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Fluorimetric determination of mercury with a water-soluble porphyrin and porphyrin-doped sol-gel films
ANALYTICA
CHIMICA ACTA ELSEVIER
Analytica Chimica Acta 304 ( 1995) 107- 113
Fluorimetric determination of mercury with a water-soluble porphyrin and porphyrin-doped sol-gel films M. Plaschke, R. Czolk F’orscilun~.~zmtrum
Karlsruhe
GmbH. Institut ftir Radiochemie,
Received 23 March 1994; revised
4 November
*,
H.J. Ache
P.O. Box 3640.
D-76021
1994: accepted 7 November
Kurl.sruhc. (Tcrmarr!
I YO4
Abstract The fluorimetric determination of mercury ions with 5,10,15,20-tetra(p-sulfonatophenyl)porphyrin in aqueous solutions and in porphyrin doped sol-gel films was investigated. Optimization of complexation conditions such as buffer components, pH range and temperature resulted in a method which enabled the determination of mercury ions in aqueous solutions within 15 min with a detection limit of 1.4 Kg/l Hg(II). The main interfering ions were found to be Cd, Zn and Pb. To develop an optochemical sensing device the porphyrin derivative was immobilized in sol-gel thin films (thickness of about 600 nm) and the sensor properties (i.e., reactivity, stability and morphology of the sensitive layers) were studied. The kind of catalyst used (acid or base) was found to influence the stability and sensitivity. Sensing films prepared with acid were stable and insensitive to mercury. Preparation of the films with alkaline catalysis resulted in mercury sensitive layers with a limited stability. Stability could be improved by covalent binding of the reagent dye. Kewordr:
Fluorimetry; Mercury; Porphyrins; Sol-gel films
1. Introduction The determination of heavy metal ions is of growing interest in environmental analysis. In contrast to well-established laboratory methods, such as atomic absorption spectrometry and voltammetry, chemical sensors enable the continuous monitoring of pollutants. Analysis will become easier, faster and inexpensive using sensor based systems. In the case of optochemical sensors (so called optrodes [ 1,2] or optodes [3]) the sensing element consists of reagent dyes immobilized in organic or inorganic matrices.
* Corresponding author. 000~-~~70/Ys/$09.50
Reaction with the analyte changes the absorption or fluorescence behaviour of the sensitive layer. Especially the use of fluorimetric indicators resulted in highly sensitive devices [4]. For the determination of metal ions different indicators immobilized in various matrices are discussed [5,6]. In our laboratory immobilized porphyrin derivatives are used to develop optochemical sensors for the determination of heavy metal ions [7,8]. Porphyrins are well-known indicators for spectrophotometric [9] and fluorimetric [lo] analysis. The complexation of heavy metal ions is reversible and therefore these dyes are suitable to be used in optochemical sensors. Furthermore, metalloporphyrins show different absorption spectra dependent on the central
6 1905 Elsevicr Science B.V. All rights reserved
.SSIII 1l003-1670~‘~4~00568-0
108
M. Plaschke et al. /Analytica Chimica Acta 304 (1995) 107-I 13
ion. For this reason the limited selectivity of the complexation could be overcome and a spectrophotometric method for the simultaneous determination of Hg, Pb and Cd was developed using multi-wavelength techniques and chemometric evaluation methods [ll]. In the present work .5,10,15,20-tetra(psulfonatophenyl)porphyrin (TPPS) was used, to develop a fluorimetric method for determination of mercury ions in aqueous solution. The influences of buffer components, pH and temperature on the complexation were studied and operation range and detection limit of the method developed as well as tolerance ratios of interfering ions were determined. For the preparation of sensing films the porphyrin was immobilized in porous sol-gel films [12] and the characteristics of the resulting layers (i.e., sensitivity and stability) was investigated. The sol-gel matrix offers several advantages over other, namely organic matrices such as photochemical stability, variability of the preparation conditions, hydrophilicity and porosity of the resulting layers (for more details see [ 131). However the preparation of films by the sol-gel process depends on many parameters (e.g., precursor structure, catalysis, evaporation), therefore the influence of the preparation conditions (i.e., catalysis of the gelation) on the morphology of the thin films was examined. To improve the stability of the sensing films binding of the dye to a water-soluble macromolecule was investigated.
2. Experimental
2.1. Reagents All chemicals were of analytical grade (Merck, Darmstadt) unless otherwise specified and used without further purification. Doubly distilled water was used throughout. To test the influence of buffer components following mixtures were used: tris(hydroxymethyl)aminomethane-HCl (Tris, 0.1 mol/l; Fluka, Neu-Ulm); 1,3-bis[tris(hydroxymethyl)methylamino]propane-HCl (Bis, 0.05 mol/l); boric acid-NaOH-citric acid and phosphoric acid (1.77 g, 6.86 g, 3.5 g/l and 124 ml, respectively, in 500 ml water); boric acid-NaOH (0.1 mol/l); glycineNaOH (0.1 mol/l) and Na,CO,-NaHCO, (0.2 mol/l).
2.2. Fluorimetric
determination
of Hg(II) in solution
500 j.~l of sample solution (various concentrations of mercury or interfering ions) were mixed in a plexiglass cuvette (Sarstedt, Niimbrecht) with 3 ml of a stock solution of TPPS (1.2 X 1Om6 mol/l TPPS in borate buffer (0.1 mol/l, pH 8; Porphyrin products, Logan, UT). The mixture was allowed to react for at least 15 min and was measured within 1 h. The fluorescence intensities were determined with a spectrofluorimeter (Shimadzu, Model RF 5000) with excitation and emission wavelengths set to 425 or 560 nm and 612 nm, respectively. 2.3. Preparation
of sensing films
Starting solutions for the sol-gel process were prepared by mixing 200 ~1 of an aqueous solution of TPPS (5 X 10m4 mol/l), 200-300 ~1 methanol, 50 ~1 HCl (0.1 mol/l) or 50 ~1 NaOH (0.1 mol/l), lo-60 ~1 of a solution of different surface active agents (hexadecyltrimethylammonium bromide (CTAB), 8 x 10-j mol/l in methanol; sodium dodecylsulfate (SDS; Fluka, Buchs), 0.02 mol/l in ethanol; Triton X-100 (Serva, Heidelberg; 70 mg/ml in methanol) and 200 ~1 tetramethoxysilane (TMOS, Petrarch Systems, Bristol, CN). The pH of the aqueous part of the starting solutions were 1.5 (acidic catalysis) and 9.5 (alkaline catalysis), respectively. The mixture was allowed to prepolymerize for 10 min and thereafter an aliquot of 150-200 ~1 of the sol was pipetted onto a rotating glass disc (2000 rpm; rotation time, 30 s). The gelled films were dried for 1 h at room temperature and 1 h at temperatures between 50 and 150°C to examine the influence of heat treatment on sensor characteristics. The sensors (glass discs with sol-gel film) were mounted as one window in a flow through cell. Sample and buffer solutions were pumped through with 6 ml/min. The morphology of the films was characterized with a scanning electron micrograph (Jeol, Model SM-840) and with a long scan profiler (Tencor, Model P 2). TPPS was covalently bound to a dextran containing amino groups (Molecular Probes, Eugene, OR) according to a procedure previously reported [14]. 150 mg of TPPS were modified into the tetrasulfochloride by reaction with 650 mg of phosphorous
M. Plaschke
et al./Analytica
Chimica
Acta 304 (1995) 107-l
13
pentachloride (60 min, 100°C). The sulfochloride was dissolved in 10 ml of toluene and 150 mg of aminodextran and 2 ml of dry pyridine were added. The reaction mixture was refluxed for 2 h. The product was washed several times with ethanol, methanol and finally crystallized from mixture of water and methanol (40:60). The dextran labeled with TPPS was water-soluble and could be immobilized in sol-gel films as mentioned above.
3. Results and discussion 3. I. Fluorimetric determination in aqueous solution
of Hg(ll) using TPPS
The complexation of Hg(I1) was found to increase the fluorescence. The excitation (Fig. la) and emission spectra of the Hg(II)-TPPS complex (Fig. lb) show two excitation bands with maximum intensity at A,,, = 425 nm and AEXZ= 560 nm and one emission band with maximum intensity at A,, = 612 nm. For the determination of Hg(I1) two pairs of excitation and emission wavelengths were possible. The green excitation band was used with respect to the availability of green LED which can serve as light source in a simple sensor system. To obtain short sensor response times it was necessary to optimize the complexation conditions. To determine the optimum parameters the time was measured being necessary to obtain 90% of maximum signal under defined conditions. The kind of buffer used (see Experimental section) was found to influence the complexation time. Best results were obtained in borate buffer solution with a complexation time of 15 min t[Hg’+] = 6.5 X lo-’ mol/l; [TPPS] = 1.2 x IO (’ mol/l; T = 20°C). Under these conditions maximum fluorescence intensity was obtained at pH 8. The temperature dependence of the complexation kinetics is shown in Table 1. Complexation of mercury ions by TPPS was accelerated with increasing temperature. Due to the slight temperature dependence in the range of 20-30°C the determination of mercury was found to be reproducible in sample solutions not having been thermostated. For constant concentration (tug = 14 pg/ll the relative standard deviation of fluorescence intensity was determined (n = 12) and the relative error was found to 1.7%.
150
400
450
01 $50 Fig.
I. (a)
t.xcitation
550
500
wavelength
650 700 600 wavelength [nm] spectrum
600
[nm]
of the
7jo
Hg(ll)-TPPS-complex
(A EM -- 612 nm). (b) Emission spectrum of the Hg(Il)-TPPScomplex (A,, = 560 nm). [TPPS] = I .3Y IO -” mol/l; [Hg(ll)]:
( 1) 0.0. (2) buffer
0.015. (3) 0.03. (3) 0.045 and (5) 0.M
mg/I;
borate
pH X.
The S-shaped calibration curve (Fig. 21 shows a linear correlation between the concentration and the fluorescence intensity in the range of 2-30 pg/l Hg(ll), which is represented by the following equation (correlation factor = 0.9976): /(cl = 1.166~ 0.748, where I = fluorescence intensity and c = concentration of mercury ( Fg/l).
110 Table 1 Temperature temperature tg,,a (min)
M. Plaschke et al./Analytica
dependence CC)
10 80
of complexation I5 43
Table 2 Interferences
kinetics
20 15
25 13
30 11
[TPPS]= 1.2X 10-e mol/l; [Hg(II)] = 3.3 x lo--’ buffer pH 8. a After this time 90% of the maximum fluorescence reached.
35 10
mol/l;
borate
intensity were
The detection limit was experimentally determined according to Kaiser [15] and the student’s t-distribution [16]. Measuring samples (n = 10) with a mercury concentration of 1.4 pg/l Hg(II) average fluorescence intensities were obtained, which correspond to a signal being 6.8 times the blank signal. Thus, the confidence level of the detection limit obtained is 99.995% according to the student’s t-distribution. The influence of the presence of other ions was tested by adding a certain amount of another ion to a mercury containing buffered solution. The concentration of interfering ion affecting a signal change of 15 + 5% was determined. According to this interference criterium the porphyrin responds most sensitively to mercury. The results are summarized in Table 2. High tolerance ratios (i.e., weak interfer-
l
Chimica Acta 304 (1995) 107-113
.
.
. .
Interfering
to the determination
,__~
concentration
+-t-
80
100
lug/l]
Fig. 2. Calibration curve ([TPPS] = 1.3X 10-O mol/l; nm, h,, = 612 nm, borate buffer pH 8).
A,, = 560
Tolerance
Acetate, carbonate, thiocyanate Citrate, phosphate, nitrate, sulfate, S&I), Rb(I) Mg(I1) Al(III), CatII), BatID As(III), Ni(I1) Ag(I), CstI), tartrate Cr(III) La(B) MntlI), FetEI), S&I) Cu(I1) PbtII) CotII), ZntII) Cd(B) EDTA
ratio
> 10000 lOOO- 10000 100-1000
60 6 3 2 1 0.5 0.3 0.1
The concentration ratio ([ion]/[Hg(II)]) of interfering ion to Hg(I1) is indicated causing variations of fluorescence intensity within a range of 15*5%. High values mean weak interference and vice versa ([Hg(II)] = 20 @g/l; [TPPS] = 1.2 X 10mh mol/l; T = 20°C).
encesl were found for complexing agents such as acetate and citrate and for main-group metal ions. The presence of some transition group metal ions and strong complexing agents such as ethylendiaminetetraacetic acid in the sample solution strongly interfered the determination of Hg(I1). For the detection of free (uncomplexed) mercury the effect of complexing agents on the determination of mercury is negligible. A competition between mercury and other heavy metal ions to the porphyrin causes the main interference of these species (e.g., Cd, Pb or Zn). In former studies [l l] the absorption spectra of different metalloporphyrins were found to vary. Thus, a simultaneous determination of Cd, Pb and Hg was enabled by spectral discrimination. Using the spectral differences depending on the central ion having been complexed the interferences mentioned above should be reducible using a statistical evaluation method (e.g., factor analysis). 3.2. Porphyrin-doped
60
of mercury in solution
ion
sol-gel films
The sol-gel process offers several advantages in preparing porous glass films especially the high purity of the starting compounds and the control of the properties of the resulting glass by variation of the process parameters [ 131. Furthermore, low process
M. Plaschke et al./Analytica
temperatures enable doping the glass with organic reagent molecules to prepare chemically sensitive layers. In the present work the influence of process parameters such as catalysis of the gelation (i.e., alkaline or acidic), heat treatment of the films and surface active agents used in the starting solutions were investigated with regard to sensing properties (i.e., leaching of the dye and sensitivity of porphyrin-doped sol-gel films). Immobilisation of the porphyrin in the glass matrix was not found to affect significantly the spectral properties of the dye. To get a higher fluorescence intensity excitation with 425 nm was used for measurements with immobilized porphyrin. 3.3. Stability of TPPS-doped
Chimica Acta 304 (1995) 107-113
0
0
-?--3
6
80
100
Fig. 4. Response to mercury. TPPS-doped sol-gel films reacted reversibly with Hg(II). Alkaline catalyscd; [H&I)]= 20 wg:/l; borate buffer pH 8; flow-rate = 6 ml/min: A,., = 42.5 nm; A,, = 612 nm.
h of preconditioning in buffer). The leaching of the immobilized dye was also influenced by the temperature of the drying step. An increase of drying temperature from 50 to 150°C (drying time, 60 min; acidic gelled films; Triton X-100 as detergent) resulted in a decreased leaching (relative fluorescence intensity after 12 days of continuous operation: 27% and 55%, respectively). The use of different surface active agents also varied stability. Starting mixtures for sensor preparation containing Triton X-100, SDS or CTAB resulted in doped sol-gel films which showed a relative fluorescence intensity of 50%, 43% and 30%, respectively, after 20 days of continuous operation (acidic gelled films). 3.4. Response
4-m
40 60 time [min]
films
The kind of catalysis used (i.e., NaOH or HCl) resulted in sensing films with different properties. For sensors prepared by acidic and alkaline gelation the leaching of the dye is shown in Fig. 3a and b. After one day of continuous operation in buffered blank solution (borate pH 8) the relative fluorescence intensity of alkaline gelled films decreased to 15% (referred to the intensity after 1 h of preconditioning in buffer) while acidic gelled films still showed 90% of relative intensity (referred to the intensity after 24
0.0
20
III
9
12
15
18
time [d] Fig. 3. Stability of porphyrin-doped sol-gel films during continuous operation in buffered blank solution. (a) Sensing films with physical encapsulated TPPS prepared with acidic catalysis. (b) Sensing films with physical encapsulated TPPS prepared with alkaline catalysis. (c) Sensing films prepared with TPPS-labeled dextran. Borate buffer pH 8; A,, = 420 nm; A,, = 612 nm.
to mercury
Unfortunately, the more stable acidic-catalyzed films were insensitive towards mercury. Otherwise less stable alkaline-catalyzed films reacted with Hg(I1) (Fig. 4). The response time of these sensing films being sampled with Hg’+ containing solutions was in the range of 20 min. Even samples containing 2 pg/l Hg(II) resulted in an accurate sensor signal (not shown). A second sampling step (right side of Fig. 4) resulted in a decreased signal indicating a significant leaching of the dye. Thus, no reproducible signals for sampling with mercury were obtained by using films that were prepared by physical encapsulation of TPPS in the sol-gel matrix as mentioned above. Therefore, covalent binding of TPPS on dextran was investigated to improve stability.
112
M. Plaschke et al./Analytica
thickness [nm]
Chimica Acta 304 (1995) 107-113
thickness [nm]
glass disc
glass disc 400
600
length[wl
200 length
400
600
WI
Fig. 5. Surface profile of the sol-gel films. (a) Acidic gelled film. To determine film thickness the film was scratched in the notch seen in the middle of the curve. (b) Alkaline gelled film.
3.5. Morphology
of the sensitive layers
The stability but insensitivity of the acidic gelled films on the one hand and the instability but sensitivity of the alkaline gelled films on the other hand were found to correlate with structural differences of the layers. Investigations with a long scan profiler showed alkaline gelled films being rough with an average thickness of 650 & 2.50 nm while acidic gelled films being relatively smooth (film thickness 600 t_ 20 nm; Fig. 5). Scanning electron micrographs show the same results (Fig. 6): alkaline gelled films were rough and grainy while acidic gelled films were smooth (not shown). This corresponds with results of the influence of catalysis on hydrolysis and condensation of silane monomers reported in literature (for more details see [13]). In the present work, acidic catalysis leads to films with densely packed Si-0-Si chains, while alkaline catalysis lead to particulate systems with reduced three-dimensional connection. These different morphologies have a strong influence on the mechanical stability of the layers. Therefore, the thickness of the layers was measured before and after continuous exposition to aqueous solution. The acidic gelled films were stable and no decrease in film thickness was observed during several weeks. The loss of dye signal within this period (see Fig. 3a) is exclusively due to insufficient immobilisation. In comparison the rough and grainy surface of the alkaline gelled films was exposed to a mechanical stress during the measurement in the flow through
with a scalpel resulting
cell which resulted in an erosion of the layer of about 1.50 nm during the first 24 h of continuous operation. This corresponds to about a quarter of the total film thickness. Thus the leaching of the dye is correlated with a mainly particle-wise erosion of the layer, which seems to be the most limiting factor with respect to the stability of these films. We assume, that acidic catalysis entraps the dye in dense and smooth films which disable the reaction with the analyte. On the other hand, the porous and grainy
Fig. 6. Scanning electron mvzrograph (magnification 500 times).
of an alkaline
gelled film
M. Plaschke et al./Aualytica
structure of the reaction of the but enforces the ical destruction
alkaline gelled films facilitates the immobilized porphyrin with Hg(II), leaching of the dye and the mechanof the layer.
3.6. immobilization
by col>alent binding
To improve the stability the sensing films were prepared using the tetrasulfochloride of TPPS which was covalently bound to dextran. The resulting TPPS-labeled macromolecule was water-soluble and could be used in the sol-gel process for film preparation. The stability of sensing films with TPPS immobilized to dextran was excellent compared to the stability obtained with films prepared by physical encapsulation of TPPS (Fig. 3~). One reason for this promising result might be the hindered diffusion of the macromolecule through the silicate network of the sol-gel film. Thus, a strongly reduced leaching of the reagent dye was obtained.
II3
Chimicct Acta 304 (109.5) 107-113
should be reducible by optimizing the preparation process. Furthermore, the sensing characteristics such as response time and sensitivity might be improved by varying process parameters. The interferences to other heavy metal ions could be decreased using multi-wavelength techniques and chemometric evaluation methods. This work is under present investigation.
References [I]
W.R. Se~tz. Anal. Chem., 56 (1084)
[2] O.S. Woltbeis,
3X7.
[.i] W. Simon, Chimia, 44 (lYY0) 305. [4] L..A. Sari
and W.R. Seitz, Anal. Chcm.. 55 (108.1) 067.
[5] 2. Zhujun and W.R.
Seitz, Anal. Chim. Acta.
171 (IYXS)
2s 1. [h] M. Lcrchi, E. Bakker. B. Rustcrholz and W. Simon. Anal. Chem.. h4 (1902)
1534.
[7] R. Czolk, .I. Reichert and H.J. Ache. Sensors Actuators 13. 7 (1992) s-lo. [8] A. Morales-Bahnik,
R. Czolk,
Sensors Actuators B, 13
4. Conclusions
IhA.
Fresenius’ Z. Anal. Chem.. 32.5 (IYXh)
J. Rcichert and H.J. Ache,
( IW3) 424.
[Y] K.L. Chcng. K. Ueno and T. Imamamura (Eds.), Handbook of Organic Analytical
Reagents, CRC Prcs\. Boa
Raton. FL.
lY82.
A very sensitive, fluorimetric method for the detection of Hg(II) ions in aqueous solutions could be developed using tetra( p-sulfonatophenyl)porphyrin as indicator. The dye was immobilized in sol-gel thin films and characteristics of the resulting sensitive layers was investigated. Alkaline catalysis of the gelation and physical encapsulation of TPPS resulted in sensitive layers. Leaching of the dye and a significant erosion of the layers were found to limit the stability to one day. The wash-out of TPPS could be overcome by covalent binding of the indicator molecule to dextran. The erosion of the sol-gel film
[IO] Tong Shen-Yang
and Sun Guo-Bin.
Acta (‘him.
Sinica. 40
of Karlsruhc,
IYYO. pp.
(198X) 815. [I I] R. Czolk,
Ph.D. thesis, University
h I-62. [12] R. Zusman,
C. Rottman.
M. Ottolcnghi
Non-Cry?t. Solids, 122 (199)) [I31 C.J. Brinker and G.W.
and D. Avnir.
.I.
107.
Scherer. Sol-Gel Science. Academic
Prcas. San Diego, CA. IYYO. [I41 H. Offenbacher.
O.S.
Actuators, Y (lY86), [IS] [lb]
Wolfheis
and E. Fiirlinger.
Sensors
73.
H. Kaiser, Z. Anal. Chem.. 2OY ( lYO5) I. R.A. Fischer and F. Yates. Statistical Ffahles for Biological. Agricultural and Medical. Oliver and Boyd. Edinburgh, lY63, p. 46.
CHIMICA ACTA ELSEVIER
Analytica Chimica Acta 304 ( 1995) 107- 113
Fluorimetric determination of mercury with a water-soluble porphyrin and porphyrin-doped sol-gel films M. Plaschke, R. Czolk F’orscilun~.~zmtrum
Karlsruhe
GmbH. Institut ftir Radiochemie,
Received 23 March 1994; revised
4 November
*,
H.J. Ache
P.O. Box 3640.
D-76021
1994: accepted 7 November
Kurl.sruhc. (Tcrmarr!
I YO4
Abstract The fluorimetric determination of mercury ions with 5,10,15,20-tetra(p-sulfonatophenyl)porphyrin in aqueous solutions and in porphyrin doped sol-gel films was investigated. Optimization of complexation conditions such as buffer components, pH range and temperature resulted in a method which enabled the determination of mercury ions in aqueous solutions within 15 min with a detection limit of 1.4 Kg/l Hg(II). The main interfering ions were found to be Cd, Zn and Pb. To develop an optochemical sensing device the porphyrin derivative was immobilized in sol-gel thin films (thickness of about 600 nm) and the sensor properties (i.e., reactivity, stability and morphology of the sensitive layers) were studied. The kind of catalyst used (acid or base) was found to influence the stability and sensitivity. Sensing films prepared with acid were stable and insensitive to mercury. Preparation of the films with alkaline catalysis resulted in mercury sensitive layers with a limited stability. Stability could be improved by covalent binding of the reagent dye. Kewordr:
Fluorimetry; Mercury; Porphyrins; Sol-gel films
1. Introduction The determination of heavy metal ions is of growing interest in environmental analysis. In contrast to well-established laboratory methods, such as atomic absorption spectrometry and voltammetry, chemical sensors enable the continuous monitoring of pollutants. Analysis will become easier, faster and inexpensive using sensor based systems. In the case of optochemical sensors (so called optrodes [ 1,2] or optodes [3]) the sensing element consists of reagent dyes immobilized in organic or inorganic matrices.
* Corresponding author. 000~-~~70/Ys/$09.50
Reaction with the analyte changes the absorption or fluorescence behaviour of the sensitive layer. Especially the use of fluorimetric indicators resulted in highly sensitive devices [4]. For the determination of metal ions different indicators immobilized in various matrices are discussed [5,6]. In our laboratory immobilized porphyrin derivatives are used to develop optochemical sensors for the determination of heavy metal ions [7,8]. Porphyrins are well-known indicators for spectrophotometric [9] and fluorimetric [lo] analysis. The complexation of heavy metal ions is reversible and therefore these dyes are suitable to be used in optochemical sensors. Furthermore, metalloporphyrins show different absorption spectra dependent on the central
6 1905 Elsevicr Science B.V. All rights reserved
.SSIII 1l003-1670~‘~4~00568-0
108
M. Plaschke et al. /Analytica Chimica Acta 304 (1995) 107-I 13
ion. For this reason the limited selectivity of the complexation could be overcome and a spectrophotometric method for the simultaneous determination of Hg, Pb and Cd was developed using multi-wavelength techniques and chemometric evaluation methods [ll]. In the present work .5,10,15,20-tetra(psulfonatophenyl)porphyrin (TPPS) was used, to develop a fluorimetric method for determination of mercury ions in aqueous solution. The influences of buffer components, pH and temperature on the complexation were studied and operation range and detection limit of the method developed as well as tolerance ratios of interfering ions were determined. For the preparation of sensing films the porphyrin was immobilized in porous sol-gel films [12] and the characteristics of the resulting layers (i.e., sensitivity and stability) was investigated. The sol-gel matrix offers several advantages over other, namely organic matrices such as photochemical stability, variability of the preparation conditions, hydrophilicity and porosity of the resulting layers (for more details see [ 131). However the preparation of films by the sol-gel process depends on many parameters (e.g., precursor structure, catalysis, evaporation), therefore the influence of the preparation conditions (i.e., catalysis of the gelation) on the morphology of the thin films was examined. To improve the stability of the sensing films binding of the dye to a water-soluble macromolecule was investigated.
2. Experimental
2.1. Reagents All chemicals were of analytical grade (Merck, Darmstadt) unless otherwise specified and used without further purification. Doubly distilled water was used throughout. To test the influence of buffer components following mixtures were used: tris(hydroxymethyl)aminomethane-HCl (Tris, 0.1 mol/l; Fluka, Neu-Ulm); 1,3-bis[tris(hydroxymethyl)methylamino]propane-HCl (Bis, 0.05 mol/l); boric acid-NaOH-citric acid and phosphoric acid (1.77 g, 6.86 g, 3.5 g/l and 124 ml, respectively, in 500 ml water); boric acid-NaOH (0.1 mol/l); glycineNaOH (0.1 mol/l) and Na,CO,-NaHCO, (0.2 mol/l).
2.2. Fluorimetric
determination
of Hg(II) in solution
500 j.~l of sample solution (various concentrations of mercury or interfering ions) were mixed in a plexiglass cuvette (Sarstedt, Niimbrecht) with 3 ml of a stock solution of TPPS (1.2 X 1Om6 mol/l TPPS in borate buffer (0.1 mol/l, pH 8; Porphyrin products, Logan, UT). The mixture was allowed to react for at least 15 min and was measured within 1 h. The fluorescence intensities were determined with a spectrofluorimeter (Shimadzu, Model RF 5000) with excitation and emission wavelengths set to 425 or 560 nm and 612 nm, respectively. 2.3. Preparation
of sensing films
Starting solutions for the sol-gel process were prepared by mixing 200 ~1 of an aqueous solution of TPPS (5 X 10m4 mol/l), 200-300 ~1 methanol, 50 ~1 HCl (0.1 mol/l) or 50 ~1 NaOH (0.1 mol/l), lo-60 ~1 of a solution of different surface active agents (hexadecyltrimethylammonium bromide (CTAB), 8 x 10-j mol/l in methanol; sodium dodecylsulfate (SDS; Fluka, Buchs), 0.02 mol/l in ethanol; Triton X-100 (Serva, Heidelberg; 70 mg/ml in methanol) and 200 ~1 tetramethoxysilane (TMOS, Petrarch Systems, Bristol, CN). The pH of the aqueous part of the starting solutions were 1.5 (acidic catalysis) and 9.5 (alkaline catalysis), respectively. The mixture was allowed to prepolymerize for 10 min and thereafter an aliquot of 150-200 ~1 of the sol was pipetted onto a rotating glass disc (2000 rpm; rotation time, 30 s). The gelled films were dried for 1 h at room temperature and 1 h at temperatures between 50 and 150°C to examine the influence of heat treatment on sensor characteristics. The sensors (glass discs with sol-gel film) were mounted as one window in a flow through cell. Sample and buffer solutions were pumped through with 6 ml/min. The morphology of the films was characterized with a scanning electron micrograph (Jeol, Model SM-840) and with a long scan profiler (Tencor, Model P 2). TPPS was covalently bound to a dextran containing amino groups (Molecular Probes, Eugene, OR) according to a procedure previously reported [14]. 150 mg of TPPS were modified into the tetrasulfochloride by reaction with 650 mg of phosphorous
M. Plaschke
et al./Analytica
Chimica
Acta 304 (1995) 107-l
13
pentachloride (60 min, 100°C). The sulfochloride was dissolved in 10 ml of toluene and 150 mg of aminodextran and 2 ml of dry pyridine were added. The reaction mixture was refluxed for 2 h. The product was washed several times with ethanol, methanol and finally crystallized from mixture of water and methanol (40:60). The dextran labeled with TPPS was water-soluble and could be immobilized in sol-gel films as mentioned above.
3. Results and discussion 3. I. Fluorimetric determination in aqueous solution
of Hg(ll) using TPPS
The complexation of Hg(I1) was found to increase the fluorescence. The excitation (Fig. la) and emission spectra of the Hg(II)-TPPS complex (Fig. lb) show two excitation bands with maximum intensity at A,,, = 425 nm and AEXZ= 560 nm and one emission band with maximum intensity at A,, = 612 nm. For the determination of Hg(I1) two pairs of excitation and emission wavelengths were possible. The green excitation band was used with respect to the availability of green LED which can serve as light source in a simple sensor system. To obtain short sensor response times it was necessary to optimize the complexation conditions. To determine the optimum parameters the time was measured being necessary to obtain 90% of maximum signal under defined conditions. The kind of buffer used (see Experimental section) was found to influence the complexation time. Best results were obtained in borate buffer solution with a complexation time of 15 min t[Hg’+] = 6.5 X lo-’ mol/l; [TPPS] = 1.2 x IO (’ mol/l; T = 20°C). Under these conditions maximum fluorescence intensity was obtained at pH 8. The temperature dependence of the complexation kinetics is shown in Table 1. Complexation of mercury ions by TPPS was accelerated with increasing temperature. Due to the slight temperature dependence in the range of 20-30°C the determination of mercury was found to be reproducible in sample solutions not having been thermostated. For constant concentration (tug = 14 pg/ll the relative standard deviation of fluorescence intensity was determined (n = 12) and the relative error was found to 1.7%.
150
400
450
01 $50 Fig.
I. (a)
t.xcitation
550
500
wavelength
650 700 600 wavelength [nm] spectrum
600
[nm]
of the
7jo
Hg(ll)-TPPS-complex
(A EM -- 612 nm). (b) Emission spectrum of the Hg(Il)-TPPScomplex (A,, = 560 nm). [TPPS] = I .3Y IO -” mol/l; [Hg(ll)]:
( 1) 0.0. (2) buffer
0.015. (3) 0.03. (3) 0.045 and (5) 0.M
mg/I;
borate
pH X.
The S-shaped calibration curve (Fig. 21 shows a linear correlation between the concentration and the fluorescence intensity in the range of 2-30 pg/l Hg(ll), which is represented by the following equation (correlation factor = 0.9976): /(cl = 1.166~ 0.748, where I = fluorescence intensity and c = concentration of mercury ( Fg/l).
110 Table 1 Temperature temperature tg,,a (min)
M. Plaschke et al./Analytica
dependence CC)
10 80
of complexation I5 43
Table 2 Interferences
kinetics
20 15
25 13
30 11
[TPPS]= 1.2X 10-e mol/l; [Hg(II)] = 3.3 x lo--’ buffer pH 8. a After this time 90% of the maximum fluorescence reached.
35 10
mol/l;
borate
intensity were
The detection limit was experimentally determined according to Kaiser [15] and the student’s t-distribution [16]. Measuring samples (n = 10) with a mercury concentration of 1.4 pg/l Hg(II) average fluorescence intensities were obtained, which correspond to a signal being 6.8 times the blank signal. Thus, the confidence level of the detection limit obtained is 99.995% according to the student’s t-distribution. The influence of the presence of other ions was tested by adding a certain amount of another ion to a mercury containing buffered solution. The concentration of interfering ion affecting a signal change of 15 + 5% was determined. According to this interference criterium the porphyrin responds most sensitively to mercury. The results are summarized in Table 2. High tolerance ratios (i.e., weak interfer-
l
Chimica Acta 304 (1995) 107-113
.
.
. .
Interfering
to the determination
,__~
concentration
+-t-
80
100
lug/l]
Fig. 2. Calibration curve ([TPPS] = 1.3X 10-O mol/l; nm, h,, = 612 nm, borate buffer pH 8).
A,, = 560
Tolerance
Acetate, carbonate, thiocyanate Citrate, phosphate, nitrate, sulfate, S&I), Rb(I) Mg(I1) Al(III), CatII), BatID As(III), Ni(I1) Ag(I), CstI), tartrate Cr(III) La(B) MntlI), FetEI), S&I) Cu(I1) PbtII) CotII), ZntII) Cd(B) EDTA
ratio
> 10000 lOOO- 10000 100-1000
60 6 3 2 1 0.5 0.3 0.1
The concentration ratio ([ion]/[Hg(II)]) of interfering ion to Hg(I1) is indicated causing variations of fluorescence intensity within a range of 15*5%. High values mean weak interference and vice versa ([Hg(II)] = 20 @g/l; [TPPS] = 1.2 X 10mh mol/l; T = 20°C).
encesl were found for complexing agents such as acetate and citrate and for main-group metal ions. The presence of some transition group metal ions and strong complexing agents such as ethylendiaminetetraacetic acid in the sample solution strongly interfered the determination of Hg(I1). For the detection of free (uncomplexed) mercury the effect of complexing agents on the determination of mercury is negligible. A competition between mercury and other heavy metal ions to the porphyrin causes the main interference of these species (e.g., Cd, Pb or Zn). In former studies [l l] the absorption spectra of different metalloporphyrins were found to vary. Thus, a simultaneous determination of Cd, Pb and Hg was enabled by spectral discrimination. Using the spectral differences depending on the central ion having been complexed the interferences mentioned above should be reducible using a statistical evaluation method (e.g., factor analysis). 3.2. Porphyrin-doped
60
of mercury in solution
ion
sol-gel films
The sol-gel process offers several advantages in preparing porous glass films especially the high purity of the starting compounds and the control of the properties of the resulting glass by variation of the process parameters [ 131. Furthermore, low process
M. Plaschke et al./Analytica
temperatures enable doping the glass with organic reagent molecules to prepare chemically sensitive layers. In the present work the influence of process parameters such as catalysis of the gelation (i.e., alkaline or acidic), heat treatment of the films and surface active agents used in the starting solutions were investigated with regard to sensing properties (i.e., leaching of the dye and sensitivity of porphyrin-doped sol-gel films). Immobilisation of the porphyrin in the glass matrix was not found to affect significantly the spectral properties of the dye. To get a higher fluorescence intensity excitation with 425 nm was used for measurements with immobilized porphyrin. 3.3. Stability of TPPS-doped
Chimica Acta 304 (1995) 107-113
0
0
-?--3
6
80
100
Fig. 4. Response to mercury. TPPS-doped sol-gel films reacted reversibly with Hg(II). Alkaline catalyscd; [H&I)]= 20 wg:/l; borate buffer pH 8; flow-rate = 6 ml/min: A,., = 42.5 nm; A,, = 612 nm.
h of preconditioning in buffer). The leaching of the immobilized dye was also influenced by the temperature of the drying step. An increase of drying temperature from 50 to 150°C (drying time, 60 min; acidic gelled films; Triton X-100 as detergent) resulted in a decreased leaching (relative fluorescence intensity after 12 days of continuous operation: 27% and 55%, respectively). The use of different surface active agents also varied stability. Starting mixtures for sensor preparation containing Triton X-100, SDS or CTAB resulted in doped sol-gel films which showed a relative fluorescence intensity of 50%, 43% and 30%, respectively, after 20 days of continuous operation (acidic gelled films). 3.4. Response
4-m
40 60 time [min]
films
The kind of catalysis used (i.e., NaOH or HCl) resulted in sensing films with different properties. For sensors prepared by acidic and alkaline gelation the leaching of the dye is shown in Fig. 3a and b. After one day of continuous operation in buffered blank solution (borate pH 8) the relative fluorescence intensity of alkaline gelled films decreased to 15% (referred to the intensity after 1 h of preconditioning in buffer) while acidic gelled films still showed 90% of relative intensity (referred to the intensity after 24
0.0
20
III
9
12
15
18
time [d] Fig. 3. Stability of porphyrin-doped sol-gel films during continuous operation in buffered blank solution. (a) Sensing films with physical encapsulated TPPS prepared with acidic catalysis. (b) Sensing films with physical encapsulated TPPS prepared with alkaline catalysis. (c) Sensing films prepared with TPPS-labeled dextran. Borate buffer pH 8; A,, = 420 nm; A,, = 612 nm.
to mercury
Unfortunately, the more stable acidic-catalyzed films were insensitive towards mercury. Otherwise less stable alkaline-catalyzed films reacted with Hg(I1) (Fig. 4). The response time of these sensing films being sampled with Hg’+ containing solutions was in the range of 20 min. Even samples containing 2 pg/l Hg(II) resulted in an accurate sensor signal (not shown). A second sampling step (right side of Fig. 4) resulted in a decreased signal indicating a significant leaching of the dye. Thus, no reproducible signals for sampling with mercury were obtained by using films that were prepared by physical encapsulation of TPPS in the sol-gel matrix as mentioned above. Therefore, covalent binding of TPPS on dextran was investigated to improve stability.
112
M. Plaschke et al./Analytica
thickness [nm]
Chimica Acta 304 (1995) 107-113
thickness [nm]
glass disc
glass disc 400
600
length[wl
200 length
400
600
WI
Fig. 5. Surface profile of the sol-gel films. (a) Acidic gelled film. To determine film thickness the film was scratched in the notch seen in the middle of the curve. (b) Alkaline gelled film.
3.5. Morphology
of the sensitive layers
The stability but insensitivity of the acidic gelled films on the one hand and the instability but sensitivity of the alkaline gelled films on the other hand were found to correlate with structural differences of the layers. Investigations with a long scan profiler showed alkaline gelled films being rough with an average thickness of 650 & 2.50 nm while acidic gelled films being relatively smooth (film thickness 600 t_ 20 nm; Fig. 5). Scanning electron micrographs show the same results (Fig. 6): alkaline gelled films were rough and grainy while acidic gelled films were smooth (not shown). This corresponds with results of the influence of catalysis on hydrolysis and condensation of silane monomers reported in literature (for more details see [13]). In the present work, acidic catalysis leads to films with densely packed Si-0-Si chains, while alkaline catalysis lead to particulate systems with reduced three-dimensional connection. These different morphologies have a strong influence on the mechanical stability of the layers. Therefore, the thickness of the layers was measured before and after continuous exposition to aqueous solution. The acidic gelled films were stable and no decrease in film thickness was observed during several weeks. The loss of dye signal within this period (see Fig. 3a) is exclusively due to insufficient immobilisation. In comparison the rough and grainy surface of the alkaline gelled films was exposed to a mechanical stress during the measurement in the flow through
with a scalpel resulting
cell which resulted in an erosion of the layer of about 1.50 nm during the first 24 h of continuous operation. This corresponds to about a quarter of the total film thickness. Thus the leaching of the dye is correlated with a mainly particle-wise erosion of the layer, which seems to be the most limiting factor with respect to the stability of these films. We assume, that acidic catalysis entraps the dye in dense and smooth films which disable the reaction with the analyte. On the other hand, the porous and grainy
Fig. 6. Scanning electron mvzrograph (magnification 500 times).
of an alkaline
gelled film
M. Plaschke et al./Aualytica
structure of the reaction of the but enforces the ical destruction
alkaline gelled films facilitates the immobilized porphyrin with Hg(II), leaching of the dye and the mechanof the layer.
3.6. immobilization
by col>alent binding
To improve the stability the sensing films were prepared using the tetrasulfochloride of TPPS which was covalently bound to dextran. The resulting TPPS-labeled macromolecule was water-soluble and could be used in the sol-gel process for film preparation. The stability of sensing films with TPPS immobilized to dextran was excellent compared to the stability obtained with films prepared by physical encapsulation of TPPS (Fig. 3~). One reason for this promising result might be the hindered diffusion of the macromolecule through the silicate network of the sol-gel film. Thus, a strongly reduced leaching of the reagent dye was obtained.
II3
Chimicct Acta 304 (109.5) 107-113
should be reducible by optimizing the preparation process. Furthermore, the sensing characteristics such as response time and sensitivity might be improved by varying process parameters. The interferences to other heavy metal ions could be decreased using multi-wavelength techniques and chemometric evaluation methods. This work is under present investigation.
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Raton. FL.
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A very sensitive, fluorimetric method for the detection of Hg(II) ions in aqueous solutions could be developed using tetra( p-sulfonatophenyl)porphyrin as indicator. The dye was immobilized in sol-gel thin films and characteristics of the resulting sensitive layers was investigated. Alkaline catalysis of the gelation and physical encapsulation of TPPS resulted in sensitive layers. Leaching of the dye and a significant erosion of the layers were found to limit the stability to one day. The wash-out of TPPS could be overcome by covalent binding of the indicator molecule to dextran. The erosion of the sol-gel film
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and Sun Guo-Bin.
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