FTIR and Synchronous Fluorescence Heterospectral Two

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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

1 ACS Paragon Plus Environment


1

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

2

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

3

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

9

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

10

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-

13

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

14

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

15

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

20

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

21

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

22

spectroscopy in studying complex interactions in environment.


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23

INTRODUCTION

24 25

Ubiquitously existing in aquatic and soil environments with various chemical

26

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

27

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

28

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

29

Carboxylic and phenolic groups in DOM are considered as the predominant

30

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

31

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

32

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

33

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

34

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

37

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

40

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

41

spectral changes and highly overlapping responses during the binding process.

42

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

43

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

44

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

45

are unclear yet.

46

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

47

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



48

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

49

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

50

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

51

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

52

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

53

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

54

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

55

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

56

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

57

areas of 2DCOS research.21-23

58

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

59

synchronous fluorescence and Fourier transform infrared (FTIR) absorption

60

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

61

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

62

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

63

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

64

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

65

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

66

combined to elucidate the copper-DOM interaction mechanisms.

67 68

EXPERIMENTAL SECTION

69 70

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

71

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

72

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

76

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

77

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

78

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

79

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

80

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

81

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

82

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

83

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

84

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

85

analysis.

86

Procedures and Parameters of Measurements. The elemental compositions of

87

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

88

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

89

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

90

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

91

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

92

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

93

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

94

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

96

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

97

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



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98

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

99

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

100

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

101

subsequent analysis.

102

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

104

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

105

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

106

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

107

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

108 109

110

= ,

(1)

A set of dynamic spectra , is defined as follows:

=

− for 1 ≤ ≤ 0 otherwise

(2)

111

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

112

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

113

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

114

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

115

$̃ = ∑! ' , N' = ) '# N' ∙

116

Ф , . = !, ∑! ∙ . #

(4)

117

Ψ , . = !, ∑! ∙ $̃ . #

(5)

+',

otherwise

(3)

118

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

119

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

120

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


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121

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

122

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

123

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

125

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

126

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

127

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

128

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

129

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

130

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

133

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

134

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

135

spectral intensity changes becomes indeterminate if only the asynchronous correlation

136

intensity is observed.

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

138

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

140

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

141

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

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Determination of Copper Binding Parameters. The fluorescence quenching

143

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

145

estimated by the following modified Stern-Volmer equation:26



146

01

01 ,0

=

2∙34 ∙54

+

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(6)

2

147

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

148

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

149

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

150

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.

153

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RESULTS AND DISCUSSION

155 156

General Spectroscopic Observations. DOM is a complex mixture of organic

157

compounds formed by decomposition of plants and animal residues by microbial

158

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

159

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

161

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

164

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

165

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

166

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

167

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

168

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

170

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

171

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

172

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

173

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

174

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

175

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

176

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

177

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

178

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

180

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

181

backbones.

182

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

183

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

184

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

185

reliable determination of the conformational changes of HA induced by copper

186

binding, enhancement of the spectral resolution and understanding the correlation

187

between the fluorescence and IR responses are needed.

188

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

189

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

190

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

191

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

192

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

193

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



194

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

195

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

196

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,

199

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

200

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

201

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

202

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

203

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-

216

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.

218

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

221

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

222

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

224

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

225

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

228

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

229

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

230

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

232

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

234

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

236

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

239

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

240

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

241

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

242

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

243

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



244

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

245

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

246

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,

248

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

249

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

251

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

252

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

253

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

254

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

255

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

256

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

257

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

259

concentration), asynchronicity was observed between two different types of

260

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

261

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

262

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

263

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

264

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

265

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

266

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

267

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

268

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

270

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

271

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

272

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

273

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

274

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

275

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

278

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

279

The degree of fluorescence quenching depends upon the hydrophobic/hydrophilic

280

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

282

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.

286

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,

289

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

292

involvment of aromatic carboxyl groups further induces the fluorescence quenching

293

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



294

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

295

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

296

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

297

structural relationship between the fluorescent groups and the corresponding IR

298

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

299

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

300

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

301

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

302

spectroscopy for further applications in exploring complex interactions in natural and

303

engineered environments.

304 305 306 307

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

308 309

ACKNOWLEDGEMENTS

310

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.

315 316

ASSOCIATED CONTENT

317

Supporting Information Available. Elemental contents of the unpurified and

318

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

320

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

321

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

322

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/.

324 325

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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)



Page 20 of 28

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



80

Proteinlike

Fulviclike

Humiclike

60

Intensity (a.u.)

Page 22 of 28

2+

[Cu ] increase

40

20

0 250

300

350

400

450

Wavelength (nm) Figure 1


500

550

Page 23 of 28


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


750


Figure 3


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Figure 4



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


0.6


Table of Contents (TOC) art


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FTIR and Synchronous Fluorescence Heterospectral Two

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