荧光及共振光散射技术对某些食品添加剂的检测及反应机理研究
摘要
食品安全是大事,涉及人们身体健康、生命安全和社会稳定。调查显示,我国部分省市的许多餐饮业存在食品添加剂违规违法案例,具体表现为：1)食品添加剂的超标、超范围使用；2)添加非食品级添加剂,出现食品添加剂质量不合格,品质差；3)使用非食品添加剂,如苏丹红。近几年,食品安全成为困扰民生的-个头疼问题,出现了诸如“苏丹红事件”、“三聚氰胺事件”、“染色馒头事件”等重大食品安全事件,引发了社会忧虑情绪。由于食品添加剂种类多,添加范围广,产品组成复杂。方便快速、灵敏度高、选择性好、准确度好、重现性好的科学检测出食品添加剂的类别、用量是一个不小的挑战。本论文主要以某些食品添加剂为主要研究对象,包括有合法和非法使用的食品着色剂：如柠檬黄(tartrazine, Tz)日落黄(sunset yellow FCF, SY)及苏丹红Ⅰ-Ⅳ(Sudan Ⅰ-Ⅳ)等。同时为了研究的需要,也选择了非食品用有机染料亚甲基蓝(methylene blue, MB)做对比研究。以这些物质作为对象,用荧光技术、共振光散射技术建立相应的测试方法；选择非法食品添加剂三聚氰胺(melamine),研究其与磷酸盐的聚沉反应特点及反应机理;选择食品添加剂没食子酸(gallic acid),研究其与高聚物聚乙烯亚胺(ethylene imine polymer, PEI)在强碱条件下的反应特点及反应机理。本文主要研究内容和成果如下：
1.基于荧光共振能量转移的快速高灵敏检测食品中的着色剂偶氮染料柠檬黄
建立了一种基于吸附竞争反应的快速、高灵敏度测定食品着色剂Tz的荧光分析方法。强荧光探针fluorescein (Fl),能被还原态石墨烯(rGO)吸附生成Fl/rGO加合物并发生荧光共振能量转移(FRET),其荧光能被显著猝灭。加入Tz后,发生竞争吸附反应,将F1从其Fl/rGO加合物中置换出来,使荧光得到恢复。建立了Tz的荧光分析方法。在优化条件下,测定Tz时的一元线性回归方程为FEETTz=0.0159cTTz+0.0554；标准曲线的相关系数(R2)为0.9955；检出限(3σ/斜率)为0.53ngmL-1。该方法用于实际样品的测定,结果令人满意。
2.基于荧光素/还原态石墨烯加合物的快速高灵敏检测有机染料通用分析方法研究
建立了一种基于吸附竞争反应的快速、高灵敏度测定有机染料的通用荧光分析方法。强荧光探针F1,能被rGO吸附生成Fl/rGO加合物并发生FRET,其荧光能被显著猝灭。实验证明,不仅阴离子染料SY,而且阳离子染料MB都能与Fl/rGO加合物发生竞争吸附反应,将F1从其F1/rGO加合物中置换出来,使荧光得到恢复。从而建立起有机染料的通用荧光分析方法。在优化条件下,测定SY、MB时的—元线性回归方程分别为FEESY=0.0142cSY-0.0427和FEEMB=0.0192CMB-0.3103；检出限(36/斜率)分别为1.03ng mL-1和1.15ng mL-1。该方法用于实际样品的测定,结果令人满意。
3.基于配体置换反应超灵敏选择性荧光检测苏丹红Ⅰ、Ⅲ
建立了一种基于配体置换反应的高灵敏度选择性测定SudanⅠ、Ⅲ的荧光分析方法。强荧光探针钙黄绿素(calcein),能与Cu(Ⅱ)反应生成Cu(Ⅱ)-calcein配合物,其荧光能被Cu(Ⅱ)显著猝灭。加入Sudan Ⅰ、Ⅲ后,发生配体置换反应,将calcein从其Cu(Ⅱ)-calcein配合物置换出来,使荧光得到恢复。实验发现,SudanⅠ、Ⅲ能同等程度地恢复Cu(Ⅱ)-calcein配合物的荧光。但是Sudan Ⅱ、Ⅳ因为具有2-甲基位阻的作用,不能和Cu(Ⅱ)-calcein配合物发生配体置换反应,即不能使Cu(Ⅱ)-calcein配合物的荧光恢复。从而实现了对SudanⅠ、Ⅲ进行个别地检测或是对其总量的检测,并且不会受到SudanⅡ、Ⅳ的干扰。在优化条件下,测定SudanⅠ、Ⅲ时的一元线性回归方程分别为FEESudanⅠ=0.0032cSudan Ⅰ-0.02613和FEESudan Ⅲ=0.0033cSudanⅢ-0.02467;标准曲线的相关系数(R2)分别为0.9984和0.9955;检出限(3σ/斜率)分别为211.3和208.5pmol L-1。该方法用于实际样品的测定,结果令人满意。
4.苏丹红Ⅰ-Ⅳ与[Cu(NH3)4]2+离子的反应机理研究及分析应用
研究了Sudan Ⅰ-Ⅳ在含氨溶液中与[Cu(NH3)4]2+相互作用机理及分析应用。实验发现Sudan Ⅱ和[Cu(NH3)4]2+反应产生了极强的共振光散射信号,而Sudan Ⅰ、Ⅲ、IV却没有出现明显的共振光散射变化信号。分析了Sudan Ⅰ-Ⅳ在含氨溶液中与[Cu(NH3)4]2+相互作用机理。紫外光谱、红外光谱、XRD-粉末衍射等分析表明,SudanⅠ、Ⅲ与[Cu(NH3)4]2+生成了稳定的螯合物,而Sudan Ⅱ、Ⅳ与[Cu(NH3)4]2+形成了离子缔合物。这种不同的结果是由于Sudan Ⅱ、Ⅳ存在2-甲基,产生了空间位阻效应,影响了它与中心离子铜的配位,所以只能通过静电引力形成离子缔合物。实验并优化了以[Cu(NH3)4]2+为探针的测定Sudan Ⅱ的实验条件,在优化条件下,测定Sudan Ⅱ时的一元线性回归方程为△IRLs=1488.6c-82.2(c,μg mL-1)；检出限(3σ/斜率)为2.1ng mL-1。该方法用于实际样品的测定,结果令人满意。
5.pH诱导的三聚氰胺与磷酸盐聚沉反应机理研究
应用共振光散射、X-单晶衍射、X-粉末衍射、傅里叶变换红外光谱以及紫外—可见光谱分析等手段系统地研究了pH诱导的三聚氰胺(Mel)与磷酸盐(Ps)聚沉反应机理。在实验条件下,三聚氰胺磷酸盐组成为2HMel-·HPO42-·3H2O,各组成成分间通过静电引力、氢键及π-π堆积作用组成了稳定的三聚氰胺磷酸盐。实验表明三聚氰胺与磷酸盐聚沉反应高度依赖于pH。pH的改变直接影响到Mel、Hmel-的组成比,从而改变其氢键及电性作用力,进而影响到三聚氰胺与磷酸盐聚沉与溶解。研究发现,聚沉过程中,三聚氰胺质子化有两种方式,发展和丰富了Peschar关于三聚氰胺磷酸盐质子转移观点,为三聚氰胺磷酸盐质子转移观点提供了有力的实验支撑。
6.强碱性条件下没食子酸和聚乙烯亚胺主客体包合反应机理研究
详细研究了在强碱性条件下(pH12.22),没食子酸与聚乙烯亚胺(PEI)形成主客体包合物的荧光光谱和吸收光谱。研究发现,没食子酸与PEI发生主客体包合作用生成具有荧光增强的主客体包合物。初步探讨了反应机理：在强碱条件下,没食子酸与PEI发生相互作用,并逐步地进入到PEI疏水性空腔形成组成比为1：1的没食子酸-PEI主客体包合物。根据包合物吸收光谱,证明了没食子酸在包合物中主要以对位醌式没食子酸离子形式存在,体系显淡黄色。在含有邻位醌式没食子酸的自氧化产物中加入PEI,橙红色的邻位醌式没食子酸会快速地向淡黄色对位醌式没食子酸转化,随后形成没食子酸-PEI主客体包合物。本研究说明没食子酸自氧化速率在PEI中能得到了有效地缓阻,PEI对没食子酸具有一定的保护作用,主客体包合物在相当长的时间里不会发生进一步地氧化。
Food safety issues that induced by the misuse of food additives have caused worldwide concern in recent years. The misuse of food additives is harmful to the pepole's health and safety which will further affect the social stability. In this thesis, some novel fluorescence and resonance light scattering methods have been developed to the dectection of some legal or illegal food additives (e.g. tartrazine, sunset yellow FCF, Sudan Ⅰ-Ⅳ). In addition, the intereaction between Sudan Ⅰ-Ⅳ and [Cu(NH3)4]2-, the mechanism of pH-induced aggregation reaction between melamine and phosphate, and the interaction between gallic acid and ethylene imine polymer in strong alkaline solution have also been studied. The main contents and some conclusions of the thesis are as follows:
1. Sensitive turn-on fluorescent detection of tartrazine based on fluorescence resonance energy transfer
We introduce a sensitive, rapid, label-free and general fluorescent method for the determination of tartrazine (Tz) by competitive binding to reduced graphene oxide (rGO) against fluorescein, and the fluorescence recovery upon fluorescein desorption from rGO can be directly spectroscopically followed. It was found that the fluorescence enhancement efficiency (FEE) is proportional to the Tz concentration over the range of2.36-236.11ng mL-1. The linear regression equations were calculated as FEETz=0.0159CTz+0.0554, with the corresponding detection limit (3σ/slope) of0.53ng mL-1. The Tz in a lemon-flavored sports drink sample was assayed with satisfactory results.
2. Fast and sensitive dye-sensor based on fluorescein/reduced graphene oxide complex
We report on a fast, sensitive, label-free and general dye-sensor platform for synthetic organic dyes detection by competitive adsorption on the reduced graphene oxide (rGO) against fluorescent dye (FD). Fluorescein (Fl) as fluorescence indicator and the cationic dye methylene blue (MB) as model analyte were employed to investigate the analytical feature of this assay platform. The anionic dye sunset yellow FCF (SY) was chosen as comparison analyte to test the generality of this strategy. Results show that rGO can bind Fl and quench the fluorescence by fluorescence resonance energy transfer, while MB can displace Fl fast from the Fl/rGO complex by competitive adsorption, inducing the fluorescence recovery which provides a quantitative readout for MB. Besides, this design was simply based on the competitive adsorption of rGO between dye and FD, so it could be generally applied to other dyes for label-free detection of a broad range of analytes, as evidenced by the comparison analyte SY. It was found that the fluorescence enhancement efficiency (FEE) is proportional to the dye concentration over the range of7.60-420.00ng mL-1MB and7.28-400.25ng mL-1SY, respectively. The linear regression equations were calculated as FEEMB=0.0192CMB-0.3103for MB and FEESY=0.0142CSY-0.0427for SY, with the corresponding detection limits (3σ/slope) of1.03ng mL-1and1.15ng mL-respectively. The MB in waste water and SY in an orange-flavored sports drink samples were assayed with satisfactory results.
3. An ultrasensitive and selective fluorescence assay for Sudan Ⅰ and Ⅲ against the influence of Sudan Ⅱ and Ⅳ
We report on an ultrasensitive and selective fluorescence assay for Sudan Ⅰ and Ⅲ against the influence of Sudan Ⅱ and Ⅳ based on ligand exchange mechanism. Calcein as a fluorescence indicator and Sudan Ⅰ-Ⅳ as model analytes were employed to investigate the analytical feature of this assay platform. Results show that the fluorescence of calcein can be efficiently quenched by Cu(Ⅱ). When the ligand exchange reaction proceeds, calcein is deprived of Cu(Ⅱ) by Sudan Ⅰ and Ⅲ, resulting in the fluorescence recovery of calcein. However, the ligand exchange reaction does not happen in the presence of Sudan Ⅱ or Ⅳ due to the2-methyl steric effects, which is favorable for selective determination of Sudan Ⅰ and Ⅲ against the influence of Sudan Ⅱ and Ⅳ. It was found that the fluorescence enhancement efficiency (FEE) against the concentration of Sudan (CSudan nmol L-1) shows a linear relationship. The calibration equations are FEESudan1=0.0032CSudan1-0.02613, and FEESudanⅢ=0.0033CSudanⅢ-0.02467over the corresponding linear range of11.25-2078.29and9.441035.78nmol L-1with the correlation coefficients (R2) of0.9984and0.9955, respectively. And the detection limits (3σ/slope) are calculated to be211.3and208.5pmol L-1for Sudan Ⅰ and Ⅲ, respectively, showing ultralow detection limit. The Sudan dye in a commercial chilli powder sample was assayed with satisfactory results.
4. Study on the reaction between Sudan Ⅰ-Ⅳ dye and [Cu(NH3)4]2+and its analytical application
Sudan Ⅰ-Ⅳ in [Cu(NH3)4]2-circumstances had different resonance light scattering (RLS) properties. Only Sudan Ⅱ-[Cu(NH3)4]2+complex shows enhanced RLS signal and the the RLS intensity was proportional to the concentration of Sudan Ⅱ in the range of0.06-1.20μg mL-1with correlation coefficient,0.9981. Linear regression equation was△IRLS=1488.6c-82.2(c, μg mL-1). The detection limit was2.1ng mL-1. The reaction mechanism has been investigated by a combination of RLS spectra, UV-absorption spectra, infrared spectra, and X-ray diffraction. The results showed that the copper chelates of Sudan Ⅰ and Ⅲ were formed while the ion-association complexes of Sudan Ⅱ and Ⅳ were formed in the presence of [Cu(NH3)4]2-
5. Mechanism of the pH-induced aggregation reaction between melamine and phosphate
The mechanism of the pH-induced aggregation reaction between melamine (Mel) and phosphate (Ps) has been studied by a combination of resonance light scattering (RLS), FTIR, UV-visible spectra, single-crystal X-ray and powder X-ray diffraction for the first time. The results confirm that the aggregation reaction between Mel and Ps is highly pH-dependent. Changes of pH dramatically influence the mol ratio of Mel to melaminium (Hmel0) and subsequently affect the synergistic manner of hydrogen bonds, electrostatic and π-π stacking interactions, resulting in the reversibility of aggregation and dissolution process. The composition of the Mel-Ps aggregates has been characterized to be2[Hmel+]·[HPO42-]·3H2O. Mechanism analysis demonstrated that there existed two different ways for the transformation of Mel into Hmel-by considering the aggregation reaction process and conditions.
6. Interaction between gallic acid and ethylene imine polymer in strong alkaline solution
The interaction between gallic acid and PEI in strong alkaline solution has been studied by a combination of UV-visible spectra and fluorescence spectra. In basic solution (pH12.22), gallic acid undergoes fast autooxidation, leading to the production of orange red solution which is non-fluorescent. Howerer, it has been changed in the presence of PEI. A strong fluorescence gallic acid-PEI complex has been formed as a light yellow one by entrapping gallic acid inside the more hydrophobic PEI box. The UV-visible spectra showed that at pH value of12.22, gallic acid in the PEI box was in the form of p-quinone. The result of the continuous variation method showed that PEI could bind to gallic acid at a molar ratio of1:1. In addition, it is dramatical that the presence of PEI slows down the autooxidation of gallic acid compared to buffer solution only.
1.基于荧光共振能量转移的快速高灵敏检测食品中的着色剂偶氮染料柠檬黄
建立了一种基于吸附竞争反应的快速、高灵敏度测定食品着色剂Tz的荧光分析方法。强荧光探针fluorescein (Fl),能被还原态石墨烯(rGO)吸附生成Fl/rGO加合物并发生荧光共振能量转移(FRET),其荧光能被显著猝灭。加入Tz后,发生竞争吸附反应,将F1从其Fl/rGO加合物中置换出来,使荧光得到恢复。建立了Tz的荧光分析方法。在优化条件下,测定Tz时的一元线性回归方程为FEETTz=0.0159cTTz+0.0554；标准曲线的相关系数(R2)为0.9955；检出限(3σ/斜率)为0.53ngmL-1。该方法用于实际样品的测定,结果令人满意。
2.基于荧光素/还原态石墨烯加合物的快速高灵敏检测有机染料通用分析方法研究
建立了一种基于吸附竞争反应的快速、高灵敏度测定有机染料的通用荧光分析方法。强荧光探针F1,能被rGO吸附生成Fl/rGO加合物并发生FRET,其荧光能被显著猝灭。实验证明,不仅阴离子染料SY,而且阳离子染料MB都能与Fl/rGO加合物发生竞争吸附反应,将F1从其F1/rGO加合物中置换出来,使荧光得到恢复。从而建立起有机染料的通用荧光分析方法。在优化条件下,测定SY、MB时的—元线性回归方程分别为FEESY=0.0142cSY-0.0427和FEEMB=0.0192CMB-0.3103；检出限(36/斜率)分别为1.03ng mL-1和1.15ng mL-1。该方法用于实际样品的测定,结果令人满意。
3.基于配体置换反应超灵敏选择性荧光检测苏丹红Ⅰ、Ⅲ
建立了一种基于配体置换反应的高灵敏度选择性测定SudanⅠ、Ⅲ的荧光分析方法。强荧光探针钙黄绿素(calcein),能与Cu(Ⅱ)反应生成Cu(Ⅱ)-calcein配合物,其荧光能被Cu(Ⅱ)显著猝灭。加入Sudan Ⅰ、Ⅲ后,发生配体置换反应,将calcein从其Cu(Ⅱ)-calcein配合物置换出来,使荧光得到恢复。实验发现,SudanⅠ、Ⅲ能同等程度地恢复Cu(Ⅱ)-calcein配合物的荧光。但是Sudan Ⅱ、Ⅳ因为具有2-甲基位阻的作用,不能和Cu(Ⅱ)-calcein配合物发生配体置换反应,即不能使Cu(Ⅱ)-calcein配合物的荧光恢复。从而实现了对SudanⅠ、Ⅲ进行个别地检测或是对其总量的检测,并且不会受到SudanⅡ、Ⅳ的干扰。在优化条件下,测定SudanⅠ、Ⅲ时的一元线性回归方程分别为FEESudanⅠ=0.0032cSudan Ⅰ-0.02613和FEESudan Ⅲ=0.0033cSudanⅢ-0.02467;标准曲线的相关系数(R2)分别为0.9984和0.9955;检出限(3σ/斜率)分别为211.3和208.5pmol L-1。该方法用于实际样品的测定,结果令人满意。
4.苏丹红Ⅰ-Ⅳ与[Cu(NH3)4]2+离子的反应机理研究及分析应用
研究了Sudan Ⅰ-Ⅳ在含氨溶液中与[Cu(NH3)4]2+相互作用机理及分析应用。实验发现Sudan Ⅱ和[Cu(NH3)4]2+反应产生了极强的共振光散射信号,而Sudan Ⅰ、Ⅲ、IV却没有出现明显的共振光散射变化信号。分析了Sudan Ⅰ-Ⅳ在含氨溶液中与[Cu(NH3)4]2+相互作用机理。紫外光谱、红外光谱、XRD-粉末衍射等分析表明,SudanⅠ、Ⅲ与[Cu(NH3)4]2+生成了稳定的螯合物,而Sudan Ⅱ、Ⅳ与[Cu(NH3)4]2+形成了离子缔合物。这种不同的结果是由于Sudan Ⅱ、Ⅳ存在2-甲基,产生了空间位阻效应,影响了它与中心离子铜的配位,所以只能通过静电引力形成离子缔合物。实验并优化了以[Cu(NH3)4]2+为探针的测定Sudan Ⅱ的实验条件,在优化条件下,测定Sudan Ⅱ时的一元线性回归方程为△IRLs=1488.6c-82.2(c,μg mL-1)；检出限(3σ/斜率)为2.1ng mL-1。该方法用于实际样品的测定,结果令人满意。
5.pH诱导的三聚氰胺与磷酸盐聚沉反应机理研究
应用共振光散射、X-单晶衍射、X-粉末衍射、傅里叶变换红外光谱以及紫外—可见光谱分析等手段系统地研究了pH诱导的三聚氰胺(Mel)与磷酸盐(Ps)聚沉反应机理。在实验条件下,三聚氰胺磷酸盐组成为2HMel-·HPO42-·3H2O,各组成成分间通过静电引力、氢键及π-π堆积作用组成了稳定的三聚氰胺磷酸盐。实验表明三聚氰胺与磷酸盐聚沉反应高度依赖于pH。pH的改变直接影响到Mel、Hmel-的组成比,从而改变其氢键及电性作用力,进而影响到三聚氰胺与磷酸盐聚沉与溶解。研究发现,聚沉过程中,三聚氰胺质子化有两种方式,发展和丰富了Peschar关于三聚氰胺磷酸盐质子转移观点,为三聚氰胺磷酸盐质子转移观点提供了有力的实验支撑。
6.强碱性条件下没食子酸和聚乙烯亚胺主客体包合反应机理研究
详细研究了在强碱性条件下(pH12.22),没食子酸与聚乙烯亚胺(PEI)形成主客体包合物的荧光光谱和吸收光谱。研究发现,没食子酸与PEI发生主客体包合作用生成具有荧光增强的主客体包合物。初步探讨了反应机理：在强碱条件下,没食子酸与PEI发生相互作用,并逐步地进入到PEI疏水性空腔形成组成比为1：1的没食子酸-PEI主客体包合物。根据包合物吸收光谱,证明了没食子酸在包合物中主要以对位醌式没食子酸离子形式存在,体系显淡黄色。在含有邻位醌式没食子酸的自氧化产物中加入PEI,橙红色的邻位醌式没食子酸会快速地向淡黄色对位醌式没食子酸转化,随后形成没食子酸-PEI主客体包合物。本研究说明没食子酸自氧化速率在PEI中能得到了有效地缓阻,PEI对没食子酸具有一定的保护作用,主客体包合物在相当长的时间里不会发生进一步地氧化。
Food safety issues that induced by the misuse of food additives have caused worldwide concern in recent years. The misuse of food additives is harmful to the pepole's health and safety which will further affect the social stability. In this thesis, some novel fluorescence and resonance light scattering methods have been developed to the dectection of some legal or illegal food additives (e.g. tartrazine, sunset yellow FCF, Sudan Ⅰ-Ⅳ). In addition, the intereaction between Sudan Ⅰ-Ⅳ and [Cu(NH3)4]2-, the mechanism of pH-induced aggregation reaction between melamine and phosphate, and the interaction between gallic acid and ethylene imine polymer in strong alkaline solution have also been studied. The main contents and some conclusions of the thesis are as follows:
1. Sensitive turn-on fluorescent detection of tartrazine based on fluorescence resonance energy transfer
We introduce a sensitive, rapid, label-free and general fluorescent method for the determination of tartrazine (Tz) by competitive binding to reduced graphene oxide (rGO) against fluorescein, and the fluorescence recovery upon fluorescein desorption from rGO can be directly spectroscopically followed. It was found that the fluorescence enhancement efficiency (FEE) is proportional to the Tz concentration over the range of2.36-236.11ng mL-1. The linear regression equations were calculated as FEETz=0.0159CTz+0.0554, with the corresponding detection limit (3σ/slope) of0.53ng mL-1. The Tz in a lemon-flavored sports drink sample was assayed with satisfactory results.
2. Fast and sensitive dye-sensor based on fluorescein/reduced graphene oxide complex
We report on a fast, sensitive, label-free and general dye-sensor platform for synthetic organic dyes detection by competitive adsorption on the reduced graphene oxide (rGO) against fluorescent dye (FD). Fluorescein (Fl) as fluorescence indicator and the cationic dye methylene blue (MB) as model analyte were employed to investigate the analytical feature of this assay platform. The anionic dye sunset yellow FCF (SY) was chosen as comparison analyte to test the generality of this strategy. Results show that rGO can bind Fl and quench the fluorescence by fluorescence resonance energy transfer, while MB can displace Fl fast from the Fl/rGO complex by competitive adsorption, inducing the fluorescence recovery which provides a quantitative readout for MB. Besides, this design was simply based on the competitive adsorption of rGO between dye and FD, so it could be generally applied to other dyes for label-free detection of a broad range of analytes, as evidenced by the comparison analyte SY. It was found that the fluorescence enhancement efficiency (FEE) is proportional to the dye concentration over the range of7.60-420.00ng mL-1MB and7.28-400.25ng mL-1SY, respectively. The linear regression equations were calculated as FEEMB=0.0192CMB-0.3103for MB and FEESY=0.0142CSY-0.0427for SY, with the corresponding detection limits (3σ/slope) of1.03ng mL-1and1.15ng mL-respectively. The MB in waste water and SY in an orange-flavored sports drink samples were assayed with satisfactory results.
3. An ultrasensitive and selective fluorescence assay for Sudan Ⅰ and Ⅲ against the influence of Sudan Ⅱ and Ⅳ
We report on an ultrasensitive and selective fluorescence assay for Sudan Ⅰ and Ⅲ against the influence of Sudan Ⅱ and Ⅳ based on ligand exchange mechanism. Calcein as a fluorescence indicator and Sudan Ⅰ-Ⅳ as model analytes were employed to investigate the analytical feature of this assay platform. Results show that the fluorescence of calcein can be efficiently quenched by Cu(Ⅱ). When the ligand exchange reaction proceeds, calcein is deprived of Cu(Ⅱ) by Sudan Ⅰ and Ⅲ, resulting in the fluorescence recovery of calcein. However, the ligand exchange reaction does not happen in the presence of Sudan Ⅱ or Ⅳ due to the2-methyl steric effects, which is favorable for selective determination of Sudan Ⅰ and Ⅲ against the influence of Sudan Ⅱ and Ⅳ. It was found that the fluorescence enhancement efficiency (FEE) against the concentration of Sudan (CSudan nmol L-1) shows a linear relationship. The calibration equations are FEESudan1=0.0032CSudan1-0.02613, and FEESudanⅢ=0.0033CSudanⅢ-0.02467over the corresponding linear range of11.25-2078.29and9.441035.78nmol L-1with the correlation coefficients (R2) of0.9984and0.9955, respectively. And the detection limits (3σ/slope) are calculated to be211.3and208.5pmol L-1for Sudan Ⅰ and Ⅲ, respectively, showing ultralow detection limit. The Sudan dye in a commercial chilli powder sample was assayed with satisfactory results.
4. Study on the reaction between Sudan Ⅰ-Ⅳ dye and [Cu(NH3)4]2+and its analytical application
Sudan Ⅰ-Ⅳ in [Cu(NH3)4]2-circumstances had different resonance light scattering (RLS) properties. Only Sudan Ⅱ-[Cu(NH3)4]2+complex shows enhanced RLS signal and the the RLS intensity was proportional to the concentration of Sudan Ⅱ in the range of0.06-1.20μg mL-1with correlation coefficient,0.9981. Linear regression equation was△IRLS=1488.6c-82.2(c, μg mL-1). The detection limit was2.1ng mL-1. The reaction mechanism has been investigated by a combination of RLS spectra, UV-absorption spectra, infrared spectra, and X-ray diffraction. The results showed that the copper chelates of Sudan Ⅰ and Ⅲ were formed while the ion-association complexes of Sudan Ⅱ and Ⅳ were formed in the presence of [Cu(NH3)4]2-
5. Mechanism of the pH-induced aggregation reaction between melamine and phosphate
The mechanism of the pH-induced aggregation reaction between melamine (Mel) and phosphate (Ps) has been studied by a combination of resonance light scattering (RLS), FTIR, UV-visible spectra, single-crystal X-ray and powder X-ray diffraction for the first time. The results confirm that the aggregation reaction between Mel and Ps is highly pH-dependent. Changes of pH dramatically influence the mol ratio of Mel to melaminium (Hmel0) and subsequently affect the synergistic manner of hydrogen bonds, electrostatic and π-π stacking interactions, resulting in the reversibility of aggregation and dissolution process. The composition of the Mel-Ps aggregates has been characterized to be2[Hmel+]·[HPO42-]·3H2O. Mechanism analysis demonstrated that there existed two different ways for the transformation of Mel into Hmel-by considering the aggregation reaction process and conditions.
6. Interaction between gallic acid and ethylene imine polymer in strong alkaline solution
The interaction between gallic acid and PEI in strong alkaline solution has been studied by a combination of UV-visible spectra and fluorescence spectra. In basic solution (pH12.22), gallic acid undergoes fast autooxidation, leading to the production of orange red solution which is non-fluorescent. Howerer, it has been changed in the presence of PEI. A strong fluorescence gallic acid-PEI complex has been formed as a light yellow one by entrapping gallic acid inside the more hydrophobic PEI box. The UV-visible spectra showed that at pH value of12.22, gallic acid in the PEI box was in the form of p-quinone. The result of the continuous variation method showed that PEI could bind to gallic acid at a molar ratio of1:1. In addition, it is dramatical that the presence of PEI slows down the autooxidation of gallic acid compared to buffer solution only.
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