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Fluorimetric determination of ascorbic acid with o-phenylenediamine
Talanta 59 (2003) 95 /99 www.elsevier.com/locate/talanta
Fluorimetric determination of ascorbic acid with ophenylenediamine Xia Wu, Yuxia Diao, Changxia Sun, Jinghe Yang , Yuebo Wang, Shuna Sun Key Laboratory for Colloid and Interface Chemistry of Education Ministry, Department of Chemistry, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, People’s Republic of China Received 28 January 2002; received in revised form 7 August 2002; accepted 23 August 2002
Abstract A simple and sensitive fluorimetric method for the determination of ascorbic acid (AA) is described. The method is based on the condensation reaction between AA and o -phenylenediamine (OPDA) in the absence of the oxidant. The fluorescence intensity is measured at excitation and emission wavelengths of 360 and 430 nm, respectively. Under optimum condition, a linear relationship is obtained between the fluorescence intensity and the concentration of AA in the range of 0.05 /40 mg ml 1. The detection limit is 0.006 mg ml 1, which is obviously lower than that of other fluorimetric methods reported. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Fluorimetric determination; Ascorbic acid (AA); o -Phenylenediamine (OPDA)
1. Introduction Ascorbic acid (AA) is essential to the health of man. Numerous analytical procedures have been included in spectrometry [1], chemiluminescence [2], HPLC [3], enzymatic method [4], flow-injection spectrophotometry [5,6] and amperometric method [7]. Recently, fluorimetric determinations of AA have been developed based on the condensation reactions of AA with o -phenylenedia-
Corresponding author. Fax: /86-931-891-2582 E-mail address: [email protected] (J. Yang).
mine (OPDA) [8], 2,3-diaminonaphthalene (DAN) [9] and 2-cyanoacetamide [10], their detection limits are 2, 0.2 and 0.03 mg ml 1, respectively. In the OPDA method, the oxidant, such as norit (carbon), 2,6-dichloroindoindophenol, N -bromosuccinimide and iodone, must be used to oxidized AA to DHAA, which then reacts with OPDA. Due to the blank effect caused by the oxidant, thus the sensitivity of OPDA method is low. In this paper, it is found that at pH 9.4, AA can react with OPDA in the absence of the oxidant and forms a condensation product that can emit strong fluorescence. The detection limit of this method is 0.006 mg ml 1, which is obviously lower than that of other fluorimetric methods reported.
0039-9140/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 3 9 - 9 1 4 0 ( 0 2 ) 0 0 4 7 5 - 7
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X. Wu et al. / Talanta 59 (2003) 95 /99
2. Experimental 2.1. Apparatus All fluorescence measurements are made on a RF-540 (Shimadzu, Japan) fluorescence spectrophotometer with 150 w Xe lamp and 1 cm quartz cell. All pH measurements are made with a pHs-2 acidity meter (Leici, Shanghai). 2.2. Reagents 2.2.1. Ascorbic acid solution AA solution is prepared by dissolving 0.100 g AA in 100 ml distilled water and stored in a brown vessel. The stock solution is 1.00 mg ml 1. 2.2.2. o -Phenylenediamine (OPDA) solution OPDA solution is prepared by dissolving 0.5 g OPDA in 100 ml 0.1 mol l 1 HCl (0.5%, w/v) and stored in a brown vessel and is preserved at 4 8C. 2.2.3. Buffer solution NH3 /NH4Cl buffer solution is prepared by dissolving 13.5 g NH4Cl and 15.75 ml ammonia spirit in 100 ml distilled water and adjusted the pH to 9.4 with 0.1 mol l 1 NaOH. 2.3. Procedure To a 25 ml test tube, solutions are added according to the following order: AA solution, 1.0 ml of 0.5% OPDA, 1.5 ml of NH3 /NH4Cl buffer solution. The mixture is diluted to 10 ml with distilled water, thoroughly mixed by shaking and then allowed to stand for 30 min. The fluorescence intensity is measured in a 1 cm quartz cell with excitation and emission wavelengths of 360 and 430 nm, respectively.
Fig. 1. Absorption Spectra (1) AA /NH3 /NH4Cl; (2) OPDA / NH3 /HN4Cl; (3) AA /OPDA /NH3 /HN4Cl; Conditions: AA, 0.01 mg ml 1; OPDA, 0.05% (w/v) 1 ml; NH3 /NH4Cl, pH 9.4 1.5 ml.
whereas AA-OPDA /NH3 /NH4Cl system has strong absorption band with the peak of 360 nm. This indicates that AA can react with OPDA in alkaline medium. The excitation and emission spectra of AA /OPDA /NH3 /NH4Cl system are shown in Fig. 2. It can be seen that the AA / OPDA /NH3 /NH4Cl system has strong fluorescence, the wavelengths of excitation and emission peaks are 360 and 430 nm, respectively, while the fluorescence intensity of the OPDA /NH3 /NH4Cl system is very low and stable. Therefore, the reagent blank of the proposed method (without the oxidant) is considerably lower than that of the OPDA method (with the oxidant) [8]. 3.2. Factors affecting the fluorescence intensity
3.1. Absorption and fluorescence spectra
3.2.1. Effect of pH The effect of pH on fluorescence intensity of the system is shown in Fig. 3. It can be seen that the fluorescence intensity is the strongest and remained stable in the range of pH 9.3 /9.5. In this study, the following buffers are examined: borateNaOH, K2HPO4, NH3 /NH4Cl and 0.1 mol l 1 NaOH. The results show that 1.5 ml of NH3 / NH4Cl buffer solution is the most suitable.
From Fig. 1 it can be seen that both AA /NH3 / NH4Cl and OPDA /NH3 /NH4Cl systems have no absorption band in the range of 330 /390 nm,
3.2.2. Effect of OPDA concentration The effect of OPDA concentration on fluorescence intensity of the system is examined. The
3. Results and discussion
X. Wu et al. / Talanta 59 (2003) 95 /99
97
Fig. 2. Fluorescence spectra: (a) excitation spectra (lex /360 nm); (b) emission spectra (lem /430 nm). (1) OPDA /NH3 /HN4Cl; (2) AA /OPDA /NH3 /NH4Cl. Conditions: AA, 0.01 mg ml 1; OPDA, 0.05% (w/v); NH3 /NH4Cl, pH 9.4 1.5 ml.
Fig. 4. Effect of OPDA concentration. Conditions: AA, 5.0 mg ml 1; NH3 /NH4Cl, pH 9.4, 1.5 ml. Fig. 3. Effect of pH. Conditions: AA, 5.0 mg ml 1; OPDA, 0.05% (w/v).
results are shown in Fig. 4. It can be seen that the suitable concentration of OPDA is 0.05%(w/v). 3.2.3. Effect of temperature It is well known that high temperature (about /40 8C) can destroy the structure of AA and
quench the fluorescence of the system. Our experiments indicate that suitable temperature is 25 8C or so.
3.2.4. Stable tests The effect of the reaction time is also studied in this experiment. The results show that at room temperature the fluorescence intensity of the system reaches a maximum in 30 min after all
98
X. Wu et al. / Talanta 59 (2003) 95 /99
Table 1 Effect of molecules and ions Ions or molecules
Tolerance limit (mg ml 1)
Vitamin B1 Vitamin B2 Vitamin B5 Na Mg2 Ca2 Fe3 Zn2 SO2 4 CO2 3 Cl
18 0.2 20 1000 480 1640 11 1365 3400 180 4400
the reagents are added and remains stable at least for 30 min. 3.2.5. Selectivity The effects of common anions, cations and vitamins on the fluorescence intensity of the system are examined for 10 mg ml 1 AA. The highest permissible mass excesses of these ions and molecules causing a 9/5% relative error in the fluorescence intensity are shown in Table 1. From Table 1, it can be seen that except vitamin B2, the other molecules and ions have little or almost no effects on the system. 3.3. Analytical applications 3.3.1. Calibration curve and detection limit The calibration curve is made according to the procedure under the optimum conditions. The results indicate that the fluorescence intensity of the system is a linear function of AA concentration
Table 2 Comparison of the methods AA found (mg ml 1) The proposed method The 2-cyanoacetamide method [7]
x9/s
1.66, 1.65, 1.62, 1.67, 1.66, 1.64 1.659/0.02 1.75, 1.76, 1.60, 1.68, 1.62, 1.67 1.689/0.07
in the range of 0.05 /40 mg ml 1. The regression coefficient is 0.9951 and the detection limit is 0.006 mg ml 1, which is lower than that of other fluorescence methods reported (Table 3). 3.3.2. Sample determination The proposed method is used to determine AA in Vitamin C tablets (Jilin Tonghua Baishan Medicine Factory of China) and compared with the 2-cyanoacetamide method [10]. The results are shown in Table 2. It can be seen that the accuracy and precision of the method are satisfactory. 3.4. Mechanism of the fluorescence reaction In the OPDA method, the oxidant must be used to oxidize AA to DHAA, which is unnecessary for this method. We think that AA will not be transferred to DHAA in the absence of the oxidant. It is very known that AA has two isomers: enol-form and keto-form. The possible mechanism is illustrated as follows:
X. Wu et al. / Talanta 59 (2003) 95 /99
99
Table 3 Comparison of sensitivity between the proposed method and other methods Method
Spectrometry Chemiluminescence Flow-injection spectrophotometric method DAN fluorimetric method 2-cyanoacetamide method OPDA fluorimetric method The proposed method a
Redox reagent
Toluidine blue Sodium hydroxide Non Non HPO3 Non
Linear calibration range (mg ml 1) 0.37a 0.001 /300a 1 /50 2 /300 0.1 /50 0.05 /40
Detection limit (mg ml 1) 2 /10 4a 0.2 0.4 0.03 2 0.006
Reference
[1] [2] [5] [8] [9] [10]
Unit is mmol l 1.
Acknowledgements Here, the keto-form of AA reacts with OPDA in alkaline medium and forms an N -heterocyclic compound with large conjugated p-bond system, which can emit strong fluorescence.
4. Conclusion In this paper, it is found that AA can react with OPDA in the absence of the oxidant and form a condensation product, which can emit strong fluorescence. The method can be used for the determination of AA. The detection limit of the proposed method is 0.006 mg ml 1, which is obviously lower than that of other fluorimetric methods reported. In addition, in the proposed method, it is unnecessary to add an oxidant to the reaction system. Therefore, this is a simple, sensitive and accurate method for the determination of AA.
This work is supported by National Science Foundations of China and Shandong Province and by Visiting Scholar Foundation of Key Lab in University.
References [1] S.H.R. Davis, S.J. Masten, Anal. Chim. Acta 248 (1991) 225. [2] T.P. Ruiz, C.M. Lozano, A. Sanz, Anal. Chim. Acta 299 (1995) 308. [3] X. Chen, M. Sato, Anal. Sci. 11 (1995) 749 /754. [4] L.T. Jin, G.Z. Zhao, Y.Z. Fang, Chem. J. Chin. Univ. 15 (1994) 189. [5] A. Jain, A. Chaurasia, K.K. Verma, Talanta 42 (1995) 779. [6] J. Fan, C.L. Ye, S.L. Feng, G. Zhang, J.J. Wang, Talanta 50 (1999) 893 /898. [7] J. Wu, J. Suls, W. Sansen, Electrochem. Commun. 2 (2000) 90 /93. [8] X. Shao, Y. Zhang, Guangpuxue Yu Guangpu Fenxi 14 (1994) 125 /127. [9] J.H. Yang, C.L. Tong, N.Q. Jie, G.L. Zhang, X.Z. Ren, J.T. Hu, Talanta 44 (1997) 855 /858. [10] J.H. Yang, Q.L. Ma, F. Huang, L.M. Sun, J. Dong, Anal. Lett. 31 (1998) 2757 /2766.
Fluorimetric determination of ascorbic acid with ophenylenediamine Xia Wu, Yuxia Diao, Changxia Sun, Jinghe Yang , Yuebo Wang, Shuna Sun Key Laboratory for Colloid and Interface Chemistry of Education Ministry, Department of Chemistry, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, People’s Republic of China Received 28 January 2002; received in revised form 7 August 2002; accepted 23 August 2002
Abstract A simple and sensitive fluorimetric method for the determination of ascorbic acid (AA) is described. The method is based on the condensation reaction between AA and o -phenylenediamine (OPDA) in the absence of the oxidant. The fluorescence intensity is measured at excitation and emission wavelengths of 360 and 430 nm, respectively. Under optimum condition, a linear relationship is obtained between the fluorescence intensity and the concentration of AA in the range of 0.05 /40 mg ml 1. The detection limit is 0.006 mg ml 1, which is obviously lower than that of other fluorimetric methods reported. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Fluorimetric determination; Ascorbic acid (AA); o -Phenylenediamine (OPDA)
1. Introduction Ascorbic acid (AA) is essential to the health of man. Numerous analytical procedures have been included in spectrometry [1], chemiluminescence [2], HPLC [3], enzymatic method [4], flow-injection spectrophotometry [5,6] and amperometric method [7]. Recently, fluorimetric determinations of AA have been developed based on the condensation reactions of AA with o -phenylenedia-
Corresponding author. Fax: /86-931-891-2582 E-mail address: [email protected] (J. Yang).
mine (OPDA) [8], 2,3-diaminonaphthalene (DAN) [9] and 2-cyanoacetamide [10], their detection limits are 2, 0.2 and 0.03 mg ml 1, respectively. In the OPDA method, the oxidant, such as norit (carbon), 2,6-dichloroindoindophenol, N -bromosuccinimide and iodone, must be used to oxidized AA to DHAA, which then reacts with OPDA. Due to the blank effect caused by the oxidant, thus the sensitivity of OPDA method is low. In this paper, it is found that at pH 9.4, AA can react with OPDA in the absence of the oxidant and forms a condensation product that can emit strong fluorescence. The detection limit of this method is 0.006 mg ml 1, which is obviously lower than that of other fluorimetric methods reported.
0039-9140/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 3 9 - 9 1 4 0 ( 0 2 ) 0 0 4 7 5 - 7
96
X. Wu et al. / Talanta 59 (2003) 95 /99
2. Experimental 2.1. Apparatus All fluorescence measurements are made on a RF-540 (Shimadzu, Japan) fluorescence spectrophotometer with 150 w Xe lamp and 1 cm quartz cell. All pH measurements are made with a pHs-2 acidity meter (Leici, Shanghai). 2.2. Reagents 2.2.1. Ascorbic acid solution AA solution is prepared by dissolving 0.100 g AA in 100 ml distilled water and stored in a brown vessel. The stock solution is 1.00 mg ml 1. 2.2.2. o -Phenylenediamine (OPDA) solution OPDA solution is prepared by dissolving 0.5 g OPDA in 100 ml 0.1 mol l 1 HCl (0.5%, w/v) and stored in a brown vessel and is preserved at 4 8C. 2.2.3. Buffer solution NH3 /NH4Cl buffer solution is prepared by dissolving 13.5 g NH4Cl and 15.75 ml ammonia spirit in 100 ml distilled water and adjusted the pH to 9.4 with 0.1 mol l 1 NaOH. 2.3. Procedure To a 25 ml test tube, solutions are added according to the following order: AA solution, 1.0 ml of 0.5% OPDA, 1.5 ml of NH3 /NH4Cl buffer solution. The mixture is diluted to 10 ml with distilled water, thoroughly mixed by shaking and then allowed to stand for 30 min. The fluorescence intensity is measured in a 1 cm quartz cell with excitation and emission wavelengths of 360 and 430 nm, respectively.
Fig. 1. Absorption Spectra (1) AA /NH3 /NH4Cl; (2) OPDA / NH3 /HN4Cl; (3) AA /OPDA /NH3 /HN4Cl; Conditions: AA, 0.01 mg ml 1; OPDA, 0.05% (w/v) 1 ml; NH3 /NH4Cl, pH 9.4 1.5 ml.
whereas AA-OPDA /NH3 /NH4Cl system has strong absorption band with the peak of 360 nm. This indicates that AA can react with OPDA in alkaline medium. The excitation and emission spectra of AA /OPDA /NH3 /NH4Cl system are shown in Fig. 2. It can be seen that the AA / OPDA /NH3 /NH4Cl system has strong fluorescence, the wavelengths of excitation and emission peaks are 360 and 430 nm, respectively, while the fluorescence intensity of the OPDA /NH3 /NH4Cl system is very low and stable. Therefore, the reagent blank of the proposed method (without the oxidant) is considerably lower than that of the OPDA method (with the oxidant) [8]. 3.2. Factors affecting the fluorescence intensity
3.1. Absorption and fluorescence spectra
3.2.1. Effect of pH The effect of pH on fluorescence intensity of the system is shown in Fig. 3. It can be seen that the fluorescence intensity is the strongest and remained stable in the range of pH 9.3 /9.5. In this study, the following buffers are examined: borateNaOH, K2HPO4, NH3 /NH4Cl and 0.1 mol l 1 NaOH. The results show that 1.5 ml of NH3 / NH4Cl buffer solution is the most suitable.
From Fig. 1 it can be seen that both AA /NH3 / NH4Cl and OPDA /NH3 /NH4Cl systems have no absorption band in the range of 330 /390 nm,
3.2.2. Effect of OPDA concentration The effect of OPDA concentration on fluorescence intensity of the system is examined. The
3. Results and discussion
X. Wu et al. / Talanta 59 (2003) 95 /99
97
Fig. 2. Fluorescence spectra: (a) excitation spectra (lex /360 nm); (b) emission spectra (lem /430 nm). (1) OPDA /NH3 /HN4Cl; (2) AA /OPDA /NH3 /NH4Cl. Conditions: AA, 0.01 mg ml 1; OPDA, 0.05% (w/v); NH3 /NH4Cl, pH 9.4 1.5 ml.
Fig. 4. Effect of OPDA concentration. Conditions: AA, 5.0 mg ml 1; NH3 /NH4Cl, pH 9.4, 1.5 ml. Fig. 3. Effect of pH. Conditions: AA, 5.0 mg ml 1; OPDA, 0.05% (w/v).
results are shown in Fig. 4. It can be seen that the suitable concentration of OPDA is 0.05%(w/v). 3.2.3. Effect of temperature It is well known that high temperature (about /40 8C) can destroy the structure of AA and
quench the fluorescence of the system. Our experiments indicate that suitable temperature is 25 8C or so.
3.2.4. Stable tests The effect of the reaction time is also studied in this experiment. The results show that at room temperature the fluorescence intensity of the system reaches a maximum in 30 min after all
98
X. Wu et al. / Talanta 59 (2003) 95 /99
Table 1 Effect of molecules and ions Ions or molecules
Tolerance limit (mg ml 1)
Vitamin B1 Vitamin B2 Vitamin B5 Na Mg2 Ca2 Fe3 Zn2 SO2 4 CO2 3 Cl
18 0.2 20 1000 480 1640 11 1365 3400 180 4400
the reagents are added and remains stable at least for 30 min. 3.2.5. Selectivity The effects of common anions, cations and vitamins on the fluorescence intensity of the system are examined for 10 mg ml 1 AA. The highest permissible mass excesses of these ions and molecules causing a 9/5% relative error in the fluorescence intensity are shown in Table 1. From Table 1, it can be seen that except vitamin B2, the other molecules and ions have little or almost no effects on the system. 3.3. Analytical applications 3.3.1. Calibration curve and detection limit The calibration curve is made according to the procedure under the optimum conditions. The results indicate that the fluorescence intensity of the system is a linear function of AA concentration
Table 2 Comparison of the methods AA found (mg ml 1) The proposed method The 2-cyanoacetamide method [7]
x9/s
1.66, 1.65, 1.62, 1.67, 1.66, 1.64 1.659/0.02 1.75, 1.76, 1.60, 1.68, 1.62, 1.67 1.689/0.07
in the range of 0.05 /40 mg ml 1. The regression coefficient is 0.9951 and the detection limit is 0.006 mg ml 1, which is lower than that of other fluorescence methods reported (Table 3). 3.3.2. Sample determination The proposed method is used to determine AA in Vitamin C tablets (Jilin Tonghua Baishan Medicine Factory of China) and compared with the 2-cyanoacetamide method [10]. The results are shown in Table 2. It can be seen that the accuracy and precision of the method are satisfactory. 3.4. Mechanism of the fluorescence reaction In the OPDA method, the oxidant must be used to oxidize AA to DHAA, which is unnecessary for this method. We think that AA will not be transferred to DHAA in the absence of the oxidant. It is very known that AA has two isomers: enol-form and keto-form. The possible mechanism is illustrated as follows:
X. Wu et al. / Talanta 59 (2003) 95 /99
99
Table 3 Comparison of sensitivity between the proposed method and other methods Method
Spectrometry Chemiluminescence Flow-injection spectrophotometric method DAN fluorimetric method 2-cyanoacetamide method OPDA fluorimetric method The proposed method a
Redox reagent
Toluidine blue Sodium hydroxide Non Non HPO3 Non
Linear calibration range (mg ml 1) 0.37a 0.001 /300a 1 /50 2 /300 0.1 /50 0.05 /40
Detection limit (mg ml 1) 2 /10 4a 0.2 0.4 0.03 2 0.006
Reference
[1] [2] [5] [8] [9] [10]
Unit is mmol l 1.
Acknowledgements Here, the keto-form of AA reacts with OPDA in alkaline medium and forms an N -heterocyclic compound with large conjugated p-bond system, which can emit strong fluorescence.
4. Conclusion In this paper, it is found that AA can react with OPDA in the absence of the oxidant and form a condensation product, which can emit strong fluorescence. The method can be used for the determination of AA. The detection limit of the proposed method is 0.006 mg ml 1, which is obviously lower than that of other fluorimetric methods reported. In addition, in the proposed method, it is unnecessary to add an oxidant to the reaction system. Therefore, this is a simple, sensitive and accurate method for the determination of AA.
This work is supported by National Science Foundations of China and Shandong Province and by Visiting Scholar Foundation of Key Lab in University.
References [1] S.H.R. Davis, S.J. Masten, Anal. Chim. Acta 248 (1991) 225. [2] T.P. Ruiz, C.M. Lozano, A. Sanz, Anal. Chim. Acta 299 (1995) 308. [3] X. Chen, M. Sato, Anal. Sci. 11 (1995) 749 /754. [4] L.T. Jin, G.Z. Zhao, Y.Z. Fang, Chem. J. Chin. Univ. 15 (1994) 189. [5] A. Jain, A. Chaurasia, K.K. Verma, Talanta 42 (1995) 779. [6] J. Fan, C.L. Ye, S.L. Feng, G. Zhang, J.J. Wang, Talanta 50 (1999) 893 /898. [7] J. Wu, J. Suls, W. Sansen, Electrochem. Commun. 2 (2000) 90 /93. [8] X. Shao, Y. Zhang, Guangpuxue Yu Guangpu Fenxi 14 (1994) 125 /127. [9] J.H. Yang, C.L. Tong, N.Q. Jie, G.L. Zhang, X.Z. Ren, J.T. Hu, Talanta 44 (1997) 855 /858. [10] J.H. Yang, Q.L. Ma, F. Huang, L.M. Sun, J. Dong, Anal. Lett. 31 (1998) 2757 /2766.