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Fluorescent reaction between ascorbic acid and DAN and its analytical application
Talanta ELSEVIER
Talanta 44 (1997) 855 858
Fluorescent reaction between ascorbic acid and D A N and its analytical application Jinghe
Y a n g ~"*, C h a n g l u n
Tong
b, N i a n q i n Jingtian
J i e ", G u i l i n g
Zhang
~', X u e z h e n
R e n ",
Hu ~
, Department o1' Chemistry Shandon~ Unirersity, .limm 250100, China b Hangzhou Ererhmg Bioteclmics, Chimt
Received 9 April 1996: accepted 11 October 1996
Abstract Fluorescent reaction between ascorbic acid (AA) and 2,3-diamino-naphthalene (DAN was studied. The experimental results showed that AA could react with DAN at p H = 10.2-10.5, and form the fluorescent heterocyclic condensation products which emitted strong fluorescence. The fluorescence intensity was measured in a I cm quartz celt with excitation and emission wavelengths of 400 and 520 rim, respectively. The relationship was obtained between the fluorescence intensity and AA concentration in the range of 2-300 tag ml ~, the regression coefficient is 0.9993. The detection limit (signal-to-noise = 2) is 0.4 pg ml ~. © 1997 Elsevier Science B.V. Kevwords: Ascorbic acid determination; Fluorescent reaction
I. Introduction M a n y different m e t h o d s for the determination of ascorbic acid (AA) have been reported in the literature. They concentrated on spectrometry [1], redox reaction [2], derivatization reaction [3], electrochemical m e t h o d [4], enzymatic m e t h o d [5], and c h r o m a t o g r a p h i c m e t h o d [6], but few fluorometric estimation m e t h o d s of A A were reported. A classic fluorometric m e t h o d with o-phenylenediamine ( O P D A ) as fluorescence reagent was used to determine A A [7]. This m e t h o d needed to treat with redox reagent beforehand and then reacted * Corresponding author.
AA with O P D A , its detection limit was 2 lag ml ~ Our experiments indicated that at the p H = 1 0 . 2 - - 1 0 . 5 , A A could react with 2,3-dia m i n o n a p h t h a l e n e ( D A N ) which didn't need pretreatment, its detection limit was 0.4 lag ml '. So the p r o p o s e d m e t h o d is m o r e simple and sensitive than O P D A method.
2. Experimental 2.1. A p p a r a t u s
All fluorescence intensities were measured on a 850 fluorescence s p e c t r o p h o t o m e t e r (Hitachi, Japan).
0039-9140'97/S17.00 .'t; 1997 Elsevier Science B.V. All rights reserved. Pll S0039-91 40(96)021 29-7
856
J. Yang et al. / Talanta 44 (1997) 855-858
2.2. Reagents
3.2. Effect o f p H
1. 2,3-diaminonaphthalene (DAN) solution (0.1%, v/v): it is prepared by dissolving 0.1 g D A N in 100 ml 0.1 mol I - ' HC1, then treated with cyclohexane, rejected organic phase, the remained water phase repeated 2 - 3 times with cyclohexane according to the above procedure. 2. Ascorbic acid solution: it is prepared by dissolving 0.100 g AA in 100 ml distilled water. The stock solution was 1.00 mg m l - t 3. Aminoacetic acid buffer solution: it is prepared by dissolving 3.8 g aminoacetic acid in 100 ml distilled water and adjusting pH to 10.3 with 6 mol 1- 1 NaOH.
The effect of pH on fluorescence intensity of the system was shown in Fig. 2. From Fig. 2, it can be seen that the fluorescence intensity is the strongest and remains stable in the range of pH = 10.2-10.5. In this paper, the following buffers were examined: Na2CO3, b o r a x - N a O H , NH3-NHaC1, K2HPO4, aminoacetic acid. The results showed that 2 ml of 0.5 tool 1- ~ aminoacetic acid buffer was the most suitable.
2.3. Procedure
To a 25 ml test tube, solutions were added according to the following order: AA solution, 0.5 ml 0.5 mol 1-1 NaOH, 1 ml 0.1% D A N solution, 2 ml aminoacetic acid buffer. The mixture was diluted to 10 ml with distilled water, thoroughly mixed by shaking and then allowed to stand for 40 min. The fluorescence intensity was measured in a 1 cm quartz cell with excitation and emission wavelengths of 400 and 520 nm, respectively.
3.3. Effect o f D A N concentration
The effect of D A N concentration on fluorescence intensity of the system was studied. The results were shown in Fig. 3. From this figure we can see that the suitable concentration of D A N was 0.01%.
i
3. Results and discussion 3.1. Fluorescence spectra
The excitation and emission spectra of AADAN-aminoacetic acid (1), DAN-aminoacetic acid (2) are shown in Fig. 1. From Fig. 1, it can be seen that the wavelength of emission peak is 520 nm, the wavelengths of excitation peak are 370, 400 nm. Since D A N itself is a strong fluorephore with excitation and emission wavelengths of 340 and 390 nm, if the excitation wavelength is 370 nm, the excess of D A N in the reaction will interfere with the emission of the fluorescence products, therefore, the excitation wavelength of 400 nm was chosen in our experiments and a low and stable reagent blank was obtained.
.A ~m) Fig. 1. Fluorescence spectra (a) excitation spectrum ("~em= 520
nm), (b) emission spectrum (2ex=400 nm). (1) AA-DANaminoacetic acid, (2) DAN-aminoaceticacid conditions: ascorbic acid: 0.2 mg ml - 1, DAN: 0.01%; aminoaceticacid: 0.5 mol l-l, 2 ml, pH=l.03.
J. Yang et al. / Talanta 44 (1997) 855-858
857
was examined, the results showed the enhancement effect was not obvious.
3.6. Calibration curve and detection limit The calibration curve was made according to the procedure in the optimum conditions. The results indicated that the fluorescence intensity of the system was a linear function of AA concentration in the range of 2-300 lag m l - 1, the regression coefficient is 0.9993, the detection limit (S/N = 2) is 0.4 lag ml
3.7. Recover)' test Fig. 2. Effect of pH. Conditions: ascorbic acid: 0.05 mg ml - ~, DAN: 0.01%; aminoacetic acid: 0.5 mol 1 - i , 2 ml.
3.4. Effect of temperature High temperature could destroy the structure o f AA and quench the fluorescence of the system. Our experiments indicted that when the temperature was higher than 50°C, the fluorescence intensity of the system was completely quenched. The suitable temperature was 30°C or so.
3.5. Stable tests The experiments indicated that at room temperature the fluorescence intensity of the system reached a maximum after 40 min and remained stable at least for 1 h. In addition, effect of surfactants of fluorescence intensity of the system
AP
Fig. 3. Effect of DAN concentration. Conditions: ascorbic acid: 0.25 mg ml-~; aminoacetic acid: 0.5 mol 1-t, 2 ml, pH = 10.3.
In the serum, recovery tests of AA were made. The results were shown in Table I. From Table 1, it can be seen that recovery ratios were upwards of 90% and serum didn't need complicated biological treatment. Therefore, the proposed method for determining AA was satisfactory.
3.8. Sample determination The proposed method was used to determine AA in Vc tablets (Jilin Tonghua Baishan Medicine Factory of China) and compared with the most commonly used OPDA method [8]. The results were shown in Table 2. From Table 2, it can be seen that the accuracy and precision of the method are satisfactory.
3.9. The characteristics oJ the proposed method In comparison with the most commonly used O P D A method, the proposed method possesses the following characteristics: 1. The detection limit is 0.4 lag m l - ~, which is obviously lower than that of O P D A method [8]. so the sensitivity of the proposed method is higher. 2. In O P D A method, the redox reagent, such as norit (carbon), 2,6-dichloroindophenol, N-Bromosuccinimide and iodone, must be used to oxidize AA to DHAA, while in this method it is unnecessary to add a redox reagent to the reaction system. We think that AA will not be transferred to D H A A in the absence of the oxidizer
858
J. Yang et al. / Talanta 44 (1997) 855-858
Table 1 Recovery test in the serum Added Vc (gg ml -I)
Found Vc (gg ml l)
X± S
Recovery (%)
20.0 100.0
18.92, 17.95, 18.33, 18.92, 18.95 94,8, 96.1, 94.3, 93.4, 95.2
18.6±0.45 94.8 ± 1.01
93.1 94.8
Table 2 Determination of samples
O P D A method.
The proposed method
OPDA method (8)
Found Vc (%)
X± S
Found Vc (%)
52.2 ± 1.5
52.4, 50.5, 50.9 53.4, 54.2
References 52.3, 50.9, 54.6 50.9, 52.3
,X ± S
52.3 ± 1.6
a n d there is a t a u t o m e r i c between enediol a n d 2-hydroxy c a r b o n y l groups in the alkaline m e d i u m . Therefore, the possible m e c h a n i s m is illustrated as follows.
Ho ~
oH
~lor-'~o
~N
Since the conjugate n - b o n d system o f the condensate p r o d u c t in the p r o p o s e d m e t h o d is larger t h a n that in the O P D A m e t h o d , the sensitivity o f the p r o p o s e d m e t h o d is higher t h a n that o f
[11 S.H.R. Davis and S.J. Masten, Anal. Chim. Acta, 248 (1991) 225. [21 B. Jaselskis and J. Nelapaty, Anal. Chem., 44 (1972) 379. [3] MJ., Deutsch and C.E. Weeks, J. Assoc. Off. Agric. Chem., 48 (1965) 1248. [4] L. Falat and H.Y. Cheng, Anal. Chem., 54 (1982) 2108. [5] J.O. Schenk, E. Miller and R.N. Adams, Anal. Chem., 54 (1982) 1452. [6] E.S. Wagner, B. Lindley and R.D. Coffin, J. Chromatogr., 163 (1979) 225.
"
%~'~<~//~"'~oH
[71 A.P. Lawrence, L.R. Donald and T.K. Peter, J. Assoc. off. Anal. Chem., 68 (1985) 1. [8] R.B. Roy, A. Conetta and J. Salpeter, J. Assoc. Off. Anal. chem., 59(1976)1244.
Talanta 44 (1997) 855 858
Fluorescent reaction between ascorbic acid and D A N and its analytical application Jinghe
Y a n g ~"*, C h a n g l u n
Tong
b, N i a n q i n Jingtian
J i e ", G u i l i n g
Zhang
~', X u e z h e n
R e n ",
Hu ~
, Department o1' Chemistry Shandon~ Unirersity, .limm 250100, China b Hangzhou Ererhmg Bioteclmics, Chimt
Received 9 April 1996: accepted 11 October 1996
Abstract Fluorescent reaction between ascorbic acid (AA) and 2,3-diamino-naphthalene (DAN was studied. The experimental results showed that AA could react with DAN at p H = 10.2-10.5, and form the fluorescent heterocyclic condensation products which emitted strong fluorescence. The fluorescence intensity was measured in a I cm quartz celt with excitation and emission wavelengths of 400 and 520 rim, respectively. The relationship was obtained between the fluorescence intensity and AA concentration in the range of 2-300 tag ml ~, the regression coefficient is 0.9993. The detection limit (signal-to-noise = 2) is 0.4 pg ml ~. © 1997 Elsevier Science B.V. Kevwords: Ascorbic acid determination; Fluorescent reaction
I. Introduction M a n y different m e t h o d s for the determination of ascorbic acid (AA) have been reported in the literature. They concentrated on spectrometry [1], redox reaction [2], derivatization reaction [3], electrochemical m e t h o d [4], enzymatic m e t h o d [5], and c h r o m a t o g r a p h i c m e t h o d [6], but few fluorometric estimation m e t h o d s of A A were reported. A classic fluorometric m e t h o d with o-phenylenediamine ( O P D A ) as fluorescence reagent was used to determine A A [7]. This m e t h o d needed to treat with redox reagent beforehand and then reacted * Corresponding author.
AA with O P D A , its detection limit was 2 lag ml ~ Our experiments indicated that at the p H = 1 0 . 2 - - 1 0 . 5 , A A could react with 2,3-dia m i n o n a p h t h a l e n e ( D A N ) which didn't need pretreatment, its detection limit was 0.4 lag ml '. So the p r o p o s e d m e t h o d is m o r e simple and sensitive than O P D A method.
2. Experimental 2.1. A p p a r a t u s
All fluorescence intensities were measured on a 850 fluorescence s p e c t r o p h o t o m e t e r (Hitachi, Japan).
0039-9140'97/S17.00 .'t; 1997 Elsevier Science B.V. All rights reserved. Pll S0039-91 40(96)021 29-7
856
J. Yang et al. / Talanta 44 (1997) 855-858
2.2. Reagents
3.2. Effect o f p H
1. 2,3-diaminonaphthalene (DAN) solution (0.1%, v/v): it is prepared by dissolving 0.1 g D A N in 100 ml 0.1 mol I - ' HC1, then treated with cyclohexane, rejected organic phase, the remained water phase repeated 2 - 3 times with cyclohexane according to the above procedure. 2. Ascorbic acid solution: it is prepared by dissolving 0.100 g AA in 100 ml distilled water. The stock solution was 1.00 mg m l - t 3. Aminoacetic acid buffer solution: it is prepared by dissolving 3.8 g aminoacetic acid in 100 ml distilled water and adjusting pH to 10.3 with 6 mol 1- 1 NaOH.
The effect of pH on fluorescence intensity of the system was shown in Fig. 2. From Fig. 2, it can be seen that the fluorescence intensity is the strongest and remains stable in the range of pH = 10.2-10.5. In this paper, the following buffers were examined: Na2CO3, b o r a x - N a O H , NH3-NHaC1, K2HPO4, aminoacetic acid. The results showed that 2 ml of 0.5 tool 1- ~ aminoacetic acid buffer was the most suitable.
2.3. Procedure
To a 25 ml test tube, solutions were added according to the following order: AA solution, 0.5 ml 0.5 mol 1-1 NaOH, 1 ml 0.1% D A N solution, 2 ml aminoacetic acid buffer. The mixture was diluted to 10 ml with distilled water, thoroughly mixed by shaking and then allowed to stand for 40 min. The fluorescence intensity was measured in a 1 cm quartz cell with excitation and emission wavelengths of 400 and 520 nm, respectively.
3.3. Effect o f D A N concentration
The effect of D A N concentration on fluorescence intensity of the system was studied. The results were shown in Fig. 3. From this figure we can see that the suitable concentration of D A N was 0.01%.
i
3. Results and discussion 3.1. Fluorescence spectra
The excitation and emission spectra of AADAN-aminoacetic acid (1), DAN-aminoacetic acid (2) are shown in Fig. 1. From Fig. 1, it can be seen that the wavelength of emission peak is 520 nm, the wavelengths of excitation peak are 370, 400 nm. Since D A N itself is a strong fluorephore with excitation and emission wavelengths of 340 and 390 nm, if the excitation wavelength is 370 nm, the excess of D A N in the reaction will interfere with the emission of the fluorescence products, therefore, the excitation wavelength of 400 nm was chosen in our experiments and a low and stable reagent blank was obtained.
.A ~m) Fig. 1. Fluorescence spectra (a) excitation spectrum ("~em= 520
nm), (b) emission spectrum (2ex=400 nm). (1) AA-DANaminoacetic acid, (2) DAN-aminoaceticacid conditions: ascorbic acid: 0.2 mg ml - 1, DAN: 0.01%; aminoaceticacid: 0.5 mol l-l, 2 ml, pH=l.03.
J. Yang et al. / Talanta 44 (1997) 855-858
857
was examined, the results showed the enhancement effect was not obvious.
3.6. Calibration curve and detection limit The calibration curve was made according to the procedure in the optimum conditions. The results indicated that the fluorescence intensity of the system was a linear function of AA concentration in the range of 2-300 lag m l - 1, the regression coefficient is 0.9993, the detection limit (S/N = 2) is 0.4 lag ml
3.7. Recover)' test Fig. 2. Effect of pH. Conditions: ascorbic acid: 0.05 mg ml - ~, DAN: 0.01%; aminoacetic acid: 0.5 mol 1 - i , 2 ml.
3.4. Effect of temperature High temperature could destroy the structure o f AA and quench the fluorescence of the system. Our experiments indicted that when the temperature was higher than 50°C, the fluorescence intensity of the system was completely quenched. The suitable temperature was 30°C or so.
3.5. Stable tests The experiments indicated that at room temperature the fluorescence intensity of the system reached a maximum after 40 min and remained stable at least for 1 h. In addition, effect of surfactants of fluorescence intensity of the system
AP
Fig. 3. Effect of DAN concentration. Conditions: ascorbic acid: 0.25 mg ml-~; aminoacetic acid: 0.5 mol 1-t, 2 ml, pH = 10.3.
In the serum, recovery tests of AA were made. The results were shown in Table I. From Table 1, it can be seen that recovery ratios were upwards of 90% and serum didn't need complicated biological treatment. Therefore, the proposed method for determining AA was satisfactory.
3.8. Sample determination The proposed method was used to determine AA in Vc tablets (Jilin Tonghua Baishan Medicine Factory of China) and compared with the most commonly used OPDA method [8]. The results were shown in Table 2. From Table 2, it can be seen that the accuracy and precision of the method are satisfactory.
3.9. The characteristics oJ the proposed method In comparison with the most commonly used O P D A method, the proposed method possesses the following characteristics: 1. The detection limit is 0.4 lag m l - ~, which is obviously lower than that of O P D A method [8]. so the sensitivity of the proposed method is higher. 2. In O P D A method, the redox reagent, such as norit (carbon), 2,6-dichloroindophenol, N-Bromosuccinimide and iodone, must be used to oxidize AA to DHAA, while in this method it is unnecessary to add a redox reagent to the reaction system. We think that AA will not be transferred to D H A A in the absence of the oxidizer
858
J. Yang et al. / Talanta 44 (1997) 855-858
Table 1 Recovery test in the serum Added Vc (gg ml -I)
Found Vc (gg ml l)
X± S
Recovery (%)
20.0 100.0
18.92, 17.95, 18.33, 18.92, 18.95 94,8, 96.1, 94.3, 93.4, 95.2
18.6±0.45 94.8 ± 1.01
93.1 94.8
Table 2 Determination of samples
O P D A method.
The proposed method
OPDA method (8)
Found Vc (%)
X± S
Found Vc (%)
52.2 ± 1.5
52.4, 50.5, 50.9 53.4, 54.2
References 52.3, 50.9, 54.6 50.9, 52.3
,X ± S
52.3 ± 1.6
a n d there is a t a u t o m e r i c between enediol a n d 2-hydroxy c a r b o n y l groups in the alkaline m e d i u m . Therefore, the possible m e c h a n i s m is illustrated as follows.
Ho ~
oH
~lor-'~o
~N
Since the conjugate n - b o n d system o f the condensate p r o d u c t in the p r o p o s e d m e t h o d is larger t h a n that in the O P D A m e t h o d , the sensitivity o f the p r o p o s e d m e t h o d is higher t h a n that o f
[11 S.H.R. Davis and S.J. Masten, Anal. Chim. Acta, 248 (1991) 225. [21 B. Jaselskis and J. Nelapaty, Anal. Chem., 44 (1972) 379. [3] MJ., Deutsch and C.E. Weeks, J. Assoc. Off. Agric. Chem., 48 (1965) 1248. [4] L. Falat and H.Y. Cheng, Anal. Chem., 54 (1982) 2108. [5] J.O. Schenk, E. Miller and R.N. Adams, Anal. Chem., 54 (1982) 1452. [6] E.S. Wagner, B. Lindley and R.D. Coffin, J. Chromatogr., 163 (1979) 225.
"
%~'~<~//~"'~oH
[71 A.P. Lawrence, L.R. Donald and T.K. Peter, J. Assoc. off. Anal. Chem., 68 (1985) 1. [8] R.B. Roy, A. Conetta and J. Salpeter, J. Assoc. Off. Anal. chem., 59(1976)1244.