Hofmeister series of ionic liquids: Kosmotropic effect of ionic liquids on the enzymatic hydrolysis of enantiomeric phenylalanine methyl ester

Article abstract of DOI:10.1016/j.tetasy.2006.01.015

The kinetic hydrolysis of enantiomeric phenylalanine methyl ester catalyzed by Bacillus licheniformis protease was performed in aqueous solutions of several hydrophilic ionic liquids (ILs). The protease enantioselectivity was found related to the kosmotropicity of individual cations and anions of ILs. The ion effectiveness in enhancing the enzyme enantioselectivity follows the Hofmeister series: kosmotropic anions and chaotropic cations stabilize the enzyme. In this application, the Hofmeister series of ILs was established in an order of decreasing effectiveness for anions: PO₄^3- > citrate^3-, CH₃COO^-, EtSO₄ ^-, CF₃COO^- > Br^- > OTs ^-, BF₄^- and for cations: [EMIM]⁺ > [BMIM]⁺ > [HMIM]⁺. The overall IL kosmotropicity was quantified by the δ value (difference in the Jones-Dole viscosity B-coefficients of anion and cation). In general, a high enzyme enantioselectivity was observed in a solution of IL with a high δ value.

Full text of DOI:10.1016/j.tetasy.2006.01.015

Tetrahedron: Asymmetry 17 (2006) 377–383
Hofmeister series of ionic liquids: kosmotropic effect of ionic
liquids on the enzymatic hydrolysis of enantiomeric
phenylalanine methyl ester
Hua Zhao,* Sophia M. Campbell, Lee Jackson, Zhiyan Song and Olarongbe Olubajo
Chemistry Program, Savannah State University, Savannah, GA 31404, USA
Received 2 December 2005; accepted 16 January 2006
Available online 20 February 2006
Abstract—The kinetic hydrolysis of enantiomeric phenylalanine methyl ester catalyzed by Bacillus licheniformis protease was per-
formed in aqueous solutions of several hydrophilic ionic liquids (ILs). The protease enantioselectivity was found related to the
kosmotropicity of individual cations and anions of ILs. The ion effectiveness in enhancing the enzyme enantioselectivity follows
the Hofmeister series: kosmotropic anions and chaotropic cations stabilize the enzyme. In this application, the Hofmeister serꢀies
of ILs was estabꢀlished in an order of decreasing effectiveness for anions: PO4 > citrate3ꢀ, CH3COOꢀ, EtSO ꢀ, CF3COO
>
3ꢀ
4
Brꢀ > OTsꢀ, BF4 and for cations: [EMIM]+ > [BMIM]+ > [HMIM]+. The overall IL kosmotropicity was quantified by the d value
(difference in the Jones–Dole viscosity B-coefficients of anion and cation). In general, a high enzyme enantioselectivity was observed
in a solution of IL with a high d value.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
number of applications.9,13–18 The enzyme activity is
related to the solvent hydrophobicity (in terms of logP
values, the partition coefficients between water and
octanol) and thermodynamic water activity (aw) as dis-
cussed in our recent review.19 Enzymes were found to
be more stable in solvents with a larger logP (>3) (such
as hexane, which has a logP of 3.9) than lower logP
(such as ethanol, which has a logP of ꢀ0.24).20 The rea-
son is because the hydrophobic solvents have a lesser
tendency of taking away the ‘essential’ (or ‘critical’)
water from the enzyme’s surface.21–23 It is known that
enzyme activity is determined by the water bound to
the enzyme (‘essential’ water, a few monolayers of
water), rather than the bulk-water content in the sys-
tem.24–26 To further examine the IL effect on protein
structures from a molecular level, the circular dichroism
(CD) spectrum of a-chymotrypsin in hydrophobic ILs
was found closer to that in water; the spectrum also re-
vealed that the b-strand of the protein secondary struc-
tures was considerably increased in ILs.15,27,28 Another
aspect to interpret the enzyme stability in ILs is based
on the observation that ILs may form the so called orga-
nized ‘nano-structures’ (hydrogen-bonded polymeric
supramolecules, just like water molecules) with polar
and nonpolar regions in solid, liquid, and solution
states, or even in the gas phase.29,30 In his review,
Dupont30 pointed out that the aqueous solution of free
Over the past several years, a great deal of excitement
has been generated by the awareness and use of ionic liq-
uids (ILs) as novel reaction media in a variety of enzy-
matic reactions.1–6 There have been some mechanistic
discussions as to why enzymes either are active or inac-
tive in certain ILs. Several factors of ILs seem to be
responsible for the enzyme activity and stability, includ-
ing IL polarity,1 hydrogen-bond basicity7,8 and anion
nucleophilicity.9 For example, Park and Kazlauskas
correlated the enzyme (Pseudomonas cepacia lipase)
activity with the IL polarity, observing higher conver-
sions in more polar ILs.10 However, many other stud-
ies5,9,11,12 have not yet established a simple correlation
between the enzyme activity and IL polarity. The other
two factors (hydrogen-bond basicity and anion nucleo-
philicity) have the same problem in terms of lacking a
general relationship between the enzyme activity and
IL properties. Therefore, the current understanding of
the IL effect on enzyme activity is still in its infancy.
In hydrophobic ILs [such as PF6 and (CF3SO2)2Nꢀ
ꢀ
salts], enzymes have shown very high stabilities in a
*
Corresponding author. E-mail: zhaoh@savstate.edu
0957-4166/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2006.01.015

378
H. Zhao et al. / Tetrahedron: Asymmetry 17 (2006) 377–383
enzyme might be embedded in the IL network, which
could protect the essential water of proteins and the
solvophobic interactions, that is critical for maintaining
the native structure.
groups are less hydrophobic, and are thus less kosmo-
tropic when compared with those containing no hydr-
oxyl groups, but with the same carbon-chain length.
Our recent studies12,44 on the protease activity in IL
aqueous solutions also revealed that enzyme stability
followed the Hofmeister series in an order of decreasing
effectiveness for anions: CH3COOꢀ, CF3COOꢀ > Clꢀ,
On the other hand, when hydrophilic ILs are dissolved
in an aqueous solution, they dissociate into individual
cations and anions. These individual ions may affect
the enzyme behavior in aqueous environments. As a
matter of fact, the effect of individual ions on protein
stabilization is well known (the Hofmeister series):31,32
kosmotropic anions and chaotropic cations stabilize
proteins, while chaotropic anions and kosmotropic cat-
ions destabilize them.33–37 Our recent review19 indicated
that the same ion effect was applicable to enzyme activ-
ity, specificity and stability as supported by numerous
experimental data. Therefore, the effect of ILs on the
enzyme activity not only relied on the overall solvent
properties (such as polarity), but also depended on the
individual ion contributions.
Brꢀ > OTsꢀ > BF4
(decreasing kosmotropicity),
ꢀ
and for cations: [EMIM]+, [BuPy]+ > [BMIM]+ >
[EtPy]+ (increasing kosmotropicity in general).
Based on the above pioneering work, we explored fur-
ther the effect of IL kosmotropicity on the enantioselec-
tivity of a protease in this study. More specifically, we
investigated the enzymatic hydrolysis of enantiomeric
phenylalanine methyl ester in aqueous solutions of var-
ious ILs. The enzyme, Bacillus licheniformis protease
(subtilisin Carlsberg), was chosen because it is cofac-
tor-independent and active in low-water environments
(ꢁ9% water/dry enzyme, wt/wt), its physiological func-
tion is to hydrolyze water-soluble proteins, and its struc-
tures, catalytic mechanism and properties are well
understood.21,45 Herein, we report the correlation
between the Hofmeister series of ILs and the enzyme
stabilization, which would enable us to use the series
as an empirical guideline in designing ILs for specific
enzymatic applications.
The structural stabilization of proteins and enzymes by
ILs has recently gained some attention. An early study
by Summers and Flowers38 investigated a room-temper-
ature IL, ethylammonium nitrate (EAN, or [EtNH3]-
[NO3]), in the protein refolding of hen egg white
lysozyme (HEWL). Their results confirmed that EAN
is a denaturant (since it is a structure-breaking electro-
lyte as suggested by its volumetric behavior during
hydration;39 meanwhile, both [EtNH3]+ (B-coefficient:40
2. Results
0.132) and NO3 (ꢀ0.043) are chaotropic anions).41
ꢀ
However, EAN was able to prevent the aggregation of
denatured protein. It was suspected that the interaction
between the ethyl group of EAN and the hydrophobic
surfaces of the protein protects EAN from intermole-
cular association. Meanwhile, electrostatic interactions
between the ions and the charged portion of the protein
could stabilize the secondary structure.42 Another recent
study examined the activity of HEWL, and the renatur-
ation of HEWL and single-chain antibody fragment
ScFvOx in 1-alkyl and 1-(x-hydroxyalkyl)-3-methylimi-
dazolium chlorides.43 The protein refolding by these ILs
was also explained as the suppression of aggregate for-
mation by these organic salts. Both the activity of
HEWL in ILs and the ability of ILs in refolding the
HEWL and ScFvOx followed the Hofmeister series (in
a decreasing order): [OH–EMIM]+ > [EMIM]+, [OH–
2.1. Effect of buffer and IL concentrations on the kinetic
hydrolysis
The enzymatic hydrolysis rate of L-phenylalanine
methyl ester is faster than that of D-ester. Figure 1 illus-
trates the effect of buffer concentration (hence the pH
value) on the hydrolysis reaction. The enantiomeric
excess (ee) of L-phenylalanine decreased with an increase
100
E
80
ee
L yield
PMIM]+ > [BMIM]+,
[OH–HMIM]+ > [HMIM]+.à
60
The cation kosmotropicity is in an increasing order
because the ion hydrophobicity increases upon lengthen-
ing of alkyl chain.41 The cations containing hydroxyl
40
20
Kosmotropes are strongly hydrated species and thus called water
‘structure-makers’. Kosmotropic ions include CH3COOꢀ, SO4
,
2ꢀ
HPO42ꢀ, Mg2+, Ca2+, Li+, H+, OHꢀ, etc. Chaotropes are weakly
hydrated species and thus called water ‘structure-breakers’. They
include SCNꢀ, Iꢀ, NO3ꢀ, BF4ꢀ, Cs+, K+, (NH2)3C+ (guanidinium),
(CH3)4N+ (tetramethylammonium), and others.
pH 7
0.05
pH 8
0.1
pH 9
0.2
0
0
0.15
0.25
à OH–EMIM, 1-(2-hydroxyethyl)-3-methylimidazolium; EMIM, 1-
ethyl-3-methylimidazolium; OH–PMIM, 1-(3-hydroxypropyl)-3-
methylimidazolium; BMIM, 1-butyl-3-methylimidazolium;
OH–HMIM, 1-(6-hydroxyhexyl)-3-methylimidazolium; HMIM,
1-hexyl-3-methylimidazolium.
NaHCO3 concentration (M)
Figure 1. Effect of buffer (NaHCO3) concentration and pH on
the enzymatic hydrolysis of phenylalanine methyl ester in 0.5 M
[EMIM][EtSO4] (40 min reaction time).

H. Zhao et al. / Tetrahedron: Asymmetry 17 (2006) 377–383
379
of NaHCO3 concentration, causing a dramatic decrease
of the enantiomeric ratio (E value). However, the high-
est yield of L-phenylalanine was observed in 0.1 M
NaHCO3 solution, where the ee retained a relatively
high value. Therefore, we selected 0.1 M NaHCO3 as
the buffer solution for the following study.
ee values being moderately high (above 80%). This find-
ing demonstrated that EtSO4 is a kosmotropic anion
ꢀ
and [EMIM][EtSO4] is an enzyme-‘friendly’ ionic sol-
vent. However, extremely high ee values (>90%) were
normally observed within 40 min of the hydrolysis time
because when the reaction time was prolonged further,
the hydrolysis rate of the L-ester was slower while that
of the D-ester became faster. Such an observation was
also reflected by the E values in Figure 3 where the high-
est E (98% ee and 42% yield for L-phenylalanine) was
achieved in 0.5 M [EMIM][EtSO4], when the reaction
time was 20 min. This E value was even higher than
those obtained in pure water, indicating that this IL con-
centration was able to activate the enzyme. Figure 3 also
indicated that with an increase of IL concentration, a
longer reaction time was needed to achieve the optimum
E value for each concentration. This was probably due
to the slow substrate dissolution causing the diffusional
limitation in viscous IL solutions (especially when the
concentration is greater than 3.0 M).
The IL, [EMIM][EtSO4], was chosen because a high
enzyme activity was observed in this organic salt
(Fig. 2). Meanwhile, this IL, known as ECOENGTM
212, showed no irritation to skin and eyes, and had a
low toxicity (LD50 value > 2000 mg/kg [rat, Wistar
strain], and EC50 value > 100 mg/L [Daphnia magna]).46
100
80
60
0 M
2.2. Effect of kosmotropicity of anions and cations on the
enantioselectivity
0.5 M
40
20
0
1.0 M
2.0 M
3.0 M
4.0 M
The effect of anion kosmotropicity on the enzyme
enantioselectivity was demonstrated in Figure 4. High
ee values were obtained in IL solutions (0.5 M) contain-
ing kosmotropic anions, such as PO43ꢀ, citrate3ꢀ
,
CH3COOꢀ, EtSO ꢀ, and CF3COOꢀ. A moderately high
0
1
2
3
4
5
4
ee was observed when the weakly chaotropic anion Brꢀ
was present, while very low enantioselectivities weꢀre
Time (hr)
Figure 2. Effect of [EMIM][EtSO4] concentration on the enantiomeric
seen when strong chaotropic anions (OTsꢀ and BF4
)
excess (ee) of L-phenylalanine.
were involved. Out of curiosity, we examined further
An inspection of Figures 2 and 3 afforded the conclusion
that the protease enantioselectivity is IL concentration
dependent. The IL concentrations investigated in this
study ranged from 0 to 4.0 M (ꢁ80%, v/v). Figure 2
illustrated that within a 5 h period, the enantioselectivity
increased as the IL concentration increased, with most
3-
PO4
200
E
ee
L yield
160
120
80
40
0
-
-
CH3CO2
CF3CO2
-
Br-
EtSO4
Citrate3-
OTs-
160
140
120
100
-
BF4
-0.093 n/a -0.033 0.137 n/a 0.246 0.333 0.495
E
80
60
40
20
Viscosity B-coefficient
Increasing kosmotropicity
Figure 4. The effect of anion kosmotropicity of ILs on the enzymatic
hydrolysis (0.5 M [EMIM]+ based ILs; 40 min reaction time; B-
coefficients from Marcus’s selections;40 n/a means the B-coefficient is
not available for that ion; the kosmotropicity of CF3COOꢀ is known
between CH3COOꢀ and Clꢀ,41 the B value of CF3COOꢀ is roughly
estimated from the NMR B0-coefficients of CF3COOꢀ (0.10) and
CH3COOꢀ (0.18)47 based on the simple assumption that the B-
coefficient is proportional to the B0-coefficients; the B-coefficient of
citrate was calculated from Ref. 48 using the simplified Jones–Dole
equation g/g0 = 1 + B · c).
4
0
3
2
1
0.5
)
M
(
c
0
Figure 3. The enantiomeric ratio (E) as a function of reaction time and
[EMIM][EtSO4] concentration.

380
H. Zhao et al. / Tetrahedron: Asymmetry 17 (2006) 377–383
the enzymatic hydrolysis in higher concentrations of
citrate based ILs (up to 2.0 M, ꢁ75% v/v IL). As shown
in Figure 5, high enantioselectivities were also achieved
in concentrated solutions of citrate based ILs; the yield
was slightly decreased with an increase of IL concentra-
tion due to the diffusional limitation in viscous solu-
tions. Figure 6 reported the effect of IL cations on the
enzymatic hydrolysis. The ee and E values decreased
with the increase of the alkyl chain length of cations.
coefficients of anion and cation) of N-alkylpyridinium
based ILs. The general relationship between the enantio-
selectivity and the d values is thus: the ee and E
increase with an increase of d value. One exception
wasꢀthe [BuPy]Cl salt, the reason behind it being that
BF4 is a much stronger chaotrope than Clꢀ while the
kosmotropicity difference between [EtPy]+ and [BuPy]+
is not significantly large.41 Unfortunately, the B-coeffi-
cients of imidazolium and many other organic cations
are not yet available for more correlations.
120
3. Discussion
E
ee
L yield
100
80
60
40
20
0
The buffer chosen for the enzymatic hydrolysis was so-
dium bicarbonate (NaHCO3). The Jones–Dole viscosity
B-coefficients for Na+ and HCO3ꢀ were 0.085 and 0.031,
respectively;40 since a large positive B value means a
strong kosmotrope while a large negative B value means
a strong chaotrope,41 both the cation and anion can be
classified as borderline ions.§ Therefore, both of them
have minimum kosmotropic effects on the enzyme activ-
ity. If a phosphate buffer was used instead, the ions pres-
ent in buffer may influence the enzyme stability since
HPO4 anion (B value:40 0.382) is a kosmotrope and
2ꢀ
ꢀ
H2PO4 anion (0.340) is considered as a borderline
ion, while Na+ and K+ (ꢀ0.009) (if present) are in the
range of borderline ions.51
0.5 (~25%)
1.0 (~40%)
2.0 (~75%)
[EMIM]3[Cirtrate] concentrations in M and %IL (v/v)
Figure 4 suggests that the protease enantioselectivity fol-
lows the Hofmeister series, that is, the kosmotropic
anions stabilize the enzyme while chaotropic anions
destabilize it. The anion ability in improving the enzyme
enantioselectivity is in an order of decreasing effec-
Figure 5. Enzymatic hydrolysis of phenylalanine methyl ester in citrate
based ILs of various concentrations (40 min reaction time).
3ꢀ
tiveness: PO4 > citrate
,
CH3COOꢀ, EtSO4
3ꢀ
ꢀ
,
100
CF3COOꢀ > Brꢀ > OTsꢀ, BF ꢀ. Figures 2 and 5 con-
E
ee
L yield
4
cluded that the enzyme enantioselectivity is still very
high in high concentrations of ILs containing kosmo-
80
ꢀ
tropic anions (such as EtSO4 and citrate). However, a
lower substrate conversion was also observed in ILs
with very strong kosmotropic anions (such as PO43ꢀ).
This could probably be explained by the reactivity–selec-
tivity principle, that is, the less the reactivity of a species,
the greater selectivity it will be.
60
40
20
0
As explained in our recent review,41 the hydrophobicity
of cations increases with the cation sizes, yielding a high-
er kosmotropicity. Since the high kosmotropic cations
destabilize the enzyme based on the Hofmeister series,
the cation ability in improving the enzyme enantio-
selectivity is in an order of decreasing effectiveness:
[EMIM]+ > [BMIM]+ > [HMIM]+ (Fig. 6).
[EMIM]+
[BMIM]+
[HMIM]+
[CF3COO]- based ILs
Increasing kosmotropicity
The d value reflects the overall kosmotropic effect of cat-
ions and anions of ILs (Fig. 7): high kosmotropicity of
anion and low kosmotropicity of cation seem desirable
for the enzyme stabilization. A balance between these
Figure 6. Effect of cation kosmotropicity of ILs on the enzymatic
hydrolysis of phenylalanine methyl ester (0.5 M [CF3COO]ꢀ based ILs;
40 min reaction time).
2.3. Correlation between the enantioselectivity and d
values
§ A different B value for HCO3 is 0.130,40 however, based on the
ꢀ
slightly negative value of structural entropy and the zero value of
DGHB (defined in the literature),50 this ion was classified as a
borderline ion.41,51
Figure 7 correlates the protease enantioselectivity with
the d values (differences in the Jones–Dole viscosity B-

H. Zhao et al. / Tetrahedron: Asymmetry 17 (2006) 377–383
381
120
100
80
60
40
20
0
E
ee
[EtPy][CF3COO]
[EtPy]Br
[BuPy]Cl
[EtPy][BF4]
[BuPy][BF4]
-0.489
-0.401
-0.321
-0.261
-0.091
(anion-cation)
δ
Figure 7. Correlation between the enantioselectivity and the difference in the Jones–Dole viscosity B-coefficients of anion and cation (d is defined40,49
P
P
as
miB(anion) ꢀ miB(cation) where mi is mole fraction of the ion; B-coefficients of anions are from Marcus’s selections;40 B-coefficients of
N-alkylpyridinium cations are from our recent review;41 reaction time is 40 min).
two could enable an optimal stabilization of biological
macromolecules (including enzymes).35,37,52 Lindsay
et al.49 correlated the reactivity of penicillin amidase for-
mulations with the d values of inorganic salts and their
mixtures (used in the lyophilization of the enzyme). Sim-
ilarly, they observed that the enzyme activity increased
with the d value.
bromide ([EMIM]Br), 1-ethyl-3-methylimidazolium
ethyl sulfate ([EMIM][EtSO4]), 1-ethyl-3-methylimi-
dazolium tosylate ([EMIM][OTs]), 1-butyl-3-methylimi-
dazolium bromide ([BMIM]Br), 1-hexyl-3-methyl-
imidazolium chloride ([HMIM]Cl), silver acetate, B.
licheniformis protease (subtilisin Carlsberg), D-phenylal-
anine methyl ester hydrochloride, L-phenylalanine
methyl ester hydrochloride, and other reagents were
purchased from the Sigma–Aldrich.
4. Conclusion
5.2. IL preparations
Both the buffer concentration (inorganic salt concentra-
tion) and the IL concentration (organic salt concentra-
tion) considerably modify the enzymatic hydrolysis
rate of enantiomeric phenylalanine methyl ester. The
protease enantioselectivity was greatly enhanced by
using IL solutions containing kosmotropic anions and
chaotropic cations because the effect of ions on the en-
zyme stabilization follows the Hofmeister series. The d
value is an empirical scale for evaluating the overall kos-
motropicity of an IL. Generally, high enzyme enantio-
selectivities were observed in ILs with high d values.
The ion ability in improving the enzyme enantioselectiv-
ity is in an order of decreasing effectiveness for anions:
Table 1 summarizes the source and appearances of the
ILs used in this study. [EMIM]3[PO4] was prepared by
the stoichiometric titration of phosphoric acid with
[EMIM][OH], which was prepared according to a litera-
ture method55 by using the anion exchange resin
(Amberliteꢁ IRA-400 Cl). [EMIM]3[citrate] was pre-
pared by mixing a [EMIM]Br aqueous solution with
slightly excess equimolar silver citrate hydrate (white
powder, insoluble) with a gentle heat; the yellow precip-
itate (AgBr) formed in the solution; the completeness
of the reaction was monitored by taking samples from
the solution and reacting with AgNO3 solution; the
precipitates (AgBr and excess silver citrate) were re-
moved when the solution was cooled by an ice bath;
the possible presence of silver citrate in the mother
liquid was checked by a [EMIM]Br solution. Other
ILs were prepared in our laboratory by the silver
metathesis method, as explained in the literatures.10,56
Charcoal was used for an effective removal of color
and impurities from all crude ILs.57 The silica gel col-
umn was further used to remove trace impurities. Water
was evaporated through a rotary evaporator under
vacuum at 50 ꢂC. [EMIM]3[PO4] is a brown and viscous
liquid, and other ILs prepared are slightly viscous and
colorless liquids. The prepared ILs were examined by
0.1 M AgNO3 and 0.1 M HCl solutions, respectively,
ꢀ
PO4 > citrate3ꢀ, CH3COOꢀ, EtSO ꢀ, CF3COO >
3ꢀ
4
Brꢀ > OTsꢀ, BF4
and for cations: [EMIM]+ >
ꢀ
[BMIM]+ > [HMIM]+. However, for different biological
applications, ions do not necessarily stabilize or destabi-
lize the enzyme in exactly the same Hofmeister
order.19,52–54
5. Experimental
5.1. Materials
N-Ethylpyridinium bromide ([EtPy]Br) and N-n-butyl-
pyridinium chloride ([BuPy]Cl) were obtained from
the Alfa Aesar Company. 1-Ethyl-3-methylimidazolium

382
H. Zhao et al. / Tetrahedron: Asymmetry 17 (2006) 377–383
Table 1. Ionic liquids (ILs) investigated in this study
IL
Molar mass (g molꢀ1
)
Appearance
Source
[EMIM]Br
191.07
236.29
170.19
224.19
282.36
197.98
522.63
428.48
219.12
252.24
202.72
280.30
188.07
221.19
194.98
171.66
223.02
Solid, slightly yellow
Sigma–Aldrich
Sigma–Aldrich
Prepared
Prepared
Sigma–Aldrich
Prepared
Prepared
Prepared
Sigma–Aldrich
Prepared
Sigma–Aldrich
Prepared
Alfa Aesar
Prepared
Prepared
Alfa Aesar
Prepared
[EMIM][EtSO4]
[EMIM][CH3COO]
[EMIM][CF3COO]
[EMIM][OTs]
[EMIM][BF4]
[EMIM]3[citrate]
[EMIM]3[PO4]
[BMIM]Br
[BMIM][CF3COO]
[HMIM]Cl
[HMIM][CF3COO]
[EtPy]Br
Viscous liquid, slightly yellow
Slightly viscous liquid, colorless
Slightly viscous liquid, colorless
Solid, white
Slightly viscous liquid, colorless
Viscous liquid, colorless
Viscous liquid, brown
Solid, slightly yellow
Slightly viscous liquid, colorless
Very viscous liquid, yellow
Slightly viscous liquid, colorless
Solid, slightly yellow
Slightly viscous liquid, colorless
Slightly viscous liquid (sometimes crystals), colorless
Solid, slightly yellow
[EtPy][CF3COO]
[EtPy][BF4]
[BuPy]Cl
[BuPy][BF4]
Slightly viscous liquid (sometimes crystals), colorless
to ensure the absence of halides and Ag+ impurities. H
NMR, FT-IR, and HPLC data confirmed that the pre-
pared ILs are free of measurable impurities including
water.
Acknowledgements
1
This project was partially supported by the NIH EAR-
DA Grant (No. 5 G11 HD 32861-08). The authors are
very thankful to the anonymous reviewer for suggesting
constructive improvements of the manuscript.
5.3. Enzymatic hydrolysis of phenylalanine methyl esters
D- (or L-) Phenylalanine methyl ester (20 mg) was dis-
solved in 2 mL solvent consisting of an IL and 0.1 M
NaHCO3 buffer (pH 8.0). Enzyme (1 mg) was added to
the reaction mixture at time zero. The reaction was
maintained at 25 1 ꢂC. The hydrolysis progress was
periodically measured by the HPLC analysis. All exper-
iments were run in duplicates. The average values were
reported. The relative errors were normally less than 5%.
References
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5.4. HPLC analysis
The samples were analyzed by a LC-10AT Schimadzu
HPLC equipped with a SPD-10A UV–vis dual wave-
length detector and a Schimadzu Premier C18 column
(150 mm · 4.6 mm, particle size 5 lm). The flow rate is
1.0 mL/min with water/MeOH ratio of 90/10. The
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´
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