Temperature Dependence of Dissolved Organic Matter Fluorescence

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Temperature Dependence of Dissolved Organic Matter Fluorescence Garrett McKay, Julie Ann Korak, and Fernando L. Rosario-Ortiz Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b00643 • Publication Date (Web): 20 Jul 2018 Downloaded from http://pubs.acs.org on July 20, 2018

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Environmental Science & Technology

Temperature Dependence of Dissolved Organic Matter Fluorescence

Garrett McKaya,, Julie. A. Koraka , Fernando L. Rosario-Ortiza* a

Department of Civil, Environmental, and Architectural Engineering, University of Colorado Boulder

TOC Art

*

Corresponding author: [email protected]; 303-492-7607 1 ACS Paragon Plus Environment


1

Abstract

2

The temperature dependence of organic matter fluorescence quantum yields (Φf) was

3

measured for a diverse set of organic matter isolates (i.e., marine aquatic, microbial

4

aquatic, terrestrial aquatic, and soil) in aqueous solution and for whole water samples to

5

determine apparent activation energies (Ea) for radiationless decay processes of the

6

excited singlet state. Ea was calculated from temperature dependent Φf data obtained by

7

steady-state methods using a simplified photophysical model and the Arrhenius equation.

8

All aquatic-derived isolates, all whole water samples, and one soil-derived fulvic acid

9

isolate exhibited temperature dependent Φf values, with Ea ranging from 5.4 to 8.4 kJ

10

mol-1 at an excitation wavelength of 350 nm. Conversely, soil humic acid isolates

11

exhibited little or no temperature dependence in Φf. Ea varied with excitation wavelength

12

in most cases, typically exhibiting a decrease between 350-550 nm. The narrow range of

13

Ea values observed for these samples when compared to literature Ea values for model

14

fluorophores (~ 5-30 kJ mol-1) points to a similar photophysical mechanism for singlet

15

excited states non-radiative inactivation across organic matter isolates of diverse source

16

and character. In addition, this approach to temperature dependent fluorescence analysis

17

provides a fundamental, physical basis, in contrast to existing empirical relationships, for

18

correcting online fluorescence sensors for temperature effects.

19

Introduction

20

Fluorescence is a widely used tool to characterize dissolved organic matter (DOM)

21

because of its relative ease of use, minimal sample preparation, small sample volume

22

requirements, and multitude of quantitative tools that have been developed for spectra

23

interpretation.1,2 DOM fluorescence spectra are often presented as three-dimensional 2 ACS Paragon Plus Environment

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excitation-emission matrices (EEMs) and are characterized by large apparent Stoke’s

25

shifts, low quantum yields, and regions of intensity often assigned to specific classes of

26

fluorophores (e.g., tryptophan-like Peak B).1,3 Despite ubiquitous use of fluorescence for

27

the study of DOM, there is still a lack of understanding of the specific fluorophores

28

present in DOM and their photophysical behaviors. Phenols, flavonoids, and coumarins

29

are moieties hypothesized to be involved in DOM fluorescence.4 Although not definitive,

30

the presence of coumarins and flavonoids in DOM is supported by the appearance of

31

molecular formulae for methoxy coumarins (C10H8O3) and flavone (C15H10O2) in high-

32

resolution mass spectra of DOM isolates. 5

33

Multiple photophysical conceptual models have been developed for describing DOM

34

fluorescence. One model proposes that electron-rich polyphenols or alkoxy phenols and

35

electron-poor aromatic ketones or quinones form intramolecular donor-acceptor

36

complexes (DA) that can absorb light to form charge-transfer (CT) excited states.3,6-12

37

This CT model postulates that these DA complexes are responsible for long wavelength

38

absorbance and fluorescence of DOM. In contrast, it has also been argued that DOM

39

optical properties are due to a superposition of non-interacting chromophores in which a

40

given optical signal is simply the sum of the contributions from individual

41

chromophores.13-15

42

Absorption of a photon by a DOM chromophore will result in a transition from the

43

ground electronic state (S0) to an excited singlet state (Sn), which quickly relaxes to the

44

ground vibrational level of the first electronic excited state (S1). The S1 state decays by

45

fluorescence (S1 → S0 + hν, kf), internal conversion (S1 → S0, kic), or intersystem

46

crossing (S1 → Tn, kisc), where internal conversion and intersystem crossing are 3 ACS Paragon Plus Environment


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radiationless processes. The fluorescence of single-fluorophore solutions of organic

48

compounds can be characterized by their fluorescence quantum yields (Φf), which

49

describes the relative importance of fluorescence compared to radiationless transitions

50

(eq. 1),

51

Φ = k f /(k f + knr ) f

(1)

52

where knr = kic + kisc represents the overall non-radiative decay rate. For DOM, values for

53

the Φf of the mixture are typically in the range of 0.005 to 0.020 indicating that strongly

54

fluorescing moieties make up only a small subset of the total chromophoric DOM

55

pool.3,8,16-19

56

Identification of the fluorophores present in DOM and the photophysical

57

mechanisms responsible for the magnitude of Φf has remained a challenge. Although past

58

work has suggested that a significant portion of DOM photophysics is due to DA

59

complexes,12 which could lead to excitation into CT states, recent work suggests that this

60

is not the primary photophysical mechanism.20

61

The temperature dependence of Φf is also a parameter that it is used to assess the

62

photophysics of single chromophores. Fluorescence intensity tends to decrease with

63

increasing temperature, due to the temperature dependence of non-radiative decay

64

processes (kic + kisc).21 Thus, the activation energies derived from these data could

65

potentially yield insight into the photophysical processes leading to deactivation of

66

singlet excited state DOM (1DOM*).

67

There have been a handful of studies examining the effect of temperature on the

68

fluorescence of DOM. 20,22-24 Baker22 showed that DOM fluorescence intensity decreased 4 ACS Paragon Plus Environment

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with increasing temperature, a result also confirmed by McKay et al.20 for a diverse

70

collection of DOM isolates. In the McKay et al.20 study, although fluorescence intensity

71

decreased with increasing temperature, normalization of the emission spectra to the

72

maximum intensity revealed that spectral shape remained unchanged, indicating that the

73

maximum emission wavelength (λem,max) is constant with temperature. This constancy in

74

λem,max was taken as evidence that fluorescence from DOM occurs from local excited

75

(LE) states as opposed to CT excited states.25 LE states refer to the singlet excited state of

76

individual moieties, whereas CT excited states refer to a singlet excited state donor-

77

acceptor complex (i.e., 1[D+A–]*).

78

There is a need for quantitative relationships explaining the temperature dependence

79

of DOM fluorescence. Data collected by in situ fluorescence probes are often corrected

80

for temperature effects, and several studies have attempted to build empirical models

81

incorporating temperature.23,24 However, there have yet to be any fundamental studies

82

into the photophysical processes controlling the quenching of DOM fluorescence by

83

increasing temperature. This information would provide insight into the photophysical

84

processes occurring in DOM and also a more fundamental basis for empirical approaches

85

used to correct fluorescence data for temperature effects.

86

In this study, we measured the temperature dependence of DOM fluorescence and

87

analyzed the data via the Arrhenius equation to calculate Ea, which represents the

88

apparent activation energy for 1DOM* non-radiative decay. Fluorescence was examined

89

over a temperature range of 10 to 60 °C for a broad range of organic matter isolates and

90

whole water samples, including terrestrial aquatic, marine aquatic, microbial aquatic, and

91

soil-derived humic substance isolates, as well as a size fractionated isolate, surface water 5 ACS Paragon Plus Environment


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and secondary treated wastewater sample. The resulting temperature dependence data

93

were compared to reported physicochemical properties in an attempt to correlate

94

photophysical observations to organic matter structure. The results in this study provide

95

both a new way to gain information about organic matter fluorescence as well as

96

fundamental insight into the photophysical processes controlling organic matter

97

fluorescence.

98

Materials and Methods

99

Samples:

100

Organic matter isolates were obtained from the International Humic Substances

101

Society and United States Geological Survey (see Table S1). Aqueous solutions were

102

prepared either by dissolving solid isolate directly into pH 7.2, 10 mM phosphate buffer

103

at a dissolved organic carbon (DOC) concentration of ~ 4 mgC L-1 or by diluting ~100

104

mgC L-1 stock solutions into purified water (≥ 18.2 MΩ•cm, Sartorius). In the latter case,

105

pH was adjusted to 7.0, monitored after dilution, and re-adjusted as necessary with 0.1 M

106

NaOH. These concentrated stocks were also used to prepare a 30% v/v solution of

107

Mississippi River Natural Organic Matter (MRNOM) in spectrophotometric grade

108

glycerol to depress the freezing point and 0-48% v/v solutions of Suwannee River Fulvic

109

Acid (SRFA) in D2O. SRFA was fractionated to obtain > 5 kDa and < 5 kDa molecular

110

weight fractions, which are denoted SRFAgt and SRFAlt, respectively. The fractionation

111

was performed using regenerated cellulose ultrafiltration membranes (Millipore, USA)

112

with a nominal molecular weight cutoff of 5 kDa.26 All solutions were filtered using

113

muffled (400 °C for 5 hr), 0.7 µm glass fiber filters and stored at 4 °C in amber glass

114

bottles until analysis.

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Suwannee River Fulvic Acid (SRFA, 2S101F), Suwannee River Humic Acid

116

(SRHA, 2S101H), Suwannee River NOM (SRNOM, 2R101N), Pony Lake Fulvic Acid

117

(PLFA, 1R109F), Mississippi River NOM (MRNOM, 1R110N), Elliot Soil Humic Acid

118

(1S102H), Pahokee Peat Humic Acid (PPHA, 1S103H), and Pahokee Peat Fulvic Acid

119

(PPFA, 2S103F) were prepared and analyzed in pH 7.2 phosphate buffer. Gulf of Maine

120

Hydrophobic Organic Acid (GMHPOA), Pacific Ocean Fulvic Acid (POFA), and Yukon

121

Hydrophobic Organic Acid (YHPOA) were prepared from ~100 mgC L-1 aqueous

122

solutions at pH 7.0. No significant difference was observed in Φf between samples

123

dissolved in pH 7.2 phosphate buffer versus water at pH 7.0 (Figure S1).

124

Two whole water samples were collected: a secondary treated wastewater from

125

Boulder, Colorado (BWW) and a surface water from the San Juan River (SJR) collected

126

near Farmington, New Mexico. Both whole water samples were filtered using muffled

127

0.7 µm glass fiber filters 5 °C in amber glass bottles until analysis.

128

Analytical methods:

129

A Sievers M5310 C (GE, USA) total organic carbon (TOC) analyzer was used to

130

measure DOC concentrations utilizing a persulfate oxidation method. pH was measured

131

with an Accumet pH meter (Fischer Scientific, USA). Absorbance was measured in

132

triplicate with a Cary-100 Bio spectrophotometer (Agilent, USA) from 800 to 200 nm in

133

1 nm increments with a 1, 5, or 10 cm path length quartz cuvette. Absorbance spectra

134

were also measured between 10 and 40 °C for selected samples using a water-jacketed 1

135

cm cuvette which allowed for temperature control to ± 1 °C. As previously reported in

136

McKay et al.20, molar extinction coefficients (in MC-1 cm-1) for SRFA, SRHA, SRNOM,

137

PLFA, MRNOM, ESHA, PPHA, and PPFA were unaffected by temperature. Because of 7 ACS Paragon Plus Environment


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this result, room temperature absorbance spectra were used for analyses of BWW,

139

GMHPOA, POFA, SJR, SRFAlt, SRFAgt, and YHPOA.

140

Fluorescence

measurements

were

performed

using

a

Fluoromax-4

141

spectrofluorometer (Horiba, USA). Emission intensity was measured between 300 nm

142

and 800 nm (PPHA and ESHA) or 300 nm and 700 nm (all other samples) in increments

143

of 2 nm at excitation wavelengths (λex) between 240 nm and 550 nm in 10 nm

144

increments. All bandpass settings were 5 nm, and integration times were 0.25 s.

145

Fluorescence intensity was corrected incorporating blank subtraction, instrument specific

146

factors, inner filter corrections, and Raman normalization as described previously.27 The

147

E2/E3 ratio, spectral slope, specific ultraviolet absorbance 254 nm (SUVA254), and

148

fluorescence index (FI) were calculated as previously described.28-30

149 150

151

Fluorescence quantum yields were calculated using quinine sulfate as a reference standard (Φf,QS = 0.51 in 0.1 N H2SO4)31 based on eq. 2.32

Φ f ( λex ) Φ f ,QS (350 nm)

∫ =

∞ 0

I DOM ( λex , λem )d λem − Absex ( λex )

− Abs ref (350 nm)

1−10

∫

1−10

− Absex

∞ 0

Iref (350 nm, λem )d λem

(2)

152

When Absex is less than ~ 0.05-0.1, the 1 −10

153

condition was met for all but two samples (PPHA and ESHA). Thus, Φf was calculated

154

using eq. 2 for PPHA and ESHA but with the linearized version of eq. (2) for the other

155

samples– i.e., eq. S4 in the Supplemental Information. Finally, it is important to note that

156

results from a previous study indicate that Φf obtained with the Fluoromax-4 are

157

systematically larger than those measured with an Aqualog spectrofluorometer at λex less

term can be linearized to Absex. This


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than 350 nm.20 This discrepancy is based on a different excitation monochromator in the

159

Fluoromax-4 that passes more stray light compared to the Aqualog. For this reason, Φf

160

values measured in this study, all of which were obtained using the Fluoromax-4, are

161

presented only at λex greater than 350 nm.

162

Temperature control was accomplished using a recirculating chiller (VWR, Model

163

1166D) and a four-position thermostatted cell holder (FL4-1011, Horiba). Control

164

experiments determined that the time necessary for a sample to reach thermal equilibrium

165

in the system was ~ 10 min. Sample temperature was measured using a VWR Traceable

166

Type K thermometer in a dummy cell filled with purified water. All cells were

167

continuously stirred, and temperature variations between the top and bottom of the cell

168

were less than 1 °C. Quoted temperatures represent an average of multiple top and

169

bottom cell measurements for each experiment, and standard deviations were less than 1

170

°C using this approach.

171

Two approaches were used to determine temperature dependent Φf values. In the

172

first approach, fluorescence spectra were measured in triplicate at five temperatures

173

between 10 and 40 °C. In the second approach, single replicates of fluorescence spectra

174

were measured at 9 temperatures between 10 and 60 °C. The latter approach resulted in

175

tighter regressions of the physical model to the data (vide infra). Furthermore, results

176

obtained using both approaches yielded similar Arrhenius parameters, justifying the

177

comparison of data obtained via different approaches.



178

Results and Discussion

179

Temperature dependence of DOM fluorescence

180

Figures 1 and 2 show representative data of fluorescence quenching due to

181

temperature. Fluorescence intensity (IDOM) decreased with increasing temperature

182

(Figures 1a, 2, S3 to S5, and S7 to S9). This result is consistent with fluorescence of

183

single-fluorophore solutions of organic compounds, including benzene and substituted

184

benzenes,21 polycyclic aromatic hydrocarbons such as naphthalene,21 tryptophan and

185

tyrosine,25,33,34 flavones,35 and coumarins,36 each of which represent plausible

186

chromophores and fluorophores within DOM.

187

Soil humic acids did not exhibit a significant decrease in IDOM over 10 to 40 °C

188

(PPHA and ESHA, Figure S5 and S6, Table S3), in contrast to all aquatic samples and

189

PPFA. To further explore this behavior, fluorescence spectra were collected for ESHA at

190

-7 and 70 °C; 70 % v/v glycerol was used to depress the freezing point of the solution.

191

IDOM decreased by 20 % between -7 and 70 °C at the λex and λem pair of 370 nm and 560

192

nm, respectively. A complete explanation of the differing behavior of ESHA in these two

193

different sets of experiments (10 to 40 °C vs. -7 and 70 °C) is unclear. One possibility is

194

that there is a limit in statistical ability to differentiate small differences in IDOM in the

195

smaller temperature range. Unfortunately, the two sets of experimental data are not

196

directly comparable because one batch (the -7 and 70 °C data) was measured in 70% v/v

197

glycerol, which decreases IDOM compared to aqueous solutions.20 Furthermore, ESHA

198

fluorescence spectra were red shifted by ~20 nm in 70% glycerol compared to ESHA in

199

phosphate buffered solution, providing an additional reason against directly comparing

200

these two sets of experiments. Importantly, isolates not derived from soils did not show


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this spectral shift in glycerol/water mixtures or in other solvents of varying polarity.20

202

Taken as a whole, the divergence in temperature dependent fluorescence behavior of soil

203

humic acids compared to the other samples examined in this study indicates distinct

204

differences in the photophysical properties of these different pools of organic matter.

205

Photophysical model

206

Quantitative information regarding the temperature dependence of fluorescence can

207

be gained by measuring fluorescence lifetimes or quantum yields at various temperatures

208

and using the following kinetic scheme,

209

DOM + hνa→ 1DOM*

(absorption, ka)

210

1

DOM* → DOM + hνf

(fluorescence, kf)

211

1

DOM* → DOM

Assuming that knr is the only temperature dependent inactivation process (i.e. kf is

212 213

not temperature dependent),21,37 an Arrhenius expression can be written for knr (eq. 3)

knr = knr0 e

214 215

(non-radiative decay, knr = kic + kisc)

− E a /RT

(3)

which when substituted into eq. 1 gives eq. 4,

Φf =

216

kf k f + knr0 e

(4)

−Ea /RT

217

Taking the inverse of both sides of eq. 4 and rearranging yields eq. 5,

218

k −E /RT 1 −1 = nr e a Φf kf

0

219

(5)

and taking the natural logarithm results in eq. 6, 11 ACS Paragon Plus Environment


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 k0  E  1  ln  −1 = ln  nr  − a Φ  k f  RT   f

221

Based on eq. 6, a plot of the natural logarithm of (Φf-1 – 1) versus T-1 should give a

222

straight line with a slope of –Ea/R and intercept of ln(knr0/kf).21 Ea represents the

223

activation energy for the temperature dependent, non-radiative decay pathways of a

224

distinct first excited singlet state, S1. knr0 represents the temperature independent portion

225

of non-radiative decay.37 If there is more than one temperature dependent inactivation

226

pathway from S1, then Ea will be influenced by each of these processes. When non-

227

linearity is observed with respect to eq. 6, it is often interpreted as evidence of multiple

228

inactivation processes occurring simultaneously.25,38-40 In addition, sometimes one

229

inactivation process dominates over others in different temperature ranges.

(6)

230

Fundamentally, Ea as stated above is meant to describe the temperature dependence

231

of non-radiative decay of the excited singlet state for a single S1 state. Due to the

232

heterogeneous nature of organic matter and the multiple 1DOM* species contributing to

233

fluorescence at a given excitation wavelength,3,41 the best term to describe these data is

234

apparent activation energy. Thus, from this point forward, the reader should assume that

235

Ea in the context of DOM represents an apparent activation energy for the non-radiative

236

decay of the many 1DOM* species in the mixture. Similarly, the intercept term,

237

ln(knr0/kf), can be used to obtain quantitative information about the ratio of the

238

temperature independent non-radiative decay rate to the rate of fluorescence. These rate

239

constants are not as frequently reported in temperature dependent studies of organic

240

fluorophores, however, a series of methyl substituted indoles were reported to have knr0

241

and kf values of ~ 107 and 109 s-1, respectively, indicating that fluorescence is much faster 12 ACS Paragon Plus Environment

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than non-radiative decay for these compounds.42 These rate constants would result in a

243

ln(knr0/kf) of ~ - 4.5. For DOM, the ln(knr0/kf) term should also be though of as an apparent

244

value.

245

Although we are applying a model mostly used for single fluorophores to DOM, the

246

fact that this model fits the data (Figure 1c) without significant deviation indicates that

247

the DOM fluorescence behaves similarly to single fluorophores solutions on a

248

phenomenological level. It is worth noting that the Arrhenius model has been used

249

previously to assess other parameters of DOM reactivity, such as photochemical

250

production and scavenging of the hydroxyl radical.43,44

251

A limitation of the model presented in eqs. 3-6 is that it based on steady-state

252

fluorescence measurements. Time-resolved measurements of select DOM isolates have

253

shown that DOM fluorescence occurs via at least three different pools of singlet states,

254

with lifetimes ranging from < 50 ps to a few ns.3 These time resolved data show that the

255

short lifetime component, which comprises the largest fraction of the time-resolved

256

signal, accounts for a small fraction of the steady-state signal. In effect, DOM

257

photophysical properties measured using steady-state fluorescence, including the Ea

258

values reported in this study, are more representative of the long-lifetime fluorescing

259

components. It should be noted, however, that nearly all data on DOM fluorescence in

260

the literature has been collected using steady-state methods, including a large portion of

261

the data used to support the CT model.45



262

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Activation energies:

263

Figure 1c shows Φf values plotted according to eq. 6 for SRFA. The linearity of the

264

data in Figure 1c indicates that the relative contribution of temperature dependent

265

inactivation processes for various 1DOM* species remains constant over 10 to 40 °C.

266

Following the same approach for the other isolates that exhibited a temperature

267

dependence, Ea and ln(knr0/kf) values were calculated and are shown in Table 1, Figure

268

S10, and Figure S11. There was a surprisingly narrow range of Ea (5.4 to 8.4 kJ mol-1)

269

values at λex = 350 nm considering the larger range of values for model organic

270

compounds in Table 1. Ea for soil humic acid isolates was not statistically different than 0

271

kJ mol-1 (Table S3).

272

There was also a narrow range of ln(knr0/kf) (6.4 to 8.7). Taking the average ln(knr0/kf)

273

value of 7.4 ± 0.6 (Table 1), the ratio of knr0/kf is about 1600, implying that non-radiative

274

decay is much more significant than fluorescence for DOM, consistent with low Φf

275

values. Φf can be calculated using eq. 1, the knr0/kf ratio, and eq. 3 if it is assumed that knr

276

≫ kf (eq. 7-9).

277

278

(7)

0 1 knr knr −Ea /RT = = e =1600×e−2.91 = 87.5 Φ kf kf f


(8)

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(9)

280

This calculation results in a Φf value of about 1 % using knr0/kf = 1600, the same order of

281

magnitude measured for most DOM isolates and whole water samples at λex = 350 nm.

282

The temperature range used in this study is most relevant to natural waters, but does

283

not cover the typically larger range explored in studies of the temperature dependence for

284

single-fluorophore solutions. This difference is important because changes in the

285

temperature dependence of Φf could be due to the relative importance of different

286

radiationless decay pathways under different temperature regimes.25,38-40 In addition,

287

studies of the temperature dependence of Φf for macromolecules indicate that non-

288

linearity in eq. 6 is sometimes observed when temperature changes modify the tertiary or

289

quaternary structure (e.g., coil vs. helical structure for collagen).46

290

In order to assess the fluorescence behavior of DOM over a wider temperature range,

291

we measured Φf at 9 temperatures between 10 and 60 °C for 7 of the 13 samples (Figure

292

S11), and data were analyzed using eq. 6. Figures 3a and 3b compare data for a select

293

isolate (MRNOM) using the 5 and 9 temperature approaches, respectively, and show that

294

linearity is maintained across a wider temperature range. The Ea values obtained at 350

295

nm using both approaches are statistically the same (7.6 ± 1.8 kJ mol-1 for 5 point

296

regression versus 7.7 ± 0.7 kJ mol-1 for 9 point regression). Furthermore, we hypothesize

297

that the linearity in Figures 3a and 3b across a wide range of temperatures provides

298

evidence against both inter- and intramolecular complexes in DOM.8,47 The range in

299

temperatures investigated could affect the structure of such assemblies, and consequently



300

result in non-linearity in Figures 3a and 3b, although additional measurements are

301

necessary to assess this hypothesis.

302

The temperature dependence of Φf for whole water samples was well described by

303

the photophysical model presented in eq. 6 (see BWW and SJR in Table 1 and Figure

304

S11). Ea values for BWW and SJR were 6.0 ± 0.5 and 6.5 ± 0.2 kJ mol-1 at λex = 350 nm,

305

respectively, lower than the majority of organic matter isolates examined. The E2/E3,

306

spectral slope, and FI values for BWW and SJR suggest that the DOM in these whole

307

water samples is of lower molecular weight than terrestrially derived organic matter

308

isolates. It is worth noting that Ea values BWW and SJR are most similar to PPFA and

309

SRHA, two isolates of high aromaticity and molecular weight, demonstrating that

310

physicochemical parameters traditionally used to describe DOM chemistry cannot be

311

used to explain the Ea data in a simple way (vide infra).

312

Figures 3c and 3d show Ea values as a function of λex for YHPOA and SJR,

313

respectively (also see Figures S12 and S13). Ea decreases with increasing λex up to about

314

500 nm. This decrease is also apparent for the other isolates for which temperature

315

dependent Φf data were measured at 9 temperatures (Figure S13).

316

Ea values for DOM isolates and whole water samples from diverse origins show a

317

similar dependence on λex. This spectral similarity suggests that there is similarity in

318

DOM fluorophore structures in samples of diverse origin. Variations in Ea as a function

319

of λex might be expected given the many potential fluorescing moieties within a given

320

DOM sample. If fluorescence from LE states is responsible for this phenomenon, the

321

consistent decrease in Ea beginning at λex ~ 350 nm between different DOM isolates and 16 ACS Paragon Plus Environment

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whole water samples shown in Figures S12 and S13 indicates a consistent group of

323

fluorophores in DOM samples of diverse source. Future research is needed to identify

324

these fluorophores in DOM and present a reconciled explanation for the decreasing Φf,

325

Ea, and fluorescence lifetimes with increasing λex.

326

Photophysical mechanism:

327 328

In this section, we present interpretations of the presented data via both the CT and superposition model. The strengths and limitations of each interpretation are provided.

329

Charge-transfer model: It is possible to interpret the generally observed decrease in

330

Ea with increasing λex using the CT model for DOM photophysics. The CT model

331

postulates that fluorescence at λex > 350 nm is a result of charge-recombination. One

332

could hypothesize that this charge-recombination approaches a barrierless transition (Ea ~

333

0 kJ mol-1) as fluorescence is increasingly due to CT excited states (and decreasingly due

334

to LE states). In addition, it could be argued that the importance of CT excited state

335

emission is not captured because our data were collected using steady-state methods.

336

However, the CT model as a whole is inconsistent with the majority of the data in this

337

study and that of McKay et al.20 First, fluorescence spectral shape is temperature

338

independent between 10-60 °C (Figures 2, S3 to S5, and S7 to S9), the opposite what

339

would be predicted if emission from CT excited states was occurring. (See McKay et al.20

340

for additional discussion regarding this point). In addition, the reasoning that increasing

341

CT character would lead to lower Ea values is contrary to the observation that the Ea

342

value for the larger molecular size SRFAgt (7.8 ± 0.5 kJ mol-1) was greater than that for

343

the smaller molecular size SRFAlt (6.9 ± 0.3 kJ mol-1), because higher molecular size

344

DOM fractions are argued to have more CT character.12 17 ACS Paragon Plus Environment


Page 18 of 36

345

Additional analyses were performed to assess the contribution of CT excited state to

346

the observed fluorescence emission. The fractional decrease in both Φf and fluorescence

347

intensity with increasing temperature were calculated (Figures S14 to S16). The CT

348

model would hypothesize that the fractional decrease in fluorescence intensity would

349

decrease as a function of increasing emission wavelength because charge-recombination

350

induced luminescence is a near barrierless process. Indeed, data for PPFA at 40 °C were

351

consistent with this hypothesis. However, fractional fluorescence decrease was constant

352

as a function of emission wavelength for all aquatic humic and fulvic acid isolates.

353

Although not conclusive (see discussion of fluorescence lifetime measurements under the

354

Photophysical Model section), these data suggest that the observed steady state emission

355

in these samples is due to LE states and not CT excited states.

356

Superposition model: Comparison of Ea values for DOM with model compounds 1

357

could yield insight into the relevant photophysical processes leading to

358

radiationless decay. Table 1 shows published Ea values for model organic compounds,

359

including aromatics, amino acids, and coumarins, as well as the Ea for the viscosity (i.e.

360

self-diffusion) of water (~17 kJ mol-1).48 Viscosity affects fluorescence intensity, because

361

solvent viscosity influences its ability to reorient around and stabilize the new dipole of

362

the 1DOM* state.49 Interestingly, the Ea values for DOM are less than the value for the

363

diffusion of solutes in water,48 indicating that the values represent intrinsic photophysical

364

processes occurring in DOM (e.g., non-radiative decay) and not just changes in solvent

365

environment.

DOM*

366

The measured Ea values for DOM are most similar in magnitude to analogous values

367

for polycyclic aromatic hydrocarbons and substituted analogues (~5-10 kJ mol-1), 18 ACS Paragon Plus Environment

Page 19 of 36


368

suggesting that these compounds could be an important group of fluorophores in DOM.

369

Ea values for polycyclic aromatic hydrocarbons are attributable to the temperature

370

dependence of intersystem crossing.21 Intersystem crossing quantum yields have been

371

measured recently for DOM, with values ranging from ~ 4 – 8 % depending on the

372

sample.50 Measuring the temperature-dependence of DOM intersystem crossing yields

373

could yield insight into the extent that this process contributes to the Ea values measured

374

in this study. The measured Ea values for DOM are lower than analogous values for

375

alkoxy substituted 4-methyl coumarins (16-30 kJ mol-1),36 which are suggested as

376

potential fluorophores within DOM.4 The Ea for tryptophan fluorescence quenching (~ 30

377

kJ mol-1) is also greater than the values observed from DOM. Although also speculative,

378

another process that could be occurring is intermolecular excited state proton transfer to

379

the solvent, H2O, or intramolecular excited state proton transfer of phenolic

380

moieties.11,51,52 For example, excited-state proton transfer for 2-napthol to H2O has an

381

activation energy of ~ 11 kJ mol-1,52 comparable in magnitude to the Ea values observed

382

for DOM. The narrow range of Ea values for DOM compared to the diversity in Ea for

383

these model fluorophores suggests similar photophysical mechanisms are responsible for

384

1

DOM* radiationless decay across organic matter of diverse origin.

385

Ea values for DOM are derived from a heterogeneous mixture, with each component

386

of the mixture having an unknown contribution to Φf and Ea at a given excitation

387

wavelength. With this in mind, determining the contribution of model fluorophores in

388

Table 1 to DOM fluorescence based on Ea for on our data is not warranted. Future studies

389

could determine the contribution of individual fluorophores by performing temperature

390

dependent studies of DOM fluorescence over excitation wavelengths that overlap with 19 ACS Paragon Plus Environment


391

model compounds (λex < 350 nm). In this approach, Ea values derived for DOM could be

392

compared more directly to model compounds, which would yield insight into

393

photophysical mechanisms leading to 1DOM* deactivation.

394

Physical quenching of

1

DOM* by solvent molecules could be an important

395

deactivation process. For example, when D2O is used as a solvent for tryptophan, a

396

decrease in Φf by a factor of about 2.5 is observed; however, the Ea for tryptophan

397

remains unchanged.33 To investigate the possibility of solvent quenching of 1DOM*, Φf

398

were measured for SRFA in varying volume percentages of D2O (0-48% v/v). Minimal

399

changes were observed in Φf with increasing concentration of D2O (Φf increased from

400

0.00205 ± 0.00003 to 0.00213 ± 0.00003 between 0 and 48% v/v D2O), unlike the trend

401

observed for tryptophan33 and other proton donating or accepting aromatic compounds in

402

previous studies.53 That Φf did not decrease with increasing D2O concentration indicates

403

that solvent quenching of 1DOM* is a non-existent or relatively unimportant pathway.

404

Another explanation of these results is that DOM fluorophores are not solvent accessible

405

and thus are not quenched by D2O.54-56 In contrast to this idea, work by McKay et al.

406

(2018) showed that DOM absorbance and fluorescence spectral shape were insensitive to

407

solvent polarity,20 in which it was hypothesized that any non-covalent (e.g. hydrophobic)

408

interactions resulting in solvent inaccessible fluorophores should be disrupted.

409

Relationship to DOM physicochemical properties:

410

Correlations between measured Ea values and these physicochemical properties were

411

examined for DOM isolates. All physicochemical properties used in this study are given

412

in Table S2. These data include parameters measured in this study as well as data from

413

the literature, including carbon-13 nuclear magnetic resonance (13C-NMR) estimates of 20 ACS Paragon Plus Environment

Page 20 of 36

Page 21 of 36


414

carbon functional group distribution,57-60 electron accepting capacity (EAC),61 and

415

electron donating capacity (EDC).62 Two of these relationships are shown in Figure 4

416

while the rest are presented in Figures S17 to S19. The correlations between Ea, aromatic

417

C, and aliphatic C abundances shown in Figure 4 are significant for this dataset and

418

consistent with one another (i.e., negative correlation with aromatic C and positive

419

correlation with aliphatic C). Although some of the correlations between Ea and DOM

420

physicochemical properties exhibited significant p-values, including those shown in

421

Figure 4, most of the correlations were heavily weighted by PLFA and PPFA, end-

422

member isolates with Ea values of 8.4 and 5.4 kJ mol-1, respectively. No correlations

423

between Ea and other physicochemical parameters were significant when PLFA and

424

PPFA are removed from the regressed data.

425

Correlations between ln(knr0/kf) and DOM physicochemical properties were also

426

examined in an attempt to gain insight into the characteristics of DOM controlling the

427

ratio of knr0/kf (Figure S17 to S19). There was a significant inverse correlation between

428

ln(knr0/kf) and Φf (Figure S18), which should be expected given the relationship between

429

Φf and knr0 in eq. 1. None of the other relationships involving ln(knr0/kf) were significant,

430

however.

431

More work is needed to establish whether these relationships are practically

432

significant, which would certainly be of value in expanding the field of DOM

433

photophysics. For example, the higher Ea for SRFAgt (7.8 ± 0.5 kJ mol-1) compared to

434

SRFAlt (6.9 ± 0.3 kJ mol-1) suggests a relationship between Ea and DOM molecular size.

435

However, this relationship does not hold across different DOM isolates, as evidenced by



436

the lack of correlation between Ea and most physicochemical properties used as

437

surrogates for DOM molecular weight (e.g., E2/E3, FI, see Figures S18).

438

There is merit in assessing these data for correlations regardless of whether the

439

relationship is significant. The similarity in Ea values points to a similar photophysical

440

process occurring for non-radiative inactivation of singlet excited DOM, which is

441

interpreted as a similar pool of compounds responsible for DOM fluorescence across

442

varying samples. Alternatively, one could argue that the similarity in values is due to a

443

non-radiative process with a similar activation barrier, namely charge-recombination

444

induced luminescence.3 Regardless of the photophysical mechanism, the lack of

445

correlation implies that the physicochemical characteristics that govern non-radiative

446

decay are independent of bulk phase DOM characteristics, unlike other reactivity

447

parameters measured for DOM (e.g., photochemical quantum yields).

448

Temperature Corrections

449

A significant conclusion from this study is that Ea values measured for non-radiative

450

deactivation of 1DOM* fell within a narrow range (5.4 to 8.4 kJ mol-1), especially

451

compared to the larger range typical of model organic fluorophores in Table 1. The

452

practical similarity of this narrow range is that an average value of Ea and ln(knr0/kf) can

453

be used to correct fluorescence data at a given temperature to a reference temperature

454

(e.g., 20 °C), reducing systematic bias due to temperature differences to less than 5% for

455

most samples (eq. 10). This temperature correction is demonstrated in Figure 5, which

456

compares the relationship between fluorescence intensities with and without correction

457

via eq. 10, where T1 is the measured temperature and T2 is the reference temperature.

458

Without temperature corrections, the bias in fluorescence intensity due to changes in 22 ACS Paragon Plus Environment

Page 22 of 36

Page 23 of 36


459

quantum yield can range from +10% at 10°C to -30% at 55°C compared to a reference

460

temperature of 20°C (Figure S23).

461

=

/

/

(10)

462

Important for online monitoring applications, this approach is robust, because

463

average values for Ea and ln(knr0/kf) can be applied across a diverse range of samples

464

independent of sample concentration. It is important to note, however, that the

465

temperature correction model in Figure 5 accounts for temperature dependent changes in

466

Φf but does not account for inner filter effects, an independent optical artifact that also

467

introduces bias into online measurements. In contrast to other purely empirical

468

approaches,23,24 this study presents a temperature correction model that is grounded in

469

theory and can reconcile the photophysics of DOM with practical applications.

470

Acknowledgments

471

Funding for this work came from the National Science Foundation grant CBET

472

#1453906. We thank the United States Geological Survey, in particular George Aiken

473

and Brett Poulin, for providing three of the isolates used in this study. We also thank

474

Anthony Kennedy for providing the San Juan River sample

475

Supplemental Information

476

Text describing quantum yield calculations, temperature dependent absorbance

477

measurements, DOM physicochemical properties, and derivation of the equation in

478

Figure 5b; Figures showing fluorescence quantum yields in buffered solution versus

479

nanopure water, DOM absorbance spectra at different temperatures, fluorescence



480

quantum yields and emission spectra at different temperatures, Arrhenius-type plots for

481

fluorescence quantum yields determined at 350 nm excitation, activation energies as a

482

function of excitation wavelength, correlations between DOM physicochemical

483

properties and activation energy and ln(knr0/kf), and temperature correction using an

484

average activation energy; Tables showing sample source and identification/acronym,

485

and sample physicochemical properties. This information is available free of charge at

486

acs.org.


Page 24 of 36

Page 25 of 36


IDOM (RU)

0.6

a)

5.0

c)

0.4 0.2

4.9

0 300

400

500

600

700

Emission Wavelength (nm)

Absorbance

0.20

b)

4.8 10 20 30 40

0.15 0.10

°C °C °C °C

4.7

0.05

E = 7.6 2.1 kJ mol

-1

a 2

R = 0.970

0 300

400

500

600

3.1

Wavelength (nm)

3.2

3.3

1000/T (K-1 )

3.4

3.5

Figure 1. Temperature dependence of a) fluorescence and b) absorbance measured between 10 and 40 °C for Suwannee River Fulvic Acid (SRFA). Fluorescence spectra in a) are measured at an excitation wavelength of 350 nm. Subplot c) shows Φf plotted according to eq. 6 at an excitation wavelength of 350 nm. The activation energy, Ea, is obtained from multiplication of the linear regression slope (red line) by the gas constant, 8.314 J mol-1 K-1. Error bars in c) represent propagated error from triplicate absorbance and fluorescence measurements on the sample and quinine sulfate reference standard. Error bars represent the 95% confidence intervals.



Page 26 of 36

λex = 310 nm

1

increasing temperature

0.6

IDOM /I DOM,max

IDOM (RU)

0.8

YHPOA

a)

0.4 0.2 0 300

400

500

600

b) YHPOA Normalized

0.75 0.5 0.25 0 300

700


400

500

600

700


λex = 350 nm

YHPOA

c)

1

IDOM /I DOM,max

IDOM (RU)

0.6 0.4 0.2

d) YHPOA Normalized

0.75 0.5 0.25

0

0 400

500

600

700

400


500

600

700


λex = 410 nm

e)

YHPOA

1

IDOM /I DOM,max

IDOM (RU)

0.15 0.1 0.05 0

f) YHPOA Normalized

0.75 0.5 0.25 0

400

500

600

700

400


500

600

700


Figure 2. Emission spectra for Yukon Hydrophobic Organic Acid (YHPOA) at 9 different temperatures between 10.5 and 56.6 °C at three excitation wavelengths: 310 nm (a-b), 350 nm (c-d), and 410 nm (e-f). (Left) Corrected emission intensity in Raman Units. (Right) Emission spectra normalized to maximum intensity at each temperature.


Page 27 of 36


4.8

a)

4.8

b) Ea = 7.72 0.71 kJ mol

-1

2

R = 0.988 4.6

4.6

4.4

4.4 E = 7.62 2.18 kJ mol -1 a

4.2 3.0

c)

3.2

3.4

4.2 3.0

3.6

1000/T (K-1 ) YHPOA

8

6

Ea (kJ mol -1 )

Ea (kJ mol -1 )

8

R2 = 0.968

4 2 0 350

400

450

500

Excitation Wavelength (nm)

3.4

3.6

1000/T (K-1 ) SJR

d)

6 4 2 0 350

550

3.2

400

450

500

550

Excitation Wavelength (nm)

Figure 3. Arrhenius-type plots for Mississippi River Natural Organic Matter (MRNOM) using a) 5 data points between 10-40 °C and b) 9 data points between 10-55 °C at an excitation wavelength of 350 nm. Error bars in a) represent propagated error from triplicate absorbance and fluorescence measurements on the sample and quinine sulfate reference standard. Subplots c) and d) show apparent activation energies, Ea, obtained using the 9 temperature approach as a function of excitation wavelength for c) Yukon Hydrophobic Organic Acid (YHPOA) and d) San Juan River (SJR). Error bars in c) and d) represent standard errors on the fitted Ea derived from eq. 6.



12.0

Page 28 of 36

a)

Ea (kJ mol -1 )

p = 0.006 10.0 8.0 6.0 4.0 10

20

b)

30

40

% aromatic C

12.0

Ea (kJ mol -1 )

p = 0.002 10.0 8.0 6.0 4.0 20

40

60

80

% aliphatic C

Figure 4. Correlation between apparent activation energy (Ea) and a) aromatic and b) aliphatic C composition as determined by 13C-NMR. Error bars represent standard error on parameters derived from eq. 6. Samples include Gulf of Main Hydrophobic Organic Acid (GMHPOA), Mississippi River Natural Organic Matter (MRNOM), Pony Lake Fulvic Acid (PLFA, Pacific Ocean Fulvic Acid (POFA), Pahokee Peat Fulvic Acid (PPFA), Suwannee River Fulvic Acid (SRFA), Suwannee River Humic Acid (SRHA), and Suwannee River Natural Organic Matter (SRNOM), Yukon Hydrophobic Organic Acid (YHPOA).


Page 29 of 36


IDOM (T1 ) (RU)

0.5

a)

0.4 0.3 0.2 Ex 350 / Em 400 Ex 350 / Em 470 Ex 350 / Em 520

0.1 0 0

0.2

0.4

0.6

Corrected I DOM (20°C) (RU)

Measured I DOM (20°C) (RU) 0.5

b)

0.4 0.3 0.2 0.1 0 0

0.2

0.4

0.6

Measured I DOM (20°C) (RU)

Figure 5. Fluorescence intensities measured at different temperatures a) without and b) with temperature corrections relative to a sample measured at a reference temperature of 20°C



Page 30 of 36

Table 1. Arrhenius parameters for dissolved organic matter (± standard error, SE) and model compounds. Isolate Solvent MRNOM 30:70% v/v glycerol:H2O MRNOM H2O PLFA H2O PPFA H2O SRFA H2O SRHA H2O SRNOM H2O SRNOMgt H2O SRNOMlt H2O GMHPOA H2O POFA H2O YHPOA H2O BWW H2O SJR H2O average ± 1 standard deviationb Compound Solvent Benzene Ethanol Toluene Ethanol Toluene Hexane p-Xylene Ethanol Napthalene Ethanol Napthalene Hexane 2-Napthol H2O Tyrosine H2O Tryptophan H2O Tryptophan H2O, D2O N-methyl tryptophan H2O, D2O Indole H2O Tyrosine 0.5 M CH3COOH 1-methyl anthroate Acetonitrile 9-methyl anthroate Ethanol 4-methyl-n-alkoxy coumarins H2O Skh-1 (citrate-soluble calf skin) 0.5 M CH3COOH Collagen 0.5 M CH3COOH H2O viscosity Ethanol viscosity Tetrahydrofuran viscosity

Ea ± SE (kJ mol-1)a 7.7 ± 0.7 7.6 ± 1.8 8.4 ± 1.7 5.4 ± 0.6 7.4 ± 2.0 7.0 ± 1.3 7.1 ± 1.4 7.8 ± 0.5 6.9 ± 0.3 7.4 ± 0.4 8.0 ± 0.3 7.4 ± 0.4 6.0 ± 0.5 6.5 ± 0.2 7.2 ± 0.7

ln(knr0/kf) ± SE

Ea (kJ mol-1) 21.2 24.1 17.4 10.6 7.72 5.79 11.0 29.7 33.9 29.3 27.2 18.3 9.10 7.60 6.20

Ref.

7.3 ± 0.3 7.6 ± 0.7 7.9 ± 0.7 6.6 ± 0.3 7.8 ± 0.8 8.7 ± 0.5 7.6 ± 0.5 7.8 ± 0.2 6.7 ± 0.1 7.4 ± 0.2 7.6 ± 0.1 6.8 ± 0.2 6.4 ± 0.2 6.7 ± 0.1 7.4 ± 0.6 21 21 21 21 21 21 63 25 25 33 33 42 46 39 39 36

16.8-34.3 46

6.20-8.40 10.3-11.4 16.8 14.3 7.60

46 c c c

Based on Φf values measured at an excitation wavelength of 350 nm, Does not include MRNOM in 30% glycerol, cCalculated using wtt-pro.nist.gov/wtt-pro/

a

b


Page 31 of 36


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Temperature Dependence of Dissolved Organic Matter Fluorescence

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