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Biosensors and Bioelectronics 22 (2006) 145–152 Potentiometric sensor for atrazine based on a molecular imprinted membranew sensing devices of th is kind have Correspondi n g a u t h o r . T e l . : + 3 9 0 3 8 2 9 8 7 580; fax: +39 0382 528 544. E-mail address: girolamo.dagostino@unipv.it (G. D’Agostino). 0956-5663/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi: 10.1016/j.bios.2006.05.014 been developed. All of them were based on the use of very thin membranes or films ( Kitade et al., 2004; Zhou et al., 2005 ), with problems due to difficulties in preparation, reproducibility and possible interferences. Recently a sensor has been developed, based on a MIP polymeric film deposited on the gate surface of an ion-sensitive field-effect transistor, obtaining good results in terms of specificity and low detection limit ( Pogorelova et al., 2002 ). Potentiometric sensors have been previously proposed for charged analytes, for example for nitrate anion based on imprinted conducting polymers ( Hutchins and Bachas, 1995 ). Atrazine is protonated, and as a consequence, positively charged in aqueous solution at sufficiently low pH, its protona- tion constant being log K = 1.7 ( Smolkova and Pacakova, 1978; Skopalova´ and Kotoucek, ˇ 1995 ). The combination of the pos- itively charged species with the imprinted membrane should produce a variation of the membrane charge. This can be mea- sured potentiometrically by a conventional ISE device, in which the membrane is placed between an internal filling solution at constant composition in contact with a reference electrode, and the sample solution ( Craggs et al., 1974 ). This configu- ration is very simple, and gives a Nernstian response to the analyte concentration, which is very convenient for quantifi- " id="pdf-obj-0-2" src="pdf-obj-0-2.jpg">
Biosensors and Bioelectronics 22 (2006) 145–152 Potentiometric sensor for atrazine based on a molecular imprinted membranew sensing devices of th is kind have Correspondi n g a u t h o r . T e l . : + 3 9 0 3 8 2 9 8 7 580; fax: +39 0382 528 544. E-mail address: girolamo.dagostino@unipv.it (G. D’Agostino). 0956-5663/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi: 10.1016/j.bios.2006.05.014 been developed. All of them were based on the use of very thin membranes or films ( Kitade et al., 2004; Zhou et al., 2005 ), with problems due to difficulties in preparation, reproducibility and possible interferences. Recently a sensor has been developed, based on a MIP polymeric film deposited on the gate surface of an ion-sensitive field-effect transistor, obtaining good results in terms of specificity and low detection limit ( Pogorelova et al., 2002 ). Potentiometric sensors have been previously proposed for charged analytes, for example for nitrate anion based on imprinted conducting polymers ( Hutchins and Bachas, 1995 ). Atrazine is protonated, and as a consequence, positively charged in aqueous solution at sufficiently low pH, its protona- tion constant being log K = 1.7 ( Smolkova and Pacakova, 1978; Skopalova´ and Kotoucek, ˇ 1995 ). The combination of the pos- itively charged species with the imprinted membrane should produce a variation of the membrane charge. This can be mea- sured potentiometrically by a conventional ISE device, in which the membrane is placed between an internal filling solution at constant composition in contact with a reference electrode, and the sample solution ( Craggs et al., 1974 ). This configu- ration is very simple, and gives a Nernstian response to the analyte concentration, which is very convenient for quantifi- " id="pdf-obj-0-4" src="pdf-obj-0-4.jpg">

Biosensors and Bioelectronics 22 (2006) 145–152

Biosensors and Bioelectronics 22 (2006) 145–152 Potentiometric sensor for atrazine based on a molecular imprinted membranew sensing devices of th is kind have Correspondi n g a u t h o r . T e l . : + 3 9 0 3 8 2 9 8 7 580; fax: +39 0382 528 544. E-mail address: girolamo.dagostino@unipv.it (G. D’Agostino). 0956-5663/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi: 10.1016/j.bios.2006.05.014 been developed. All of them were based on the use of very thin membranes or films ( Kitade et al., 2004; Zhou et al., 2005 ), with problems due to difficulties in preparation, reproducibility and possible interferences. Recently a sensor has been developed, based on a MIP polymeric film deposited on the gate surface of an ion-sensitive field-effect transistor, obtaining good results in terms of specificity and low detection limit ( Pogorelova et al., 2002 ). Potentiometric sensors have been previously proposed for charged analytes, for example for nitrate anion based on imprinted conducting polymers ( Hutchins and Bachas, 1995 ). Atrazine is protonated, and as a consequence, positively charged in aqueous solution at sufficiently low pH, its protona- tion constant being log K = 1.7 ( Smolkova and Pacakova, 1978; Skopalova´ and Kotoucek, ˇ 1995 ). The combination of the pos- itively charged species with the imprinted membrane should produce a variation of the membrane charge. This can be mea- sured potentiometrically by a conventional ISE device, in which the membrane is placed between an internal filling solution at constant composition in contact with a reference electrode, and the sample solution ( Craggs et al., 1974 ). This configu- ration is very simple, and gives a Nernstian response to the analyte concentration, which is very convenient for quantifi- " id="pdf-obj-0-8" src="pdf-obj-0-8.jpg">

Potentiometric sensor for atrazine based on a molecular imprinted membrane

G. D’Agostino , G. Alberti, R. Biesuz, M. Pesavento

Dipartimento di Chimica Generale, Universita` degli Studi di Pavia, Via Taramelli 12, 27100 Pavia, Italy

Received 24 January 2006; received in revised form 21 April 2006; accepted 4 May 2006 Available online 3 July 2006

Abstract

A molecular imprinted polymer (MIP) membrane for atrazine, not containing macropores, was synthesized and implemented in a potentiometric sensor. It is expected to work like a solid ISE (where the specific carrier are the imprinted sites) the specific carrier being the imprinted site. The active ion is the protonated atrazine, positively charged. To form this species the determination is carried out in acidic solution at pH lower than 1.8, in which atrazine is prevalently monoprotonated. At these conditions the membrane potential increases with atrazine concentration over a wide concentration range (3 × 10 5 to 1 × 10 3 M). The slope of the function E versus log c is about 25 mV/decade, showing that the atrazine form sorbed on MIP is the biprotonated one. The detection limit is determined by the relatively high concentration of atrazine released by the membrane in the sample solution at the considered conditions. It seems to be independent of the atrazine concentration in the internal solution of the sensor, but it depends on the acidity of the solution. The response time is less than 10 s and the sensor can be used for more than 2 months without any divergence. © 2006 Elsevier B.V. All rights reserved.

Keywords: Molecular imprinted polymer; Potentiometric sensor; Atrazine; Ion selective electrode

1. Introduction

The molecular imprinted polymers for triazinic herbicides, particularly atrazine, were the subject of many investigations in recent years with interesting application in chemical analysis. MIPs are used for solid phase extraction (SPE) and chromato- graphic separation (Matsui et al., 1995, 1997; Muldoon and Stanker, 1997; Olsen et al., 1998; Takeuchi et al., 1999; Lanza and Sellergren, 1999; Bjarnason et al., 1999; Ferrer et al., 2000). The use of these polymers for the recognition in chemical sen- sors has been also proposed. Some authors prepared polymeric membranes for conductimetric and capacimetric (Piletsky et al., 1995, 1998; Sergeyeva et al., 1999; Panasyuk-Delaney et al., 2001), for piezoelectric detection (Pogorelova et al., 2002), and other signal transduction methods, for example through the bulk acoustic wave and fluorescence (Liang et al., 2000; Jenkins et al., 2001) Despite the relatively simple transduction of the poten- tiometric signal, only a few sensing devices of this kind have

Corresponding author. Tel.: +39 0382 987 580; fax: +39 0382 528 544. E-mail address: girolamo.dagostino@unipv.it (G. D’Agostino).

0956-5663/$ – see front matter © 2006 Elsevier B.V. All rights reserved.

doi:10.1016/j.bios.2006.05.014

been developed. All of them were based on the use of very thin membranes or films (Kitade et al., 2004; Zhou et al., 2005), with problems due to difficulties in preparation, reproducibility and possible interferences. Recently a sensor has been developed, based on a MIP polymeric film deposited on the gate surface of an ion-sensitive field-effect transistor, obtaining good results in terms of specificity and low detection limit (Pogorelova et al., 2002). Potentiometric sensors have been previously proposed for charged analytes, for example for nitrate anion based on imprinted conducting polymers (Hutchins and Bachas, 1995). Atrazine is protonated, and as a consequence, positively charged in aqueous solution at sufficiently low pH, its protona- tion constant being log K a = 1.7 (Smolkova and Pacakova, 1978;

Skopalova´ and Kotoucek, ˇ

1995). The combination of the pos-

itively charged species with the imprinted membrane should produce a variation of the membrane charge. This can be mea- sured potentiometrically by a conventional ISE device, in which the membrane is placed between an internal filling solution at constant composition in contact with a reference electrode, and the sample solution (Craggs et al., 1974). This configu- ration is very simple, and gives a Nernstian response to the analyte concentration, which is very convenient for quantifi-

  • 146 G. D’Agostino et al. / Biosensors and Bioelectronics 22 (2006) 145–152

146 G. D’Agostino et al. / Biosensors and Bioelectronics 22 (2006) 145–152 Scheme 1. cation. Moreover

Scheme 1.

cation. Moreover it reduces potential interferences, for example those from redox active substances (Hutchins and Bachas, 1995), or generally from substances active at the transductor. In the present work, a molecular imprinted polymeric mem- brane for atrazine was prepared, The membrane was formed directly at the end of a small Teflon tube which can be filled with a solution at constant composition, in contact with an inter- nal reference electrode, as in a classical potentiometric cell for ion selective electrode (ISE). (Craggs et al., 1974), like that depicted in the Scheme 1 reported below. This preparation method for potentiometric sensors for charged species is here applied because it is simple and straightforward. The affinity of the protonated form of atrazine for the imprinted sites in the MIP membrane can be different from that of the neutral atrazine, which is the template. For this reason the effect of the solution acidity is particularly investigated in the present work.

2. Experimental

  • 2.1. Materials

Methacrylic acid [79-41-4] and ethylene glycol dimethacry- late [97-90-5] (Sigma–Aldrich)) were distilled in vacuum prior to use in order to remove stabilizers. Atrazine [1912-24- 9] (Sigma–Aldrich)) and 2,2 -azobisisobutyronitrile [78-67-1] (AIBN) (Sigma–Aldrich), were of reagent grade and were used without any further purification. All other chemicals were of analytical reagent grade and the solutions were prepared with ultrapure water (Milli-Q).

  • 2.2. Preparation of the MIP membrane

The molecular imprinted polymer membranes for atrazine was prepared from a reagent mixture obtained by mixing 48 l of methacrylic acid, 95.2 l of ethylene glycol dimethacrylate, 25 mg of atrazine and 28 mg of AIBN. The mixture was uni- formly dispersed by sonication (sonic bath model Transsonic T420—Elma) and then deaerated with nitrogen for 10 min. Ethy- lene glycol dimethacrylate acts not only as cross-linker, but also as solvent. A 25 l aliquot of reagent mixture was placed in a home made Teflon device that controls the shape of the liq- uid during the polymerization. This device was introduced in an oven (model M710) at 70 C for 17 h. A glassy membrane is obtained with 5 mm diameter, tightly fixed at the end of a small Teflon tube 10 mm long. This is directly used for assembling the sensor without any further manipulation of the membrane. The thickness of the membrane can be varied by placing different amounts of reagent mixture into the Teflon device for the poly- merization. The template was removed by washing the mem- brane successively in 10 ml of a methanol/acetic acid solution (4:1, v/v, of 98% methanol and pure acetic acid) for three times, each time for 1 h, then in 10 ml of water/acetic acid (5:1) for three times for 1 h and finally in 10 ml of pure water for three times for

1 h. The membrane was conditioned in 3.7 × 10 4 mol l 1 aque- ous atrazine solution (pH 1.5, HCl solution, KCl 0.02 mol l 1 ) for 16 h. In some cases the membrane was treated in 0.1 mol l 1 HCl before conditioning.

  • 2.3. Sensor construction and potential measurements

The Teflon tube bearing the membrane at the end was filled with a solution containing 3.7 × 10 4 mol l 1 atrazine, HCl 0.03 M (pH 1.5) and saturated with KCl. A Ag/AgCl reference electrode was contacted with the internal filling solution to trans- duce the potentiometric signal. All the potential measurements were carried out at 25.0 C (±0.1 C) in the cell as shown in Scheme 1.

Test solution: HCl pH 1.5, KCl 2 × 10 2 mol l 1 , atrazine; Membrane: MIP membrane.

The cell potential was measured at different atrazine con- centration in the test solution in the range 1.7 × 10 7 to 1.7 × 10 4 mol l 1 . All pH adjustments were made with HCl 37% or NaOH 50%. The liquid junction potential E j at the interface between the filling solution of the double junction reference electrode, 0.34 mol l 1 KNO 3 , and the test solution at the usual composi- tion depends on the proton activity according to the experimental relation: E j = 24a H = 24γ H c H . The correction for the liquid junction potential was done when appropriate.

3. Results and discussion

  • 3.1. Potentiometric response of the MIP membrane

The molecular imprinted polymer membrane was prepared from a reagent mixture containing only methacrylic acid as the functional monomer. Not any porogen solvent was used, in order to avoid the formation of macropores through which a rapid diffusion of atrazine across the membrane would take place. A large fraction of cross-linker, EDMA, was employed, 88%, and in this way a rigid membrane, resistant to stress, and well sealed to the Teflon tube, was obtained. Some examples of the potentiometric response of MIP mem- branes are displayed in Fig. 1. Increasing concentrations of atrazine (c add ) were added to the test solution. The potential increases immediately after the atrazine addition at concentrations higher than around 3 × 10 5 M, in the case of the membrane conditioned as described in the experimental part. For comparison the poten- tiometric response obtained with a similar membrane, but not previously conditioned in atrazine solution, is reported in Fig. 1. It shows that in the first point, at c add = 1 × 10 7 mol l 1 , a long time is required to reach the equilibrium potential. Besides, the slope of the function E versus log c add increases with the con-

G. D’Agostino et al. / Biosensors and Bioelectronics 22 (2006) 145–152

147

G. D’Agostino et al. / Biosensors and Bioelectronics 22 (2006) 145–152 147 Fig. 1. Cell potential

Fig. 1. Cell potential at different atrazine concentrations in function of time. Conditions reported in Scheme 1. Atrazine concentration 1.7 × 10 7 to 1.7 × 10 4 mol l 1 . Membrane 0.5 mm thick. ( ) MIP membrane conditioned; (*) MIP membrane non conditioned; (–) NIP membrane conditioned.

centration, being in any case much lower than that obtained with the conditioned membrane. Probably when the concentration of atrazine in the membrane is low, as it is in the not conditioned membrane, some atrazine is sorbed from the test solution into the membrane, producing a potential decrease. It has been pre- viously reported, for example in the case of a potentiometric sensor for nitrate (Hutchins and Bachas, 1995), that the sensor conditioning, reduces the potential drift due to the diffusion of the template in the bulk of the membrane and shortens the equi- libration time, even in the case of relatively thick membranes, as seen in Fig. 1. A membrane synthesized, washed and conditioned like the MIP membrane but in the absence of template (NIP membrane)

gave a much lower potential variation in function of the atrazine concentration, as seen in Fig. 1. This indicates that atrazine inter- acts only weakly with the NIP membrane because of the absence of specific molecular interaction sites. Besides, the potential of the not imprinted membrane is considerably lower than that of the MIP membrane, for example 24 mV instead of 99 mV. The experiments reported in Fig. 1 were carried out in acidic solution, HCl 0.031 mol l 1 (pH 1.5) containing KCl 0.02 mol l 1 . Atrazine is at least partially protonated at pH 1.5, according to the protonation constant reported in the litera- ture (Smolkova and Pacakova, 1978; Skopalova´ and Kotoucek, ˇ 1995), while it is not protonated at neutral pH. Indeed not any potentiometric response was obtained in neutral 0.02 mol l 1 KCl. This shows that the membrane potential depends on the concentration of charged species, similarly to the usual ion selec- tive electrodes (Bard and Faulkner, 1980). The effect of the proton concentration will be further discussed below. Other membranes were treated with 0.1 mol l 1 HCl, after the usual washing with acetic acid/methanol, and before the condi- tioning procedure. The E versus log c add straight line is shown in Table 1. While the slope was similar to that obtained for the membranes not treated with HCl 0.1 M, E 0 and E (p) were about 40 mV lower. In this case, 2 days were required for condition- ing in 3.6 × 10 4 mol l 1 atrazine solution. The lower measured potential could be related to the higher protonation of the car- boxylic groups in the membranes treated in more acidic solution. Anyhow the potentiometric response of the membranes treated with 0.1 mol l 1 HCl to increasing atrazine concentration was similar to that of the not treated ones both in terms of slope and detection limit. After the HCl treatment, the selectivity of the membrane was improved, as it will be shown below. A lower potential was measured using a thicker membrane, 1 mm instead of 0.5 mm, as seen in Fig. 2. The equilibration time was near to that of the thinner membranes, only a few seconds, suggesting that the potentiometric equilibrium is established at the interface membrane–aqueous solution in the case of the con- ditioned membranes. The membrane potential was constant at 0.1 mV after equili- bration. The sensors can be used for at least 1 month with no modifi- cation of the potentiometric response. The sensors were stored

Table 1 Standardization curve of different atrazine MIP membranes, at pH 1.5, in KCl 0.02, and results from Eq. (3)

Standardization line E = E add + Nlog c add

0

E (p)

(mV)

DL

(10 5 M)

Linearized Eq. (1) Slope

Ord. origin

E 0 (mV)

K

pot Atr,j c j (10 5 )

Membrane

0.5 mm

Membrane

1 mm

Membrane 0.5 mm, treated with 0.1 M HCl Membrane 0.5 mm, atrazine concentration internal solution c = 5 × 10 5 M Average of four different membranes 0.5 mm

E = 211(3) + 24.2(7)log c E = 135(2) + 32.2(5)log c E = 171(4) + 22(1)log c

E = 177(5) + 26(1)log c

99

3.5

62.7

61.0

E = 209(12) + 23(3)log c

106(1)

2.3

5.0

1.2

3.2

3(1)

3.4(1) × 10 7 1.3(1) × 10 4 5.8(2) × 10 6

2.8(2) × 10 6

1.9(1) × 10 3

0.5(1)

85(7)

31(1)

228.2

121.3

205.8

196.4

3.5(2)

4.1(9)

1.5(4)

1.1(0)

8(2) × 10 7

1.2(4) × 10 3

239(3)

1.9(8)

  • 148 G. D’Agostino et al. / Biosensors and Bioelectronics 22 (2006) 145–152

148 G. D’Agostino et al. / Biosensors and Bioelectronics 22 (2006) 145–152 Fig. 2. Standardization curves

Fig. 2. Standardization curves of atrazine at different conditions. ( ) Membrane 0.5 mm thick, pH 1.5; (×) membrane 0.5 mm thick, pH 7; ( ) membrane 1 mm thick, pH 1.5.

in aqueous solution containing 3.6 × 10 4 mol l 1 atrazine, 0.02 mol l 1 KCl and 0.031 mol l 1 HCl. Atrazine diffuses through the MIP membrane because of the concentration difference at the two interfaces of the membrane. The diffusion was checked in the following way: the Teflon tube carrying the membrane, previously conditioned in a con- centrated atrazine solution, was filled with 0.1 ml of atrazine solution 3.6 × 10 4 mol l 1 , containing KCl 0.02 mol l 1 and HCl 0.031 mol l 1 , and the membrane was contacted with 1 ml of solution at the same composition, but not containing atrazine. The contact was maintained for 48 h at room temperature. After this time, the concentration of atrazine in the outer solution was 5 × 10 6 mol l 1 . The determination was made spectropho- tometrically at λ = 225 nm, at which the molar absorptivity of atrazine is 2.07 × 10 4 cm 1 l mol 1 . This demonstrates that actually some atrazine diffuses from the inner to the outer solu- tion. The diffusion rate is relatively low, since the concentration in the inner and outer solution is still different after 48 h. This is favorable for the potentiometric determination, and is due in part to the absence of macropores in the membrane. The diffusion can affect the detection limit.

  • 3.2. Selectivity of the atrazine MIP membrane

The effect of increasing concentrations of simazine (6- chloro-N2,N4-diethyl-1,3,5-triazine-2,4-diamine), a chlorinated triazine with a structure very similar to atrazine, was evaluated for a MIP membrane washed only with methanol/acetic acid and for a membrane further treated with 0.1 mol l 1 HCl solu- tion before conditioning in concentrated atrazine solution. The standardization curve obtained for the membrane only treated in methanol/acetic acid is reported in Table 2, it is similar to that obtained for atrazine (see Table 1). On the contrary, there was not any potential variation for simazine concentration in the case of the membrane treated with HCl 0.1 M. The results are also reported in Table 2 for comparison. The same holds for two other triazines: ametryne (N2-ethyl-N4-isopropyl-6-methylthio- 1,3,5-triazine-2,4-diamine), which contains a –SCH 3 group instead of a chlorine atom, and has protonation properties differ- ent from atrazine (log K a = 4.1), and 2-hydroxy-atrazine bearing an –OH instead of –SCH 3 or chlorine, which is the principal degradation product of atrazine. Evidently the membrane is more specific for atrazine after treatment in HCl 0.1 mol l 1 .

  • 3.3. Response of the MIP membrane based sensor to

atrazine concentration

Some typical Nernstian plots, E versus log c add are reported in Fig. 2. At pH 7, the slope is always near to zero, while at pH 1.5 the curve is composed of two straight lines, one of which, at lower atrazine concentration, has slope near to zero and the other has a positive slope near to 25 mV/decade. The slope and the ordinate at the origin determined by fitting the linear part of the calibra- tion plot to the Nerst equation are reported in Table 1, together with the potential corresponding to the part with slope equal to zero (E (p) ). The detection limit (DL), i.e. the intercept of the two straight lines (Buck and Lindner, 1994) is also reported. Four different membranes were synthesized and used at similar con-

Table 2 Standardization curve of atrazine MIP membranes 0.5 mm thick, in KCl 0.02 M and pH 1.5, for different triazines

 

Standardization line

0

E (p)

DL

(10 5 M)

Linearized Eq. (1)

E = E add + Nlog c add

(mV)

Slope (10 7 )

Ord. origin

E 0 (mV)

ΣK

pot Atr,j c j (10 5 )

Simazine membrane washed in methanol/

E = 223(4) + 29(1)log c

95.6

4.65

3.75

Not significantly different from 0

  • 230 Not significantly different from 0

acetic acid Simazine Membrane washed in methanol/ acetic acid and treated in HCl 0.1 M

E = 81(1) + 4.0(2)log c

62.70

2-OH atrazine membrane washed in methanol/ acetic acid and treated in HCl 0.1 M

E = 68.5(2) + 0.64(5)log

66.6

Ametrine membrane washed in methanol/ acetic acid and treated in HCl 0.1 M

E = 68.2(1) + 0log c

68.1

G. D’Agostino et al. / Biosensors and Bioelectronics 22 (2006) 145–152

149

ditions in a short time (2 days) to investigate the reproducibility of the membranes. The average values obtained for the param- eters are reported in Table 1, with the corresponding standard deviation, which is relatively low. The slope (N) of the Nernstian part of the standardiza- tion curve E versus log c add is similar for all the considered membranes, and is near to 25 mV/decade. The same slope was obtained with an ISFET/MIP based sensor, with a differ- ent imprinted polymer (Pogorelova et al., 2002). According to the classical model for ISE this should indicate that a dou- bly charged ion is responsible for the potentiometric signal. However atrazine is not doubly protonated at the considered con- ditions, as calculated from the protonation constant of atrazine reported in the literature, log K a = 1.7 (Smolkova and Pacakova, 1978; Skopalova´ and Kotoucek, ˇ 1995). Only 63% of atrazine in solution is monoprotonated at pH around 1.5. E 0 is noticeably different in some of the experiments reported in Table 1. For example, when an internal solution at lower atrazine concentration is used, a lower potential is obtained. This variation corresponds to the concentration difference in the inter- nal solution. The detection limit is only slightly affected by the atrazine concentration in the internal solution, probably because of the very slow diffusion rate. Considering that interfering cations may be present, and that the potentiometrically active form of atrazine is ionic, say z- charged or z-protonated, the following relationship can be used for modeling the potentiometric response of the electrode:

E

=

E 0 + N

E

=

E 0 +

N

N =

RT

nF

ln([H z A z+ ] + K

pot

Atr,j c z/z I

j

)

ln

c

add

A + K

α H z

pot

Atr,j c z/z I

j

(1)

where z is the charge of the species which actually distributes

pot

between the two phases, H z A z+ . K Atr,j is the atrazine/jion (with charge z j ) potentiometric selectivity coefficient, and α H z A is the ratio of the total atrazine concentration in the solution phase to the concentration of the z-protonated form (side reaction coeffi- cient of protonated atrazine):

α H z A =

β za [H] z

β za [H] z

c Atr = [H z A z+ ] = [H z A z+ ] β za [H]

z

β za

[H]

β za =

[H z A z+ ]

[H]

z [A]

(2)

β za is the z-protonation constant of atrazine, and the summa- tion is extended from z = 0 to n (n is the maximum protona- tion number), and β 0a = 1. Charges are omitted for simplicity. The concentration of the active species depends on the total atrazine concentration, and on the acidity of the solution. Possi- ble interfering species are protons and atrazine released by the membrane, the concentration of which, indicated by the sym- bol c r , is unknown. Its potentiometric selectivity coefficient is

K

pot Atr,Atr = 1.
pot
Atr,Atr = 1.

Eq. (1) may be written in the following linearized way, to differentiate the two terms in the logarithm, one depending on

the atrazine concentration added to the solution and the other independent of it, but possibly depending on the atrazine con- centration released by the membrane:

10 E/N = 10 E 0 /N

c

add

A + K

α H z

pot

Atr,j c z/z I

j

(3)

N is assumed to be 29.5 mV/decade at 25 C, near to the experimental value. The results obtained by the linearization procedure are also reported in Table 1. The logarithm of the slope of the linearized function is E 0 N log α H z A , equal to that obtained directly from the function E versus log c add (E 0 ). It is also reported in Table 1 for comparison. The differences are due to the slope of the func- tion, which is slightly different from 29.5, as seen in the second column of Table 1. The term accounting for the interferences, expressed as the

pot

summation K Atr,j c j , obtained from the parameters of Eq. (3), is reported in Table 1. It is similar in the different experiments, because the composition of the solutions was the same. It is slightly lower in the case of lower atrazine concentration in the internal solution. The detection limit, defined as the intercept of the two straight lines in the E versus log c Atr plot, is relatively high. On the basis of the experiments described in this paragraph the interfering substances responsible for this high detection limit cannot be identified.

3.4. Effect of proton concentration on the sensor response

Some curves E versus log c add obtained with the same mem- brane, but at different acidity of the test solution are reported in Fig. 3. It can be seen that the potential is higher at higher acidity. At pH 4, the potential does not increase at increasing atrazine concentration, while at lower pH the curve E versus log c add is composed of two straight lines. The parameters of the part of the curve E versus log c add with slope different from zero are reported in Table 3, together with those obtained from Eq. (3). At the pH of the experiments, the liquid junction potential variation

is low, so that not any correction was made.

G. D’Agostino et al. / Biosensors and Bioelectronics 22 (2006) 145–152 149 ditions in a short

Fig. 3. Standardization curves of atrazine at different acidities. Membrane 0.5 mm thick, treated in HCl 0.1 M. ( ) pH 1.0; ( ) pH 1.45; ( ) pH 1.8; (*) pH 4.0.

  • 150 G. D’Agostino et al. / Biosensors and Bioelectronics 22 (2006) 145–152

Table 3 Standardization curve of atrazine MIP membrane 0.5 mm thick, treated in 0.1 M HCl, in KCl 0.02 at different pH, and results from Eq. (3)

Standardization line E = E add + Nlog c add

0

E (p) (mV)

DL (10 5 M)

Linearized Eq. (1) Slope (10 7 )

Ord. origin (10 2 )

E 0 (mV)

K

pot Atr,j c j (10 5 )

pH

pH

pH

1.00

1.40

1.85

E = 218(10) + 23(3)log c E = 214(6) + 25(1)log c E = 198(9) + 24.2(2)log c

115

104

96

3.30

4.90

6.60

15(1)

5.9(4)

2.4(2)

23(6)

5(2)

4(1)

241

229

218

1.5(4)

0.9(4)

1.5(4)

The slope of the curve E versus log c add is almost indepen- dent of the pH, while the ordinate at the origin (E 0 ) decreases with increasing pH. This can be ascribed to the protonation of atrazine, if the z-protonated form, is that potentiometrically active. Its concentration becomes higher at higher proton activ- ity, so that the corresponding potential should also increase, as experimentally observed. If the completely deprotonated form prevails, E 0 depends on the solution pH as here reported

E 0 = E 0 + N log β za N z pH

The plot of E 0 versus pH is actually a straight line, with these parameters obtained by least square method:

E 0 = 269.2(6) 27.3(4)pH

The expected slope is 59.16 mV/decade, since z corresponds to the charge of the ionic form of atrazine. This is the same effect observed in the case of the E versus log c atr plot. The difference between the constant potential E (p) and E 0 is constant at different pH, around 105 mV, a value welll cor- responding to Nlog(DL). It can be deduced that protons do not influence significantly the detection limit. The other pos- sible interfering ion is the atrazine released by the membrane, or diffusing from the internal solution to the sample solution,

the potentiometric selectivity coefficient of which, K

pot Atr,Atr , is
pot
Atr,Atr , is

equal to 1, as atrazine is the primary ion. So the interference term represents the concentration released by the membrane at the interface membrane/aqueous solution. This is a reasonable value compared to the detection limit, and is near to that reported in Table 1, obtained at fixed pH. Since the detection limit increases slightly at decreasing H + concentration, it is possible that more atrazine is released in solution at higher pH. This could explain why at sufficiently high pH, for instance at pH 4, the membrane potential does not increase at increasing atrazine concentration. The effect of the acidity on the potentiometric response of the atrazine MIP membrane is seen also in Fig. 4, in which the poten- tial in function of the solution pH is reported for different mem- branes, conditioned in the usual way but not containing atrazine in the test solution, so that the measured potential corresponds to E (p) . The cell potential reported in ordinates is corrected for the liquid junction potential variation at pH lower than around 1.2, as reported in the experimental part. At pH lower than 2.0 the potential increases at decreasing pH, while at higher pH it is constant. The potential versus pH function for pH lower than around 2 is a straight line with slope near to 23 mV/decade, and ordinate at the origin approximately 135 mV. One of these lines, obtained from one of the experiments in Fig. 4, is here given

as an example: E = 136(1)–24(1)pH (R 2 = 0.991). These results are in good agreement with those obtained in the experiments reported above, and the explanation is that the concentration of protonated atrazine, which is the potentiometrically active form, is higher at higher acidity. The effect of the pH variation on a NIP membrane is com- pletely different, as shown in Fig. 4 for comparison. Here the potential increases at increasing pH, contrary to what observed in the case of the MIP membrane. This could indicate that protons easily diffuse through the membrane not containing atrazine, while their mobility is reduced in presence of atrazine. This increases the conductivity of the membrane. It will be seen below that actually the resistance of the membrane is lower at higher atrazine concentration in the solution phase.

3.5. Electrical characterization of the membrane

The MIP membrane was characterized by the impedance spectroscopy technique (De Marco and Pejcic, 2000) at different atrazine concentration in the external solution. The instrument utilized was a Solartron SI 1260 impedance/gain-phase ana- lyzer, in the 0.1 Hz–10 MHz frequency range. The usual cell, that reported in Scheme 1, was investigated. The impedance spectrum shows that only one resistance and capacitance in parallel configuration constitute the equivalent circuit of the electrochemical system considered, to be attributed to the poly- meric material. The electrical capacitance of the membrane was 13.4 pF, and does not significantly vary in function of the atrazine concentration. The membrane electrical resistance at different atrazine con- centration in the external solution is reported in Fig. 5. The membrane resistance is relatively low, and decreases at concen- tration of atrazine higher than 3 × 10 5 mol l 1 , which is near

150 G. D’Agostino et al. / Biosensors and Bioelectronics 22 (2006) 145–152 Table 3 Standardization curve

Fig. 4. Dependence of the potential of cell 1 on the pH of the test solution. No added atrazine. ( , , ) MIP membrane 0.5 thick; (*) NIP membrane 0.5 thick.

G. D’Agostino et al. / Biosensors and Bioelectronics 22 (2006) 145–152

151

G. D’Agostino et al. / Biosensors and Bioelectronics 22 (2006) 145–152 151 Fig. 5. Resistance of

Fig. 5. Resistance of the conditioned MIP membrane at different atrazine con-

centration obtained by impedence spectroscopy.

to the detection limit of the potentiometric determination. The variation of the resistance is almost linear at the considered con- centrations. The decrease of the membrane resistance with the atrazine concentration is probably sufficiently high to reduce the slope of the Nernst function with respect to 59.16 mV/decade.

4. Conclusions

A potentiometric sensor for atrazine was developed, based on a molecular imprinted polymer membrane. It works as an ion selective electrode highly specific for atrazine. The mem- brane was rigid enough to bear the filling solution in contact with the internal reference electrode. To obtain a good potentio- metric response, the membrane must be previously conditioned. An interesting characteristics of the potentiometric sensor is that a short time, only a few seconds, is required to reach the equi- librium potential. Since the potential variation is produced by the charged species, the solution pH must be lower than pH 1.7 to ensure the protonation of atrazine,. Protons do not interfere according to an ion exchange mechanism, but they have an influence on the measured potential, since the concentration of the proto- nated species of atrazine increases with increasing acidity. The slope of the potentiometric curve E versus log c add is lower than expected for a monocharged ion. This is probably due to the variation of the electrical resistance of the membrane with the atrazine concentration. Atrazine is sorbed more strongly at low pH, at which the carboxylic groups in the polymeric membrane are completely protonated, as demonstrated by the fact that the detection limit slightly decreases with pH. This is probably the reason for which at pH higher than approximately 1.7 the mem- brane potential does not change with the atrazine concentration. The detection limit is around 2 × 10 5 mol l 1 , and it appears to be determined by the distribution coefficient of atrazine between the acidic aqueous solution and MIP. Thus the best way to lower the detection limit is using membranes with stronger sorbing properties at the considered conditions.

Acknowledgment

This work was financially supported by the project FIRB01 (MURST, Italy).

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