American Journal of Analytical Chemistry, 2011, 2, 605-611
doi:10.4236/ajac.2011.25068 Published Online September 2011 (http://www.SciRP.org/journal/ajac)
Determination of Mercuric Ion in Water Samples with a
LED Exciting and CCD Based Portable Spectrofluorimeter
Arsenio Muñoz de la Peña1*, María Isabel Rodríguez-Cáceres1, Diego Bohoyo Gil1,
María del Carmen Mahedero1, María del Carmen Hurtado-Sánchez1, Reyes Babiano2
1Department of Analytical Chemistry, University of Extremadura, Badajoz, Spain
2Department of Organic and Inorganic Chemistry, University of Extremadura, Badajoz, Spain
Received March 29, 2011; revised June 3, 2011; accepted June 15, 2011
Abstract
The fluorescent characteristics of a fluorimetric chemosensor for mercuric ion, Hg2+, employing a synthe-
sized Rhodamine 6G derivative, have been analyzed. For that, a portable spectrofluorimeter composed of a
515 nm LED as excitation source, two fiber-optics and a CCD camera as detector, has been used, intended
for “in situ” analysis. A highly selective Rhodamine based probe for Hg2+, that is water soluble and gives a
positive response upon analyte binding, is reported. The reagent is bearing a monothiospirolactone group in a
Rhodamine 6G architecture and the thiol atom served for the direct attack of thiophilic Hg2+. The fluores-
cence enhancement is attributed to the spirolactone ring opening and the coordination of two sulphur atoms
to Hg2+ giving a 2:1 reagent: Hg2+ stoichiometry complex.
Keywords: Chemosensor, LED, Mercuric Ion, Water Analysis
1. Introduction
Mercury is widely distributed in the air, water and soil,
and is considered by the Environmental Protection Agency
(EPA) to be a highly dangerous element because of its
severe inmunotoxic, genotoxic and neurotoxic effects [1].
It is generated by volcanic emission and in several hu-
man activities as gold mining, combustion of solid waste
or burning of fossil fuels. The great time of residence of
mercury in vapour state and his easy oxidation to inor-
ganic species of Hg2+ soluble in water have caused envi-
ronmental Hg2+ levels. Mercury easily passes through
biological membranes such as skin, respiratory and gas-
trointestinal tissues. When absorbed in human body,
mercury causes damage to the central nervous and endo-
crine systems as well as neurological irreversible dam-
ages. The toxicity of mercury is known to be highly de-
pendent on its chemical form: organomercury is gener-
ally more toxic than inorganic mercury salts [2].
Recently, a variety of selective and sensitive fluores-
cent Hg2+ chemosensors have been developed based on
fluorescein derivatives [3-6], NO2S2-donor macrocyle [7],
naphthalimide [8,9], BODIPY [10-12] or Rhodamine
derivatives [13-23].
BODIPY (boron dipyrromethene) fluorophores have
been widely used for the determination of Hg(II). Those
fluorophores have high absorption coefficient (ε > 50000
M–1 cm–1), high fluorescence quantum yield (Φ > 0.5),
and high photostability [24]. Polyamide receptor photo-
electron transfer (PET) based fluorescent sensor mole-
cules could be used to detect Hg2+ ions with either fluo-
rescence off-on response or fluorescence colour change
[8]. J. Wang et al. used a series of polyamide receptors
incorporating two, three and four amide arms to the
BODIPY fluorophore. They observed that a clear emis-
sion turn-on response when as low as 2 ppb (content
limit in drinking water set by EPA) of Hg(II) are present.
Thus, the sensor is practical as Hg2+ ion “annunciator”
for drinking water [10]. On the other hand, J. Du et al.
synthesized a highly selective PET fluorescent sensor for
Hg2+ containing a BODIPY fluorophore and a NS2O2
pentachelating receptor. With this sensor the Hg2+ could
be detected in a wide pH range [11]. Also, thiacrown and
crown ethers have been appended to BODIPY fluoro-
phores [12].
Rhodamines are classic dyes/fluorophores whose photo-
chemical properties have already been well studied. Be-
cause of their low cost, long-wavelength (> 500 nm) ab-
sorption/emission and high molar absorption coefficient
and quantum yield, these fluorophores are usually util-

606 A. M. de la PEÑA ET AL.
ized as reporting groups in routine optical analysis. The
metal ion sensing behaviour of these rhodamine-based
optical sensors is very interesting. Typically, in the ab-
sence of metal ions, the sensor molecules prefer their
spirolactam ring-closed state, which shows little absorp-
tion or fluorescence in the visible range (400 - 700 nm).
However, upon the addition of specific metal ions, the
chelating or reaction of metal ions with sensor molecules
will simultaneously open the spirolactam ring and make
the sensors converted into their ring-opened state, which
is highly absorbent and fluorescent above 500 nm [25].
Y.-K. Yang et al. have developed a rhodamine-based
fluorescent and colorimetric chemodosimeter for the
rapid detection of Hg ions in aqueous media. The selec-
tivity of the system for mercuric ion over other metal
ions is remarkably high, and its sensitivity is below 2 ppb
in aqueous solution [13]. On the other hand, a rhodamine
thiospirolactone chemosensor was developed by X.-Q.
Zhan et al. [14] for the selective and sensitive reversible
sensing of Hg2+. W. Shi and H. Ma develop a rhodamine
B thiolactone for Hg2+ in neutral solution with good
linearity between 0.5 and 5 μM and detection limit 20
nM [15]. S.-K. Ko et al. develop a rhodamine-based mo-
lecular probe for in vivo monitor mercury ions in living
cells and vertebrate organisms in aqueous media. The
results of the experiments shown that the chemosensor
responds selectively to mercury ions over other metal
ions and it is possible to detect the accumulation of mer-
cury ions in zebrafish tissue and organs [16]. A rhoda-
mine B thiohydrazide assay has been developed by H.
Zheng et al. in which Hg(II) could be detected fluorimet-
rically at least down to 5.0 × 10–8 M [17]. Finally, H.N.
Kim et al. developed a method to determine mercury in
vivo in the nematode Caenorhabditis elegans, previously
incubated with Hg(ClO4)2 in concentrations of Hg2+
comprised between 1 nm and 1 µM [19].
Moreover, Rhodamine has been employed to deter-
mine organic derivatives of mercury such as methylmer-
cury. This compound is produced by aquatic microor-
ganism from inorganic mercury and is one of the most
toxic species of mercury. Methylmercury passes through
biological membranes causing severe damage to various
tissues and organs humans. So, Yang et al. [20] have
been developed a method to determine this compound
with a rhodamine hydrazide derivative. The analysis is
based in the desulfurization of the derivative in presence
of methylmercury and formation of the cyclic fluorescent
compound, and the method was applied in zebrafish. The
same chemosensor was used by Muñoz de la Peña et al.
for the determination of Hg2+ in water and fish samples
[21,22].
Recently, derivatives for determination of mercury
have been developed from rhodamine in combination
with pyrene [23]. The use of pyrene provided a deriva-
tive with excellent spectroscopic properties such as high
molar absorption coefficients and high fluorescence quan-
tum yields.
The semiconductor light sources, as laser diodes (LDs)
and ultrabright light emitting diodes (LEDs), are a huge
improvement in fluorescence applications. LED is a
semiconductor p-n junction device, which emits light in a
narrow spectrum, produced by a form of electrolumines-
cence [26]. LEDs are cheap and commercially available
from 350 nm to the near infrared. LEDs with different
colours and white light have been developed. High-
brightness LEDs are becoming increasingly important
because of their potential applications. Light is generated
from the active region of LED spontaneously in all di-
rections and escape from LED die. Commercially manu-
factured LEDs consists of a small point like light emit-
ting active area typically ≈ 1 mm2 surrounded by high
refractive-index semiconductor surface shape [27]. A
general overview of LDs and LEDs is given by Landgraf
[28]. Among other applications, different LEDs and filter
combinations have been tested for the analysis of crude
oil fluorescence.
In the work described here, a LED based spectro-
fluorimeter combined with fiber-optics and a CCD de-
tector has been tested, in order to check its selectivity
and sensitivity for the determination of mercuric ion in
aqueous samples, using a highly selective synthesized
monothiospirolactone Rhodamine 6G derivative.
2. Experimental Procedures
2.1. Synthesis and Characterization of
Rhodamine-Derivative I
Rhodamine derivative I was synthesized by a similar
procedure, which has been already reported [29]. Rho-
damine 6 G (R6G) (3 g, 6.3 mmol) was added to the so-
lution of NaOH (4.8 g, 0.12 mol) in ethanol (30 mL) and
water (60 mL). After refluxed for 12 h, the reaction mix-
ture was cooled and ethanol was evaporated in vacuo,
then adjusted pH to 6 - 7 using hydrochloric acid (2 M).
The formed precipitate was filtered, washed several
times with water and dried to give 2.88 g Rhodamine-
derivative II as a red solid (yield 96%).
To a stirred solution of II (2 g, 4.8 mmol) in 1,2-di-
chloroethane (15 mL), phosphorus oxychloride (3 mL)
was added dropwise. After refluxed for 4 h, the reaction
mixture was cooled and evaporated in vacuo. The crude
acid chloride was dissolved in THF (6 mL), and the re-
sulting solution was then added dropwise to a mixed so-
lution of thiourea (1.52 g, 20 mmol) and triethylamine
(12 mL) in THF (50 mL)/water (10 mL) at room tem-
perature. After stirring over night, the solvent was re-

A. M. de la PEÑA ET AL.
607
moved under reduced pressure. Then, 50 mL of water
was added, and the formed precipitate was filtered. The
precipitate was washed several times with water and
dried. The crude product was purified by silica-gel col-
umn chromatography with CH2Cl2 as eluent, affording
1.0 g of Rhodamine-derivative I (yield 50%).
The structure of Rhodamine-derivative I was con-
firmed by 1H NMR and 13C NMR. 1H NMR’s study of
Rhodamine-derivative I structure agrees with the as-
signed structure. At low fields there are four protons
(7.89, 7.55, 7.49 and 7.18 ppm) of the ring of ben-
zothiolane-2-one with their expected multiplicity, fol-
lowed by two singlets (6.55 and 6.36 ppm), correspond-
ing to the protons of the xanthene ring, and the reso-
nances of the groups N-Et (3.23 ppm and 1.34 ppm) and
Me (1.98 ppm). A second set of signals appears in the 1H
NMR spectrum, probably due to the balance with the
opened structure, which appears in approximately 20%
in chloroform. The spectrum of 13C NMR shows also
relevant signals, as the resonance at 62.8 ppm, that is
assigned to the quaternary carbon of the spiranic bridge,
and at 197.90 ppm, assigned to CO-S.
2.2. Chemicals and Reagents
All chemical and solvents used in this study were of
analytical grade. Ultra pure water was provided by use of
a Millipore Milli-Q system (Millipore, Bedford, MA,
USA).
Mercury chloride (II) was obtained from Panreac. The
standard stock solution of mercury (II) (1 mM) and the
HEPES buffer (Sigma) 0.02 M (pH 7.4) were prepared in
ultra pure water. The Rhodamine-derivative I (10–3 M)
was prepared in acetonitrile. Diluted solutions of the
stock solutions were prepared by appropriate dilution.
Standard Reference Material 1641d was purchased
from NIST, containing 1.557 ± 0.020 mg/kg of Hg2+.
2.3. Apparatus
UV-Vis measurements were performed using a Spectro-
photometer Varian Model Cary 50 Bio, equipped with a
Xenon lamp.
For the studies of the influence of physical and chemi-
cal variables on the reaction, fluorescence measurements
were performed using a Fluorescence Spectrophotometer
Varian Model Cary Eclipse, equipped with a Xenon flash
lamp. Excitation and emission bandwidths of 2.5 nm
were used in these studies. Intensity was measured at 554
nm (excitation at 527 nm), which is the emission maxi-
mum of the complex Rhodamine-derivative I-Hg2+. All
measurements were performed in 10 mm quartz cells at
10˚C, by use of a thermostatically controlled cell holder
and a Selecta Model 382 thermostatically controlled wa-
ter-bath.
On the other hand, a portable instrument AvaSpec-
2048-USB2-SPU was used. The instrument is equipped
with a Cerny-Turner symmetric monocromator, with 75
mm of focal distance, and a CCD detector of 2048 pixels
connected to an USB2 interface. A Prizmatix Black-
LED-515, exciting at 515 nm (spectrum half width 36
nm), equipped with a filter Omega Optical 525AF45
(XF1074), to suppress the emission of LED further than
525 nm, was employed. The AvaSoft Full Software was
used for data acquisition and analysis. All measurements
were carried out after 5 minutes of turning on the LED to
ensure a constant operating temperature.
1H and 13C NMR spectra were recorded on a Bruker
400 AC/PC instrument at 400 and 100 MHz, respectively
in CDCl3. Assignments were confirmed by homo- and
hetero-nuclear double-resonance, DEPT (distortionless
enhancement by polarization transfer). TMS was used as
the internal standard (δ = 0.00 ppm) and all J values are
given in Hz. A Crison MicropH 501 meter was used for
pH measurements.
2.4. General Procedure for Fluorescence
Measurements
In a fluorescence cell, 30 μL of Rhodamine-derivative I
in acetonitrile (10–3 M) were placed. Later, an aliquot of
the mercuric ion stock solution (1 mM) to give the final
concentration desired is added, followed by 1.5 mL of
HEPES buffer (0.02 M, pH 7.4). Then, the solution was
diluted with ultra pure water to complete 3 mL, and the
fluorescence was measured at 554 nm (excitation at 527
nm) in the Varian spectrofluorimeter or at 555 nm (exci-
tation at 515 nm) in the portable instrument.
For the determination of mercury (II) at low concen-
trations, a mercury (II) stock solution of 10 µM was pre-
pared, by diluting the original stock solution, and proper
amounts of this solution were used in the analytical pro-
cedure. In those cases, fluorescence measurements were
carried out after addiction of Hg2+ for 5 min.
2.5. Procedure for Determination of Mercury(II)
in Certified Samples
In a fluorescence cell, 30 μL of Rhodamine-derivative I
in acetonitrile (10–3 M) was placed. Later, an aliquot of
the certified sample (Standard Reference Material 1641d)
to give the final concentration desired is added (155 μL),
followed by 1.5 mL of HEPES buffer (0.02 M, pH 7.4),
and ultra pure water to complete 3 mL. The fluorescence
was measured at 555 nm (excitation at 515 nm) in the
portable instrument.

608 A. M. de la PEÑA ET AL.
3. Results and Discussion
3.1. Conventional Spectrofluorimeter
At first, the spectral characteristics of the Rhodamine-
derivative I were studied. The compound is red-colored
but is non-fluorescent, probably due to its stable “spiro-
lactam form” [25]. The absorbance spectra of the Rho-
damine-derivative I in absence and in the presence of 10
μM of Hg2+ is shown in Figure 1. The absorption spec-
trum of Rhodamine-derivative I is showing a high in-
crement of its absorbance in the presence of Hg2+, giving
absorption maxima at 500 nm and 525 nm. On the other
hand, the fluorescence emission spectra of Rhodamine-
derivative I, in the absence and presence of different
amounts of mercury (II), are shown in Figure 2. It can be
seen that the Rhodamine-derivative I displays no obvious
spectral characteristics in its emission spectra. However,
a significant enhancement of fluorescence, with an exci-
tation maximum at 527 nm, and an emission maximum
at 554 nm was observed, when mercury (II) was added.
A spectral red shift of 7 nm was observed in the emission
maxima with the increase of the concentration of mer-
cury (II).
The effect of temperature was studied between the
range 5˚C - 40˚C. It could be observed a quenching of
fluorescence when the temperature increases. Also, the
effect of Rhodamine-derivative I concentration on the
fluorescence intensity was investigated. Figure 3 shows
that a maximum fluorescence enhancement was observed
200 300 400 500 600 700
Wavelength (nm)
0
0.1
0.2
0.3
0.4
Absorbance
1
2
Figure 1. Absorbance spectra of Rhodamine-derivative I in
the absence (1) and in the presence (2) of Hg2+. [Rhoda-
mine-derivative I] = [Hg2+] = 10 μM.
540 560 580 600 620 640
Wavelength (nm)
0
40
80
120
160
200
Fluo
r
escence Intensity (a.u.)
Spectrum [Hg
2+
] nM
1 0
2 200
3 300
4 400
5 500
6 600
1
2
3
4
5
6
Figure 2. Emission spectra of Rhodamine-derivative I in the
absence and the presence of different amounts of Hg2+.
[Rhodamine-derivative I] = 10 μM; λexc= 527 nm.
0
40
80
120
Fluo
0 10203040
r
escence Int ensity (a.u.)
[Rhodamine-der i vative I] x 10
-6
M
Figure 3. Influence of the concentration of Rhodamine-
derivative I on the fluorescence intensity of the complex.
[Hg2+] = 10 μM.
when 10 μM of Rhodamine-derivative I reacted with 10
μM of mercury (II) in HEPES buffer 7.4 for 5 min, so 10
μM was chosen for further experiments.
Also, the addition order was studied being Rhoda-
mine-derivative I + Hg2+ + HEPES buffer + water the
one which shows maximum fluorescence intensity. The
proposed binding mode between Rhodamine-derivative I
and Hg2+ is shown in Scheme 1. The large fluorescence

A. M. de la PEÑA ET AL.
609
enhancement can be attributed to a ring-opening process
of the spirothiolactone ring, and the complexation with
Hg2+ in a 2:1 stoichiometric ratio. The evidences of the
binding mode were previously reported, based in ESI-
MS spectra and X-ray analysis [29].
The 2:1 stoichiometry was also confirmed by the clas-
sical method of Job.
An interference study was performed to evaluate the
selectivity of the reagent. Cu2+, Ni2+, Zn2+, Cd2+, Co2+,
Cs+, Li+, Ca2+, Mn2+, K+, Na+, Sr2+, Fe2+, Rb+, Al3+, Mg2+,
Cr3+ and Pb2+ did not interfere at 100 μM concentration
level. Only Ag+ gave a small fluorescence signal at that
level.
3.2. Portable Spectrofluorimeter
(LED-fluorescence)
In order to obtain high excitation efficiency, the emission
wavelength of LED had to match the excitation wave-
length of the complex Rhodamine-derivative I: Hg2+.
Therefore, the excitation wavelength of the LED should
be chosen carefully. As it could be seen in Figure 2, in
the conventional spectrofluorimeter, the complex pro-
duced high fluorescence with a maximum excitation at
527 nm and a maximum emission at 554 nm. Because
the fluorescent complex has only a small Stoke’s shift
(λem – λexc = 27 nm), if the maximum wavelength of LED
approaches the fluorescence emission of the complex,
then their spectra overlap phenomenon will be serious.
For this reason, a LED with a maximum at 515 nm and a
525 nm interference filter were used, in order to avoid
overlapping of the spectra. Figure 4 shows the emission
spectra of the complex recorded in the portable spectro-
fluorimeter at optimum conditions.
According to the procedure developed for the conven-
tional spectrofluorimeter, the calibration curve for the
determination of mercury (II) was constructed in the
portable instrument, under the optimum conditions. The
linear range was 0 - 600 nM, with a correlation coeffi-
cient of R2 = 0.9968 (n = 15). The regression parameters
are summarized in Table 1.
The detection limit, based on the definition by IUPAC
[30] (LOD = 3 Sb / m), was found to be 28 nM from 10
blank solutions and 32 nM, according to the Clayton
criterium, taking into account the probabilities of false
positive and false negative type errors [31]. The relative
standard deviation (R.S.D.) for ten repeated measure-
ments of 400 ng·mL–1 of mercury (II) was 1.4%.
OHN
S
O
H
N
OHN
S
O
N
HgO
S
O N
H
N
Hg
2+
2+
Scheme 1. Proposed binding mode between Rhodamine-derivative I and Hg2+.
520 560 600 640 680
Wavelength (nm )
0
5000
10000
15000
20000
Fluorescence Intensity (a.u. )
Rhodamine-derivative I
Rhodamine-derivative I + Hg
2+
Figure 4. Emission spectra of the complex formed between Rhodamine-derivative I and Hg2+ recorded in the portable in-
strument. [Rhodamine-derivative I] = [Hg2+] = 10 μM; λexc= 515 nm.

610 A. M. de la PEÑA ET AL.
Table 1. Regression parameters for the determination of mercury (II) in the portable instrument.
Portable
Linear range (nM) 0 - 600
Slope (nM) 5561
Intercept 16.3
St. Dev. of slope 87
St. Dev. of Intercept 0.3
Linearity (%) 98.4
RSD (%) (n = 10) 1.4
R2 0.9968
LOQ (IUPAC), nM 93
LOD (IUPAC), nM 28
LOD* (Clayton,  =  = 0.05), nM 32
*LOD according to the criterium of Clayton et al. (Ref. 31)
The proposed method was validated by application to
the certified sample Standard Reference Material 1641d.
Three replicates were analyzed and satisfactory results
were found with the portable instrument, suggesting the
potential application of the reagent for the better analysis
of environmental water samples.
4. Conclusions
In conclusion, the Rhodamine 6G monothiolactone de-
rivative was synthesized and characterized. Its absorp-
tiometric and fluorimetric properties, in absence and in
the presence of Hg2+, were established, and a calibration
study was performed and validated by using of a certified
sample reference material. A suitable method for “in
situ” analysis of Hg2+ was developed, using a portable
instrument, composed of a 515 nm LED as excitation
source, two fiber optics and a CCD camera as detector.
The high selectivity of the method towards other metal
ions was corroborated by an interference study.
5. Acknowledgements
This work was supported by the Ministerio de Ciencia e
Innovación of Spain (Project CTQ2011-25388) and the
Junta de Extremadura (Consolidation Project GR10033
of Research Group FQM003 co-financed by European
FEDER Funds).
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