Effects of room temperature ionic liquids on fluorescence characteristics of 17β-estradiol and its derivative

Article abstract of DOI:10.1007/s10895-011-0853-z

Previous reports have seldom concerned about the RTILs (Room temperature ionic liquids), and their effects on derivatization reaction or derivatives. In this study, we reported that the effects of four different RTILs, i.e., [EMIM]PF₆, [BMIM]PF₆, [HMIM]PF₆ and [OMIM]PF₆, on fluorescence spectra of 17 β-estradiol (E2), and its derivatization solvent dansyl chloride (DNSCl) and the derivative. [BMIM]PF₆ had a significant quenching effect on the fluorescence intensity of E2, suggesting the formation of [BMIM]PF₆/E2 complexes and possible buried E2 molecular in a more hydrophobic microenvironment. The estimated Stern-Volmer quenching constant (Ksv=0.3519) proved that E2 quenching caused by [BMIM]PF₆ was a dynamic quenching process. Four RTILs, with different alkyl chain-length in imidazolium cation, resulted in different quenching intensities to E2 as follows: [EMIM]PF₆ > [BMIM]PF ₆ > [HMIM]PF₆ > [OMIM]PF₆. At 5 mg L ^-1 of DNSCl, [BMIM]PF₆, [HMIM]PF₆ and [OMIM]PF₆ increased the fluorescence intensities of E2 in water by 8.5, 7.6 and 6.1 times, respectively, and a 37-nm hypsochromic shift occurred. The fluorescence intensity for [BMIM]PF₆-extracted derivative of E2 increased more than two times compared with that for the control. In conclusion, this study demonstrated that above four hexafluorophosphate salt ionic liquids could be used in derivatization reaction to enhance fluorescent sensitivity in E2 trace residual analysis. Springer Science+Business Media, LLC 2009.

Full text of DOI:10.1007/s10895-011-0853-z

J Fluoresc (2011) 21:1643–1648
DOI 10.1007/s10895-011-0853-z
ORIGINAL PAPER
Effects of Room Temperature Ionic Liquids on Fluorescence
Characteristics of 17β-estradiol and its Derivative
Xuedong Wang & Yanyan Li & Xiwei Du & Zhenkun Lin &
Changjiang Huang
Received: 25 September 2010 /Accepted: 1 February 2011 /Published online: 18 February 2011
#
Springer Science+Business Media, LLC 2011
Abstract Previous reports have seldom concerned about the
RTILs (Room temperature ionic liquids), and their effects on
derivatization reaction or derivatives. In this study, we
reported that the effects of four different RTILs, i.e.,
[EMIM]PF6, [BMIM]PF6, [HMIM]PF6 and [OMIM]PF6,
on fluorescence spectra of 17 β-estradiol (E2), and its
derivatization solvent dansyl chloride (DNSCl) and the
derivative. [BMIM]PF6 had a significant quenching effect
on the fluorescence intensity of E2, suggesting the
formation of [BMIM]PF6/E2 complexes and possible
buried E2 molecular in a more hydrophobic microenvi-
ronment. The estimated Stern-Volmer quenching constant
(Ksv=0.3519) proved that E2 quenching caused by
[BMIM]PF6 was a dynamic quenching process. Four
RTILs, with different alkyl chain-length in imidazolium
cation, resulted in different quenching intensities to E2 as
derivatization reaction to enhance fluorescent sensitivity in
E2 trace residual analysis.
.
Keywords Room temperature ionic liquids Fluorescence
.
.
.
intensity 17β-estradiol Dansyl chloride Derivative
Introduction
As a kind of new “green” solvents and soft materials, and an
alternative to the conventional and environmentally detri-
mental volatile solvents, room temperature ionic liquids
(RTILs) possess several attractive features, including low-
melting temperature (below 373 K), negligible vapor
pressure, excellent thermal stability, strong ability to dissolve
many chemicals, high electrical conductivity, wide electro-
chemical window, and ability of repetitive utilization [1]. All
these properties lead RTILs to a wide application in
material synthesis [2], chemical reactions [3], separations
[4], electrochemistry [5] and formation of ordered molec-
ular assembly [6]. RTILs were also reconstructed for some
specific applications in chemistry [7, 8]. In instrumental
analytical area, RTILs were used as stationary phase in gas
chromatography [9], and as mobile phase additives in
capillary electrophoresis and fluorescence analysis [10, 11].
However, the mechanisms underlying the interaction
between ionic liquids and fluorescent substances still
remain under investigation, especially for the weak fluo-
rescent chemicals, which may hinder the complete and most
efficient utilization of these “green” solvents in fluorescent
analysis of organic chemicals.
follows: [EMIM]PF6 > [BMIM]PF6 > [HMIM]PF6
>
[OMIM]PF6. At 5 mg L−1 of DNSCl, [BMIM]PF6,
[HMIM]PF6 and [OMIM]PF6 increased the fluorescence
intensities of E2 in water by 8.5, 7.6 and 6.1 times,
respectively, and a 37-nm hypsochromic shift occurred. The
fluorescence intensity for [BMIM]PF6-extracted derivative of
E2 increased more than two times compared with that for the
control. In conclusion, this study demonstrated that above
four hexafluorophosphate salt ionic liquids could be used in
Xuedong Wang and Yanyan Li contributed equally to this work.
:
:
:
X. Wang Y. Li Z. Lin C. Huang (*)
Institute of Watershed Science and Environmental Ecology,
Wenzhou Medical College, Chashan Campus,
Wenzhou, Zhejiang 325035, People’s Republic of China
e-mail: cjhuang5711@163.com
In recent years, phenolic estrogens such as estrone (E1),
17β-Estradiol (E2), ethnylestradiol (EE2) and estradiol
(E3), have been frequently studied as a group of environ-
mental phenolic estrogens due to their structural and
X. Du
The Second Affiliated Hospital of Xi’an Medical University,
Xi’an, Shanxi 710038, People’s Republic of China

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J Fluoresc (2011) 21:1643–1648
estrogenic similarities [12]. As a consequence of its large
use in our daily life, E2 has made an important impact on
our environment, including on animals and human health
[13]. Fluorescence determination is a direct, simple and
quick method for E2 residual analysis. However, due to the
weak fluorescent property, E2 need to be derivatized with
organic derivatization solvents prior to its fluorescent
analysis. To the best of our knowledge, no data are
available on interaction between ionic liquids and E2, and
on application of ionic liquids in fluorescent analysis of E2.
Therefore, the aim of this study is to evaluate the effect of
RTILs on fluorescent behavior of E2 and its derivative, and
to lay foundation for the application of RTILs in weak
fluorescent chemicals.
Methods
The stock standard solution of E2 (1,000 mg L−1) was
prepared in methanol and stored at −20 °C before use. The
working solutions were obtained by appropriate dilution of
the stock standard solution with relevant solvents or the ionic
liquids. To investigate the effect of RTILs on fluorescence
intensity of E2, precalculated amount of RTILs was directly
added to the aqueous solutions containing E2. Samples were
transferred into 1 cm2 quartz cuvettes at 25 °C for
fluorescence analysis. Fluorescence spectra were acquired
on a model RF-5301PC spectrofluorometer (Shimadzu
Corporation, Kyoto, Japan) with a 75 W Xenon arc lamp
as the excitation source and single-grating monochromators
as wavelength selection devices with slit width of 3 nm. All
emission spectra were corrected for emission monochroma-
tor response and were background subtracted using appro-
priate blanks. All fluorescence data were measured in 1 cm2
quartz cuvettes at 25 °C unless otherwise stated.
Experimental
Materials
To study on the effects of RTILs on E2 derivative with
DNSCl, the standard solution of E2 (5 mg L−1) in methanol
was first dried under a gentle nitrogen flow at 40 °C. The
dried residue was redissolved in 200 μL of sodium
bicarbonate buffer (0.1 M, pH 10) by vortexing for 1 min.
This solution was then mixed with 200 μL of dansyl
chloride in acetone (1 mg mL−1) and vortexed for 1 min.
The derivatization reaction was allowed to proceed for
5 min at 60 °C, and the derivative was detected immedi-
ately or extracted by 200 μL [BMIM]PF6. The fluorescence
data for two treatments, non-extracted and extracted by
[BMIM]PF6, were compared to analyze the effects of
RTILs on the derivative of E2.
Reference compounds of 17 β-estradiol (E2), dansyl
chloride (DNSCl) and RTILs: 1-ethyl-3-methylimidazolium
hexafluorophosphate [EMIM]PF6, 1-butyl-3- methylimidazo-
lium hexafluorophosphate [BMIM]PF6, 1-hexyl-3-methylimi-
dazolium hexafluorophosphate [HMIM]PF6 and 1-octyl-3-
methylimidazolium hexafluorophos- phate [OMIM]PF6
with stated purities of 99.0% were all purchased from
Sigma-Aldrich (St. Louis, MO, USA). The previous
standard compounds were used as received. Ultrapure
water used in the study was purified with a Millipore
Milli-Q plus system (Bedford, MA, USA). Methanol and
acetone were LC-grade.
Fig. 1 The fluorescence spectra
of E2 in different concentra-
tions of [BMIM]PF6
1
6
*1 (0 mM), 2 (5 mM), 3 (10 mM), 4 (25 mM), 5 (50 mM), 6 (100 mM)

J Fluoresc (2011) 21:1643–1648
1645
1.6
1.4
series of concentrations (1, 5, 10, 20, 50 and 100 mM) in
methanol, and then prepared 50 mg L−1 of E2 with the
above mixed solvents. As shown in Fig. 1, the fluorescence
intensity of E2 decreased gradually with the increase of
[BMIM]PF6 concentrations from 0 to 100 mM. The
fluorescence intensity was about 815.7 in absence of
[BMIM]PF6, while it decreased to 330.9 in the presence
of [BMIM]PF6 at 100 mM. Therefore, [BMIM]PF6 had a
significant quenching effect on the fluorescence of E2,
which indicates the formation of [BMIM]PF6/E2 com-
plexes. Also it suggests that E2 molecular is likely buried in
a more hydrophobic microenvironment. However, a signif-
icant hypsochromic or bathochromic shift of the emission
maxima was not observed in the quenching processes.
Indeed, our results were in agreement with Fletcher’s
observation which demonstrated the steady-state emission
behavior of six polycyclic aromatic hydrocarbons along
with the fluorescence quenching within [BMIM]PF6 [15].
However, the effect of ionic liquid on fluorescent behavior
of organic chemical is a debating problem. In some cases,
ionic liquids may significantly improve the fluorescence
intensity of organic matters. Deng et al. reported that,
[BMIM]PF6 had a prominent synergistic action to its
fluorescence when the concentration of Pyronine B ranged
from 1×10−7 to 1×10−4 mol L−1. The similar action also
occurred in the case of phenanthroline and rhrodamine [16].
y = 0.3519x - 0.435
R2 = 0.92
1.2
1
0.8
0.6
0.4
0.2
0
0.005
0.01
0.25
0.05
0.1
-0.2
C[BMIM]PF6
Fig. 2 Stern-Volmer plot for the binding of [BMIM]PF6 to estradiol
Results and Discussion
Effects of [BMIM]PF6 Different Concentrations
on Fluorescence Spectra of E2
It has been reported that different concentrations of RTILs
in solvents result in complex interplay due to the
physicochemical properties of the solvents (e.g. viscosity,
static dielectric constant, refractive index, density, polariz-
ability, etc.), which may further affect steady-state emission
behavior of the analytes [14]. At ambient conditions,
[BMIM]PF6 is much more viscous than the organic
solvents such as methanol and acetonitrile. The viscosity
of acetonitrile is 0.37 mN s m−2 at 20 °C, while the
viscosity of [BMIM]PF6 is reported to be 330 mN s m−2 at
the same temperature. Considering the vast difference
between the viscosity values of [BMIM]PF6 and of the
common organic solvents, we diluted [BMIM]PF6 to a
Fluorescence Quenching Mechanism of [BMIM]PF6
Fluorescence quenching has been widely used to reveal the
combined position of quenchers to the fluorophore. There
exist two fluorescence quenchings, i.e., static and dynamic
Fig. 3 The fluorescence spectra
of E2 in different kinds of
RTILs
1 [EMIM]PF6, 2 [BMIM]PF6, 3 [HMIM]PF6, 4 [OMIM]PF6

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Table 1 The excitation wave-
length (EX/nm) and emission
wavelength (EM/nm) of
different concentrations of
E2 in different ionic liquids
Ionic liquid concentrations
C(mM)
[EMIM]PF6
[BMIM]PF6
[HMIM]PF6
[OMIM]PF6
EX
EM
EX
EM
EX
EM
EX
EM
0
282
281
281
281
281
281
307
306
307
307
307
306
282
282
282
281
281
283
307
307
307
307
308
308
282
282
282
281
282
282
307
306
308
307
308
307
282
282
282
282
281
282
307
307
306
307
307
308
5
10
25
50
100
“EX/EM” denotes the maximum
excitation or emission wave-
length, respectively
quenching. Static quenching refers to the formation of a
ground state fluorophore–quencher complex which does
not emit a photon. Dynamic quenching refers to the
formation of an excited state fluorophore–quencher com-
plex [17]. Dynamic quenching processes are described by
the Stern–Volmer equation:
of ionic liquids and interactions between ionic liquids and
fluorophores. The fluorescence enhancement or quenching
effects depends on the type and concentration of fluorescent
substances and features of ionic liquids [16]. To demon-
strate the impact of ILs on fluorescence intensity of E2,
four types of ILs with different alkyl chain-length in
imidazolium cations were selected: [EMIM]PF6, [BMIM]
PF6, [HMIM]PF6 and [OMIM]PF6. Similar to [BMIM]PF6,
[EMIM]PF6, [HMIM]PF6 and [OMIM]PF6 all showed
quenching effects on fluorescence intensities of E2. On
the other hand, the length of alkyl chain had an impact on
the quenching ability of ILs, which were as in the following
Io
¼ 1 þ Ksv½Qꢀ ¼ 1 þ KqΓ0½Qꢀ
ð1Þ
I
where Io and I are fluorescence intensities with or without
the presence of a quencher, and Ksv and Kq are the Stern–
Volmer quenching constant and the bimolecular quenching
constant, respectively; Γ0 is the lifetime of fluorophore in the
absence of a quencher. The results of Io/I against concen-
trations of [BMIM]PF6 were shown in Fig. 2. A fair-to-good
linear correlation coefficient (r2=0.9200) was observed,
suggesting the compliance of the Stern-Volmer relation. As
reported by Geng et al. [17], the Stern–Volmer quenching
constant for dynamic quenching was less than 1.0×103 L/mol.
Therefore, the 17 β-estradiol quenching (Ksv=0.3519)
caused by [BMIM]PF6 in this study was dynamic quenching.
order: [EMIM]PF6 > [BMIM]PF6 > [HMIM]PF6
>
[OMIM]PF6 (Fig. 3). The shorter alkyl chain in imidazolium
cations displayed the higher quenching intensities for E2,
which possibly resulted from the different microenvironment
formed by varying length of alkyl chain. As summarized in
Table 1, the maximum excitation wavelength of these four
ILs ranged from 281–282 nm, and the maximum emission
wavelength varied between 306 and 308 nm, suggesting that
no hypsochromic or bathochromic shift with different ILs.
Effect of Ionic Liquids (ILs)
Fluorescence Intensities of DNSCl in Water and ILs
Ionic liquids affect the fluorescence spectra by transforming
the features and varying fluorescence intensities. The
changes of spectra are associated with the unique features
As reported by Deng et al., the interaction of strong or weak
fluorescent substances with ionic liquids had significant
difference. Therefore, fluorescent properties of organic
Table 2 The comparison of fluorescence behavior of dansyl chloride (DNSCl) in water solution and ionic liquids
DNSCl concentrations
DNSCl - Water
5 mg L−1
10 mg L−1
25 mg L−1
50 mg L−1
100 mg L−1
EX/EM
FI
318/498
16.585
320/497
35.098
320/497
77.257
320/494
111.009
321/496
125.115
ND
DNSCl - [BMIM]PF6
DNSCl - [HMIM]PF6
EX/EM
FI
332/461
141.750 (8.5)
331/463
262.391 (7.5)
330/465
565.593 (7.3)
334/463
873.386 (7.9)
EX/EM
FI
331/460
332/459
332/460
335/462
336/465
125.396 (7.6)
332/458
248.878 (7.1)
331/456
533.759 (6.9)
334/458
844.366 (7.6)
334/462
917.220 (7.3)
341/460
DNSCl - [OMIM]PF6
EX/EM
FI
101.342 (6.1)
219.213 (6.2)
435.903 (5.6)
778.874 (7.0)
808.405 (6.5)
“EX/EM” denotes the maximum excitation or emission wavelength, respectively
“FI” indicates the fluorescence intensity

J Fluoresc (2011) 21:1643–1648
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Fig. 4 The fluorescence spec-
trum of dansyl chloride in
[BMIM]PF6 (a) and water
solution (b)
a
b
chemicals may lead to varying effects within ionic liquids.
As a weak fluorescent substance, E2 is often derivatized with
derivatization solvents prior to fluorescent analysis. Dansyl
chloride, a type of strong fluorescent substance, is often used
to derivatize E2 to enhance fluorescence response [18–20].
Therefore, DNSCI was selected as a representative of
strong fluorescent substances to study the effects of ionic
liquids. As shown in Table 2, [BMIM]PF6, [HMIM]PF6
and [OMIM]PF6 in 5 mg L−1 of DNSCl increased the
fluorescence intensity of E2 by 8.5, 7.6. and 6.1 times,
respectively, compared with those in water. Similarly,
[BMIM]PF6, [HMIM]PF6 and [OMIM]PF6 in 50 mg L−1
of DNSCI increased the intensity by 7.9, 7.6 and 7.0 times,
respectively. These results demonstrated that DNSCl had an
enhancement effect on the fluorescence intensity of E2.
However, the increase of DNSCl concentration from 5 to
100 mg L−1 did not lead to significant change of RTILs
effects on E2 intensity, which ranged from 6.1 to 8.5. The
maximum emission peak for DNSCl was at 498 nm in
water samples, while it was at 461 nm in [BMIM]PF6,
indicating the occurrence of 37-nm hypsochromic shift
(Fig. 4). In conclusion, [BMIM]PF6 had enhancement
effects for strong fluorescence chemicals and quenching
effects for weak fluorescence chemicals. In line with our
findings, Fletcher et al. also observed that the emission
from alternant PAHs was quenched while that from
nonalternant PAHs was not [15].
Effect of [BMIM]PF6 on the Derivative of E2
As shown in Fig. 5, E2-Dansyl was the reaction product
combined from E2 and DNSCl by oxygen atom. Conse-
quently, the derivatization product possessed higher fluo-
rescence intensity than its parent compound E2. After
derivatization, the fluorescence intensities of the derivative
extracted with [BMIM]PF6 was 256.06, while that of the
non-extracted derivative,was 806.59. The fluorescence
intensity for [BMIM]PF6-extracted derivative increased
more than two times compared with that for nonionic
liquid-extracted derivative. The emission maxima was
Fig. 5 The derivatization reaction of E2 with dansyl chloride (DNSCl)

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J Fluoresc (2011) 21:1643–1648
480 nm in nonionic liquid-extracted derivative, while it was
465 nm in [BMIM]PF6-extracted derivative, revealing an
occurrence of 15-nm hypsochromic shift. This result further
proved that [BMIM]PF6 had an enhancement effect on
strong fluorescent derivatives, but quenching effect on
weak ones. To the best of our knowledge, this is the first
report on the effects of ionic liquids on fluorescence
intensities of derivatives of phenolic estrogens. We are
currently synthesizing the purity standards of derivatization
product of phenolic estrogens to further study the effects of
ionic liquids and to apply ionic liquids with enhancement
fluorescence effects in fluorescent or chromatographic
analysis of phenolic estrogens at trace levels.
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In the absence of RTILs, the fluorescence intensity of
E2, at fortified level of 50 mgL-1, was quenched by
40.5% in the presence of [BMIM]PF6 (100 mM) than in
the absence. [BMIM]PF6 had significant quenching effect
on the fluorescence of E2, demonstrating the formation of
[BMIM]PF6/E2 complexes and possible submerse of E2
molecular in a more hydrophobic microenvironment. The
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Acknowledgments This work was jointly funded by Key Project of
Science and Technology Department of Zhejiang Province
(2008C03001-2), Key Project of Science and Technology Department
of Wenzhou City (S20060023), Natural Science Foundation of China
(21077079), International Cooperation Project of Wenzhou City
(H20090079) and Key project of Natural Science Foundation of
Zhejiang Province (Z2080266).