The fluorescence quenching method using laser-induced nanosecond time-resolved fluorescence (LITRF) combined with parallel factor (PARAFAC) analysis was developed for in situ investigation of the interactions of two typical polycyclic aromatic hydrocarbons (PAHs),pyrene (Pyr) and phenanthrene (Phe) either singly or in a mixture with Aldrich humic acid (HA).The concentration of free Pyr and Phe cannot be determined directly at the same time because the LITRF spectra of Pyr,Phe and HA are overlapping at the excitation wavelength of 266 nm.The fluorescent interference of HA can be eliminated quickly and effectively,while the fluorescence intensity and fluorescence decay curves of Pyr and Phe can be acquired by LITRF-PARAFAC analysis.The binding characteristics of PAHs with HA were described with Freundlich isothermal model.Nonlinear binding (n<1) and competitive sorption of Pyr and Phe on HA were verified,which showed that the linearity increased (n tend to be 1) and the single point binding coefficient decreased after the addition of Pyr or Phe.The degree of competition was depended on the concentrations of cosolutes,and competition was much stronger in the case of relatively high competitor concentrations.In addition,the results indicated that the binding characteristics of single component Pyr and Phe with HA obtained with LITRF-PARAFAC quenching method were in accordance with conventional fluorescence quenching method.The quenching mechanism for Pyr and Phe by HA was primarily static quenching which was also verified by quenching rate constant and fluorescence lifetime analyses.The LITRF-PARAFAC quenching method could be used for in situ investigate the interactions between mixture of PAHs and HA,which could be applied to predict and evaluate the environmental behavior and ecological risk of PAHs in real time.%基于平行因子(PARAFAC)分析的激光诱导纳秒时间分辨荧光(LITRF)猝灭法原位研究典型多环芳烃(PAHs)芘(Pyr)和菲(Phe)单独及混合状态下与Aldrich腐植酸(HA)相互作用.实验表明,在266 nm激发波长下,Pyr,Phe及HA三者的LITRF光谱相互重叠,无法直接同时测定混合组分中游离Pyr和Phe荧光强度.利用PARAFAC分析可快速有效消除HA荧光干扰,获取混合溶液LITRF光谱中Pyr和Phe荧光强度及各自荧光衰减曲线,并以Freundlich吸附等温模型描述PAHs与HA结合特性.结果验证了Pyr和Phe与HA以非线性形式结合(n<1);Pyr和Phe共存时,两者与HA结合存在竞争关系.加入竞争吸附质后,Pyr和Phe与HA的吸附等温线非线性程度减弱(n趋近于1),单点结合系数下降,且竞争强度随竞争吸附质浓度增加而增强.此外,LITFR-PARAFAC猝灭法与传统荧光猝灭法所得单组分Pyr和Phe与HA结合特性无显著差异.猝灭速率常数及荧光寿命分析反映出Pyr和Phe与HA间荧光以静态猝灭形式为主.LITFR-PARAFAC猝灭法可快速原位研究混合PAHs与HA相互作用,有利于原位预测和评估PAHs的环境行为及其生态风险.
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