trp, and dl-Tyr by
CE. Buffer: 30 mm [C6mim]ACHTUNTGRENUNG[Pro], 15 mm Cu(Ac)2, and MeOH (30% v/v)
in water (pH 4.0). Other conditions were the same as indicated in
Figure 4.
Figure 6. Effect of buffer pH on enantioseparation of dl-Phe in CE.
Buffer: 20 mm [C6mim]ACHTUNGTRENNUNG[Pro], 10 mm Cu(Ac)2, and MeOH (30% v/v) in
water. The buffer pH was adjusted by an aqueous solution HCl or NaOH
to the desired value. Other conditions were the same as indicated in
Figure 4. Note: In the case of pH 3.0, the concentration of d and l enan-
tiomer are 1 and 0.5 mm, respectively, thus indicating that their migration
order is d before l.
in HPLC, it is competitive with or even better than the pre-
viously reported results for enantioseparation of underivat-
ized amino acids by ligand-exchange CE.[60,61]
Discussion
the pH increases from 3.0 to 6.0. When at pH 7.0 or higher,
no effective enantioseparation was achieved. In addition, be-
cause a much higher concentration of CuII was used in CE
than in HPLC (15 mm vs. 0.5 mm), the precipitates were ob-
served at pH>7.0 in CE buffer, whereas they were absent
even at pH 9.9 in the HPLC mobile phase. On the other
hand, lower pH causes longer migration times and lower ef-
ficiency, that is, by varying the pH from 5.0 to 3.0, the effi-
ciency of d- and l-Phe decreases rapidly from 148100 and
132500 platesmÀ1 to 26200 and 21900 platesmÀ1, respective-
ly. To compromise between high enantioselectivity and high
efficiency, pH 4.0 was used in CE. Interestingly, the same
pH was also used in a previous study that used l-Pro and l-
hydroxyproline as chiral ligands.[63]
From the above results, we realize that AAILs are good al-
ternatives as chiral ligands for coordinating with metal ions
in ligand-exchange chiral separations. Interestingly, AAILs
show different performances in HPLC and CE. This pro-
vides a potential approach to gain a better insight into the
separation mechanism. It should be noted that the enantio-
selectivity in HPLC is evidently higher than that in CE,
even though a much higher concentration of AAILs was
used in CE. This result implies that the separation is facili-
tated more effectively by different retention of amino acid–
CuII complexes and uncomplexed amino acids in HPLC
than by their different electrophoretic mobilities in CE. No-
tably, the AAIL cation greatly affects the separation in
HPLC (as shown in Figure 1): the longer the alkyl chain the
AAIL cation has, the better the obtained resolution. Such a
significant impact was not expected initially, because the
AAILs should be ionized in aqueous solution and their cat-
ions should not be involved in the coordination with CuII.
The potential way for AAIL cations to exert such an impact
is for the alkylimidazolium cations and l-Pro to form ion
pairs on the surface of the stationary support (as illustrated
in Scheme 1A). The ternary copper complexes are attached
onto the stationary-phase surface by means of the ion-pair
formation, thus yielding an ion-exchange type of retention
for the dl enantiomers. The enantiomer that has a larger
complex stability constant will have a stronger retention on
the column. This retention can be enhanced by increasing
the alkyl chain length of [Cnmim] because of the enhance-
We have also investigated the effects of CuII concentra-
tion. As expected, the highest enantioselectivity was ob-
tained when the molar ratio of AAIL to CuII ions was 2:1.
Changing the CuII to CoII shows no enantioselectivity for
the racemic Phe, probably because the complexes formed
with CoII are too stable.[64] When changing the CuII to NiII,
the Rs is only 0.29. ZnII was also tested, but it greatly sup-
pressed the EOF[65] and no useful data were obtained. Con-
clusively, CuII is the best metal ion to serve as the coordina-
tion center. This result is consistent with the previous report
using an amino acid–CuII system.[64,66]
Figure 7 shows the electropherograms of the tested amino
acid enantiomers by CE. Their corresponding separation
data are given in Table 1. As listed, Rs ranges from 1.34 to
4.27. Although this resolution is somewhat lower than that
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S. Yao et al.
ry (as illustrated in Scheme 1B). The longer the alkyl chain
the AAIL cation has, the stronger the retention the capillary
wall causes to the amino acid–CuII complex, and thus the
better the resolution obtained. Similar to that in HPLC, this
hypothesis is also supported by the fact that the combined
use of l-Pro and [Cnmim][Br] does not yield a comparable
separation to l-Pro ILs. However, the open-tubular station-
ary phase should be a small part in the CE separation mech-
anism. Thus, the alkyl chain length of the AAIL cation
exerts a similar but less pronounced impact on the separa-
tion than that in HPLC. From the above discussion, we can
realize that it is the formation of alkylimidazolium cations
and l-Pro ion pairs on the surface of the support that makes
the AAILs superior to conventional amino acids in chiral
ligand exchange chromatography. However, the AAIL
cation plays a less essential role in CE than in HPLC due to
their different separation mechanisms. This also is the
reason that HPLC shows better enantioselectivity than CE.
It is also interesting to discuss the distinct effects of pH in
HPLC and CE. This difference can be readily interpreted
based on the above separation mechanisms. In HPLC, in-
creasing the eluent pH makes l-Pro more anionic and thus
is favorable to l-Pro interaction with alkylimidazolium cat-
ions and the formation of ion pairs on the support surface.
Thus, the enantioselectivity increases with the eluent pH
ranging from 3.9 to 7.9. A superfluously high pH (i.e., over
7.9) decreases the enantioselectivity probably due to the hy-
dration of CuII ions and the augmentation of the ternary
complex stability.[70] In CE, the separation is mainly depen-
dent on the difference in charge-to-mass ratio between
amino acid–CuII complex and uncomplexed amino acid.[70]
Under acidic conditions, the uncomplexed amino acid is
more cationic than the neutral amino acid–CuII complex and
thus migrates before the latter.[63,71] Increasing the buffer pH
makes the amino acid less cationic and thus minimizes the
difference in electrophoretic mobilities between the ternary
mixed complex and the uncomplexed amino acid. Therefore,
the enantioselectivity deteriorates with an increase in pH.
Scheme 1. Hypothetical schemes showing the retention mechanisms of
[Cu(AAIL)(l-amino acid)] complexes on A) the HPLC column and
B) the capillary wall. The dots between l-Pro and alkylimidazolium cat-
ions represent the formation of ion pairs. Note: d-amino acid is shown in
an uncomplexed form, but actually it can also form complexes with CuII
and AAIL, albeit with a smaller stability constant than l-amino acid.
ACHTUNGTRENNUNG
ment of the hydrophobic interaction with the C18 column.
Therefore, the resolution increases with the alkyl chain
length of [Cnmim] in HPLC.
This hypothesis is also supported by two other facts: first,
the retention time of the enantiomers increases with the
alkyl chain length, as the retention of [Cnmim] is associated
with the retention of the amino acid–CuII complexes by
means of ion-pairing; second, the combined use of l-Pro
and [Cnmim][Br] yields a poorer separation than [Cnmim]-
ACHTUNGTRENNUNG
[Pro], for the presence of [Br]À interferes the formation of
À
ion pairs. Note that the pK1 ( COOH) of l-Pro is 1.99. At
pH above this value, the ion pairing of alkylimidazolium cat-
ions and l-Pro is feasible. Furthermore, in all the cases, the
d enantiomer is eluted before the l enantiomer (as shown in
Figure 3), suggesting that the stability of [Cu
amino acid)] is higher than [Cu(l-Pro IL)(d-amino acid)].
This is consistent with the previously reported results upon
[Cu
(l-Pro)(dl-amino acid)].[67–69]
In CE, the separation is generally driven by different elec-
trophoretic mobilities of amino acid–CuII complex and un-
complexed amino acid.[70] At pH 4.0, the tested amino acids
have net positive charges, and thus they migrate faster than
their neutral complexes with CuII.[63,71] Figure 7 shows that d
migrate faster than l enantiomers, thereby indicating that
ACHTUNGTREN(NUNG l-Pro IL)(l-
G
Conclusion
E
In this paper, we show for the first time the application of
AAILs in chiral separation by HPLC and CE. AAILs cou-
pled with CuII were used as chiral selectors for the enantio-
separation of racemic amino acids based on the chiral ligand
exchange principle. In both of the two techniques, AAILs
have shown higher enantioselectivity than conventionally
used amino acid ligands. This superiority is thought to be re-
lated to the ion pairing of alkylimidazolium cations and l-
Pro on the surface of the stationary support or capillary
wall. The AAIL cation plays a more essential role in HPLC
than in CE due to the different separation mechanisms of
these two techniques. This study not only shows that AAILs
are good ligands for ligand-exchange chiral separation tech-
niques, but also reveals the tremendous application potential
of AAILs.
[Cu
[Cu
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
the results from HPLC. On the other hand, the capillary dy-
namically coated with alkylimidazolium cations can be re-
garded as an open-tubular stationary phase.[21] Thus, the
alkyl chain length of [Cnmim] can also affect the separation,
probably by means of the formation of ion pairs of alkylimi-
dazolium cations and l-Pro IL on the surface of the capilla-
9894
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Chem. Eur. J. 2009, 15, 9889 – 9896

Amino Acid Ionic Liquids
FULL PAPER
Experimental Section
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Chemicals: AAIL 1-alkyl-3-methylimidazolium l-proline salts ([Cnmim]-
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ACHTUNGTRENNUNG
ported in the literature.[44] Their structure and purity were examined by
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in the Supporting Information. l-Proline (l-Pro), dl-phenylalanine (dl-
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system (Shimadzu, Kyoto, Japan) consisting of two LC-20AT pumps, a
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at (25Æ0.1)8C using a thermostat.
Enantioseparation of racemic amino acids by HPLC: An aqueous solu-
tion containing AAILs and Cu(Ac)2 (molar ratio 2:1) was used as an iso-
cratic elution solvent. MeOH (15% v/v) was added into the mobile
phase to adjust the retention time of analytes unless otherwise noted.
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amino acids have native fluorescence, fluorescence detection was adopted
to reduce the background noise. The lex/lem for dl-Phe, dl-Trp, dl-Tyr,
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Received: April 14, 2009
Published online: August 18, 2009
9896
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Chem. Eur. J. 2009, 15, 9889 – 9896