一个催化氧化四甲基联苯胺荧光测定超痕量铁的新方法

doi:10.3964/j.issn.1000-0593(2019)10-3066-05

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摘要：铁是一种必需的微量元素，它在生命过程中起着重要的作用，但是摄入过多的三价铁会使机体的载氧能力下降，引起不稳定血红蛋白病以及高铁血红蛋白症等疾病。无论是从人类健康还是环境保护角度出发，探究简便、快速、灵敏度高和选择性好的检测Fe（Ⅲ）的分析新方法很有意义。荧光分析是一种优异的分子光谱分析方法，具有灵敏度高、选择性强、操作简单等特点，在重金属离子的检测方面也取得了较好的进展，目前利用荧光法测定Fe³⁺也有报道，但有的灵敏度不高，有的选择性不好，有的试剂毒性较大。报道了一种简单、快速、灵敏检测Fe（Ⅲ）的四甲基联苯胺（TMB）荧光分析新方法。在pH 4.5 Tris-HCl缓冲液及35 ℃水浴条件下，H₂O₂氧化无毒易得的四甲基联苯胺（TMB）这一反应较慢；当有痕量Fe（Ⅲ）存在时，它催化过氧化氢（H₂O₂）氧化TMB生成具有较强荧光活性的TMB氧化产物（TMB_ox），用激发波长280 nm激发，TMB_ox在405 nm处有一个较强的荧光峰，且在一定的范围内，随着Fe（Ⅲ）浓度的增大，其荧光强度线性增强。采用单变量变换法优化了荧光分析条件，选择Tris-HCl缓冲溶液的pH为4.5，其浓度为3.3×10^-4 mol·L^-1，TMB浓度为3.0×10^-5 mol·L^-1，H₂O₂浓度为6.0×10^-6 mol·L^-1，在35 ℃条件下反应35 min。在选定条件下，Fe³⁺浓度在0.027～400 nmol·L^-1范围内，随着Fe³⁺浓度的增大，体系在405 nm处的荧光信号线性增强，其线性方程为ΔF_{405 nm}=2.31c+5.0，线性相关系数R²为0.985，其检出限为0.008 nmol·L^-1。考察了共存物质对测定200 nmol·L^-1 Fe（Ⅲ）的影响。结果表明，当相对误差在±10%之内，20 μmol·L^-1的HCO^-₃，K⁺，SO^2-₄，NH⁺₄，Mn²⁺，Na⁺，Cu²⁺，Al³⁺，Zn²⁺，F^-，Mg²⁺，Ba²⁺，Ca²⁺，Co²⁺，NO^3-，NO^2-，10 μmol·L^-1的CO^2-₃，Cr⁶⁺，2 μmol·L^-1的Hg²⁺，BSA不干扰测定。表明该法具有较好的选择性。据此，建立了一个简单、快速、灵敏高、选择性高的测定Fe（Ⅲ）的荧光分析新方法。按以下步骤制备了乳制品的样品溶液，准确吸取1.4 mL乳制品加入600 μL乙酸（V/V=3％），于10 000 r·min^-1下离心3 min，然后吸取离心上清液1mL加入48 μL 2.5 mol·L^-1 NaOH定容至2 mL，于10 000 r·min^-1下离心3 min，最后吸取1 mL上清液稀释至5 mL得到样品溶液。然后采用该催化荧光分析新方法测定了牛奶样品中Fe（Ⅲ）含量，结果令人满意, 其相对标准偏差为0.29%～0.41%，回收率为94.6%～108.0%。

关键词：铁离子；催化；3,3’,5,5’-四甲基联苯胺；荧光

Abstract：Iron is an essential trace element, which plays an important role in the life process, but excessive intake of ferric iron will reduce the oxygen carrying capacity of the body, causing unstable hemoglobin disease and methemoglobinosis. Whether from the point of view of human health or environmental protection, it is of great significance to explore a simple, rapid, sensitive and selective method for the determination of Fe(Ⅲ). Fluorescence analysis is an excellent method of molecular spectroscopy. It has the characteristics of high sensitivity, good selectivity and simple operation. It has also made good progress in the detection of heavy metal ions. At present, the determination of Fe³⁺ by fluorescence method has been reported, but some of them have low sensitivity, poor selectivity and toxicity of organic reagent. In this article, a simple, rapid and sensitive fluorescence method for the determination of Fe(Ⅲ) has been developed, using tetramethylbenzidine (TMB) fluorescence reagent. In pH 4.5 Tris-HCl buffer solution at 35 ℃, the reaction of H₂O₂-TMB was slow. When Fe (Ⅲ)was added, it catalyzed strongly H₂O₂ oxidization of TMB to form strong oxidized product TMB_ox with strong fluorescence. Using excited wavelength of 280 nm, TMB_ox exhibited a strong fluorescence peak at 405 nm, and the fluorescence intensity increased linearly with the increase of Fe(Ⅲ) concentration in a certain range. The fluorescence analysis conditions were optimized by univariate transformation. The pH of Tris-HCl buffer solution was 4.5, its concentration was 3.3×10^-4 mol·L^-1, the concentration of TMB was 3.0×10^-5 mol·L^-1, the concentration of H₂O₂ was 6.0×10^-6 mol·L^-1, and the reaction time was 35 min at 35 ℃. Under the selected conditions, the fluorescence signal of the system increased linearly at 405 nm with the increase of Fe³⁺ concentration in the range of 0.027～400 nmol·L^-1. The linear equation is F_{405 nm}=2.31c+50.0, the linear correlation coefficient R² is 0.985, and the detection limit is 0.008 nmol·L^-1. According to the procedure, the influence of coexistent substances on the determination of 200 nmol·L^-1 Fe³⁺ was tested, with a relative error of ±10%. Results indicated that 100 times HCO^-₃，K⁺，SO^2-₄，NH⁺₄，Mn²⁺，Na⁺，Cu²⁺，Al³⁺，Zn²⁺，F^-，Mg²⁺，Ba²⁺，Ca²⁺，Co²⁺，NO^3-，NO^2-, 50 times CO^2-₃，Cr⁶⁺, 10 times Hg²⁺，BSA did not interfere with the determination. It showed that this Fluorescence method had good selectivity. Thus, a simple, rapid, sensitive and highly selective fluorescence method for the determination of Fe (Ⅲ) was developed. Sample solution of dairy products was prepared by the following steps: accurately absorbd 1.4 mL dairy products with 600 L acetic acid (V/V=3%), centrifugated for 3 min at 10 000 r·min^-1, then took the centrifugal supernatant 1 mL with 48 L 2.5 mol·L^-1 NaOH, mixed well, centrifugated for 3 min at 10 000 r·min^-1, and finally piped 1 mL the supernatant, and diluted to 5 mL to get the sample solution. Then the new catalytic fluorescence method was used to determine the content of Fe(Ⅲ) in milk samples, with satisfactory results. The relative standard deviation was 0.29%～0.41%, and the recovery was 94.6%～108.0%.

Key words：Iron ion; Catalysis; 3,3’,5,5’-tetramethylbenzidine; Fluorescence

收稿日期: 2018-09-08 修订日期: 2019-01-10

中图分类号:

O657.3

基金资助: 国家自然科学基金项目(21465006, 21477025, 21467001)资助

通讯作者: 梁爱惠，蒋治良 E-mail: zljiang@mailbox.gxnu.edu.cn; ahliang2008@163.com

作者简介: 李丹，1996年生，广西师范大学环境与资源学院硕士研究生 e-mail: 244108362@qq.com

引用本文:

李丹，梁爱惠，蒋治良. 一个催化氧化四甲基联苯胺荧光测定超痕量铁的新方法[J]. 光谱学与光谱分析, 2019, 39(10): 3066-3070.
LI Dan, LIANG Ai-hui, JIANG Zhi-liang. Fluorescence Detection of Ultratrace Fe³⁺ Ions Based on Its Catalysis of the New Indicator Reaction between TMB and H₂O₂. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2019, 39(10): 3066-3070.

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