中国科技论文在线 http://www.paper.edu.cn

Talanta 55 (2001) 1055–1058 www.elsevier.com/locate/talanta Flow injection determination of polyhydroxy phenols using luminol–ferricyanide/ferrocyanide system Jianxiu Du, Yinhuan Li, Jiuru Lu *

Department of Chemistry, Shaanxi Normal Uni6ersity, Xi’an 710062, People’s Republic of China

Received 17 January 2001; received in revised form 7 May 2001; accepted 15 May 2001

Abstract

It was found that the weak chemiluminescence produced from the reaction of polyhydroxy phenols with luminol in alkaline solution could be strongly enhanced by ferricyanide and ferrocyanide. Based on this found, a new flow injection chemiluminescence method is proposed for the determination of four polyhydroxy phenols: pyrogallol, phlorglucinol, quinol and resorcinol. The detection limits of the method are 0.03 mgml−1 pyrogallol, 0.03 mgml−1 phlorglucinol, 0.04 mgml−1 quinol, and 0.02 mgml−1 resorcinol. The possible mechanism of CL reactions is also discussed briefly. © 2001 Elsevier Science B.V. All rights reserved.

Keywords: Chemiluminescence; Flow injection; Polyhydroxy phenols

1. Introduction CL reaction was used to determine activity of superoxide dismutase [3]. We found that plorglu- The oxidation of pyrogallol by molecular oxy- cinol, quinol and resorcinol can also generate gen and is accompanied by a weak CL with luminol in alkaline solution. This weak chemiluminescence in alkaline solution. This weak CL reaction can be effectively catalyzed by CL reaction can be increased by several en- ferricyanide and ferrocyanide, which has been hancers, such as formaldehyde [1] and 1-ethyl-3- used as a catalyst for the CL reaction of luminol (3-dimethylaminopropyl)-carbodiimide [2]. It was with reducing agents [4,5]. Based on these obser- also reported that pyrogallol can react with lumi- vations, a new flow injection CL method is pro- nol to produce weak CL in alkaline solution, this posed for the determination of four polyhydroxy phenols: pyrogallol, phlorglucinol, quinol and re- * Corresponding author. Fax: +86-29-530-7025. sorcinol. A brief discussion on the CL reaction E-mail address: [email protected] (J. Lu). was also given.

0039-9140/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S0039-9140(01)00452-0 转载 中国科技论文在线 http://www.paper.edu.cn 1056 J. Du et al. / Talanta 55 (2001) 1055–1058 2. Experimental the standard stream, producing CL. The concen- tration of polyhydroxy phenols was quantified by 2.1. Apparatus the CL intensity.

The flow injection system used in this work is shown in Fig. 1. A peristaltic pump was used to 3. Results and discussion deliver all solutions at an equal flow rate of 2.0 ml min−1 for each line. PTFE tubing (0.8 mm i.d.) was used to connect all components in the flow 3.1. Optimum of the experimental conditions system. Injection was made using a six-way injec- tion valve equipped with a 40 ml sample loop. The Zero point four microgram per millilitre pyro- CL signal produced in the flow cell was detected gallol solution was used to optimize the experi- mental conditions. with an R456 Photomultiplier tube (Hamamatsu) and recorded with an IBM-compatible computer The influence of the flow rate on the CL reac- employing an IFFL-D flow-injection CL analysis tion was examined in the range of 0.7–3.5 ml −1 system software (Xi’an Ruike Electronic Equip- min . The maximum signal-to-blank ratio was −1 ment Corporate, Xi’an, China). obtained at a flow rate of 2.0 ml min . So, this flow rate was selected. 2.2. Reagents The influence of the concentration of luminol on the CL reaction was examined in the range of −4 −3 −1 All reagents were obtained from Xi’an Chemi- 1.0×10 –2.0×10 mol l . As the concen- cal Reagent Factory except luminol, which was tration of luminol increasing, the CL signal of homemade. The 1.0 mg ml−1 standard solutions pyrogallol and the blank value both increased. of pyrogallol, phlorglucinol, quinol and resorcinol The signal-to-blank ratio reached maximum when −4 −1 were freshly prepared by dissolving 0.1000 g of 8.0×10 mol l luminol was used. So, 8.0× −4 −1 each in water and diluting with water to 100 ml, 10 mol l luminol was selected. respectively. These standard solutions were stored The influence of the concentration of sodium in the refrigerator and protected from light. The in the luminol solution on the CL −1 testing solutions were prepared by appropriate reaction was examined from 0.3 to 0.7 mol l . dilution of these standard solutions with water The CL signal of pyrogallol and the blank value before used. The 8.0×10−4 mol l−1 luminol both decreased with raising of the concentration solution was prepared into 0.5 mol l−1 sodium of sodium hydroxide. The CL reaction got the hydroxide solution and contained 0.25 mol l−1 potassium ferrocyanide. The 0.022 mol l−1 potas- sium ferricyanide solution was prepared by dis- solving 1.81 g of in water and diluting with water to 250 ml. All the reagents used were of analytical grade; doubly de-ionized water was used for the preparation of solutions.

2.3. Procedures

As shown in Fig. 1, flow line a, b, c were con- Fig. 1. Schematic diagram of the flow system for determina- nected with luminol solution, potassium ferri- tion of polyhydroxy phenols: (a) luminol solution; (b) potas- sium ferricyanide solution; (c) standard solution. P, peristaltic solution and standard solution, pump; V, six-way injection valve; F, flow cell; PMT, photo- respectively. Forty microlitre of mixture of lumi- multiplier tube; HV, high voltage; COM, computer; W, waste nol and potassium ferricyanide was injected into solution. 中国科技论文在线 http://www.paper.edu.cn J. Du et al. / Talanta 55 (2001) 1055–1058 1057

Table 1 fold comparing with in the absence of potassium Relative CL intensities for 0.4 mgml−1 polyhydroxy phenols ferricyanide and potassium ferrocyanide.

Species Relative CL intensities 3.2. Calibration cures and detection limits

Pyrogallol 17.6 Phloroglucinol 19.5 Under the optimum conditions, four polyhy- Quinol 13.5 droxy phenols were determined independently. Resorcinol 41.5 Table 1 lists the relative CL intensities obtained for the polyhydroxy phenols compared at the same concentration of pyrogallol. The parameters maximum signal-to-blank ratio when 0.5 mol l−1 of the calibration cures and calculated detection sodium hydroxide was used. So, 0.5 mol l−1 limits are shown in Table 2. The relative standard sodium hydroxide was chosen. deviations for 11 replicate measurements of four Pyrogallol, phlorglucinol, quinol and resorcinol polyhydroxy phenols were 1.3–2.2%. can react with luminol to produce weak CL in alkaline solution. The CL reaction can be cata- 3.3. Interference studies lyzed by potassium ferricyanide, but blank signal is very high in the absence of potassium ferro- In order to assess the selectivity of the proposed cyanide because potassium ferricyanide can also method, the influence of some common inorganic oxidize luminol to generate strong CL in alkaline ions on the CL reaction was examined by prepar- solution. Shevlin and Neufeld have reported that ing solutions containing 0.4 mgml−1 pyrogallol. the CL reaction of luminol with potassium ferri- The tolerance of foreign species was taken as the cyanide can be inhibited by potassium ferro- largest concentration yielding an error of less than cyanide [6]. So, if there was a suitable 95% in the CL signal of pyrogallol. No interfer- concentration of potassium ferricyanide and con- ence has been found when including up to a centration of potassium ferrocyanide in the reac- 100-fold Na+,K+,Ca2+,Mg2+,Zn2+,Al3+, tion, they can not only efficiently catalyze the CL 2− − − − 3− 2− SO4 ,NO3 ,Cl ,Br ,PO4 ,C2O4 , 10-fold reaction of luminol with polyhydroxy phenols, + 3+ 2+ 3+ 2+ 2+ 2+ NH4 ,Fe ,Fe ,Cr ,Co ,Mn ,Ni , but also strongly decrease the blank signal arising 2− 2− − 2− CO3 ,SO3 ,NO2 , five-fold S , and equal from the reaction of luminol with potassium ferri- amount of Cu2+. cyanide. The 0.015–0.025 mol l−1 potassium fer- ricyanide and 0.10–0.30 mol l−1 potassium 3.4. Nature of the CL reaction ferrocyanide was examined. The maximum signal- to-blank ratio was obtained at 0.022 mol l−1 The standard solution, the luminol solution and potassium ferricyanide and 0.25 mol l−1 potas- the potassium ferricyanide solution were purged sium ferrocyanide. At the selected concentrations with nitrogen or oxygen for 5 min. When the of potassium ferricyanide and potassium ferro- dissolved oxygen was removed from all solutions cyanide, the CL reaction of polyhydroxy phenols by the flow of nitrogen, the CL intensities de- with luminol could be enhanced more than 50- creased 15–40% for polyhydroxy phenols. In con-

Table 2 Linear ranges and detection limits for four polyhydroxy phenols

Species Linear ranges (mgml−1) Slope Intercept Correlation coefficient Detection limits (mgml−1)

Pyrogallol 0.1–6 3.03 6.54 0.9990 0.03 Phloroglucinol 0.1–50 5.84 −2.19 0.9995 0.03 Quinol 0.1–1 1.55 7.30 0.9995 0.04 Resorcinol 0.06–1 9.87 5.09 0.9975 0.02 中国科技论文在线 http://www.paper.edu.cn

1058 J. Du et al. / Talanta 55 (2001) 1055–1058 trast, when all solutions were saturated with the “superoxide radical flow of oxygen, the CL intensities increased 40– K3Fe(CN)6-K4Fe(CN)6 superoxide radical+luminol “ 90% for polyhydroxy phenols. These results indi- cated that the CL reaction required dissolved excited aminophthalate oxygen. excited aminophthalate“aminophthalate+hw It was reported that superoxide radical could be produced from the photochemical reaction of ri- boflavin in alkaline solution [7], and the superox- 4. Conclusion ide radical can oxidize luminol to produce CL in alkaline solution [8]. In order to examine the role Based on the catalytic effect of ferricyanide and of potassium ferricyanide and potassium ferro- ferrocyanide on the weak CL reaction of polyhy- cyanide on the CL reaction of superoxide radical droxy phenols with luminol in alkaline solution, a with luminol, they were added into the reaction new flow injection CL method is proposed for the system. The CL intensity of reaction of riboflavin determination of these polyhydroxy phenols. The (UV-light irradiation)–luminol system was proposed method is sensitive, rapid, and could be strongly enhanced in the presence of potassium applied to the determination of reported polyhy- ferricyanide and potassium ferrocyanide. The re- droxy phenols in pharmaceutical preparation and sults showed that potassium ferricyanide and environmental water after separation. potassium ferrocyanide can catalyze the CL reac- tion of luminol with superoxide radical in alkaline solution. References It was reported that superoxide radical could be generated from the auto-oxidation reaction of [1] N.J. Kearney, E.S. Bridgeland, R.A. Jewsbury, N.D. pyrogallol in alkaline solution [9]. Therefore, it Martin, S.J. Kelly, S.R. Korn, Anal. Commun. 33 (1996) was assumed that pylorglucinol, quinol and resor- 241. [2] Y. Ohba, M. Yamashita, M. Nakazono, L. Ma, K. cinol also having analogous structure could re- Zaitsu, Anal. Sci. 16 (2000) 979. duce dissolved oxygen to superoxide radical in [3] A.G. Guo, Z.Y. Wang, Zhiwushenglixue Tongxun 3 alkaline solution. Alwarthan and Lu have also (1989) 54 indicated that the formation of superoxide radical [4] H. Kubo, A. Toriba, Anal. Chim. Acta 353 (1997) 345. was an important intermediate process in the CL [5] H. Kubo, M. Saitoh, S. Murase, T. Inomata, Y. Yoshimura, H. Nakazawa, Anal. Chim. Acta 389 (1999) reaction of luminol with reducing agents [10,11]. 89. In presence of potassium ferricyanide and potas- [6] P.B. Shevlin, H.A. Neufeld, J. Org. Chem. 35 (1970) 2178. sium ferrocyanide, the CL reaction of superoxide [7] Q. Chen, Chinese Medicine Study Methodology, People radical with luminol in alkaline solution was cata- Health Press, Beijing, 1993, p. 953. lyzed and the reaction equilibrium of polyhydroxy [8] Y.X. Li, R.Z. Fang, Prog. Biochem. Biophys. 10 (1983) 59. phenols with dissolved oxygen was destroyed. [9] S. Marklund, G. Marklund, Eur. J. Biochem. 47 (1974) Based on the discussion described above, the 469. possible reaction mechanism was suggested as [10] A.A. Alwarthan, A. Townshend, Anal. Chim. Acta 196 following: (1987) 135. [11] J.R. Lu, X.R. Zhang, Z.J. Zhu, Z.J. Zhang, M.Q. Li, Chem. J. Chin. Univ. 12 (1991) 1595. polyhydroxy phenols+O2(aq)+NaOH