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A kinetic method for the determination of thiourea by its catalytic effect in micellar media
Spectrochimica Acta Part A 72 (2009) 327–331
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
A kinetic method for the determination of thiourea by its catalytic effect in micellar media Shahryar Abbasi a,∗ , Hossein Khani a , Mohammad Bagher Gholivand b , Ali Naghipour a , Abbas Farmany a , Freshteh Abbasi c a b c
Chemistry Department, Faculty of Science, Ilam University, Ilam, Iran Chemistry Department, Faculty of Science, Razi University, Kermanshah, Iran Chemistry Department, Faculty of Science, Azad University of Ilam, Ilam, Iran
a r t i c l e
i n f o
Article history: Received 2 July 2008 Received in revised form 6 September 2008 Accepted 26 September 2008 Keywords: Thiourea Janus green Catalytic Spectrophotometry Micellar medium
a b s t r a c t A highly sensitive, selective and simple kinetic method was developed for the determination of trace levels of thiourea based on its catalytic effect on the oxidation of janus green in phosphoric acid media and presence of Triton X-100 surfactant without any separation and pre-concentration steps. The reaction was monitored spectrophotometrically by tracing the formation of the green-colored oxidized product of janus green at 617 nm within 15 min of mixing the reagents. The effect of some factors on the reaction speed was investigated. Following the recommended procedure, thiourea could be determined with linear calibration graph in 0.03–10.00 g/ml range. The detection limit of the proposed method is 0.02 g/ml. Most of foreign species do not interfere with the determination. The high sensitivity and selectivity of the proposed method allowed its successful application to fruit juice and industrial waste water. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Thiourea (TU) has various industrial, agricultural and analytical applications. This material is widely used in photography as a fixing agent and also removes stains from negative. In agriculture, it is used as fungicides, herbicides and rodenticides [1] and also to decrease the content of nitrifying bacteria in soil [2]. TU is also used for induction of early ripening in several fruits [3]. In analytical chemistry TU is used as a spectrophotometric reagent for the determination of several metals [4]. TU is also used as a reagent for copper electrolytes refinery [5]. The toxic effects of TU seem to arise from a disturbance of carbohydrate metabolism and could result in chronic goitrogenic and other glandular difficulties in humans. Furthermore, TU and its derivatives have also been screened as allergenic and carcinogenic factors [6]. Therefore, its determination, taking into account the detection limit must be considerably below 76 ppb. A method for the determination of TU in waste water meets three main problems: (1) The samples of waste water examined contain a large number of organic components and some of these are present in relatively large amounts, at ppm level or even more.
∗ Corresponding author. Fax: +98 841 2227022. E-mail address: sh [email protected] (S. Abbasi). 1386-1425/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.saa.2008.09.029
(2) TU is very soluble in water and practically insoluble in typical extraction solvent such as diethyl ether and chloroform or methylene chloride. Its extraction is therefore not possible. For the same reason, solid phase extraction does not work, as only a small fraction can be adsorbed on the extraction cartridge. (3) TU is poorly volatile and is not easily analyzed by gas chromatography [7]. Various methods have been proposed for the determination of TU such as titrimetry with iodine [8], N-bromosuccinimdie [9] or haloamines [10], Raman spectroscopy [11], spectrophotometry [12,13], polarography [14], voltammetry [4,15], high performance liquid chromatography [16,17], kinetic methods [18], ion selective electrode potentiometry [19], FTIR [20], flow injections methods [1,21,22], amperometric methods [23–25], chemiluminescence [4] and tandem mass spectrometry [7]. However, most of these methods are either insensitive [5,7,10,11,19,23], inselective [1,3,8,22] or time consuming [3,8,12,22]. The current paper describes an original method for the highly sensitive, selective and precise determination of trace levels of TU based on its catalytic effect on the oxidation of janus green with potassium iodate (KIO3 ) in the micellar medium. The micelle has low surface tension and strong soluble power [26], so it can enhance the rate of reaction [27]. The method was conveniently applied to the determination of TU in fruit juice and industrial waste water.
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S. Abbasi et al. / Spectrochimica Acta Part A 72 (2009) 327–331
2. Experimental 2.1. Apparatus A double beam UV–vis spectrophotometer Scinco model S-3100 (South Korea) with 10 mm quartz cell was used for the recording the absorption spectra. A photometer (Metrohm, Model 662) was used to measure the absorbance change at 617 nm. A thermostat water bath was used to keep the reaction temperature at 45 ◦ C. Eppendorf Vary-pipettes (10–100 and 100–1000 l) were used to deliver accurate volume. All glassware and storage bottles were soaked in 10% HNO3 overnight and thoroughly rinsed with water prior to use. 2.2. Reagents and chemical All chemical reagents were of analytical grade and were purchased from Merck (Darmstadt, Germany), Aldrich (Milwaukee, WI, USA) or Fluka (Buchs, Switzerland). All solutions were made in doubly distilled water. Stock standard solution of thiourea (1000 g/ml) was prepared by dissolving 0.1 g of thiourea in water and diluting to 100 ml in a volumetric flask. Working standard solutions were obtained by appropriately diluting the stock solution before use. Janus green solution (1 × 10−3 M) was prepared by dissolving 0.0511 g of it in 100 ml water. Iodate stock’s solution (0.02 M) was prepared by dissolving the required amount of potassium iodate in water. A phosphoric acid solution (0.5 M) was prepared from 85% purity reagent. Triton X-100 solution (5 g/l) was prepared by dissolving 5 g of Triton X-100 in water and diluting to 1 l in a volumetric flask. 2.3. Recommended procedure Into a 25 ml volumetric flask, 1 ml phosphoric acid (0.5 M), 0.6 ml janus green (1 × 10−3 M) and appropriate amount of thiourea (0.03–10.00 g/ml) were transferred. Then 1.9 ml of Triton X-100 and 2.7 ml of KIO3 (0.02 M) were added and solution was diluted
Fig. 1. Structure of janus green (3-diethylamino-7-(4-dimethylaminophenylazo)-5phenylphenazinium chloride).
Then 1 ml of HCl (1 M) was added and solution was filtered again and centrifuged for 15 min in 3500 rpm. 5 ml of final solution was transferred to volumetric flask, diluted to 50 ml and analyzed following the recommended procedure. 3. Results and discussion Janus green (Fig. 1) is a basic dye of mono-azo group used in cytology for specific supravital staining of mitochondria [36]. It is cationic dye, which tends to form ion pair with bulky anions, so it is used as an excellent carrier for the transport of copper as Cu(SCN)4 2− ion through a bulk liquid membrane [37]. Janus green can be oxidized with strong oxidizing agents such as BrO3 − and IO3 − at slow reaction [29]. Thiourea can increase the rate of this reaction at trace levels. The mechanism of the janus green oxidation seems to contribute to the following reactions in its simplest form IO3 − + H+ + janus green (Red) → janus green (OX)
(1)
6H2 NCSNH2 + IO3 − + 6H+ → 3[CS(NH2)2]2 2+ + I− + H2 O
(2)
IO3 − + 5I− + 6H+ → 3I2 + 3H2 O
(3)
(4)
(5) with water to 25 ml, shaken and left in (45 ± 0.5) ◦ C bath water for 15 min. The absorbance of this catalyzed reaction was labeled as As and measured at 617 nm. The same procedure was repeated without addition of thiourea to get the blank signal (uncatalyzed reaction) and the signal was labeled as Ab . The calibration graph was constructed by plotting A = Ab − As vs. thiourea concentration. 2.4. Procedure for real samples analysis Fruits were purchased from a supermarket and juices were obtained by squeezing them [3]. The juices were filtered using Whatman 42 paper (semi-analytical) and centrifuged for 15 min at 3500 rpm. After refluxing (for removing the ascorbic acid interference [28]) for 10 min, 1 ml of this solution was transferred to a volumetric flask, diluted to 50 ml and analyzed following the recommended procedure. A collected waste water sample from texture factory was filtered using Whatman 42 paper (semi-analytical), 1 ml of H2 SO4 (1 M) was added and the solution was shaken well.
where Red is reduced form and OX is the oxidized form of janus green. Because reaction (1) is very slow, in the absence of thiourea, IO3 − oxidized janus green to produce weak reduce in absorbance of dye. When thiourea was added to this system, reactions (2) [11], (3) and (5) were fast. The H2 NCSIN⊕ H2 generated in situ in step (4) increased the rate of oxidation of janus green. For finding the optimum experimental condition, the effect of acidity, IO3 − concentration, janus green concentration, volume of Triton X-100, temperature and time on the reaction rate were studied. 3.1. The absorption spectra As shown in Fig. 2, the absorption spectra, both catalyzed (Fig. 2b–d) and unanalyzed (Fig. 2a), reach their maximum at 617 nm reaction systems, and their difference also reaches its maximum at 617 nm. As a result, we selected 617 nm as the measuring wavelength.
S. Abbasi et al. / Spectrochimica Acta Part A 72 (2009) 327–331
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Fig. 2. Variation of absorbance spectra with thiourea concentration. Condition: (a) Acid phosphoric (0.5 M): 1 ml, janus green (1 × 10−3 M): 0.5 ml, Triton X-100 (5 g/l): 2.0 ml, KIO3 (0.02 M): 2.0 ml. (b) a + 4.00 g/ml thiourea. (c) a + 7.00 g/ml thiourea. (d) a + 10.00 g/ml thiourea.
3.2. Effect of reaction medium Several reaction media such as sulfuric acid (H2 SO4 ), hydrochloric acid (HCl), phosphoric acid (H3 PO4 ), boric acid (H3 BO3 ), sodium hydroxide (NaOH), ammonium-chloride-ammonia (NH4 Cl-NH3 ), potassium acid phthalate-NaOH, acetic acid-sodium acetate (CH3 COOH-CH3 COONa), hexamethylenetetraamine-hydrochloric acid ((CH2 )6 N4 -HCl), sodium borate (Na2 B4 O7 ), citric acid-NaOH and sodium bicarbonate-sodium hydroxide (NaHCO3 -NaOH) had been tested in several temperatures. From the experiment results it can be seen that A reached its maximum in the medium of phosphoric acid at room temperature. Therefore a solution of 0.5 M phosphoric acid was prepared. The effect of acidity on A was tested and the results are given in Fig. 3. Experimental results show that when 1 ml of acid is used A has a maximum of sensitivity. Therefore we selected 1 ml of phosphoric acid as optimum acidity value. 3.3. Effect of oxidant The effects of five oxidants such as hydrogen peroxide, ammonium peroxodisulfate, potassium iodate, potassium bromate and potassium periodate were tested on A. When potassium iodate (KIO3 ) was used as the oxidant, A reached its maximum. The effect of volume of KIO3 0.02 M (0.2–3.5 ml) on A had been tested and the results are given in Fig. 4 that shows by increasing the dosage of iodate up to 2.7 ml the sensitivity (A = Ab − As ) increased, whereas greater dosage of iodate caused decreasing sensitivity. This effect
Fig. 3. Effect of acidity on the A value. Condition: Thiourea: 2 g/ml, janus green (1 × 10−3 M): 1 ml, KIO3 (0.02 M): 2 ml, Triton X-100 (5 g/l): 2 ml, time: 15 min and temperature: 45 ◦ C.
Fig. 4. Effect of oxidant volume on the A value. Condition: Thiourea: 2 g/ml, phosphoric acid (0.5 M): 1 ml, janus green: 1 ml, Triton X-100: 2 ml, time: 15 min and temperature: 45 ◦ C.
is due to the fact that blank reaction rate becomes faster and sensitivity decreases. So 2.7 ml of 0.02 M of KIO3 was selected as the optimum experimental condition. 3.4. Effect of indicator When the other reaction factors were fixed, the effect of janus green (1 × 10−3 M) dosage on A was studied. When the dosage of janus green increased, A increased correspondingly; when volume of janus green was more than 0.6 ml, A would decrease, thus 0.6 ml was selected as the optimum dosage of indicator. The results are shown in Fig. 5.
Fig. 5. Effect of indicator on the A value. Condition: Thiourea: 2 g/ml acid phosphoric: 1 ml KIO3 : 2.7 ml, Triton X-100: 2 ml, time: 15 min and temperature: 45 ◦ C.
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Fig. 6. Effect of Triton X-100 volume on the A value. Conditions: Thiourea: 2 g/ml, phosphoric acid: 1 ml, janus green: 0.6 ml, KIO3 : 2.7 ml, time: 15 min and temperature: 45 ◦ C.
3.5. Effect of micellar medium The reactors were studied in two different conditions: (1) in micellar medium; (2) without micellar medium. The results show that the A reaches to its maximum in micellar medium. Therefore, effects of seven kinds of surfactants were tested on A: Triton X-114, Tween-20, cetyl trimethyl ammonium bromide (CTAB), cetyl pyridine bromide (CPB), Triton X-100, tetradecyl pyridine bromide (TPB) and 2,2 -pyridine. We found that Triton X100 in the concentration of 5 g/l was the best one. Fig. 6 shows when the volume of Triton X-100 was 1.9 ml A reached its maximum, thus 1.9 ml Triton X-100 was the optimum experimental factor.
Fig. 8. Effect of time on the A value. Conditions: Thiourea: 2 g/ml, phosphoric acid: 1 ml, janus green: 0.6 ml, KIO3 : 2.7 ml, Triton X-100: 1.9 ml and temperature: 45 ◦ C.
0.012 in the range of 15–60 min. Therefore, 15 min was selected as the optimum reaction time. 4. Calibration graph, detection limit and precision Under the optimum experimental conditions, the difference in absorbance between blank and sample varied linearly with the concentration of thiourea in the range of 0.03–10.00 g/ml and fitted the equation: A = 0.0605C + 0.0510 with a square correlation coefficient (r2 ) of 0.9997, where C is the thiourea concentration expressed in g/ml. The detection limit calculated from three times the standard deviation of the blank divided by the slope of the calibration graph was 0.02 g/ml. The relative standard deviation (n = 6) was 3.11% for the determination of 0.1 g/ml and 2.22% for the determination of 6.0 g/ml of thiourea. 5. Interference study
3.6. Effect of temperature By changing the temperature and studying its effect on A, according to the results shown in Fig. 7, we can see that A is linearly increased when temperature increases to 45 ◦ C. The A almost has a constant value in the temperature range of 45–55 ◦ C. When the temperature is higher than 55 ◦ C, A decreases terribly with the rising temperature. However, in our experiment 45 ◦ C was taken as the best temperature for reaction. 3.7. Effect of time As shown in Fig. 8, when the reaction time is between 0 and 15 min, A is linearly correlating to the reaction time. After 15 min the change in A is very little with time, when the time is longer than 60 min A will be constant and have no tendency to increase. The A reaches its maximum in 60 min. The change in A is only
The effects of various interfering species, which may accompany thiourea in fruit juice and industrial waste water, were studied using 1.0 g/ml thiourea. These species tolerated at reasonably high Table 1 Interference study for the determination of thiourea. Species
Tolerance limit
Zn2+ , Cd2+ , K+ , Mn2+ , Ba2+ , NO3 − , EDTA, fructose Mn4+ , methanol, ethanol, glucose, tartaric acid Cl− , ClO4 − , H2 PO4 − Ni2+ Bi3+ , urea Na+ , Al3+ Cr3+ , Ca2+ Citric acid Mg2+ , CH3 COO− , C2 O4 2− , PO4 3− Li+ NH4 + , SO4 2− Br− , HCO3 − , Cu2+ , BrO3 − Fe3+ Fe2+ , F− , NO2 − , S2 O3 2− Pb2+ a (400)b SCN− c (500) I− d , S2 O3 2− e (400) Ascorbic acidf , CN− g (600) Hg2+ a , Ag+ h (300)
2000 1200 1000 900 800 600 500 400 220 180 140 60 30 20 12 5 4 2 0.5
a
Masking with 0.1% EDTA [30,31]. The numbers in brackets indicate the tolerance limit after removal the interference. c Can be removed by adding the KI solution [22]. d Can be removed by adding the 0.035% mercuric nitrate [32]. e Can be eliminated by adding the MnO2 [33]. f Can be removed by refluxing the sample for few minutes [1]. g Can be removed by adding the NiNO3 [34]. h Masking with dithizone [35]. b
Fig. 7. Effect of temperature on the reaction speed. Conditions: Thiourea: 2 g/ml, phosphoric acid: 1 ml, janus green: 0.6 ml, KIO3 : 2.7 ml, Triton X-100: 1.9 ml and time: 15 min.
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Table 2 Recovery of thiourea in real samples. Average recovery (%)
Relative error (%)
Referencea method (n = 5)
0.02 0.03 0.03 0.04
104.0 101.7 100.8 99.3
1.64 0.85 0.37 0.40
1.00 ± 0.03 3.60 ± 0.02 8.14 ± 0.05 10.00 ± 0.06
± ± ± ±
0.02 0.07 0.08 0.13
94.0 97.4 98.9 96.1
1.84 2.09 0.98 1.33
1.06 ± 0.05 3.63 ± 0.05 8.03 ± 0.10 10.09 ± 0.09
1.04 3.56 7.91 9.85
± ± ± ±
0.03 0.05 0.06 0.09
104.0 101.7 98.9 98.5
3.33 1.47 0.76 0.92
1.10 ± 0.02 3.63 ± 0.06 8.02 ± 0.07 10.03 ± 0.05
1.73 2.66 5.69 10.17
± ± ± ±
0.08 0.09 0.10 0.13
– 99.3 100.0 129.2
4.36 3.30 1.77 1.26
1.90 ± 0.06 2.78 ± 0.08 5.84 ± 0.06 10.26 ± 0.09
Sample
Amount added
Amount found (n = 4)
Orange juice (g/ml)
1.0 3.5 8.0 10.0
1.04 3.56 8.05 9.93
± ± ± ±
Lemon juice (g/ml)
1.0 3.5 8.0 10.0
0.94 3.41 7.91 9.61
Tangerine juice (g/ml)
1.0 3.5 8.0 10.0
Waste water (g/ml) a
0.05 1.0 4.0 8.0
Standard pulse polarographic method, Ref. [2].
concentration show high selectivity of the proposed method. The maximum tolerable concentration of foreign species is shown in Table 1, where the tolerance limit was defined as the concentration of foreign species that produced a change in the A less than 5%. According to the results, except Ag+ and Hg2+ , no ion interferes at the same level of thiourea, so the method has good selectivity. 6. Application Because of its high sensitivity and selectivity, the proposed method was directly applied to the determination of fruit juice and industrial waste water samples. Thiourea was determined in real samples using standard addition method. The recovery results for the analysis of some real samples are shown in Table 2. 7. Conclusion A simple, relatively rapid, low cost, highly sensitive and selective method is proposed for the determination of trace levels of thiourea based on its catalytic effect on the oxidation of janus green in phosphoric acid media and presence of Triton X-100. The method does not require any separation or pre-concentration steps and was directly applied to the determination of trace levels of thiourea in fruit juice and industrial waste water. The result in Table 2 shows that the method is accurate and gives good recoveries of added thiourea. As can be seen, the assay results for all the real samples were in good agreement with the values that were obtained by standard pulse polarographic method [2]. The advantageous sensitivity and selectivity make proposed method a favorable competitor to the previously published methods for the determination of thiourea. Acknowledgements The authors are grateful to the Ilam University Research Council for financial support of the project. We thank the referee for his helpful comments.
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Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
A kinetic method for the determination of thiourea by its catalytic effect in micellar media Shahryar Abbasi a,∗ , Hossein Khani a , Mohammad Bagher Gholivand b , Ali Naghipour a , Abbas Farmany a , Freshteh Abbasi c a b c
Chemistry Department, Faculty of Science, Ilam University, Ilam, Iran Chemistry Department, Faculty of Science, Razi University, Kermanshah, Iran Chemistry Department, Faculty of Science, Azad University of Ilam, Ilam, Iran
a r t i c l e
i n f o
Article history: Received 2 July 2008 Received in revised form 6 September 2008 Accepted 26 September 2008 Keywords: Thiourea Janus green Catalytic Spectrophotometry Micellar medium
a b s t r a c t A highly sensitive, selective and simple kinetic method was developed for the determination of trace levels of thiourea based on its catalytic effect on the oxidation of janus green in phosphoric acid media and presence of Triton X-100 surfactant without any separation and pre-concentration steps. The reaction was monitored spectrophotometrically by tracing the formation of the green-colored oxidized product of janus green at 617 nm within 15 min of mixing the reagents. The effect of some factors on the reaction speed was investigated. Following the recommended procedure, thiourea could be determined with linear calibration graph in 0.03–10.00 g/ml range. The detection limit of the proposed method is 0.02 g/ml. Most of foreign species do not interfere with the determination. The high sensitivity and selectivity of the proposed method allowed its successful application to fruit juice and industrial waste water. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Thiourea (TU) has various industrial, agricultural and analytical applications. This material is widely used in photography as a fixing agent and also removes stains from negative. In agriculture, it is used as fungicides, herbicides and rodenticides [1] and also to decrease the content of nitrifying bacteria in soil [2]. TU is also used for induction of early ripening in several fruits [3]. In analytical chemistry TU is used as a spectrophotometric reagent for the determination of several metals [4]. TU is also used as a reagent for copper electrolytes refinery [5]. The toxic effects of TU seem to arise from a disturbance of carbohydrate metabolism and could result in chronic goitrogenic and other glandular difficulties in humans. Furthermore, TU and its derivatives have also been screened as allergenic and carcinogenic factors [6]. Therefore, its determination, taking into account the detection limit must be considerably below 76 ppb. A method for the determination of TU in waste water meets three main problems: (1) The samples of waste water examined contain a large number of organic components and some of these are present in relatively large amounts, at ppm level or even more.
∗ Corresponding author. Fax: +98 841 2227022. E-mail address: sh [email protected] (S. Abbasi). 1386-1425/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.saa.2008.09.029
(2) TU is very soluble in water and practically insoluble in typical extraction solvent such as diethyl ether and chloroform or methylene chloride. Its extraction is therefore not possible. For the same reason, solid phase extraction does not work, as only a small fraction can be adsorbed on the extraction cartridge. (3) TU is poorly volatile and is not easily analyzed by gas chromatography [7]. Various methods have been proposed for the determination of TU such as titrimetry with iodine [8], N-bromosuccinimdie [9] or haloamines [10], Raman spectroscopy [11], spectrophotometry [12,13], polarography [14], voltammetry [4,15], high performance liquid chromatography [16,17], kinetic methods [18], ion selective electrode potentiometry [19], FTIR [20], flow injections methods [1,21,22], amperometric methods [23–25], chemiluminescence [4] and tandem mass spectrometry [7]. However, most of these methods are either insensitive [5,7,10,11,19,23], inselective [1,3,8,22] or time consuming [3,8,12,22]. The current paper describes an original method for the highly sensitive, selective and precise determination of trace levels of TU based on its catalytic effect on the oxidation of janus green with potassium iodate (KIO3 ) in the micellar medium. The micelle has low surface tension and strong soluble power [26], so it can enhance the rate of reaction [27]. The method was conveniently applied to the determination of TU in fruit juice and industrial waste water.
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2. Experimental 2.1. Apparatus A double beam UV–vis spectrophotometer Scinco model S-3100 (South Korea) with 10 mm quartz cell was used for the recording the absorption spectra. A photometer (Metrohm, Model 662) was used to measure the absorbance change at 617 nm. A thermostat water bath was used to keep the reaction temperature at 45 ◦ C. Eppendorf Vary-pipettes (10–100 and 100–1000 l) were used to deliver accurate volume. All glassware and storage bottles were soaked in 10% HNO3 overnight and thoroughly rinsed with water prior to use. 2.2. Reagents and chemical All chemical reagents were of analytical grade and were purchased from Merck (Darmstadt, Germany), Aldrich (Milwaukee, WI, USA) or Fluka (Buchs, Switzerland). All solutions were made in doubly distilled water. Stock standard solution of thiourea (1000 g/ml) was prepared by dissolving 0.1 g of thiourea in water and diluting to 100 ml in a volumetric flask. Working standard solutions were obtained by appropriately diluting the stock solution before use. Janus green solution (1 × 10−3 M) was prepared by dissolving 0.0511 g of it in 100 ml water. Iodate stock’s solution (0.02 M) was prepared by dissolving the required amount of potassium iodate in water. A phosphoric acid solution (0.5 M) was prepared from 85% purity reagent. Triton X-100 solution (5 g/l) was prepared by dissolving 5 g of Triton X-100 in water and diluting to 1 l in a volumetric flask. 2.3. Recommended procedure Into a 25 ml volumetric flask, 1 ml phosphoric acid (0.5 M), 0.6 ml janus green (1 × 10−3 M) and appropriate amount of thiourea (0.03–10.00 g/ml) were transferred. Then 1.9 ml of Triton X-100 and 2.7 ml of KIO3 (0.02 M) were added and solution was diluted
Fig. 1. Structure of janus green (3-diethylamino-7-(4-dimethylaminophenylazo)-5phenylphenazinium chloride).
Then 1 ml of HCl (1 M) was added and solution was filtered again and centrifuged for 15 min in 3500 rpm. 5 ml of final solution was transferred to volumetric flask, diluted to 50 ml and analyzed following the recommended procedure. 3. Results and discussion Janus green (Fig. 1) is a basic dye of mono-azo group used in cytology for specific supravital staining of mitochondria [36]. It is cationic dye, which tends to form ion pair with bulky anions, so it is used as an excellent carrier for the transport of copper as Cu(SCN)4 2− ion through a bulk liquid membrane [37]. Janus green can be oxidized with strong oxidizing agents such as BrO3 − and IO3 − at slow reaction [29]. Thiourea can increase the rate of this reaction at trace levels. The mechanism of the janus green oxidation seems to contribute to the following reactions in its simplest form IO3 − + H+ + janus green (Red) → janus green (OX)
(1)
6H2 NCSNH2 + IO3 − + 6H+ → 3[CS(NH2)2]2 2+ + I− + H2 O
(2)
IO3 − + 5I− + 6H+ → 3I2 + 3H2 O
(3)
(4)
(5) with water to 25 ml, shaken and left in (45 ± 0.5) ◦ C bath water for 15 min. The absorbance of this catalyzed reaction was labeled as As and measured at 617 nm. The same procedure was repeated without addition of thiourea to get the blank signal (uncatalyzed reaction) and the signal was labeled as Ab . The calibration graph was constructed by plotting A = Ab − As vs. thiourea concentration. 2.4. Procedure for real samples analysis Fruits were purchased from a supermarket and juices were obtained by squeezing them [3]. The juices were filtered using Whatman 42 paper (semi-analytical) and centrifuged for 15 min at 3500 rpm. After refluxing (for removing the ascorbic acid interference [28]) for 10 min, 1 ml of this solution was transferred to a volumetric flask, diluted to 50 ml and analyzed following the recommended procedure. A collected waste water sample from texture factory was filtered using Whatman 42 paper (semi-analytical), 1 ml of H2 SO4 (1 M) was added and the solution was shaken well.
where Red is reduced form and OX is the oxidized form of janus green. Because reaction (1) is very slow, in the absence of thiourea, IO3 − oxidized janus green to produce weak reduce in absorbance of dye. When thiourea was added to this system, reactions (2) [11], (3) and (5) were fast. The H2 NCSIN⊕ H2 generated in situ in step (4) increased the rate of oxidation of janus green. For finding the optimum experimental condition, the effect of acidity, IO3 − concentration, janus green concentration, volume of Triton X-100, temperature and time on the reaction rate were studied. 3.1. The absorption spectra As shown in Fig. 2, the absorption spectra, both catalyzed (Fig. 2b–d) and unanalyzed (Fig. 2a), reach their maximum at 617 nm reaction systems, and their difference also reaches its maximum at 617 nm. As a result, we selected 617 nm as the measuring wavelength.
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Fig. 2. Variation of absorbance spectra with thiourea concentration. Condition: (a) Acid phosphoric (0.5 M): 1 ml, janus green (1 × 10−3 M): 0.5 ml, Triton X-100 (5 g/l): 2.0 ml, KIO3 (0.02 M): 2.0 ml. (b) a + 4.00 g/ml thiourea. (c) a + 7.00 g/ml thiourea. (d) a + 10.00 g/ml thiourea.
3.2. Effect of reaction medium Several reaction media such as sulfuric acid (H2 SO4 ), hydrochloric acid (HCl), phosphoric acid (H3 PO4 ), boric acid (H3 BO3 ), sodium hydroxide (NaOH), ammonium-chloride-ammonia (NH4 Cl-NH3 ), potassium acid phthalate-NaOH, acetic acid-sodium acetate (CH3 COOH-CH3 COONa), hexamethylenetetraamine-hydrochloric acid ((CH2 )6 N4 -HCl), sodium borate (Na2 B4 O7 ), citric acid-NaOH and sodium bicarbonate-sodium hydroxide (NaHCO3 -NaOH) had been tested in several temperatures. From the experiment results it can be seen that A reached its maximum in the medium of phosphoric acid at room temperature. Therefore a solution of 0.5 M phosphoric acid was prepared. The effect of acidity on A was tested and the results are given in Fig. 3. Experimental results show that when 1 ml of acid is used A has a maximum of sensitivity. Therefore we selected 1 ml of phosphoric acid as optimum acidity value. 3.3. Effect of oxidant The effects of five oxidants such as hydrogen peroxide, ammonium peroxodisulfate, potassium iodate, potassium bromate and potassium periodate were tested on A. When potassium iodate (KIO3 ) was used as the oxidant, A reached its maximum. The effect of volume of KIO3 0.02 M (0.2–3.5 ml) on A had been tested and the results are given in Fig. 4 that shows by increasing the dosage of iodate up to 2.7 ml the sensitivity (A = Ab − As ) increased, whereas greater dosage of iodate caused decreasing sensitivity. This effect
Fig. 3. Effect of acidity on the A value. Condition: Thiourea: 2 g/ml, janus green (1 × 10−3 M): 1 ml, KIO3 (0.02 M): 2 ml, Triton X-100 (5 g/l): 2 ml, time: 15 min and temperature: 45 ◦ C.
Fig. 4. Effect of oxidant volume on the A value. Condition: Thiourea: 2 g/ml, phosphoric acid (0.5 M): 1 ml, janus green: 1 ml, Triton X-100: 2 ml, time: 15 min and temperature: 45 ◦ C.
is due to the fact that blank reaction rate becomes faster and sensitivity decreases. So 2.7 ml of 0.02 M of KIO3 was selected as the optimum experimental condition. 3.4. Effect of indicator When the other reaction factors were fixed, the effect of janus green (1 × 10−3 M) dosage on A was studied. When the dosage of janus green increased, A increased correspondingly; when volume of janus green was more than 0.6 ml, A would decrease, thus 0.6 ml was selected as the optimum dosage of indicator. The results are shown in Fig. 5.
Fig. 5. Effect of indicator on the A value. Condition: Thiourea: 2 g/ml acid phosphoric: 1 ml KIO3 : 2.7 ml, Triton X-100: 2 ml, time: 15 min and temperature: 45 ◦ C.
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Fig. 6. Effect of Triton X-100 volume on the A value. Conditions: Thiourea: 2 g/ml, phosphoric acid: 1 ml, janus green: 0.6 ml, KIO3 : 2.7 ml, time: 15 min and temperature: 45 ◦ C.
3.5. Effect of micellar medium The reactors were studied in two different conditions: (1) in micellar medium; (2) without micellar medium. The results show that the A reaches to its maximum in micellar medium. Therefore, effects of seven kinds of surfactants were tested on A: Triton X-114, Tween-20, cetyl trimethyl ammonium bromide (CTAB), cetyl pyridine bromide (CPB), Triton X-100, tetradecyl pyridine bromide (TPB) and 2,2 -pyridine. We found that Triton X100 in the concentration of 5 g/l was the best one. Fig. 6 shows when the volume of Triton X-100 was 1.9 ml A reached its maximum, thus 1.9 ml Triton X-100 was the optimum experimental factor.
Fig. 8. Effect of time on the A value. Conditions: Thiourea: 2 g/ml, phosphoric acid: 1 ml, janus green: 0.6 ml, KIO3 : 2.7 ml, Triton X-100: 1.9 ml and temperature: 45 ◦ C.
0.012 in the range of 15–60 min. Therefore, 15 min was selected as the optimum reaction time. 4. Calibration graph, detection limit and precision Under the optimum experimental conditions, the difference in absorbance between blank and sample varied linearly with the concentration of thiourea in the range of 0.03–10.00 g/ml and fitted the equation: A = 0.0605C + 0.0510 with a square correlation coefficient (r2 ) of 0.9997, where C is the thiourea concentration expressed in g/ml. The detection limit calculated from three times the standard deviation of the blank divided by the slope of the calibration graph was 0.02 g/ml. The relative standard deviation (n = 6) was 3.11% for the determination of 0.1 g/ml and 2.22% for the determination of 6.0 g/ml of thiourea. 5. Interference study
3.6. Effect of temperature By changing the temperature and studying its effect on A, according to the results shown in Fig. 7, we can see that A is linearly increased when temperature increases to 45 ◦ C. The A almost has a constant value in the temperature range of 45–55 ◦ C. When the temperature is higher than 55 ◦ C, A decreases terribly with the rising temperature. However, in our experiment 45 ◦ C was taken as the best temperature for reaction. 3.7. Effect of time As shown in Fig. 8, when the reaction time is between 0 and 15 min, A is linearly correlating to the reaction time. After 15 min the change in A is very little with time, when the time is longer than 60 min A will be constant and have no tendency to increase. The A reaches its maximum in 60 min. The change in A is only
The effects of various interfering species, which may accompany thiourea in fruit juice and industrial waste water, were studied using 1.0 g/ml thiourea. These species tolerated at reasonably high Table 1 Interference study for the determination of thiourea. Species
Tolerance limit
Zn2+ , Cd2+ , K+ , Mn2+ , Ba2+ , NO3 − , EDTA, fructose Mn4+ , methanol, ethanol, glucose, tartaric acid Cl− , ClO4 − , H2 PO4 − Ni2+ Bi3+ , urea Na+ , Al3+ Cr3+ , Ca2+ Citric acid Mg2+ , CH3 COO− , C2 O4 2− , PO4 3− Li+ NH4 + , SO4 2− Br− , HCO3 − , Cu2+ , BrO3 − Fe3+ Fe2+ , F− , NO2 − , S2 O3 2− Pb2+ a (400)b SCN− c (500) I− d , S2 O3 2− e (400) Ascorbic acidf , CN− g (600) Hg2+ a , Ag+ h (300)
2000 1200 1000 900 800 600 500 400 220 180 140 60 30 20 12 5 4 2 0.5
a
Masking with 0.1% EDTA [30,31]. The numbers in brackets indicate the tolerance limit after removal the interference. c Can be removed by adding the KI solution [22]. d Can be removed by adding the 0.035% mercuric nitrate [32]. e Can be eliminated by adding the MnO2 [33]. f Can be removed by refluxing the sample for few minutes [1]. g Can be removed by adding the NiNO3 [34]. h Masking with dithizone [35]. b
Fig. 7. Effect of temperature on the reaction speed. Conditions: Thiourea: 2 g/ml, phosphoric acid: 1 ml, janus green: 0.6 ml, KIO3 : 2.7 ml, Triton X-100: 1.9 ml and time: 15 min.
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Table 2 Recovery of thiourea in real samples. Average recovery (%)
Relative error (%)
Referencea method (n = 5)
0.02 0.03 0.03 0.04
104.0 101.7 100.8 99.3
1.64 0.85 0.37 0.40
1.00 ± 0.03 3.60 ± 0.02 8.14 ± 0.05 10.00 ± 0.06
± ± ± ±
0.02 0.07 0.08 0.13
94.0 97.4 98.9 96.1
1.84 2.09 0.98 1.33
1.06 ± 0.05 3.63 ± 0.05 8.03 ± 0.10 10.09 ± 0.09
1.04 3.56 7.91 9.85
± ± ± ±
0.03 0.05 0.06 0.09
104.0 101.7 98.9 98.5
3.33 1.47 0.76 0.92
1.10 ± 0.02 3.63 ± 0.06 8.02 ± 0.07 10.03 ± 0.05
1.73 2.66 5.69 10.17
± ± ± ±
0.08 0.09 0.10 0.13
– 99.3 100.0 129.2
4.36 3.30 1.77 1.26
1.90 ± 0.06 2.78 ± 0.08 5.84 ± 0.06 10.26 ± 0.09
Sample
Amount added
Amount found (n = 4)
Orange juice (g/ml)
1.0 3.5 8.0 10.0
1.04 3.56 8.05 9.93
± ± ± ±
Lemon juice (g/ml)
1.0 3.5 8.0 10.0
0.94 3.41 7.91 9.61
Tangerine juice (g/ml)
1.0 3.5 8.0 10.0
Waste water (g/ml) a
0.05 1.0 4.0 8.0
Standard pulse polarographic method, Ref. [2].
concentration show high selectivity of the proposed method. The maximum tolerable concentration of foreign species is shown in Table 1, where the tolerance limit was defined as the concentration of foreign species that produced a change in the A less than 5%. According to the results, except Ag+ and Hg2+ , no ion interferes at the same level of thiourea, so the method has good selectivity. 6. Application Because of its high sensitivity and selectivity, the proposed method was directly applied to the determination of fruit juice and industrial waste water samples. Thiourea was determined in real samples using standard addition method. The recovery results for the analysis of some real samples are shown in Table 2. 7. Conclusion A simple, relatively rapid, low cost, highly sensitive and selective method is proposed for the determination of trace levels of thiourea based on its catalytic effect on the oxidation of janus green in phosphoric acid media and presence of Triton X-100. The method does not require any separation or pre-concentration steps and was directly applied to the determination of trace levels of thiourea in fruit juice and industrial waste water. The result in Table 2 shows that the method is accurate and gives good recoveries of added thiourea. As can be seen, the assay results for all the real samples were in good agreement with the values that were obtained by standard pulse polarographic method [2]. The advantageous sensitivity and selectivity make proposed method a favorable competitor to the previously published methods for the determination of thiourea. Acknowledgements The authors are grateful to the Ilam University Research Council for financial support of the project. We thank the referee for his helpful comments.
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