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Journal of Luminescence 131 (2011) 838842

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Journal of Luminescence
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Application of BoxBehnken design in the optimization of catalytic behavior of a new mixed chelate of copper (II) complex in chemiluminescence reaction of luminol
Tahereh Khajvand, Mohammad Javad Chaichi n, OmLeila Nazari, Hamid Golchoubian
Department of Chemistry, University of Mazandaran, P.O. Box 47416-95447, Babolsar, Iran

a r t i c l e i n f o
Article history: Received 9 June 2010 Received in revised form 20 October 2010 Accepted 15 November 2010 Available online 15 December 2010 Keywords: BoxBehnken design Luminol Hydrogen peroxide Fluorescein Complex (N-(2-(2aminoethylamino)ethyl)-1H-pyrrole-2carboxamide-Cu(II))

a b s t r a c t
In this work, we observed an enhancement of chemiluminescence (CL) emission of luminol when a new mixed chelate of copper complex (N-(2-(2-aminoethylamino)ethyl)-1H-pyrrole-2-carboxamide-Cu(II)) was mixed with a solution containing luminol in methanol/water. The BoxBehnken design matrix and response surface methodology (RSM) have been applied to design the experiments to evaluate the interactive effects of the three most important operating variablesluminol (10 410 2 M), uorescein (10 510 3 M) and hydrogen peroxide (13 M) concentrations on the CL emission of luminol. The total 15 experiments were conducted in the present study towards the construction of a quadratic model. Independent variables luminol and hydrogen peroxide have signicant value P o 0.0001, which indicates the importance of these variables in the CL emission of luminol. Values of Prob 4 F less than 0.0500 indicate that model terms are signicant for the CL emission of luminol. The regression equation coefcients were calculated and the data tted to a second-order polynomial equation for CL emission of luminol. The new introduced inorganic catalyst of luminol CL reaction can be effect more than that of the common ones such as potassium hexacyanoferrate (III) and copper (II) acetate. & 2010 Elsevier B.V. All rights reserved.

1. Introduction Chemiluminescence (CL) has attracted considerable attention as a versatile and highly sensitive detection tool within diverse elds such as biology, biotechnology, bioimaging and analytical technology [1]. Luminol is oxidized by strong oxidants in the presence of a catalyst such as peroxidase, metal ions, or metal complexes to produce chemiluminescence, leading to its use in a variety of analytical methods [2]. The oxidation of luminol (3-aminophthalhydrazide) in alkaline medium is one of the most efcient CL reactions. Luminol oxidation leads to the formation of an aminophthalate ion in an excited state, which emits light when returning to the ground state. The quantum yield of the reaction is low ( E 0.01) and the emission spectrum shows a maximum at 425 nm [3]. The mechanism of luminol oxidation by H2O2 is well established [4]. The chemiluminescent reaction of luminol with hydrogen peroxide catalyzed by hemin and some enzymes such as peroxidase and microperoxidase that is widely used in biochemistry and clinical chemistry [5]. However, enzymes are expensive, and their solutions are unstable. An alternative is to seek an understanding of the enzyme behavior and thus provide a

Corresponding author. Tel./fax: +98 112 5342350. E-mail address: (M.J. Chaichi).

substitute for such enzymes. In the past decade, several catalysts have been tested for H2O2 decomposition, which can be classied into two categories, (1) homogeneous and (2) heterogeneous catalysts. For both homogeneous and heterogeneous catalysts, transition metals play very important roles since catalytic activity varies with the different transition metals doped in the catalysts [6]. Certain transition metals such as iron (II) and copper (II) can catalyze the CL reaction of luminol in the usual aqueous media [710]. Generally, chelating reagents make unavailable free metal ions for the catalysis of CL due to the formation of stable metal complexes [11]. The produced CL emission is strongly enhanced when different metal ions (Co(II), Cu(II), Cr(III), etc.) are used as a catalyst, being applied with an analytical purpose for the sensitive detection of these metal ions [1214]. In this investigation, (N-(2-(2-aminoethylamino)ethyl)-1Hpyrrole-2-carboxamide-Cu(II)) (CuAEPc) was synthesized and used as a new catalyst for the CL reaction of luminol and hydrogen peroxide. We observed an enhancement in CL emission when CuAEPc was mixed with a solution containing luminol in methanol/ water. The reaction mechanism between CuAEPc and hydrogen peroxide are shown. After an oxidized form of the catalyst is formed, it reacts with the luminol anion LH and the native form of the catalyst can be regenerated (Scheme 1). The new copper complex has been used as a catalyst for the oxidation of luminol. It has been reported that copper (II) complexes possess catalyze-like activity. The effect of the new copper

0022-2313/$ - see front matter & 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jlumin.2010.11.015

T. Khajvand et al. / Journal of Luminescence 131 (2011) 838842


In the present study, the 2-level 3-factor BoxBehnken experimental design is applied to investigate and validate parameters affecting the CL emission of luminol. Luminol (10 410 2 M), uorescein (10 510 3 M) and hydrogen peroxide concentrations (13 M) on CL emission of luminol are variable input parameters.

2. Experimental 2.1. Reagents and solutions A new copper (II) complex with a tetradentate unsymmetrical ligand was prepared by one-pot condensation of methyl-2-pyrrole carboxylate, diethylenetriamine and copper (II) sulfate (Scheme 2). The complex was characterized by elemental analysis, electronic and IR spectral, as well as X-ray crystal structure determination. The X-ray structure of the molecule reveals the copper (II) center to be in a square planar environment through coordination by two nitrogen atoms of the amine, one amide nitrogen atom and one nitrogen atom of the pyrrole moieties. The copper (II) complex is neutral due to deprotonation of the amide and pyrrole groups [17]. The stock solution was prepared by dissolving 0.00256 g of CuAEPc in 10.0 ml of water, giving a molar concentration of 1 mM. A stock solution of hydrogen peroxide (30%, v/v, commercially available) was prepared by appropriate dilution of 30% solution with water. Luminol (Fluka) was dissolved in 0.10 M NaOH solution to give a 0.025 M stock standard solution. The stock solution of uorescein (0.001 M) was prepared in basic methanol and protected from light. All of the reagents used were of analytical grade. The water used was deionized and distilled. 2.2. Chemiluminescence measurements The chemiluminescence intensity was measured with the home made apparatus equipped with a model BPY47 photocell (Leybold, Huerth, Germany). The apparatus was connected to a personal computer via a suitable interface (Micropars, Tehran, Iran).

Scheme 1.

Fig. 1. Comparison of catalytic activity of Cu(II) complex and other metal catalysts in presence of H2O2. Reaction was performed using uorescein (5 10 4 M), luminol (10 4 M), H2O2 (1.6 10 4 M) and catalyst (10 mM).

complex on luminol CL was evaluated in the presence of H2O2 and compared with the already-known catalysts including Cu(OAc)2, K3[Fe(CN)6], hemin and Cu(II)-chloride complex. It should be noted that the new copper complex exhibited catalytic activity lower than that of the hemin system, which is a potent catalyst for luminol CL in the presence of H2O2 (Fig. 1). In conventional multifactor experiments, optimization is usually carried out by varying a single factor while keeping all other factors xed at a specic set of conditions. Moreover, this approach is time consuming and ignores the combined interactions between physicochemical parameters [15]. To solve this problem, response surface methodology (RSM) can be employed as an interesting strategy to implement process conditions that drive to optimal response by performing a minimum number of experiments. RSM is a combination of mathematical and statistical techniques used for developing, improving and optimizing the processes, and used to evaluate the relative signicance of several affecting factors even in the presence of complex interactions. Recently, this method has been used to determine optimum parameters in different processes [16,8].

Scheme 2.


T. Khajvand et al. / Journal of Luminescence 131 (2011) 838842

Experiments were carried out in a light-tight attened bottom glass cell of 15 mm diameter at room temperature. This study design, requiring a total of 15 experimental runs (formulation combinations), was generated and analyzed using MINITAB 13. 2.3. Procedures Solution A was made with 50 ml of luminol, 25 ml of uorescein in methanol and 10 ml of CuAEPc in methanol were rst mixed in a 10.0 ml calibrated tube, then diluted with water to the mark and mixed thoroughly. Solution A was transferred to the instrument quartz cuvette via polypropene syringes. Then 100 ml of hydrogen peroxide solution in methanol was injected into the quartz cuvette and the chemiluminescence spectrum was recorded. 2.4. BoxBehnken design The BoxBehnken design is used in order to optimize the number of experiments to be carried out to ascertain the possible interactions between the studied parameters and their effects on the CL emission of luminol. The BoxBehnken design is a spherical, revolving design; it consists of a central point and the middle points of the edges of the cube circumscribed on the sphere [18]. It is a three level fractional factorial design consisting of a full 22 factorial seeded into a balanced incomplete block design. It consists of three interlocking 22 factorial designs having points, all lying on the surface of a sphere surrounding the center of the design. It has been applied in the optimization of several chemical and physical processes, and the number of experiments are decided accordingly [19]. In the present study, the BoxBehnken experimental design is applied to investigate and validate the treatment process parameters affecting CL emission of luminol. Fluorescein (X1), Luminol (X2) and hydrogen peroxide (X3) concentrations are input variable parameters, while CuAEPc, 1 mM, was kept as a constant input parameter. The interval of the allowed values for these factors was deduced from the preliminary tests carried out (Table 1). The factor levels were coded as 1 (low), 0 (central point or middle) and 1 (high). Fluorescein (10 510 3 M), luminol (10 410 2 M) and hydrogen peroxide (13 M) concentrations on CL emission of luminol are variable input parameters. The CL emission intensity of luminol, Y was designed as a response of the studied system. For this response (Y), a polynomial model of the second degree is established to quantify the inuence of the variables I b0 b1 X1 b2 X2 b3 X3 b12 X1 X2 b13 X1 X3
2 2 2 b23 X2 X3 b11 X1 b22 X2 b33 X3

3. Results and discussion 3.1. Statistical analysis In this work the combined effects of uorescein, luminol and hydrogen peroxide concentration at various levels for CL emission of luminol were monitored (Fig. 2). Table 2 shows the data resulting from the experiments of the effect of the three variables on the CL emission of luminol. The experimental results were analyzed through a RSM design to obtain an empirical model for the best response. The predicted results by the model are shown in Table 2. The estimated response seems to have a functional relationship only in a local region or near the central points of the model. The quadratic model was used to explain the mathematical relationship between the independent variables and dependent responses.
Table 2 BoxBenhken matrix for optimization of chemical factors. SE expected 2.723 2.723 2.723 1.815 2.723 2.723 2.723 2.723 2.723 2.723 2.723 1.815 2.723 1.815 2.723

Experiment Fluoresein Luminol H2O2 Experimental Expected 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 0 1 0 0 0 1 1 1 1 1 1 0 1 0 0 1 1 0 0 1 0 0 0 1 0 1 0 1 0 1 1 0 1 0 1 1 1 1 0 1 0 0 0 0 1 105.01 90.20 72.11 60.21 72.59 86.24 35.33 98.00 30.01 41.00 40.02 58.60 88.44 60.04 10.07 108.25 89.12 72.00 59.33 72.00 85.12 37.33 95.62 30.87 41.87 42.37 59.33 85.62 59.33 6.75

Table 3 Test of signicance for regression coefcients. Coefcient Coefcient value 59.333 3.375 25.375 24.375 1.458 1.208 4.208 2 1.5 7.25 Standard error 1.185 1.111 1.111 1.111 1.639 1.639 1.639 1.572 1.572 1.572 Signicance level (P) o 0.0001 0.02 o 0.0001 o 0.0001 0.414 0.439 0.05 0.259 0.384 0.006

b0 b1 b2 b3 b11 b22 b33 b12 b13 b23

where X1, X2 and X3 are the independent variables representing uorescein, luminol and hydrogen peroxide concentrations, respectively; b0 is a constant; b1, b2 and b3 are the coefcients translating the linear weight of X1, X2 and X3, respectively; b12, b13 and b23 are the coefcients translating the interactions between the variables; and b11, b22 and b33 of the coefcients translating the quadratic inuence of X1, X2 and X3. Linear and second-order polynomials were tted to the experimental data to obtain the regression equations.
Table 1 Experimental design levels of chosen variables. Variable (M) Fluorescein (X1) Luminol (X2) H2O2 (X3) Low ( ) 10 10 4 1

Middle (0) 5 10 5 10 3 2

High (+ ) 10 3 10 2 3

Fig. 2. CL intensity as a function of time for reaction of uorescein (5 10 4 M), luminol (10 4 M) and H2O2 (1.6 10 4 M).

T. Khajvand et al. / Journal of Luminescence 131 (2011) 838842


Table 4 ANOVA results for quadratic equation for CL intensity of luminal. Source Regression Residual error Lack-of-t Pure error Total DF 9 5 3 2 14 Sum of squares 10722.6 49.4 46.8 2.7 10772.6 Mean square 1191.40 9.88 15.58 1.33 F-ratio 120.55 11.69 P o 0.0001 0.0800 Prob 4 F Signicant Not signicant

120 Experimental CL intensity 100

R2 = 0.9954

80 60 40 20 0 0 20 40 60 80 100 120 Expected CL intensity

Fig. 3. Relation between experimental and expected CL intensity (Eq. (2)).

Fig. 5. Estimated response surface for [H2O2] 0.

Fig. 4. Estimated response surface for [uorescein] 0.

The coefcients values of Eq. (1) were calculated and tested for their signicance and are listed in Table 3. The P values are used as a tool to check the signicance of each coefcient, which in turn may indicate the pattern of the interactions between the variables. The smaller the value of P, the more signicant the corresponding coefcient. It can be seen from this table that the linear coefcients (X2 and X3) were highly signicant (P 0.0001). A quadratic coefcient (X23 and X33) and the linear coefcients (X1) may be slightly signicant (P 0.05). The other term coefcients (X1X2, X1X3, X11 and X22) are not signicant. The function representing the relationship among luminol, H2O2 and uorescein concentrations and the chosen response is
2 2 1:208X2 Y 59:333 3:750X1 25:375X2 24:875X3 1:458X1 2 4:208X3 2:000X1 X2 1:500X1 X3 7:250X2 X3

Fig. 3 shows the representation of the observed value (experimental) according to the predicted values (calculated). The good correlation between the measured values and those predicted by the model conrms the quality of this model. In addition, the model gives R2 value of 0.9957 and an adjusted R2 value of 0.987. These values conrm that the equation of the model is highly reliable. This indicates also that the model terms are signicant at 95% of probability level. The statistical signicance of the ratio of mean square variation due to regression and mean square residual error was tested using the analysis of variance (ANOVA). ANOVA is a statistical technique that subdivides the total variation in a set of data into component parts associated with specic sources of variation for the purpose of testing hypotheses on the parameters of the model [20]. According to analysis of variance (Table 4), it was shown that the predictability of the model is at 95% condence interval. The ANOVA of these responses demonstrated that the model is highly signicant as is evident from the value of Fstatistic (the ratio of mean square due to regression to mean square to real error; Fmodel 120.55) and a very low probability value (P o 0.0001). The value of probability P o 0.01 indicates that the model is considered statistically signicant [21]. The non-signicant lack-of-t (more than 0.05) showed that the quadratic model is valid for present study. Non-signicant lack-of-t is good for data tness in the model.

3.2. Effect of variables on CL intensity Using the experimental design, the combined effects of the three variables can be predicted, which is difcult to observe in conventional methods. The surface plot shows the increase in CL intensity with an increase in luminol and H2O2 concentration. The corresponding surface responses (Figs. 46) show that the optimum values for luminol and [H2O2] concentrations are out of the experimental


T. Khajvand et al. / Journal of Luminescence 131 (2011) 838842

surface method and in particular a BoxBenhken design. The results obtained from the present study revealed that RSM was a suitable method to optimize the experimental variables that affecting on the CL emission of luminol. Analysis of variance showed a high coefcient of determination value (R2 0.995), ensuring a satisfactory adjustment of the second-order regression model with the experimental data.

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Fig. 6. Estimated response surface for [luminol] 0.

region while the optimum value for uorescein concentration is in the experimental region. However, concentrations of 10 4 M of luminol and 2 M of H2O2 were chosen to be suitable for the purposes of the study, because higher concentrations of luminol and H2O2 could produce the saturation of the detector due to the high CL intensity of the blank signal obtained (Figs. 46).

4. Conclusion In this work, we observed an enhancement in CL emission when the copper complex (N-(2-(2-aminoethylamino)ethyl)-1H-pyrrole-2-carboxamide-Cu(II)) was mixed with a solution containing luminol in methanol/water. In this investigation, CuAEPc was synthesized and used as a new catalyst for the CL reaction of luminol and hydrogen peroxide in the presence of uorescein. The inuence of operating parameters such as uorescein, luminol and hydrogen peroxide concentrations was studied using a response