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FTIR and Synchronous Fluorescence Heterospectral Two-Dimensional Correlation Analyses on the Binding Characteristics of Copper onto Dissolved Organic Matter Wei Chen, Nuzahat Habibul, Xiao-Yang Liu, Guo-Ping Sheng, and Han-Qing Yu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es5049495 • Publication Date (Web): 22 Jan 2015 Downloaded from http://pubs.acs.org on February 5, 2015

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Environmental Science & Technology

FTIR and Synchronous Fluorescence Heterospectral Two-Dimensional Correlation Analyses on the Binding Characteristics of Copper onto Dissolved Organic Matter

Wei Chen, Nuzahat Habibul, Xiao-Yang Liu, Guo-Ping Sheng, Han-Qing Yu* CAS Key Laboratory of Urban Pollutant Conversion, Department of Chemistry, University of Science and Technology of China, Hefei, 230026, China

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Dissolved organic matter (DOM) is known to form strong complexes with heavy

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metals and thus governs the distribution, toxicity, bioavailability, and the ultimate fate

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of heavy metals in the environment. The relevant aspects of metal-organic interactions

4

remain unclear because the metal binding functionalities in DOM are substantially

5

non-uniform and the availability of the models is limited. In this work, two-

6

dimensional correlation spectroscopy (2DCOS) integrated with synchronous

7

fluorescence and infrared absorption spectroscopy was used to explore the binding

8

process of copper to DOM. A series of heterogeneous binding sites in humic acid

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(HA), a representative DOM, and the subsequent subtle changes of these sites within

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the molecular interactions were elucidated by 2DCOS method. The band assignments

11

and the correspondence between the results obtained by two spectral probes

12

(synchronous fluorescence and infrared absorption spectra) were verified by hetero-

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2DCOS. Our results showed that, during the copper binding process, the carboxyl and

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polysaccharide groups gave the fastest responses to copper binding. Then,

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fluorescence quencing of fluorescent humic-like moieties occurred with vibrational

16

change of the related functionalities, i.e., phenolic and aryl carboxylic groups, which

17

further induces the fluorescence quenching of fulvic-like fractions. Finally, small

18

amounts of amide and aliphatic groups participated in the copper binding after the

19

fluorescence of the protein-like fraction decreased. With these promising results, a

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comprehensive picture of structural changes of HA during copper binding process was

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developed, highlighting the superior potential of 2D-heterospectral correlation

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spectroscopy in studying complex interactions in environment.

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INTRODUCTION

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Ubiquitously existing in aquatic and soil environments with various chemical

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functional groups,1-3 dissolved organic matter (DOM) is known to form strong

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complexes with heavy metals, thus determining the distribution, toxicity,

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bioavailability, and the ultimate fate of heavy metals in the environment.4-6

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Carboxylic and phenolic groups in DOM are considered as the predominant

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coordination sites with metal ions.7, 8 Some recent reports, however, have suggested

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the presence of other types of metal coordination arising from the heterogeneous

32

distribution of the metal binding sites in DOM.9, 10

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Approaches studying the metal-binding characteristics of DOM are quite diverse,

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including size exclusion chromatography, equilibrium dialysis, differential absorbance

35

titration, and fluorescence excitation-emission matrix coupled with PARAFAC

36

analysis.6, 11-13 Optical spectroscopy is the most frequently used tool for metal-DOM

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interaction analysis owing to its characteristic responses to the abundant light

38

absorbing and emitting groups in metal-DOM complexes. Models like NICA-

39

Donnan14 or Stockholm Humic Model,15 and chemometric methods like PARAFAC

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analysis,10 have been employed to enhance the spectral resolution to reveal the subtle

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spectral changes and highly overlapping responses during the binding process.

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However, since the metal binding functionalities in DOM are substantially non-

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uniform, the availability of models is limited. Therefore, some aspects of metal-

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organic interactions, such as the copper binding affinities of functional groups in HA,

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are unclear yet.

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Two-dimensional correlation spectra (2DCOS) is capable of resolving overlapped

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peaks by extending spectra along the second dimension as well as providing

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information about the relative directions and sequential orders of structural variations.

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Thus, it has been recently applied to probe the interaction mechanisms of some

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environmental-related substances.9, 16-18 The major probes used in 2DCOS are UV-Vis,

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fluorescence and IR, each of which gives particular molecular information about the

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binding process. 2D hetero-spectral correlation analysis integrates two different types

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of spectra for a system obtained by using multiple spectroscopic probes under the

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same external perturbation.19, 20 Correlation between the two complementary spectral

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signals can help to better understand the structural variation caused by the

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perturbation. For these appealing features, hetero-2DCOS is among the most active

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areas of 2DCOS research.21-23

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In the present work, by using a novel hetero-2DCOS analysis which integrates

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synchronous fluorescence and Fourier transform infrared (FTIR) absorption

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spectroscopy, we aim to provide an in-depth understanding about the heterogeneous

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binding characteristics of copper onto DOM at the molecular level. This might, to the

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best of our knowledge, be the first time to use such a new hetero-2DCOS method to

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study the metal-DOM interactions. Humic acid (HA) was used as the representative

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DOM and copper concentration was used as the external perturbation. The 2D

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fluorescence, 2D IR and fluorescence/IR hetero-2DCOS were compared and

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combined to elucidate the copper-DOM interaction mechanisms.

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EXPERIMENTAL SECTION

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Sample Preparation. Commercial HAs (Sigma-Aldrich Co., USA) were purified by

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dissolving in NaOH solution (pH = 13.0) with subsequent filtration and acidifying the

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filtrate with HCl to pH 1.0. The precipitate was collected by filtration, washed

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extensively with 0.1 M HCl and freeze-dried. AR-grade CuCl2, NaOH and HCl were

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purchased from Sinopharm Chemical Reagent Co., China, and were used as received.

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The purified HAs were dissolved and diluted to a concentration of 12.5 mg/L (pH

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7.0). Cu2+ titrations were carried out by adding appropriate volumes of 1 mM CuCl2

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stock solution into 20 mL HA solution to generate a series of samples containing Cu2+

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concentration in a range of 0-160 µM. The final volume was 25 mL and thus the HA

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concentration was fixed at 10 mg/L. Background ionic strength was maintained by

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adding 0.04 M KBr (IR grade). The pH was maintained constant by adjusting both

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HA and Cu2+ stock solution to pH 7.0, and the mixed solution was fine tuned with 0.1

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M NaOH or HCl solution. Each solution was shaken for 12 h at 25 oC to ensure

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coordination equilibrium. Then, 5 mL of each solution was analyzed by synchronous

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fluorescence spectroscopy and the rest was freeze-dried for further FTIR spectroscopy

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analysis.

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Procedures and Parameters of Measurements. The elemental compositions of

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HA samples were analyzed using an elemental analyzer (vario EL cube, Elementar

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Co., Germany). Synchronous fluorescence spectra of HA solutions with increasing

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copper concentration were obtained by an average of 3 scans using a luminescence

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spectrometer (Perkin–Elmer LS-55, USA). Excitation and emission slits were both

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adjusted to 10 nm, and the excitation wavelengths ranging from 250 to 550 nm were

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used in 0.5 nm increment at a scan rate of 1000 nm/min with a constant offset ∆λ of

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60 nm. 60 nm was chosen as the offset to provide a higher fluorescence intensity and

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a better resolution compared with other offsets (Figure S1).9 50 mg of the freeze-dried

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HA samples with different copper concentration were ground, homogenized and

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pressed under the irradiation of an infrared lamp to eliminate the influence of moisture

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in samples. Their transmission IR spectra were recorded on a Vertex 70 spectrometer

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(Bruker Co., Germany) with a DTGS detector, and each spectrum was obtained after

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32 scans with 4 cm-1 resolution. The IR spectra were smoothed, baseline-corrected

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and finally transformed to absorbance spectra using OPUS 5.5 software for the

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subsequent analysis.

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2DCOS Analysis. To obtain the structural variation information of copper-bound

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HA, 2DCOS was employed using synchronous fluorescence and FTIR spectra with

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copper concentration as the external perturbation. The 2DCOS were produced

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according to the method of Noda and Ozaki.24 The analytical spectral changes of y(x,

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C), as a function of a spectral variable (x, here representing λ or v) and an external

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variable (C, copper concentration) at m evenly spaced points, can be represented as:

108 109

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  = ,  

(1)

A set of dynamic spectra  ,  is defined as follows:

   = 

   −   for 1 ≤  ≤  0 otherwise

(2)

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where   = ! ∑! #   , denoting the reference spectrum, typically the m variable

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averaged spectrum. A computational method based on the discrete Hilbert-Noda

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transform is used to generate a pair of correlation intensity maps, i.e., synchronous (Ф)

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and asynchronous (Ψ) correlation spectra: 0 if  = *

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$̃   = ∑! '  , N' = ) '# N' ∙

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Ф , .  = !, ∑!    ∙   .  #

(4)

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Ψ , .  = !, ∑!    ∙ $̃  .  #

(5)

+',

otherwise

(3)

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Spectral coordinates, intensities and signs of correlation peaks appearing on 2D

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spectra can be interpreted by a set of well-established principles.24, 25 Synchronous

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spectra, corresponding to the real part of the cross correlation function, consist of

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auto-peaks located along the main diagonal of the map and cross-peaks located at the

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off-diagonal positions. Higher susceptibility of intensity changes to the perturbation

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generates greater positively signed auto-peak intensities in the synchronous spectra.

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Cross-peaks suggest the coordinated changes of spectral intensities observed at two

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different spectral variables (x1, x2). A positive cross peak indicates the same direction

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of the intensities change at the corresponding spectral coordinates, while a negative

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value suggests an opposite direction. Asynchronous spectra, the imaginary part of the

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cross correlation function, show cross-peaks exclusively. Their signs reveal the

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sequential order of the dynamics of spectral intensity variations induced by the

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perturbation. Same signs of the spectral coordinate (x1, x2) in both the synchronous

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and asynchronous maps indicate that the spectral intensity change at x1 occurs

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predominantly before that of x2 along the perturbation variable axis. This order is

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reversed when signs are opposite. The spectral changes occur simultaneously if only

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the synchronous correlation intensity is observed, and the temporal relationship of

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spectral intensity changes becomes indeterminate if only the asynchronous correlation

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intensity is observed.

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The spectra of each sample for 2DCOS analysis have subtracted the background

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signal (without addition of copper) first. The data set was then transformed into a new

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spectral matrix suitable for 2DCOS maps using 2D Shige software (Kwansei-Gakuin

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University, Japan). Synchronous fluorescence 2DCOS, IR 2DCOS and synchronous

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fluorescence/IR hetero-2DCOS maps were plotted using Origin 8.5 software.

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Determination of Copper Binding Parameters. The fluorescence quenching

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titration method was employed to quantify the copper binding characteristics of

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fluorescent fractions of HA, and the parameters related to copper binding were

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estimated by the following modified Stern-Volmer equation:26

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01

01 ,0

=

2∙34 ∙54

+

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(6)

2

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where F, F0, f, and CM represent the measured fluorescence intensity, the initial

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fluorescence intensity, the fraction of the initial fluorescence that corresponds to the

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binding fluorophores, and the Cu2+ concentration, respectively. The conditional

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stability constant KM can be obtained by plotting F0/ (F0-F) against 1/CM. The

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fluorescence wavelengths were selected with a consideration of the pronounced peaks

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from the 2DCOS maps.

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RESULTS AND DISCUSSION

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General Spectroscopic Observations. DOM is a complex mixture of organic

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compounds formed by decomposition of plants and animal residues by microbial

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activity, which contains carboxyl, phenol, quinonyl, ester, ketone, hydroxyl, amino

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and other functional groups. Elemental analysis and structural characterization of the

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purified HA are given in Table S1 and Figure S2. Figure 1 shows the change of

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synchronous fluorescence of HA with the addition of Cu2+. Three fluorescence

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regions, corresponding to the wavelength ranges of 250-300 nm, 300-380 nm and

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380-550 nm, could be roughly assigned to protein-like, fulvic-like and humic-like

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fluorescence fractions, respectively.27 The fluorescence intensity of protein-like

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fractions was relatively low, compared with that of the sharp fulvic-like peaks and the

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broad humic-like shoulders. An increase in copper concentration caused a higher

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extent of fluorescence quenching for all the fractions, indicating the occurrence of

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electronic structural changes in the fractions by forming complexes with copper.

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The predominant infrared spectral characteristics of HA occurred in the range of

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1750-750 cm-1, where almost all the vibrational information of HA backbones could

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be identified. The spectral shift of HA upon addition of copper was shown in Figure

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2(a), in which the characteristic bands changed to various degrees with variation of

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copper concentration. The effect of copper to the structural variation of HA was

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illustrated more clearly in differential IR absorbance spectra in Figure 2(b), in which

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each of the spectra was subtracted by the spectra without copper addition and the

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trend for the spectral change was amplified. Even so, some of the IR bands remained

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strongly overlapped with uncertain assignments. It can be seen that the intensities of

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these IR bands diminished with increasing copper concentration, except for the bands

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centered at 800 cm-1. These observations indicated that during the binding process

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with copper, the vibrational structural changes varied in the fractions of HA

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backbones.

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The one-dimensional synchronous fluorescence and FTIR spectra failed to give

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detailed information on the exact binding characteristics of HA to copper, owing to

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the strong overlapped absorbances caused by the diverse groups of HA. To achieve a

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reliable determination of the conformational changes of HA induced by copper

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binding, enhancement of the spectral resolution and understanding the correlation

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between the fluorescence and IR responses are needed.

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Correlation Analysis. 2DCOS helps to enhance the spectral resolution by

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spreading the spectrum in a second dimension, thus facilitating the deconvolution of

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overlapping peaks. Spectral features of different molecular origins that make similar

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contributions to the spectra in the static case may behave differently (either delayed or

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accelerated) in the case of a dynamic change. Such differences can be distinguished

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by 2DCOS. The synchronous 2DCOS map for the synchronous fluorescence spectra

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of HA in the region 280-500 nm (Figure 3a) showed three predominant auto-peaks

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centered at 445, 380 and 350 nm with a small peak at 292 nm identified from the

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cross-peaks. Compared with one dimensional fluorescence spectra (Figure 1), the

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2DCOS gave peaks responsible for fluorescent fractions of HA with a higher

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resolution. Intensities of peaks decreased in the order of 445, 380, 350 and 292 nm,

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suggesting that the fluorescence of humic-like fractions were more susceptible to

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copper addition, while the fluorescence of protein-like fractions were less sensitive,

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and the fulvic-like fractions could be divided into two components (380 and 350 nm).

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All the cross-peaks were positive, indicating that the spectral changes proceeded in

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the same direction as the variation of copper ion concentration.

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The asynchronous map provided additional useful information about the

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sequential relationship between two spectral origins during copper binding. The

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fluorescence intensity changes at one wavelength raises or lags those at other

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wavelengths. As shown in Figure 3b and Table S2, the cross-peaks located at the

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upper-left corner of the asynchronous map all exhibited negative signs, which

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suggested that, according to Noda’s rule, the change followed the order of 445 > 380,

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350 > 292 nm. This suggested that copper bound to HA fractions in the following

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sequence: humic-like fraction → fulvic-like fraction → protein-like fraction.

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The various functional groups of HA always lead to overlapping IR peaks,

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resulting in ambiguous band assignments confusing the conformational analysis of

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HA. 2DCOS is a promising tool to solve this problem. Figure 4 showed the copper-

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induced 2D-IR-COS of HA. The synchronous map gave seven predominant auto-

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peaks centered at 1685, 1620, 1360, 1100, 1020, 850, 780 cm-1 and a small peak at

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1260 cm-1 (Figure 4a). The band of 1380 cm-1 was shown to change most significantly.

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In contrast, the smallest change was observed for bands at 1100 and 850 cm-1. It could

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be seen that the the signs of the cross-peaks in the synchronous map were all positive,

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except for ones associated with 850 and 780 cm-1. This indicated that conformational

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changes associated with most IR bands underwent the same direction, while those

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associated with 850 and 780 cm-1 bands proceeded reversely.

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The peak at 1685 cm-1 splitted into two parts at 1720 and 1660 cm-1 respectively

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in the asynchronous map (Figure 4b), and the peak at 1360 cm-1 was overlapped with

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peaks at 1400 and 1350 cm-1. The 1720 and 1260 cm-1 singals can be attributed to the

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C=O streching and C-O streching (or OH deformation) of carboxylic acid groups,

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while the bands at 1350 and 1020 cm-1 corresponded to the aliphatic C-H deformation

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and C-OH streching, and the bands at 850 and 780 cm-1 corresponded to the aromatic

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C-H deformation.2 The detailed assignments of bands and signs of their cross-peaks in

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the asynchronous map were shown in Table 1. Using the sequential order rules, it

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could be concluded that the structural change sequence of HA backbones by copper

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binding followed the order: carboxyl → C-O of polysaccharides → phenolic groups

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→ aryl → amide → aliphatic groups. The 2D-IR-COS results suggested that not only

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the fluorescent groups, but also some non-fluorescent groups (like polysaccharides) of

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HA were involved in the binding process of copper with HA, and the binding

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affinities of hydrophilic sites were higher than that of hydrophobic sites.

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Fluorescence/IR Hetero-2DCOS. To verify the band assignments as well as the

238

correspondence between the two complementary tehcniques, fluorescence and IR

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spectra that represent different parts of HA structure, we employed a 2D

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heterospectral fluorescence/IR correlation analysis. The hetero-2D spectra could serve

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as a visual aid to help describe the structural change of HA during the copper binding.

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Figure 5 shows the synchronous and asynchronous maps of the hetero-2DCOS,

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where the IR frequency and the fluorescence shift were ploted on the x-axis and y-axis

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respectively. Remarkably, four narrow cross-peaks, located in the IR regions of 1650,

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1350, 850 and 780 cm-1 and the corresponding fluorescence region of 450-350 nm,

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appeared in the synchronous map (Figure 5a). The former two peaks showed positive

247

signs while the latter two were negative. These results indicated that the carboxyl,

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phenolic and aryl groups of HA were the basic fluorescent units for humic-like and

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fulvic-like fractions. In the asynchronous map (Figure 5b), the cross-peaks were

250

divided into different parts, and the humic-like fraction (445 nm) was related to the

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phenolic (1380 cm-1) and aryl (1620, 850, 780 cm-1) groups, while the fulvic-like

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fraction (380-350 nm) was related to the carboxyl (1720, 1260 cm-1), phenolic (1400

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cm-1) and aryl groups (1620, 850 cm-1). Two new cross-peaks were also observed in

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the asynchronous map at 1660 and 1350 cm-1 with the fluorescence peak at 290 nm,

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corresponding to the fluorescence region of protein-like fraction. This suggested that

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the protein-like fraction was related to the amide and aliphatic groups.

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As a result of the difference in IR and fluorescence spectra probing the tinily

258

different aspects of molecular responses toward an identical perturbation (copper

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concentration), asynchronicity was observed between two different types of

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spectroscopic data of the same species. In the asynchronous map, there were two

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negative signs at coordinates (1620 cm-1, 445 nm) and (1380 cm-1, 445 nm), and two

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positive signs at (850 cm-1, 445 nm) and (780 cm-1, 445 nm), indicating that the

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fluorescence response of humic-like fraction occurred before the spectral changes of

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phenolic, aryl, and Ar-H groups detected by IR. On the other hand, the positive signs

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of fluorophore coordinates at 380-350 nm and 290 nm suggested that the fluorescence

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change of fulvic-like fraction occurred after the vibrational changes of carboxyl,

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phenolic and aryl groups but before that of Ar-H groups, and the fluorescence change

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of protein-like fractions occurred after the vibrational changes of amide and aliphatic

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groups. This order of intensity changes are in good agreement with that obtained by

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2D-fluorescence and 2D-IR correlation studies.

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To quantitatively confirm our interpretation of the 2DCOS results, copper binding

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parameters were calculated by modified Stern–Volmer equation, which has been

273

widely used for characterizing the metal binding behavior of DOM, at the

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wavelengths corresponding to the main peaks of the 2DCOS maps (Figure 6). The

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calculated values of the conditional stability constants (Table 2) were comparable to

276

those reported in similar studies. And they decreased in the order 445 > 380 > 350

277

> 292 nm, supporting our aforementioned interpretation of the fluorescence 2DCOS

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maps. In addition, the fraction of the initial fluorescence also decreased in this trend.

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The degree of fluorescence quenching depends upon the hydrophobic/hydrophilic

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nature and the origin of HA. The relative bigger Km of the fulvic-like and humic-like

281

fractions of HA might be attributed to the high contents of phenolic and aromatic

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carboxylic groups, which could form highly stable ring structures with copper. These

283

results suggested that the 2DCOS was a promising tool to readily capture subtle

284

sequential differences in the spectral variations as well as to probe the structural

285

responses with external factors.

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Significance of This Work. In the present work, a comprehensive picture about

287

the structural changes of DOM in copper binding process is established according to

288

the 2DCOS results at molecular scales. As copper ions are added to interact with HA,

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the carboxyl and polysaccharide groups give the fastest responses, then the

290

fluorescent humic-like groups participate in the fluorescence queching, followed by

291

vibrational changes of related groups, i.e., phenolic and aryl groups. Subsequently, the

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involvment of aromatic carboxyl groups further induces the fluorescence quenching

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of fulvic-like fraction. Finally, the small amounts of amide and aliphatic groups

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become participated in the copper binding process, after which the fluorescence of

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protein-like fraction decreases. Hetero-2DCOS offers an unique insight to understand

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the transformation of HA in the presence of metals in aquatic systems and clarify the

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structural relationship between the fluorescent groups and the corresponding IR

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groups in HA in the copper binding process. This study might open a door to visit the

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geochemical cycling process of metals in natural environments. On the other hand,

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elucidation of the nature of interactions between HA and copper shown in this work

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demonstrates the great potential of using the 2D-heterospectral correlation

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spectroscopy for further applications in exploring complex interactions in natural and

303

engineered environments.

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AUTHOR INFORMATION *Corresponding author: Prof. Han-Qing Yu, Fax: +86 551 63601592; E-mail: [email protected]

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ACKNOWLEDGEMENTS

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We thank the Natural Science Foundation of China (51129803), Hefei Center for

311

Physical Science and Technology (2012FXZY005) and the Program for Changjiang

312

Scholars and Innovative Research Team in University and the Collaborative

313

Innovation Center of Suzhou Nano Science and Technology of the Ministry of

314

Education of China for the support of this study.

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ASSOCIATED CONTENT

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Supporting Information Available. Elemental contents of the unpurified and

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purified HA samples (Table S1), sign of each cross-peak in the synchronous (Ф) and

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asynchronous (Ψ, in the brackets) maps from the synchronous fluorescence spectra of

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HA with copper binding (Table S2), excitation-emission matrix of HA samples and

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synchronous spectra recorded with different ∆λ values (Figure S1), FTIR spectra for

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HA samples (Figure S2) and possible assignment for each band. This information is

323

available free of charge via the Internet at http://pubs.acs.org/.

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Table 1. 2D-IR-COS Results on the Assignment and Sign of Each Cross-Peak in Synchronous (Ф) and Asynchronous (Ψ, in the brackets) Maps of HA with Copper Binding position

sign

assignment

(cm-1) 1720

carboxyl vC=O

1660

amide vC=O

1620

aromatic vC=C

1400

phenolic vC-O, δO-H

1350

aliphatic δC-H

1100

polysaccharides vC-O

850

Ar-H, vC-H

1720 1660 1620

1400

1350

1100

850

+

+(+)

+(+)

+(+)

+(+)

+(+)

-(-)

+

+(-)

+(-)

+(+)

+(-)

-(-)

+

+(-)

+(+)

+(-)

-(-)

+

+(+)

+(-)

-(-)

+

+(-)

-(-)

+

-(-) +

(signs were obtained in the upper-left corner of the maps, + positive, - negative)

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Table 2. Copper Binding Parameters Calculated by the Modified SternVolmer Equation fraction

peak (nm)

f

LogKM

R*

protein-like

292

0.542

4.61

0.987

350

0.769

4.99

0.994

380

0.771

5.09

0.992

445

1

5.26

0.999

fulvic-like

fumic-like *

Correlation coefficients (R).

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Figure captions

Figure 1. Changes in the synchronous fluorescence spectra of 10 mg/L HA at pH 7.0 upon addition of copper with concentrations of 0, 1, 2, 5, 10, 20, 40, 60, 80, 100, 120, 160 µM. The dashed vertical lines shows the protein-like, the fulvic-like, and the humic-like regions. Each spectrum was averaged by three scans

Figure 2. FTIR absorbance spectra (a) and differential IR absorbance spectra (b) of the freeze-dried HA samples with varying copper concentrations. The spectra were an average of 32 scans

Figure 3. Synchronous (a) and asynchronous (b) 2D correlation maps generated from the synchronous fluorescence spectra of HA with increasing copper ion concentration in the region 280-500 nm

Figure 4. Synchronous (a) and asynchronous (b) 2D IR correlation maps from a FTIR analysis of HA with copper binding

Figure 5. 2D heterospectral correlation plots generated from the dynamic synchronous fluorescence and IR spectra of HA upon copper addition: (a) synchronous map; (b) asynchronous map

Figure 6. Modified Stern-Volmer plots for the fluorescence quenching of HA fractions by copper. Four wavelengths at 292, 350, 380 and 445 nm were selected corresponding to the peaks in the 2DCOS map in Figure 3a

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80

Proteinlike

Fulviclike

Humiclike

60

Intensity (a.u.)

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2+

[Cu ] increase

40

20

0 250

300

350

400

450

Wavelength (nm) Figure 1

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500

550

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2+

[Cu ] 160 µM 120 µM 100 µM 80 µM 60 µM 40 µM 20 µM 10 µM 5 µM 2 µM 1 µM 0 µM

IR Absorbance

(a)

1750

1500

1250

1000 -1

750

Differential Absorbance

Wavenumber (cm ) (b)

1750

160 µM 120 µM 100 µM 80 µM 60 µM 40 µM 20 µM 10 µM 5 µM 2 µM 1 µM

1500

1250

1000 -1

Wavenumber (cm )

Figure 2

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Figure 3

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Figure 4

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Figure 5

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15

F0/(F0-F)

10

292 nm 350 nm 380 nm 445 nm Modified Stern-Volmer model

5

0 0.0

0.2

0.4 2+

-1

1/[Cu ] (µM )

Figure 6

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Table of Contents (TOC) art

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FTIR and synchronous fluorescence heterospectral two-dimensional correlation analyses on the binding characteristics of copper onto dissolved organic matter.

Dissolved organic matter (DOM) is known to form strong complexes with heavy metals and thus governs the distribution, toxicity, bioavailability, and u...
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