- Email: [email protected]
Flow–injection chemiluminescence determination of ascorbic acid by use of the cerium(IV)–Rhodamine B system
Analytica Chimica Acta 464 (2002) 289–293
Flow–injection chemiluminescence determination of ascorbic acid by use of the cerium(IV)–Rhodamine B system Yongjun Ma a , Min Zhou a , Xiaoyong Jin a , Baozhu Zhang a , Hui Chen a,∗ , Naiyun Guo b a b
Institute of Chemistry, Northwest Normal University, Lanzhou 730070, China Department of Cardiac–Renal-Disease, West Hospital, Lanzhou 730070, China
Received 11 December 2001; received in revised form 18 February 2002; accepted 17 May 2002
Abstract A highly sensitive flow–injection (FI) method with chemiluminescence (CL) detection is used for the determination of l-ascorbic acid. The method is based on the CL reaction of Rhodamine B with cerium(IV) in sulfuric acid media. l-Ascorbic acid is suggested to be a catalyst utilized in the energy-transferred excitation process. The proposed procedure allows quantitation of l-ascorbic acid in the range 3.8 × 10−13 to 1.0 × 10−10 mol l−1 with a correlation coefficient of 0.9998 (n = 5) and relative standard deviation (R.S.D.) of 0.92% (n = 11) at 1.0 × 10−11 mol l−1 . The detection limit (3 × blank) was 1.0 × 10−13 mol l−1 . The method is successfully used to determine l-ascorbic acid in fresh vegetables. The possible mechanism of the chemiluminescence in the system is discussed. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Chemiluminescence; l-Ascorbic acid; Cerium(IV); Rhodamine B; Flow–injection analysis; Vegetables
1. Introduction Ascorbic acid (Vitamin C) has a recommended daily intake of 60 mg. It occurs naturally in most fruits and vegetables and has a number of physiological roles. It participates in numerous biological events concerning electron transport reactions, hydroxylations and oxidative catabolism of aromatic amino acids. Because of its importance, there has been considerable interest in alternative methods of determining the ascorbic acid content of food products. A number of studies have focused on utilizing titrimetric [1], spectrophotometric [2], kinetic [3], electroanalytical [4], fluorimetric [5] and liquid chromatographic [6] methods. ∗ Corresponding author. E-mail address: [email protected] (H. Chen).
These procedures may not always offer the required detectability. In some cases, time-consuming procedures and expensive instrumentation are involved. Compared with the techniques mentioned earlier, the main attractions of chemiluminescence (CL) for analytical applications are excellent sensitivity over a wide linear dynamic range and low limits of detection that can be achieved in comparison with fluorescence because no external excitation source is required and, therefore, the background signal is very low. Analytical procedures applying CL measurements in flow–injection (FI) systems combine the advantages of instrumental simplicity, rapidity (normally 0.1–10 s) and reproducibility in signal detection, and being appropriate for on-line analyses. CL has been extensively employed for the determination of ascorbic acid, mostly conducted in basic media and using
0003-2670/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 0 2 ) 0 0 4 8 3 - X
290
Y. Ma et al. / Analytica Chimica Acta 464 (2002) 289–293
luminol [7–10] or lucigenin [11,12] as reagent. As is well known ascorbic acid and its primary oxidation product, dehydroascorbic acid (DHAA), are not stable in basic media. Hence, Agater and Jewsbury [13] determined the ascorbic acid content in pharmaceuticals and juices with a linear range of 5 × 10−4 to 1.0 mmol l−1 , by measurement of the CL from direct oxidation with permanganate in an acidic medium. As D-ascorbic acid is not naturally present in foodstuffs, the main aim of this work is to develop a simple and reproducible CL-based FI method that can apply a new CL system, cerium(IV) with Rhodamine B in sulfuric acid for the routine determination of l-ascorbic acid in vegetables. Other ions or compounds normally present along with ascorbic acid do not interfere.
2. Experimental 2.1. Reagents All chemicals were of analytical grade. An aqueous stock standard solution of l-ascorbic acid (1.0 × 10−3 mol l−1 ) was freshly prepared in twice distilled water. The cerium(IV) solution (0.05 mol l−1 ) was prepared by dissolving Ce(SO4 )2 ·4H2 O in 0.2 mol l−1 H2 SO4 . A 100 mg l−1 Rhodamine B solution was prepared by dissolving Rhodamine B in twice distilled water.
Corporation, China). This system consisted of two peristaltic pumps, provided with PTFE tubing (0.5 mm i.d.) and various end-fittings and connectors. Sample injection (150 l) was by sample loop injection valve. CL emission was measured using a detector in which a sensitive photomultiplier tube (PMT) was operated at 750 V. No wavelength selection was involved. 2.3. Procedure Working standard solutions containing l-ascorbic acid in the range 3.8 × 10−13 to 1.0 × 10−10 mol l−1 were prepared by diluting a fresh stock standard solution. The CL signal was measured by using the FI manifold shown in Fig. 1. Rhodamine B (100 mg l−1 ) and working standard solutions were merged into a single stream during passage through a mixing tube (300 mm); 0.03 mol l−1 cerium(IV) and 0.2 mol l−1 H2 SO4 were merged similarly through a mixing tube (300 mm) into the sample loop (150 l), which then joined the former stream and was finally injected into the CL detector through a mixing tube (100 mm). Peak height CL emission was measured.
3. Results and discussion 3.1. Kinetic curve
2.2. Apparatus The FI system, as shown in Fig. 1, was an IFFL-D FI–CL analysis system (Xi’an Record Electric Ltd.
The CL emission intensity with respect to time was investigated. The signal profile is shown in Fig. 2, indicating that the luminescence reaction was rapid.
Fig. 1. FI manifold for ascorbic acid determination: Ce(IV), 0.03 M in 0.2 M H2 SO4 ; Rhodamine B (RhB), 100 mg l−1 ; P1, P2, peristaltic pump; D, CL detector; volume of flow cell 150 l.
Fig. 2. Kinetic curve.
Y. Ma et al. / Analytica Chimica Acta 464 (2002) 289–293
291
The CL signal was detected ca. 1 s after injection of solutions and the intensity reached a maximum ca. 5 s later. The intensity then became weaker and was almost zero after ca. 25 s. 3.2. Optimization A series of experiments were conducted to establish the optimum analytical conditions. The parameters optimized included chemical variables, such as the concentrations of the reagents used for the CL reaction, and physical variables, including total flow rate. Fig. 4. Effect of H2 SO4 concentration.
3.3. Effect of cerium(IV) concentration The effect of Ce(IV) concentration on the CL signal was examined over the range 1 × 10−4 to 5 × 10−2 mol l−1 in 0.2 mol l−1 H2 SO4 . Maximum intensity was observed at 3 × 10−2 mol l−1 Ce(IV), as shown in Fig. 3. Therefore, 3 × 10−2 mol l−1 Ce(IV) in 0.2 mol l−1 H2 SO4 was used for all the following work. 3.4. Effect of H2 SO4 concentration The effect of sulfuric acid concentration on the sensitivity was studied. The highest CL signal was observed at 0.2 mol l−1 H2 SO4 , as shown in Fig. 4, which was therefore chosen to prepare and dilute the Ce(IV) solution and for use as a carrier.
Fig. 3. Effect of cerium(IV) concentration.
3.5. Effect of Rhodamine B concentration The effect of the Rhodamine B concentration on the CL signal was investigated. The results in Fig. 5 show that 100 mg l−1 Rhodamine B solution provided maximum CL intensity. Lower concentrations of Rhodamine B gave decreased CL emission, and higher concentrations produced self-absorption of the emitted radiation. Therefore, 100 mg l−1 of Rhodamine B was adopted for further use. 3.6. Effect of flow rate The flow rate of each stream was optimized. In general, the intensity increased with increasing flow rate. However, a total flow rate of 11 ml min−1
Fig. 5. Effect of Rhodamine B concentration.
292
Y. Ma et al. / Analytica Chimica Acta 464 (2002) 289–293
Table 1 Maximum ratio of interferent to ascorbic acid concentration which gives <5% change in response to ascorbic acid
that the main interferences came from some strong reductive species.
Species added
Maximum tolerable mole ratioa
3.9. Application
Na+ , K+ , Cu2+ , Zn2+ , Ca2+ , Mg2+ , NH4 + , Cl− , SO4 2− , NO3 − , NO2 − , Br− , starch, ethanol Glucose Sucrose, Fe2+ Al3+ Sn(II) SO3 2−
1000
Four vegetables were bought fresh from the local market. They were weighed, crushed and centrifuged with about 0.2% (w/v) calcium chloride solution, used to decrease interference of oxalic acid in vegetables, until a clear liquid was obtained. The filtrate was diluted to a suitable concentration for the assay by the proposed FI–CL method. The accuracy of the method was checked by carrying out recovery studies (Table 2). When known amounts of l-ascorbic acid standard solution were added to the dilute juices, quantitative recoveries (98.2–103.0%) were obtained.
a
130 50 40 10 2
1000 is greatest ratio tested.
(5.5 ml min−1 for each channel) was recommended because of greater precision and economy in the use of reagents.
4. CL mechanism
3.7. Calibration Under the optimum conditions described previously, the calibration graph concentration of log l-ascorbic acid versus peak height was linear in the range 3.8 × 10−13 to 1.0 × 10−10 mol l−1 (r = 0.9998, n = 5). The relative standard deviation (R.S.D.) for 1.0 × 10−11 mol l−1 l−ascorbic acid measurement was 0.92% (n = 11). The detection limit, defined as three times the S.D. for the reagent blank signal, was 1.0 × 10−13 mol l−1 ascorbic acid. 3.8. Interferences A range of organic and inorganic compounds, which could be present at significant levels in vegetables, was investigated in the determination of 1.0 × 10−11 mol l−1 l-ascorbic acid (Table 1). A substance was considered not to interfere when the effect on the peak height was <5%. The results showed
It was found that the reaction of Ce(IV) with Rhodamine B in an acidic medium could produce significant CL. Thus, Rhodamine B was used in this work as an illuminant, similar to luminol in basic media, instead of as sensitizer [14]. Furthermore, fluorescence and infrared spectroscopy showed that the wavelength of this CL reaction was in agreement with that of the reaction of Ce(IV) solution with Rhodamine B solution (420 nm). Additionally, it has been reported that ascorbic acid gives little luminescence in cerium(IV) solution in acidic media [8], and, it was found that there was no CL in the absence of Rhodamine B when l-ascorbic acid was oxidized into DHAA in this procedure. Accordingly, it should be pointed out that DHAA did not participate in the CL reaction in this procedure. In this system, however, the CL signal of the reaction of Ce(IV) with Rhodamine B was enhanced by l-ascorbic acid. Rajanna et al. [15] has already studied the mechanism involved in the ascorbic acid reduction
Table 2 Determination of l-ascorbic acid in vegetables Vegetables
Found (mg g−1 )
R.S.D. (%)
Added (mg)
Recovered (mg g−1 )
Recovery (%)
Celery Cabbage Cole Long crooked squash
0.293 0.472 0.136 0.236
4.0 1.1 1.0 2.3
0.200 0.200 0.200 0.200
0.492 0.660 0.346 0.447
99.7 98.2 103.0 102.5
Y. Ma et al. / Analytica Chimica Acta 464 (2002) 289–293
of Ce(IV) in H2 SO4 media. They thought the mechanism for the ascorbic acid reduction of Ce(IV) in this medium could be the reaction of Ce(IV) with ascorbic acid (H2 A) involving rapid complexation of the metal ion by the reductant (H2 A) followed by the inner sphere transfer of one electron, so that a radical HA• was generated. The mechanism of the CL reaction in the Ru(II)(phen)3 2+ –ascorbic acid–Ce(IV) system has also been discussed [16]. Based on the phenomena in this work and the mechanism mentioned earlier, the possible mechanism of the CL reaction could be explained as follows: Ce(IV)
RhB → [• RhB− ]∗ox → [• RhB− ]ox + hν
293
the main interferents were not observed. In addition, good accuracy and precision as required for routine quality control were obtained. Furthermore, the system might be applied to make a CL sensor or probe for determination of l-ascorbic acid since the method has such a low detection limit.
Acknowledgements We are grateful to Natural Scientific Foundation of Gansu province, China (Project ZS001-A24-054-Y) for supporting the research work.
H2 SO4
(λ = 420 nm) Ce(IV)
H2 A → HA• H2 SO4
Ce(IV)
[• RhB− ]ox +HA• → [• RhB− ]∗ox + H+ + A H2 SO4
where RhB is Rhodamine B, ox the oxidized form, hν the CL emission, H2 A the ascorbic acid; and [• RhB− ]∗ox is the Rhodamine B illuminant with negative oxygen ion.
5. Conclusions The proposed FI method has been applied to determine l-ascorbic acid in sulfuric acid at very low concentration by using the signal enhancement of the CL reaction of cerium(IV) with Rhodamine B. The method has a linear range of 3.8 × 10−13 to 1.0 × 10−10 mol l−1 , and a detection limit of 1.0 × 10−13 mol l−1 , which is far lower than those assays mentioned in the introduction. In the determination of real samples, obvious effects on CL signal from
References [1] M.A. Koupparis, P. Anagnostopoulou, H.V. Malmstadt, Talanta 32 (1985) 411. [2] E. Luque-Perez, A. Rios, M. Valcarcel, Fresenius’ J. Anal. Chem. 366 (2000) 857. [3] S.M. Sultan, N.I. Desai, Talanta 45 (1998) 1061. [4] J. Wang, T. Golden, Anal. Chim. Acta 217 (1989) 343. [5] M.J. Deutsch, C.E. Weeks, J. Assoc. Off. Anal. Chem. 48 (1965) 1284. [6] X. Wang, S. Gao, J. Yu, Shipin Kexue 20 (1999) 52. [7] M.K. Jong, Y.L. Huang, D.S. Rolf, Anal. Lett. 23 (1990) 2273. [8] A.A. Alwarthan, Analyst 118 (1993) 639. [9] Y. Weiping∗ , L. Baolin, Z. Zhujun, T. Guanghui, Fenxi Huaxue 24 (1996) 579. [10] Z. Zhang, W. Qin, Talanta 43 (1996) 119. [11] T. Pérez-Ru´ız, C. Mart´ınez-Lozano, A. Sanz, Anal. Chim. Acta 308 (1995) 299. [12] T. Hasebe, T. Kawashima, Anal. Sci. 12 (1996) 773. [13] I.B. Agater, R.A. Jewsbury, Anal. Chim. Acta 356 (1997) 289. [14] J.S. Lancaster, P.J. Worsfold∗ , A. Lynes, Analyst 114 (1989) 1659. [15] K.C. Rajanna, Y.R. Rao, P.K. Saiprakash∗ , Ind. J. Chem. 17A (1979) 66. [16] H. Zhike, M. Rongming, L. Qingyao, Y. Ximao, Z. Yun’e, Huaxue Xuebao 54 (1996) 1003.
Flow–injection chemiluminescence determination of ascorbic acid by use of the cerium(IV)–Rhodamine B system Yongjun Ma a , Min Zhou a , Xiaoyong Jin a , Baozhu Zhang a , Hui Chen a,∗ , Naiyun Guo b a b
Institute of Chemistry, Northwest Normal University, Lanzhou 730070, China Department of Cardiac–Renal-Disease, West Hospital, Lanzhou 730070, China
Received 11 December 2001; received in revised form 18 February 2002; accepted 17 May 2002
Abstract A highly sensitive flow–injection (FI) method with chemiluminescence (CL) detection is used for the determination of l-ascorbic acid. The method is based on the CL reaction of Rhodamine B with cerium(IV) in sulfuric acid media. l-Ascorbic acid is suggested to be a catalyst utilized in the energy-transferred excitation process. The proposed procedure allows quantitation of l-ascorbic acid in the range 3.8 × 10−13 to 1.0 × 10−10 mol l−1 with a correlation coefficient of 0.9998 (n = 5) and relative standard deviation (R.S.D.) of 0.92% (n = 11) at 1.0 × 10−11 mol l−1 . The detection limit (3 × blank) was 1.0 × 10−13 mol l−1 . The method is successfully used to determine l-ascorbic acid in fresh vegetables. The possible mechanism of the chemiluminescence in the system is discussed. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Chemiluminescence; l-Ascorbic acid; Cerium(IV); Rhodamine B; Flow–injection analysis; Vegetables
1. Introduction Ascorbic acid (Vitamin C) has a recommended daily intake of 60 mg. It occurs naturally in most fruits and vegetables and has a number of physiological roles. It participates in numerous biological events concerning electron transport reactions, hydroxylations and oxidative catabolism of aromatic amino acids. Because of its importance, there has been considerable interest in alternative methods of determining the ascorbic acid content of food products. A number of studies have focused on utilizing titrimetric [1], spectrophotometric [2], kinetic [3], electroanalytical [4], fluorimetric [5] and liquid chromatographic [6] methods. ∗ Corresponding author. E-mail address: [email protected] (H. Chen).
These procedures may not always offer the required detectability. In some cases, time-consuming procedures and expensive instrumentation are involved. Compared with the techniques mentioned earlier, the main attractions of chemiluminescence (CL) for analytical applications are excellent sensitivity over a wide linear dynamic range and low limits of detection that can be achieved in comparison with fluorescence because no external excitation source is required and, therefore, the background signal is very low. Analytical procedures applying CL measurements in flow–injection (FI) systems combine the advantages of instrumental simplicity, rapidity (normally 0.1–10 s) and reproducibility in signal detection, and being appropriate for on-line analyses. CL has been extensively employed for the determination of ascorbic acid, mostly conducted in basic media and using
0003-2670/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 0 2 ) 0 0 4 8 3 - X
290
Y. Ma et al. / Analytica Chimica Acta 464 (2002) 289–293
luminol [7–10] or lucigenin [11,12] as reagent. As is well known ascorbic acid and its primary oxidation product, dehydroascorbic acid (DHAA), are not stable in basic media. Hence, Agater and Jewsbury [13] determined the ascorbic acid content in pharmaceuticals and juices with a linear range of 5 × 10−4 to 1.0 mmol l−1 , by measurement of the CL from direct oxidation with permanganate in an acidic medium. As D-ascorbic acid is not naturally present in foodstuffs, the main aim of this work is to develop a simple and reproducible CL-based FI method that can apply a new CL system, cerium(IV) with Rhodamine B in sulfuric acid for the routine determination of l-ascorbic acid in vegetables. Other ions or compounds normally present along with ascorbic acid do not interfere.
2. Experimental 2.1. Reagents All chemicals were of analytical grade. An aqueous stock standard solution of l-ascorbic acid (1.0 × 10−3 mol l−1 ) was freshly prepared in twice distilled water. The cerium(IV) solution (0.05 mol l−1 ) was prepared by dissolving Ce(SO4 )2 ·4H2 O in 0.2 mol l−1 H2 SO4 . A 100 mg l−1 Rhodamine B solution was prepared by dissolving Rhodamine B in twice distilled water.
Corporation, China). This system consisted of two peristaltic pumps, provided with PTFE tubing (0.5 mm i.d.) and various end-fittings and connectors. Sample injection (150 l) was by sample loop injection valve. CL emission was measured using a detector in which a sensitive photomultiplier tube (PMT) was operated at 750 V. No wavelength selection was involved. 2.3. Procedure Working standard solutions containing l-ascorbic acid in the range 3.8 × 10−13 to 1.0 × 10−10 mol l−1 were prepared by diluting a fresh stock standard solution. The CL signal was measured by using the FI manifold shown in Fig. 1. Rhodamine B (100 mg l−1 ) and working standard solutions were merged into a single stream during passage through a mixing tube (300 mm); 0.03 mol l−1 cerium(IV) and 0.2 mol l−1 H2 SO4 were merged similarly through a mixing tube (300 mm) into the sample loop (150 l), which then joined the former stream and was finally injected into the CL detector through a mixing tube (100 mm). Peak height CL emission was measured.
3. Results and discussion 3.1. Kinetic curve
2.2. Apparatus The FI system, as shown in Fig. 1, was an IFFL-D FI–CL analysis system (Xi’an Record Electric Ltd.
The CL emission intensity with respect to time was investigated. The signal profile is shown in Fig. 2, indicating that the luminescence reaction was rapid.
Fig. 1. FI manifold for ascorbic acid determination: Ce(IV), 0.03 M in 0.2 M H2 SO4 ; Rhodamine B (RhB), 100 mg l−1 ; P1, P2, peristaltic pump; D, CL detector; volume of flow cell 150 l.
Fig. 2. Kinetic curve.
Y. Ma et al. / Analytica Chimica Acta 464 (2002) 289–293
291
The CL signal was detected ca. 1 s after injection of solutions and the intensity reached a maximum ca. 5 s later. The intensity then became weaker and was almost zero after ca. 25 s. 3.2. Optimization A series of experiments were conducted to establish the optimum analytical conditions. The parameters optimized included chemical variables, such as the concentrations of the reagents used for the CL reaction, and physical variables, including total flow rate. Fig. 4. Effect of H2 SO4 concentration.
3.3. Effect of cerium(IV) concentration The effect of Ce(IV) concentration on the CL signal was examined over the range 1 × 10−4 to 5 × 10−2 mol l−1 in 0.2 mol l−1 H2 SO4 . Maximum intensity was observed at 3 × 10−2 mol l−1 Ce(IV), as shown in Fig. 3. Therefore, 3 × 10−2 mol l−1 Ce(IV) in 0.2 mol l−1 H2 SO4 was used for all the following work. 3.4. Effect of H2 SO4 concentration The effect of sulfuric acid concentration on the sensitivity was studied. The highest CL signal was observed at 0.2 mol l−1 H2 SO4 , as shown in Fig. 4, which was therefore chosen to prepare and dilute the Ce(IV) solution and for use as a carrier.
Fig. 3. Effect of cerium(IV) concentration.
3.5. Effect of Rhodamine B concentration The effect of the Rhodamine B concentration on the CL signal was investigated. The results in Fig. 5 show that 100 mg l−1 Rhodamine B solution provided maximum CL intensity. Lower concentrations of Rhodamine B gave decreased CL emission, and higher concentrations produced self-absorption of the emitted radiation. Therefore, 100 mg l−1 of Rhodamine B was adopted for further use. 3.6. Effect of flow rate The flow rate of each stream was optimized. In general, the intensity increased with increasing flow rate. However, a total flow rate of 11 ml min−1
Fig. 5. Effect of Rhodamine B concentration.
292
Y. Ma et al. / Analytica Chimica Acta 464 (2002) 289–293
Table 1 Maximum ratio of interferent to ascorbic acid concentration which gives <5% change in response to ascorbic acid
that the main interferences came from some strong reductive species.
Species added
Maximum tolerable mole ratioa
3.9. Application
Na+ , K+ , Cu2+ , Zn2+ , Ca2+ , Mg2+ , NH4 + , Cl− , SO4 2− , NO3 − , NO2 − , Br− , starch, ethanol Glucose Sucrose, Fe2+ Al3+ Sn(II) SO3 2−
1000
Four vegetables were bought fresh from the local market. They were weighed, crushed and centrifuged with about 0.2% (w/v) calcium chloride solution, used to decrease interference of oxalic acid in vegetables, until a clear liquid was obtained. The filtrate was diluted to a suitable concentration for the assay by the proposed FI–CL method. The accuracy of the method was checked by carrying out recovery studies (Table 2). When known amounts of l-ascorbic acid standard solution were added to the dilute juices, quantitative recoveries (98.2–103.0%) were obtained.
a
130 50 40 10 2
1000 is greatest ratio tested.
(5.5 ml min−1 for each channel) was recommended because of greater precision and economy in the use of reagents.
4. CL mechanism
3.7. Calibration Under the optimum conditions described previously, the calibration graph concentration of log l-ascorbic acid versus peak height was linear in the range 3.8 × 10−13 to 1.0 × 10−10 mol l−1 (r = 0.9998, n = 5). The relative standard deviation (R.S.D.) for 1.0 × 10−11 mol l−1 l−ascorbic acid measurement was 0.92% (n = 11). The detection limit, defined as three times the S.D. for the reagent blank signal, was 1.0 × 10−13 mol l−1 ascorbic acid. 3.8. Interferences A range of organic and inorganic compounds, which could be present at significant levels in vegetables, was investigated in the determination of 1.0 × 10−11 mol l−1 l-ascorbic acid (Table 1). A substance was considered not to interfere when the effect on the peak height was <5%. The results showed
It was found that the reaction of Ce(IV) with Rhodamine B in an acidic medium could produce significant CL. Thus, Rhodamine B was used in this work as an illuminant, similar to luminol in basic media, instead of as sensitizer [14]. Furthermore, fluorescence and infrared spectroscopy showed that the wavelength of this CL reaction was in agreement with that of the reaction of Ce(IV) solution with Rhodamine B solution (420 nm). Additionally, it has been reported that ascorbic acid gives little luminescence in cerium(IV) solution in acidic media [8], and, it was found that there was no CL in the absence of Rhodamine B when l-ascorbic acid was oxidized into DHAA in this procedure. Accordingly, it should be pointed out that DHAA did not participate in the CL reaction in this procedure. In this system, however, the CL signal of the reaction of Ce(IV) with Rhodamine B was enhanced by l-ascorbic acid. Rajanna et al. [15] has already studied the mechanism involved in the ascorbic acid reduction
Table 2 Determination of l-ascorbic acid in vegetables Vegetables
Found (mg g−1 )
R.S.D. (%)
Added (mg)
Recovered (mg g−1 )
Recovery (%)
Celery Cabbage Cole Long crooked squash
0.293 0.472 0.136 0.236
4.0 1.1 1.0 2.3
0.200 0.200 0.200 0.200
0.492 0.660 0.346 0.447
99.7 98.2 103.0 102.5
Y. Ma et al. / Analytica Chimica Acta 464 (2002) 289–293
of Ce(IV) in H2 SO4 media. They thought the mechanism for the ascorbic acid reduction of Ce(IV) in this medium could be the reaction of Ce(IV) with ascorbic acid (H2 A) involving rapid complexation of the metal ion by the reductant (H2 A) followed by the inner sphere transfer of one electron, so that a radical HA• was generated. The mechanism of the CL reaction in the Ru(II)(phen)3 2+ –ascorbic acid–Ce(IV) system has also been discussed [16]. Based on the phenomena in this work and the mechanism mentioned earlier, the possible mechanism of the CL reaction could be explained as follows: Ce(IV)
RhB → [• RhB− ]∗ox → [• RhB− ]ox + hν
293
the main interferents were not observed. In addition, good accuracy and precision as required for routine quality control were obtained. Furthermore, the system might be applied to make a CL sensor or probe for determination of l-ascorbic acid since the method has such a low detection limit.
Acknowledgements We are grateful to Natural Scientific Foundation of Gansu province, China (Project ZS001-A24-054-Y) for supporting the research work.
H2 SO4
(λ = 420 nm) Ce(IV)
H2 A → HA• H2 SO4
Ce(IV)
[• RhB− ]ox +HA• → [• RhB− ]∗ox + H+ + A H2 SO4
where RhB is Rhodamine B, ox the oxidized form, hν the CL emission, H2 A the ascorbic acid; and [• RhB− ]∗ox is the Rhodamine B illuminant with negative oxygen ion.
5. Conclusions The proposed FI method has been applied to determine l-ascorbic acid in sulfuric acid at very low concentration by using the signal enhancement of the CL reaction of cerium(IV) with Rhodamine B. The method has a linear range of 3.8 × 10−13 to 1.0 × 10−10 mol l−1 , and a detection limit of 1.0 × 10−13 mol l−1 , which is far lower than those assays mentioned in the introduction. In the determination of real samples, obvious effects on CL signal from
References [1] M.A. Koupparis, P. Anagnostopoulou, H.V. Malmstadt, Talanta 32 (1985) 411. [2] E. Luque-Perez, A. Rios, M. Valcarcel, Fresenius’ J. Anal. Chem. 366 (2000) 857. [3] S.M. Sultan, N.I. Desai, Talanta 45 (1998) 1061. [4] J. Wang, T. Golden, Anal. Chim. Acta 217 (1989) 343. [5] M.J. Deutsch, C.E. Weeks, J. Assoc. Off. Anal. Chem. 48 (1965) 1284. [6] X. Wang, S. Gao, J. Yu, Shipin Kexue 20 (1999) 52. [7] M.K. Jong, Y.L. Huang, D.S. Rolf, Anal. Lett. 23 (1990) 2273. [8] A.A. Alwarthan, Analyst 118 (1993) 639. [9] Y. Weiping∗ , L. Baolin, Z. Zhujun, T. Guanghui, Fenxi Huaxue 24 (1996) 579. [10] Z. Zhang, W. Qin, Talanta 43 (1996) 119. [11] T. Pérez-Ru´ız, C. Mart´ınez-Lozano, A. Sanz, Anal. Chim. Acta 308 (1995) 299. [12] T. Hasebe, T. Kawashima, Anal. Sci. 12 (1996) 773. [13] I.B. Agater, R.A. Jewsbury, Anal. Chim. Acta 356 (1997) 289. [14] J.S. Lancaster, P.J. Worsfold∗ , A. Lynes, Analyst 114 (1989) 1659. [15] K.C. Rajanna, Y.R. Rao, P.K. Saiprakash∗ , Ind. J. Chem. 17A (1979) 66. [16] H. Zhike, M. Rongming, L. Qingyao, Y. Ximao, Z. Yun’e, Huaxue Xuebao 54 (1996) 1003.