Fluorescence enhancement of europium(III) perchlorate by benzoic acid on bis(benzylsulfinyl)methane complex and its binding characteristics with the bovine serum albumin (BSA)

Article abstract of DOI:10.1016/j.saa.2013.09.135

A novel ligand with double sulfinyl groups, bis(benzylsulfinyl)methane L, was synthesized by a new method. Its novel ternary complex, EuL _2.5×L′·(ClO₄)₂×5H ₂O, has been synthesized [using L as the first ligand,

Full text of DOI:10.1016/j.saa.2013.09.135

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 118 (2014) 972–980
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Fluorescence enhancement of europium(III) perchlorate by benzoic acid
on bis(benzylsulfinyl)methane complex and its binding characteristics
with the bovine serum albumin (BSA)
⇑
Jing Zhang, Wen-Xian Li , Bo-Yang Ao, Shu-Yan Feng, Xiao-Dong Xin
College of Chemistry and Chemical Engineering, Inner Mongolia University, Hohhot 010021, People’s Republic of China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
ꢀ
ꢀ
ꢀ
ꢀ
Two new rare earth complexes highly
stables were synthesized for the first
time in this research.
The Eu emission intensities of both
rare earth complexes were high, with
long lifetimes.
The introduction of the second
organic ligand benzoic acid enhanced
the fluorescence intensity.
The binding interactions of Eu (III)
complexes and bovine serum albumin
3
+
(BSA) were studied by fluorescence
spectra.
The synthesis scheme of L
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 March 2013
Received in revised form 22 September 2013
Accepted 29 September 2013
Available online 8 October 2013
A novel ligand with double sulfinyl groups, bis(benzylsulfinyl)methane L, was synthesized by a new
0
method. Its novel ternary complex, EuL2.5ꢁL ꢁ(ClO
4
)
2
ꢁ5H
2
O, has been synthesized [using L as the first
0
ligand, and benzoic acid L as the second ligand], and characterized by elemental analysis, molar conduc-
1
tivity, coordination titration analysis, FTIR, TG–DSC, H NMR and UV–vis. In order to study the effect of
the second ligand on the fluorescence properties of rare-earth sulfoxide complex, a novel binary complex
EuL2.5ꢁ(ClO
4
)
3
ꢁ3H
2
O has been synthesized. Photoluminescent measurement showed that the first ligand L
Keywords:
Bis(benzylsulfinyl)methane
Benzoic acid
Eu (III) complexes
Fluorescence
3+
could efficiently transfer the energy to Eu ions in the complex. Furthermore, the detailed luminescence
analyses on the rare earth complexes indicated that the ternary Eu (III) complex manifested stronger fluo-
rescence intensities, longer lifetimes, and higher fluorescence quantum efficiencies than the binary Eu
0
(III) materials. After introducing the second ligand L , the fluorescence emission intensities and fluores-
Phosphorescence
BSA
cence lifetimes of the ternary complex enhanced more obviously than the binary complex. This illus-
trated that the presence of both the first ligand L and the second ligand L could sensitize fluorescence
0
intensities of Eu (III) ions. The fluorescence spectra, fluorescence lifetime and phosphorescence spectra
were also discussed. To explore the potential biological value of Eu (III) complexes, the binding interac-
tion among Eu (III) complexes and bovine serum albumin (BSA) was studied by fluorescence spectrum.
The result indicated that the reaction between Eu (III) complexes and BSA was a static quenching proce-
dure. The binding site number, n, of 0.60 and 0.78, and binding constant, K
a
, of 0.499 and 4.46 were cal-
0
culated according to the double logarithm regression equation, respectively for EuL2.5ꢁL ꢁ(ClO
4 2 2
) ꢁ5H O and
EuL2.5ꢁ(ClO
4
)
3
ꢁ3H
2
O systems.
Ó 2013 Elsevier B.V. All rights reserved.
⇑
Corresponding author. Tel.: +86 471 4990061 60311.
E-mail address: nmglwx@163.com (W.-X. Li).
1
386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.saa.2013.09.135

J. Zhang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 118 (2014) 972–980
973
Introduction
monitored on SDTQ600 differential scanning calorimeter and ther-
mal gravimetric analyzer. FT-IR spectra were recorded on KBr disk
by using NEXUS 670 FT spectrometer in 4000–400 cm region.
Trivalent lanthanide ions (Ln3+) with organic–inorganic ligands
have dramatically improved the application of luminescent lantha-
nide ions in chemosensors [1], polymer-based devices [2,3], phos-
ꢂ1
The ultraviolet spectra (190–400 nm) of the ligands and the ter-
nary complex were recorded on a Shimadzu UV-265 spectropho-
tometer and acetone was used as a reference and solvent
phors, lasers and optical amplifiers [4–6], and bioimaging probes
7–9]. However, luminescence of Ln3 originated from electronic
+
(concentrations: 1 ꢃ 10 mol L ). 1H NMR spectra were mea-
ꢂ5
ꢂ1
[
transitions between the 4f orbital and these transitions are
strongly forbidden by the parity selection rules, which lead to
low absorption coefficient [10,11]. Some organic ligands such as
sulfoxides are well known to be efficient sensitizers for the lumi-
nescence of lanthanide ions, whose rare earth complexes could
provide distinct advantages, such as high quantum yields, superior
luminescence, high thermodynamic stability and good solubility
6
sured by Bruker AC-300 instrument in DMSO-d . The fluorescence
spectra were determined by FLS920 fluorescence photometer and
the excitation and emission slit width of the complexes was
2 nm. The phosphorescence spectra were measured by F-4500 FL
spectrophotometer at room temperature, and the excitation and
emission slit width was 5.0 and 10.0 nm and excitation wavelength
was 300 and 347 nm. Fluorescent decay curves were determined
by FLS920 Combined Steady State and Lifetime Spectrometer. Fluo-
rescence measurements of complexes with BSA were recorded on a
[
12,13].
The lanthanide complexes with carboxylic acids have been
studied in considerable detail because they show higher thermal,
luminescent stabilities and coordination ability for practical appli-
cation than other lanthanide complex systems [14,15]. As a result,
we choose benzoic acid as the second organic ligand in ternary Eu
Shimadzu
temperature.
RF-5301PC
spectrofluorophotometer
at
room
Synthesis of the novel ligand
(
III) complex in this paper. On the other side, Eu (III) was chosen as
metal ion, because it has excellent luminescence properties mak-
ing it an ideal probe in spectroscopy-based speciation [16]. The
purpose of this study was to search out new fluorescence materi-
als, and to explore the effect of the second ligand (L ) on the fluo-
rescence properties of rare-earth complexes. So it was reported
The synthesis route of the sulfoxide was expressed in Fig. 1.
The synthesis of the sulfide
The sulfide was synthesized according to the method of Shriner
et al. [21].
0
[
17] that the solid ternary complex of europium perchlorate with
Sodium hydroxide was dissolved in alcohol. The mechanical
stirrer was started and benzyl mercaptan was added in a slow
but steady stream. Then dibromomethane was run in drop by drop.
The mixture was refluxed, with stirring, on a steam-bath for one
and one-half hours. The hot solution was immediately poured on
ice powder. A light yellow solid was precipitated, filtered and dried
in vacuum. The sulfide was a light yellow crystalline substance
which was purified by recrystallization from alcohol. Yield:
0
L and L . In order to make a comparison, we report here the synthe-
sis and spectroscopic study of the binary complex. The fluores-
cence properties of the Eu (III) complexes were discussed and
their phosphorescence spectra and fluorescence emission mecha-
nisms were also investigated.
As it is well known, the albumin is the richest protein in blood
circulatory system, which can combine with many materials and
play an important role in transporting protein [18]. BSA has been
studied as a model protein in the group due to its structural homol-
ogy with human serum albumin (HSA). In addition, the two trypto-
phan residues in the molecule induce to the intrinsic fluorescence
of BSA [19]. So the reaction of the rare earth complexes with BSA
was investigated based on fluorescence spectroscopy. The results
demonstrated complexes caused the fluorescence quenching of
BSA via a static quenching procedure, changing the structures of
protein accordingly.
6
6
0–70%, mp: 46–48 °C. Anal. calcd. For C15
16 2
H S : C, 69.23%; H,
.154%; S, 24.616%; found: C, 69.20%; H, 6.353%; S, 24.32%.
Synthesis of the sulfoxide
Bis(benzylthio)methane was dissolved in acetic acid, then 30%
hydrogen peroxide was added to it at once. The mixture was stir-
red continuously at room temperature for 24 h. After the reaction,
the mixture was extracted with ether until the pH of mixture was
7
.0. Then a white solid was precipitated, filtered and dried in vac-
uum. Yield: 80%, mp: 212–214 °C. Anal. calcd. For C15
1.64%; H, 5.479%; found: C, 61.33%; H, 5.485%.
H
16
2
S O
2
: C,
6
Experiment
Synthesis of the ternary Eu (III) complex
Reagents and apparatus
0
4
mmol the novel ligand L and 1 mmol the second ligand L
The purity of lanthanide oxide exceeds 99.99%. The rare earth
were dissolved in ethyl ether solution. So did 3 mmol Eu(ClO
4
)
3
.
(
(
III) perchlorates were prepared by dissolving their oxide
ꢂ1
During the stirring, Eu(ClO4)3 ethyl ether solution was added to
ethyl ether solution containing ligands. After a few minutes a
white precipitate was formed. The mixture was stirred for 0.5 h
99.99%) in HClO
4
(2.0 mol L ). All other chemicals were of analyt-
ical reagent grade. The stock solution of BSA (purity 99%) was
ꢂ5
ꢂ1
prepared to be the concentration of 1.0 ꢃ 10 mol L by dissolv-
ing it in buffer solution at pH 7.4, and kept it in the dark at 0–4 °C.
By means of dissolving Eu (III) complexes in a small amount of
ꢂ4
DMF, then diluting them to be the concentration of 1.0 ꢃ 10
ꢂ1
mol L with DMF, respectively, the working solutions of Eu (III)
complexes were obtained. The Trise-HCl buffer solution (pH 7.4)
ꢂ1
was given via adding dropwise HCl (0.1 mol L ) to Tris solution
ꢂ1
(
0.1 mol L ), which used double distilled water. Elemental analy-
sis was carried out on a HANAU analyzer. Rare earth contents of
the complexes were determined by EDTA titration using Xylenol-
orange as an indicator [20]. Conductivity measurements were
ꢂ3
ꢂ1
made by using a 10 mol L solution in DMF on a DDS-11D con-
ductivity meter at room temperature. The thermal behavior was
Fig. 1. The synthesis scheme of L.

9
74
J. Zhang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 118 (2014) 972–980
and precipitate was filtered. The products were washed with ethyl
ether for several times, and then dried in drying oven. It was pow-
der (yield > 90%).
Preparation of the binary rare-earth complex
An ether solution containing 3 mmol rare earth perchlorates
was added drop by drop in an ether solution containing 5 mmol
ligand L. After a few minutes a white precipitate was formed.
The mixture was stirred for 0.5 h and precipitate was filtered.
The products were washed with ether for several times, and then
dried in drying oven. It was white powder (yield > 90%).
BSA binding experiments
To a 5 mL of calorimetric tube, 2 mL BSA stock solution and
Fig. 3. Infrared absorption spectrum of L.
1
0
0 lm L of Eu (III) complexes working solution (concentration:
–25 ꢃ 10 mol L ) was added in turn. The mixture was incu-
ꢂ7
ꢂ1
bated for 5 min at room temperature. Subsequently, quenching of
the emission intensity of BSA at 347 nm was monitored using the
Eu (III) complexes as quenchers with the increased concentration.
Fluorescence spectra were recorded from 300 to 450 nm at an exci-
tation wavelength of 296 nm.
Results and discussion
Properties of the complexes
Elemental analysis and molar conductivity values were pre-
sented in Table 1, and the composition of the ternary complex
0
was EuL2.5ꢁL ꢁ(ClO
4
)
2
ꢁ5H
2
O. The binary complex conformed to
EuL2.5(ClO
4
)
3
ꢁ3H
2
O. The molar conductivity values of the com-
plexes in DMF were in good accordance with the formula as 1:2
0
Fig. 4. Infrared absorption spectrum of L .
Table 1
2
ꢂ1
Composition analysis (%) and molar conductivities (S cm mol ) of the rare earth
complexes (25 °C).
electrolytes [22]. Those two complexes were white powder, which
were stable under atmospheric conditions, and soluble in DMF and
DMSO.
Complexesa
M
Anal. calcd. (found) (%)
m
k (a)
2
ꢂ1
(
S cm mol )
C
H
RE
EuL2.5ꢁ(ClO
4
)
3
ꢁ3H
2
O
1231.4 36.24
3.89
11.78
151.3
178.2
(
36.45) (3.73) (12.23)
TG–DSC studies
0
EuL2.5ꢁL ꢁ(ClO
4 2
) ꢁ5H
2
O
1293.1 40.92 4.18 12.32
(
41.33) (4.26) (11.76)
The TG–DSC analyses were carried out up to 1000 °C in N
2
at a
heating rate of 10 °C min . The TG–DSC curves of EuL2.5ꢁL ꢁ(ClO
5H O was showed in Fig. 2. The TG curve of Eu (III) complex
a
ꢂ
ꢂ1
0
L = C
6
5
H CH
2
SOCH
2
2
SOCH C
6
H
5
, L’ = C
6
H
5
COO .
4 2
)
ꢁ
2
0
0
Fig. 2. The TG–DSC curves of EuL2.5ꢁL ꢁ(ClO
4
)
2
ꢁ5H
2
O.
Fig. 5. Infrared absorption spectrum of the ternary complex. EuL2.5ꢁL ꢁ(ClO
4
2
) ꢁ5H
2
O.

J. Zhang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 118 (2014) 972–980
975
Table 2
Some main IR data of ligand and the rare earth complexes.
Table 3
Chemical shift data of 1H NMR Spectra (1 ꢃ 10ꢂ6).
ꢂ1
0
Assignment (cm
)
L
L
Eu (III) complex
1H NMR
Ligand and complexes
L
m
m
m
OAH (H
C@C (C
CAH (C
2
O)
–
–
–
3415
1619
3062
770
ACH
3.311–4.143 4.246–4.452 7.279–7.379
s, 2 H) (s, 4 H) (m, 10 H)
3.381–4.153 4.239–4.452 7.288–7.371
s, 2 H) (s, 4 H) (m, 10 H)
3.839–4.145 4.247–4.454 7.278–7.383
s, 2 H) (s, 4 H) (m, 10 H)
2
A
ACH
2
A(ꢃ2)
AC
6
H
5
A(ꢃ2)
6
H
5
)
1622
3058
770
6
H
5
)
3066
709
681
–
–
–
–
–
–
(
d
6 5
CAH (C H )
EuL2.5ꢁ(ClO
EuL2.5ꢁC
4
)
3
ꢁ3H
2
O
7
00
702
(
m
m
S@O
1038
2961
1404
3027
1451
–
1019
2966
1402
3030
1454
1080
624
ꢂ
6
H
5
COO ꢁ(ClO
4 2 2
) ꢁ5H O
CAH (C
6
H
5
ACH
ACH
CAH (ASOACH
2
ASOA)
ASOA)
ASOA)
(
d
CAH (C
6
H
5
2
m
2
d
CAH (ASOACH
2
ASOA)
ꢂ
m
ClAO (ClO4
)
–
–
–
–
Infrared spectra
ꢂ
m
m
S
(COO )
1416
1550
1402
1663
ꢂ
as(COO )
Ligand L and the ternary Eu (III) complex were determined by IR
spectrometer and the significant information was presented in
Figs. 3–5 and Table 2.
ꢂ1
Fig. 5 showed the disappearance of the band at the 1038 cm
ꢂ1
showed that the first mass loss occurred in the range 35.7–188.0 °C
and mass loss percentage was 6.77%. The result was consistent well
with the release of the five water molecules (6.97%). The compar-
atively low temperature for water loss indicated that they were
crystal water. At the same time, DSC curve showed small endother-
mic peak at 111.2 °C when losing water. In addition, there were
two exothermic peaks in DSC curve, which were attributed to the
(mS@O) and the existence of band at 1019 cm
(mS@O), which indi-
cated that Eu (III) ions were bonded with oxygen atom in sulfonyl
group. The characteristic absorption bands of benzene appeared at
approximately 3058 cm
(dCAH) and 700 cm (dCAH) in Fig. 3 and they were also found in
ꢂ1
ꢂ1
ꢂ1
(mCAH), 1622 cm
(mC@C), 770 cm
ꢂ1
the spectra of ternary Eu (III) complex.
0
The
m
asðCOO
ꢂ
Þ
and
m
D
sðCOO
m
ꢂ
Þ
absorption of L occurred at 1550 and
0
ꢂ1
ꢂ1
decomposition of the ligands L and L , as well as two obvious
1416 cm , and the
value was 134 cm in Fig. 4. The
m
asðCOO
ꢂ1
ꢂ
Þ
weight loss occurred on the TG curve. The final product was found
and
m
sðCOO
ꢂ
Þ
absorption bands appeared at 1663 and 1402 cm in
ꢂ1
3
to be EuCl(CO ), near 1000 °C. The total weight loss of the complex
the ternary Eu (III) complex and the
than that of L , which could be deduced that carboxyl group was
coordinated with Eu (III) ions by monodentate type [23]. When
D
m
value was 261 cm larger
0
was closely corresponding to the calculated values. The results
were consistent with element analyses.
0
Fig. 6. The UV absorption spectra of (a) L, (b) L , and (c) the Eu (III) complex.

9
76
J. Zhang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 118 (2014) 972–980
Fig. 7. Fluorescent excitation and emission spectra of the ternary complex
0
EuL2.5ꢁL ꢁ(ClO
4
)
2
ꢁ5H
2
O.
Fig. 9. Fitted curve of (a) binary Eu (III) complex and (b) ternary Eu (III) complex,
recorded at room temperature, setting the Eu3 emission and excitation wavelength
at 612 and 394 nm, respectively.
+
2
72 nm, respectively. There was one absorption band at 268 nm
in the absorption spectra of (c) the ternary Eu (III) complex. There
was no obvious shift in the complex, indicating that the conjugated
system of benzoic acid was not influenced obviously after coordi-
nating with the rare earth ions.
1
Fig. 8. Fluorescent excitation and emission spectra of the binary complex
H NMR spectra
EuL2.5ꢁ(ClO
)
4 3
ꢁ3H
2
O.
The 1H NMR spectra data of L and its complexes in DMSO-d
6
1
ꢂ
could be seen from Table 3. The H NMR of the ligand showed
the proton resonance of two phenyl group peaks was at
d7.279 ꢄ 7.379 ppm, and the integral intensities showed that it
had ten protons. The proton resonance of two methylene group
peaks was at d4.246 ꢄ 4.452 ppm, the integral intensities of peaks
showed that it had four protons. There was also one methylene
group in the ligand and the proton resonance of its peaks was at
d3.311 ꢄ 4.143 ppm, and the integral intensities showed that it
had two protons. And the ratio of resonance peaks area was 10:2:4.
ClO4 was not coordinated, it was Td symmetry and there were two
ꢂ
absorption bands. When ClO4 was coordinated, it was C3v symme-
try and there were five absorptions [24,25]. In the spectra of the
ternary Eu (III) complex, two absorption bands could be seen
clearly at approximately 1080 and 624 cm . Therefore, ClO4 was
Td symmetry. In terms of molar conductivities, it could be sug-
gested that none of ClO4 were bonded with Eu (III) ions.
ꢂ1
ꢂ
ꢂ
1
UV absorption spectra
In the H NMR spectrum of the ternary Eu (III) complex, the pro-
ton resonance peaks of phenyl and methylene group were very
clear, which had shifted at different degree. The reason may be
concerned with coordinate effect. A broad proton resonance peak
was found between d7.278 and d7.383 ppm, which belonged to
0
The UV absorption spectra of (a) L, (b) L , and (c) the ternary Eu
(
III) complex were recorded in Fig. 6 and DMF was used as a refer-
ence and solvent. In Fig. 6(a) and (b), the major band of p–p elec-
tronic transition in phenyl group was observed at 266 nm and
*
0
the phenyl group absorption peak of L overlapped with that of L .
Table 4
Comparison of fluorescent emission spectra data of Eu (III) binary and ternary complex.
Complex
kex (nm)
k
em (nm)
I (a.u.)
Energy state transitions
5
? 7
?
EuL2.5ꢁ(ClO
4
)
3
ꢁ3H
2
O
394
394
612
612
8153
34,510
D
D
0
0
F
F
2
2
ꢂ
5
7
EuL2.5ꢁC
6
H
5
COO ꢁ(ClO
4 2
) ꢁ5H
2
O

J. Zhang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 118 (2014) 972–980
977
Fig. 11. The phosphorescence spectrum of L.
nary complexes, which were attributed to the characteristic
5
7
D
0
– F
J
(J = 0, 1, 2, 3, 4) transitions of Eu (III) at about 579, 592,
5
7
6
12, 650, 698 nm, and the
0 2
D – F transition of Eu (III) presented
the higher emission intensity at about 612 nm.
Under the same conditions, the intensity of the strongest fluo-
rescence emission peak of Eu (III) ion was 8153 a.u. in the Eu
(
III)-sulfoxide binary system. It is worth mentioning that the fluo-
rescence emission intensity peak of the Eu (III) ion was 34,510 a.u.
in the Eu (III)-sulfoxide–benzoic acid ternary system. It was 4.23
times as great as that of the binary system. In the ternary complex,
0
L and L could play a mutually synergistic part in the energy trans-
fer process of the ligand to Eu (III) ion. The luminescence of the Eu
Fig. 10. The fluorescence decay curves of (a) binary Eu (III) complex and (b) ternary
Eu (III) complex, recorded at room temperature, setting the Eu emission and
excitation wavelength at 612 and 394 nm, respectively.
3
+
(
III) ion in the ternary system was more enhanced than that of the
binary systems.
Luminescence decay times (s) and emission quantum efficiency (g)
Table 5
Photoluminescent data of the compounds.
The fluorescence decay curves of the binary complex and the
ternary complex were measured. Fig. 9 showed the fitted curve
of (a) binary Eu (III) complex and (b) ternary Eu (III) complex.
Fig. 10 showed the decay curves of (a) binary Eu (III) complex
and (b) ternary Eu (III) complex. The fluorescence lifetime values
of the Eu (III) complexes were calculated by the double exponential
ꢂ
EuL2.5ꢁ(ClO
) ꢁ3H
4 3 2
O
EuL2.5ꢁC
H
6 5
COO ꢁ(ClO
4
2
) ꢁ5H
2
O
t
t
t
t
t
00
01
02
03
04
17,271
16,892
16,340
15,385
14,327
2298
17,271
16,863
16,340
15,385
14,327
7920
34,510
4.357
4.518
50
224.841
16.448
231.395
527.203
0.641
2
1
2
2
I
I
I
01
02
02/I01
mode ½
s
¼ ðA
1
s
þ A
2
s
Þ=ðA
1
s
1
þ A
2
s
2
Þꢅ. From the results, the fluo-
s, and the
s.
According to the emission spectrum and the lifetime of the Eu
8153
rescence lifetime of the ternary complex was 641.07
l
3.548
12.470
50
183.388
8.624
69.984
324.467
0.434
fluorescence lifetime of the binary complex was 434.14
l
A
A
A
A
A
A
s
00
01
02
03
04
r
3+
5
first excited level (
s
,
D
0
), the emission quantum efficiency (
Eu excited state can be calculated. There is a hypothesis
that only nonradiative and radiative processes are substantially in-
g) of
5
3+
the D
0
5
volved in the depopulation of the D
follows [26]:
0
statea,
g
can be confirmed as
1
/s
2304
1560
g
%
14.08
33.80
A
þ Anr
r
g
¼ A
ð1Þ
r
where A
respectively. A
rates A0J for each
r
and Anr are radiative and nonradiative transition rates,
can also be obtained by summarizing the radiative
Fluorescence spectra
r
5
7
3+
0 J
D ? F (J = 0–4) transitions of Eu .
The excitation and emission slit width of the complexes was
nm. The fluorescence excitation and emission spectra of euro-
X
2
A
r
¼
A
0J ¼ A00 þ A01 þ A02 þ A03 þ A04
ð2Þ
pium perchlorate ternary and binary complexes were measured
at room temperature. According to the fluorescence spectra of
the complexes reported in Figs. 7 and 8 and Table 4, we could
see that the ternary complex had excellent fluorescence properties.
The excitation spectra of the two Eu (III) complexes were ob-
tained by monitoring their emissions at 612 nm; the maximum
peaks were at 394 nm. The excitation spectra of the two complexes
were similar (shown in Figs. 7 and 8). There were five emission
peaks in the fluorescence emission spectra of the binary and ter-
5
7
The branching ratio for the D ? F transitions can be ne-
0
5,6
glected as they are not measured experimentally, whose influence
5
can be elided in the depopulation of the D0 excited state. Since
5
7
D ? F belongs to the isolated magnetic dipole transition, it is
0
1
practically independent of the chemical environments around the
3
+
Eu ion, and thus can be considered as an internal reference for
the whole spectrum, the experimental coefficients of spontaneous
emission, A0J can be calculated according to the equation [27–30].

9
78
J. Zhang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 118 (2014) 972–980
5
? 7F
5
? 7F
intensities of the D
01 and 0J = 1/k
energy barrier and can be determined from the emission bands of
0
1
and D
0
J
transitions (J = 0–4) with
m
m
0J
(
m
J
) energy centers, respectively. m
0J refers to the
3
+
5
7
Eu ’s D
0
? F
J
emission transitions. The emission intensity, I, ta-
ken as integrated intensity S of the
can be defined as below:
5
7
D
0
? F0–4 emission curves,
I
iꢂj ¼ h
-
iꢂj
A
iꢂj
N
i
ꢆ Siꢂj
ð4Þ
5
) and final levels (7F0–4), respec-
where i and j are the initial ( D
tively, iꢂj is the transition energy, Aiꢂj is the Einstein’s coefficient
of spontaneous emission, and N emit-
is the population of the 5
0
x
i
D
0
ting level. On the basis of reference [32–36], the value of
ꢂ1
A
(
01 ꢆ 50 s and the lifetime (
r
s), radiative (A ), and nonradiative
Anr) transition rates are related through the following equation:
Atot ¼ 1=
s
¼ A
r
þ Anr
ð5Þ
0
Fig. 12. The phosphorescence spectrum of L .
On the basis of the above discussion, the quantum efficiencies of
the two kinds of europium complexes can be determined, as shown
in Table 5. From the equation of , it can be seen the value mainly
g
g
depends on the values of two quantum: one is lifetimes and the
other is I02/I01. As can be clearly seen from Table 5, the quantum
0
efficiencies of EuL2.5ꢁL ꢁ(ClO
4
)
2
ꢁ5H
2
O (g = 33.80%) is higher than
that of EuL2.5ꢁ(ClO
4
)
3
ꢁ3H
2
Oꢁ(14.08%).
Phosphorescence properties of ligands and luminescence mechanism
0
The phosphorescence spectra of ligands L and L were recorded
using a F-4500 FL spectrophotometer in solid state and are shown
in Figs. 11 and 12. The excitation and emission slit width of the
complexes was 5.0 nm and 10.0 nm, and excitation wavelength
was 300 nm and 347 nm, respectively.
According to the energy transfer and intra-molecular energy
transfer mechanism [37,38], intra-molecular energy transfer effi-
ciency chiefly lied on two energy transfer processes: one was the
transitions from the triplet state energy level of ligand to the ex-
cited states of the Eu (III) ion by Dexter’s resonant exchange inter-
action [39], the other was just an inverse energy transfer process
by the thermal deactivation mechanism [40]. Fig. 11 shows that
two bands could be seen clearly at 519 and 471 nm which corre-
Fig. 13. Triplet state of ligands and excited state of the Eu (III) ion.
sponded to the triplet state energy level of ligand
L T
1
A
0J ¼ A01ðI0J=I01Þð
m
01
=
m
0J
Þ
ð3Þ
ꢂ1
ꢂ1
(
2
19,268 cm ) and T (21,231 cm ), respectively. The triplet state
5
Here, A0J is the experimental coefficient of spontaneous emis-
sion. A01 is the Einstein’s coefficient of spontaneous emission be-
tween the
energy level T was appropriately higher than D0 of Eu (III) ion
(17,241 cm ). Fig. 12 showed that the triplet state energy level
of the second ligand L was at 500 nm (T : 20,000 cm ) and 470
1
(T : 21,276 cm ), which was also higher than D0 of Eu (III) ion.
1
ꢂ1
5
7
0
ꢂ1
D
0
and
F
1
energy levels. In vacuum, A01 as a value of
ꢂ1
ꢂ1
5
1
4.65 s , when an average index of refraction n equal to 1.506
2
ꢂ1
was considered, the value of A01 can be determined to be 50 s
approximately (A01 = n A01(vac)) [31]. I01 and I0J are the integrated
These results are shown in Fig. 13, which also shows that the trip-
3
0
let state energy level T of the ligands L and L was higher than the
1
0
O on fluorescence spectrum of BSA (Room temperature, pH = 7.4, kex = 296 nm). a–f, c(BSA) = 1.0 ꢃ 10ꢂ5
Fig. 14. Effect of (a) EuL2.5ꢁL ꢁ(ClO
4
)
2
ꢁ5H
2
O, (b) EuL2.5ꢁ(ClO
)
4 3
ꢁ3H
2
ꢂ1
ꢂ7
ꢂ1
mol L , c(a and b) (ꢃ10 mol L ): 0, 5.0, 10.0, 15.0, 20.0 and 25.0, respectively.

J. Zhang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 118 (2014) 972–980
979
0
Fig. 15. The Stern–Volmer curves of BSA quenched by (a) EuL2.5ꢁL ꢁ(ClO
)
4 2
ꢁ5H
2
O and (b) EuL2.5ꢁ(ClO
4 3
) ꢁ3H
2
O.
Table 6
Fluorescence quenching constants of complexes-BSA system.
-0.6
-0.8
-1.0
a
Complexes
pH
K
SV
K
q
R2
4
ꢂ1
12
ꢂ1 ꢂ1
(ꢃ10 L mol
)
(ꢃ10 L mol
6.97
7.32
s )
0
EuL2.5ꢁL ꢁ(ClO
4
)
2
ꢁ5H
2
O
7.4 6.97
7.32
0.99854
0.99834
EuL2.5ꢁ(ClO
4
)
3
ꢁ3H
2
O
excited state energy level 5
D of Eu (III). It was deduced that two
0
ligands could absorb energy effectively and transfer energy to the
Eu (III) ion, and enhanced the fluorescence emission intensity.
Therefore, the type of luminescence of Eu (III) ion was L ? M.
-
6.4
-6.2
-6.0
lg [Q]
-5.8
-5.6
Because both of the triplet state energy level T
1
of L and T
1
of the
of Eu (III), they
energy level of the Eu (III)
0
5
L was higher than the excited state energy level D
0
could be matched better with the 5
D
0
-
-
-
-
0.6
ions. In the Eu (III)-sulfoxide–benzoic acid ternary system, L and
b
0
L were able to transfer energy to the Eu (III) ion together. There-
fore, in the ternary complex, the two ligands could play a mutually
synergistic part in the energy transfer process of the ligands to the
Eu (III) ion, making the fluorescence emission of the Eu (III) ion in-
crease exponentially in the ternary complex. All these suppositions
have been confirmed by experiment.
0.8
1.0
1.2
BSA binding studies
Quenching mechanism of fluorescence of BSA by Eu (III) complexes
At the excitation wavelength of 296 nm, the fluorescence spectra
of BSA with varying concentrations of Eu (III) complexes are shown
in Fig. 14. The naturally fluorescence of BSA at around 347 nm was
gradually quenched along with the increasing concentration of Eu
-
6.4
-6.2
-6.0
lg [Q]
-5.8
-5.6
0
Fig. 16. Double log plots of (a) EuL2.5ꢁL ꢁ(ClO
)
4 2
ꢁ5H
2
O and (b) EuL2.5ꢁ(ClO
)
4 3
ꢁ3H
2
O
quenching effect on BSA fluorescence.
(
III) complexes. Results above indicate that there are strong interac-
tions and radiationless energy transfer between Eu (III) complexes
and protein [41–43]. In given conditions (such as pH 7.4 and room
temperature), fluorescence quenching may result from ground
complex formation, energy transfer and dynamic quenching pro-
cesses [44]. Dynamic quenching is a process that the fluorophore
and the quencher come into contact during the lifetime of the ex-
cited state, whereas static quenching refers to fluorophore–quench-
er complex formation [43]. Fluorescence quenching data can be
analyzed by the Stern–Volmer equation [45]:
1
0
ꢂ1 ꢂ1
scattering collision quenching constant is 2.0 ꢃ 10 mol
Fig. 15 shows the Stern–Volmer plots of F /F versus [Q], the values of
SV and correlation coefficient R are outlined in Table 6. The results
show the values of are much larger than that of the maximum
s
[47].
0
2
K
j
q
scattering collision quenching constant, which indicate that the
main quenching mechanism of BSA by Eu (III) complexes should
be a static quenching procedure [43].
F
0
=F ¼ 1 þ KSV ½Qꢅ ¼ 1 þ
s
0
j
q
½Qꢅ
ð1Þ
Binding constants and binding sites
where F and F are the fluorescence intensities in the absence and
0
As we have known from results above, the fluorescence quench-
ing coming from Eu (III) complexes formation between protein and
quencher is static quenching process, hence the equilibrium be-
tween free and bound molecule can be expressed by the following
double logarithm regression equation [48]:
presence of quencher, respectively. KSV is the Stern–Volmer quench-
ing constant. [Q] is the concentration of quencher. is the average
fluorescence lifetime of biomolecule at about 10 s [46]. is the
biomolecule quenching rate constant and the value of the maximum
s
0
ꢂ8
j
q

9
80
J. Zhang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 118 (2014) 972–980
Table 7
Binding constants K
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