Combined Effects of pH and Borohydride Reduction on Optical

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Combined effects of pH and borohydride reduction on the optical properties of humic substances (HS): A comparison of optical models Tara Marie Schendorf, Rossana Del Vecchio, Marla Bianca, and Neil V. Blough Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.9b01516 • Publication Date (Web): 07 May 2019 Downloaded from http://pubs.acs.org on May 8, 2019

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Environmental Science & Technology

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Combined effects of pH and borohydride reduction on the optical properties of humic

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substances (HS): A comparison of optical models

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Authors Tara Marie Schendorf,† Rossana Del Vecchio,*,‡ Marla Bianca,† and Neil V. Blough*,†. †Department

of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742, United States ‡Earth System Science Interdisciplinary Center, University of Maryland, College Park, Maryland 20742, United States

Submitted to Environmental Science & Technology

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* Corresponding authors:

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Rossana Del Vecchio

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email: [email protected], telephone: 001 301-405-0337

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Neil V. Blough (to whom correspondence should be addressed)

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email: [email protected], telephone: 001 301-405-0051

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Number of pages: 31 Number of figures: 4 Number of tables: 0 Number of words: 8134 (5734 text + 2400 figures)

1 ACS Paragon Plus Environment


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Abstract The combined effects of pH and borohydride reduction on the optical properties of a

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series of humic substances and a lignin model was examined to probe the molecular moieties and

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interactions that give rise to the observed optical properties of these materials. Increasing the pH

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from 2 to 12 produced significantly enhanced absorption across the spectra of all samples, with

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distinct spectral responses observed over pH ranges attributable to the deprotonation of

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carboxylic acids and phenols. Borohydride reduction substantially attenuated the broadband

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absorption enhancements with pH, clearly indicating that the loss of absorption due to

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ketone/aldehyde reduction is coupled with the pH-dependent increase in absorption due to

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deprotonation of carboxylic acids and phenols. These results cannot be easily explained by a

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superposition of the spectra of independently absorbing chromophores (superposition model),

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but are readily interpretable within a charge transfer (CT) model. Changes of fluorescence

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emission with pH for both untreated and borohydride reduced samples suggest that a pH-

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dependent structural reorganization of the HS may also be influencing the fluorescence emission.

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Independent of optical model, these results demonstrate that chemical tests targeted to specific

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moieties can identify distinct structural differences among HS sources, as well as provide insight

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to the molecular moieties and interactions that produce the observed optical and photochemical

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properties.


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Introduction Chromophoric dissolved organic matter (CDOM) and humic substances (HS), a constituent

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of the CDOM, absorb and emit light across the UV and visible wavelengths and play a major

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role in the optical and photochemical properties of natural waters. Despite the widely recognized

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importance of CDOM and HS in many environmental processes,1-8 the structural basis of their

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optical and photochemical properties along with the structures of the light-absorbing moieties

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contributing to these properties are still not fully understood. An electronic interaction model

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was proposed previously to explain the rather unusual absorption and emission properties of HS

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and the “humic-like” component of the CDOM.9-12 In this model, the short-wavelength

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absorbance (≤ 375 nm) arises in part from individual electron donor (D) and acceptor (A)

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chromophores, while the longer wavelength near-UV and visible absorption and emission arises

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increasingly from optical charge transfer (CT) transitions between the D and A leading to spectra

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that are not a simple sum of the spectra of the isolated chromophores. Phenolic and possibly

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substituted aromatic carboxylic acid groups have been proposed to represent some of the

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principle electron donors within CDOM/HS, while carbonyl-containing structures, such as

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aromatic ketones, aldehydes, and quinones, have been proposed as the likely primary electron

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acceptors formed upon partial oxidation of lignin10-13 and possibly other hydroxy-aromatic

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(phenolic) precursors.11-13 Although several studies have questioned the importance of CT

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interactions and have promoted the idea that the optical properties are a simple superposition of

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independently absorbing and emitting compounds,14-18 a large body of evidence suggests that the

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CT model can largely explain the rather unusual optical and photochemical properties of these9-

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12, 19-26

and other materials.27-30



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The donor and acceptor moieties proposed within this model are known to be present in

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CDOM/HS based on a variety of analytical techniques9, 31-42, with a number of these moieties

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contributing significantly to the optical and photochemical properties. Reduction by sodium

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borohydride of carbonyl-containing species within CDOM, particularly (aromatic)

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ketones/aldehydes, produces the preferential and primarily irreversible loss of visible absorption

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combined with significantly enhanced, blue-shifted fluorescence emission, results explainable

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within the CT model.9-13, 28-30, 43, 44 Reduction also significantly decreases the rates of sensitized

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phenol loss and 1O2 formation, consistent with the role of aromatic ketones/aldehyde triplets in

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these processes.22, 45-47 The anoxic reduction by sodium dithionite of quinones within HS/CDOM

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produces small and reversible losses of visible absorption combined with little or no change in

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fluorescence emission, results that are also explainable within the CT model. 13

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Korshin and co-workers 38, 39 and others 27 have shown that the UV-visible absorption of

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several HS increases with increasing pH. Absorption difference spectra, acquired by subtracting

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the spectra obtained at higher pH from a low pH reference (i.e. pH=2), has revealed the existence

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of two predominant types of absorption changes with pH, one associated with deprotonation of

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moieties having pKa’s in the range of carboxylic acids (~3 to 6) and the other with the

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deprotonation of moieties having pKa’s in the range of phenols (~8 and above). Distinct spectral

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responses are observed over these two pH ranges, with much broader and larger increases in

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absorption across the near-UV and visible wavelengths over the phenolic pKa range.

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Here, we examine the pH dependence of the absorption and emission properties of

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different HS spanning aquatic, microbial and terrestrial sources as well as a lignin model in both

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their original and sodium borohydride (NaBH4) reduced forms. We show that the results are very

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difficult to explain within a superposition model 14-16 but are readily interpretable within a CT


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model. In particular, while all untreated samples show broadband increases in the near-UV and

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visible absorption with increasing pH, particularly above pH 8 associated with phenol

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deprotonation, the increase in visible absorption is substantially attenuated in the borohydride-

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reduced samples, consistent with the loss of charge transfer interactions between donors

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(substituted aromatic carboxylic acids and phenol moieties) and carbonyl acceptors (aromatic

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ketones/aldehydes) due to the removal of the latter by borohydride reduction. The gain in the

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emission with increasing pH occurs primarily at lower pH (< 7) associated with carboxylic acids

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and may result from a structural re-organization; this gain is enhanced and blue-shifted upon

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borohydride-reduction consistent with the loss of the carbonyl-containing electron acceptors.

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Material and Methods

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Materials. Suwannee River fulvic and humic acid (SRFA; Lot 2S101F and SRHA; Lot

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2S101H), Pony Lake fulvic acid (PLFA; Lot 1R109F), Leonardite humic acid (LHA; Lot

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1S104H-5), and Elliot Soil humic acid (ESHA; Lot 1S102H) were obtained from the

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International Humic Substances Society (IHSS). Alkali-extracted and carboxylated lignin (LAC;

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Lot 19714 DS) was obtained from Sigma-Aldrich. Sodium borohydride (NaBH4; Lot 083480B)

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was obtained from Fisher Scientific. Sephadex G-10 (Lot SLBF7082V) was obtained from

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Sigma-Aldrich. Fluka Analytical Trace Select sodium hydroxide solution (NaOH; 30%; Lot

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BCBK1724V) was obtained from Sigma-Aldrich. ACS grade perchloric acid (HClO4; Lot

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117272) was obtained from Fisher Scientific. Quinine sulfate (QS) was obtained from Sigma-

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Aldrich. Water was obtained from a Milli-Q Plus purification system (Millipore). Tumeric

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Analytical Test Strips were obtained from Scientific Equipment of Houston (Lot 4012).

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Apparatus. A Shimadzu UVPC 2401 spectrophotometer was employed to acquire UVvisible absorption spectra. The spectrophotometric performance of this instrument was



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previously characterized by Cartisano et al.48 An Aminco-Bowman AB-2 and a FluoroMax4

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luminescence spectrometer were employed for the fluorescence measurements (monochromator

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excitation and emission band-passes set to 4 nm). A Thermoscientific micro pH electrode

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coupled to an Orion 4 Star pH ISE bench top meter was employed for the pH measurements.

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Optical Measurements. Absorption spectra were recorded using 1-cm quartz cuvettes

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over the range from 190 to 820 nm against air and were MQ subtracted afterward. Changes in

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absorption were calculated relative to a reference pH spectrum as absorption difference spectra

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(Delta A) and as Fractional A as follows

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Delta A = ApHx-ApHy

Eq. 1

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Fractional A = ApHx/ApH2

Eq. 2

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where ApHx and ApHy are the absorption spectra recorded at pH x and y, respectively, and ApH2 is

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the absorption spectrum recorded at pH 2.

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The spectral slope, S (nm-1), was calculated by fitting the data to an exponential function over the 300-700 nm range as follows A() = A(0) e-S(-0)

Eq. 3

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where A is the absorbance at a certain wavelength () and 0 is the reference wavelength (355

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nm), as previously reported.49 Changes in S over different pH ranges (% S) were calculated

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relative to the pH y value as follows,

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% S = [1- (Sx/Sy)]*100

Eq. 4

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where Sx and Sy are the S values at any pH (x) and at the reference pH y, respectively.

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Fluorescence emission spectra (EEMs) were collected over the 240 to 600 nm excitation range

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every 10 nm, while the absorbance was kept at < 0.05-0.1 OD at the excitation wavelength

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(λexc).11, 43 Emission spectra were collected at increasing concentrations with increasing


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excitation wavelengths to enhance emission at long excitation wavelengths; scans were then

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normalized to the lower concentration (as reported in the figures) and merged into a single

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EEMs. Emission spectra were MQ subtracted and corrected for the instrument response applying

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correction factors provided by the manufacturer. Difference emission spectra (Delta F) were

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obtained as follows

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Delta F = F(pHx) - F(pHy)

Eq. 5

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where F(pHx) the emission spectrum at a certain pH x, and F(pHy) is the spectrum obtained at

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the reference pH (3 or 7, as specified). Emission quantum yields were obtained relative to QS as

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previously reported.11

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Borohydride (NaBH4) reductions. Three milliliters of 100 mg/L concentrations of

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SRFA, SRHA, PLFA, LHA, ESHA and LAC, in MQ adjusted to pH 7 were transferred to 1-cm

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cuvettes. Solid NaBH4 was added at a 25-fold mass excess (25x) of NaBH4 to HS. SRFA was

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also reduced at the 5-fold, 25-fold, and 50-fold mass excess to test whether the extent of

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reduction influenced the pH dependence of the optical properties. Samples were treated in the

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dark under aerated conditions. Due to the large amounts of borate remaining after the reduction

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process, a G-10 column was used to adjust the pH back down to approximately 7 and remove the

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excess borate 50.

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pH Titration. The pH meter was calibrated daily prior to running any titrations. HClO4

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(0.25 M) and NaOH (0.25 M and 0.125 M) were used to control the pH of the HS solutions.38

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The untreated and the reduced samples were brought down to pH 2 using the HClO4 (0.25 M)

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and their absorbance was recorded. NaOH (0.25 M) was then added to the pH 2 samples to

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increase the pH to 12, while recording the absorbance every ~ 1-pH unit interval. Although some

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humic acids may precipitate at pH’s as low as 2, we observed no optical evidence of



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precipitation (scattering), most likely due to the relatively low concentrations of the HS

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employed and short incubation times (~ 5 min) at pH 2. Fluorescence emission spectra were

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recorded at pH 3-7-10. All samples were corrected for dilution from the acid and base additions

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prior to processing the data.

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Results

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Optical properties at neutral pH. The optical absorption and emission properties of the

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untreated and borohydride-reduced HS and LAC have been mostly reported elsewhere9, 50 but are

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further provided in the supporting information for this set of experiments along with the

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chemical properties of the HS examined (Figs. S1-S5; Tables S1-S3). Briefly, all samples

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exhibited an approximately exponential decrease in absorption with increasing wavelength, with

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values of the spectral slope, S, increasing in the order ESHA

Combined Effects of pH and Borohydride Reduction on Optical

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