Reduction-induced molecular signature of humic substances: structural evidence for optical changes

Abstract

The redox behavior of humic substances (HS) has drawn significant attention. It has been recently reported that aquatic HS after NaBH₄ reduction exhibit a loss in UV absorption but enhanced and blue-shifted fluorescence emission. This unique property was proven to also apply to terrestrial HS in the current study. To further understand the underlying relationship between the molecular structural changes and the optical changes, multiple techniques were employed to obtain solid spectroscopic evidence for Aldrich humic acid. Attenuated total reflectance infrared spectroscopy, Raman spectroscopy and solid-state ¹³C nuclear magnetic resonance spectroscopy were used together to demonstrate that the alcohol-like moieties increased while the carbonyl moieties decreased in HS molecules during reduction, which is responsible for optical properties. In addition, the obtained results could explain the change in molecular size as evidenced by dynamic light scattering. The integrated results presented in this study unambiguously provide solid spectroscopic evidence for the charge transfer model, which is a relatively new concept framework for understanding HS.

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Introduction

As an important component of natural organic matter (NOM), humic substances (HS) are ubiquitous in nature and play a critical role in various environmental processes. It is well-known that HS, having considerable chromophores, absorb light across the ultraviolet and visible spectrum and in turn affect aquatic photosystems and the chemical reactions of other substances as well as their environmental behavior.^1–4 Moreover, as an essential aspect, the optical properties of HS have been studied in recent decades.^4–11 Recently, the redox properties of HS have become a major focus for evaluating their potential to affect the fate of redox-sensitive contaminants.^1,6,12–14

A unique optical phenomenon of aquatic HS was reported recently.^6,15 After reduction with borohydride, the absorption spectra of HS exhibited some loss across the ultraviolet and visible regions, while fluorescence emission was substantially enhanced (2–3 fold increase) and blue-shifted. In addition, it was proven that the optical properties of aquatic HS and chromophoric dissolved organic matter (CDOM) can not arise from a simple superposition of the spectra of independent chromophores,^7,16 nor can they be directly attributed to quinones or hydroquinones.⁶

Therefore, a charge transfer model, accounting for the optical properties of NOM, was proposed in recent years.^5–7,16,17 The hypothesis attributes the optical properties of HS to a vast number of donor–acceptor charge-transfer complexes, which form within and between the individual molecules in HS. It suggests that electron donor moieties, such as poly-hydroxylated aromatics or phenols, and acceptor moieties, such as quinones or carbonyls, co-localize randomly on the molecular chains of HS and can form donor–acceptor charge-transfer complexes by intramolecular charge transfer transitions.

First, further solid foundation and spectroscopic evidence for this theoretical model remains scarce until now.¹⁸ The coupling of optical measurements with spectroscopic techniques should provide clear complementary results and make the theoretical model more reliable. Thus, to study the structural mechanism of these distinct optical changes at the molecular level is of environmental significance and is necessary.

Second, definite characterization of the structure of HS is elusive despite promising developments in advanced spectroscopic techniques.^19–23 Moreover, relatively few studies have focused on the relationship between the structural features and the optical properties of HS. Therefore, a definite and consistent interpretation for the underlying structural basis of the rather unique optical features of HS is urgently required.

Contrary to studies that solely focus on characterizing the complete structure of HS or explore only its optical features, the current study attempts to reveal the underlying consistency between the optical properties and the structural changes, i.e. a reduction-induced molecular signature of HS. Attenuated total reflectance infrared (ATR-IR) spectroscopy and Raman spectroscopy along with ¹³C solid-state nuclear magnetic resonance (NMR) techniques were employed to investigate the structural changes of HS after NaBH₄ reduction with an increase in emission.^6,15 The obtained results provide solid spectroscopic evidence of the structural changes responsible for the optical changes of HS and support unambiguously the charge transfer model—a relatively new concept framework for understanding HS.

Materials and methods

Materials

Elliott soil humic acid (ESHA) and Pony Lake fulvic acid (PLFA) were acquired from the International Humic Substances Society (IHSS). Aldrich humic acid (Aldrich HA) was obtained from Aldrich. Sodium borohydride and hydrochloric acid were obtained from Alfa Aesar. Dialysis membranes were obtained from American spectrum-labs. All solutions for the experiments were prepared in Milli-Q water.

NaBH₄ reduction

3 mL of 20 mg L⁻¹ Aldrich HA solution was transferred into a quartz cuvette, and then a slight excess of solid NaBH₄ (2 mg) was added to the cuvette. Higher concentrations of Aldrich HA were reduced with a larger amount of NaBH₄ (according to the above proportion). The reduction process was considered to be complete when no further changes were observed in the absorption spectra. Reduction with NaBH₄ resulted in an increase of the pH of the solution from 7 to 10 due to the reduction of H⁺ to H₂. To observe whether pH influenced the optical properties of HS, when required, a small amount of diluted hydrochloric acid was added to adjust the pH back to the original after NaBH₄ reduction.

For ATR-IR spectroscopic, Raman spectroscopic and NMR experiments, dialysis membranes were employed to remove the excess NaBH₄ and reduce the salt concentration in the reduced Aldrich HA solution.^19,24 As control experiments, the original Aldrich HA solution was treated with the same procedure. Dialysis of Aldrich HA was performed using a 100 Da molecular weight cut-off (MWCO) and 34 mm flat width membrane. The dialysis membrane was stored in 0.05% sodium azide solution and thoroughly washed prior to use. The solution of Aldrich HA that was treated or not treated with NaBH₄ was added to the dialysis membrane, which was then closed by two clamps on both sides. Then, the dialysis solution was immersed in water in a 1000 mL beaker and stirred gently at room temperature for 48 h. After dialysis, the retentate was removed and lyophilized for 48 h. Finally, the lyophilized samples were ground and stored in a vial kit at 4 °C.

The total organic carbon (TOC), which was used to determine the organic loss during the dialysis, was measured with a Tekmar Dohrmann Apollo 9000 TOC analyzer. For the 100 Da dialysis membrane used in this study, the organic loss was 6.4% for the reduced samples compared to the original samples.

Optical Measurements

UV-visible absorption spectra and fluorescence spectra of the untreated and reduced Aldrich HA solution (20 mg L⁻¹) were measured in a 1 cm quartz cuvette (3 mL) using a Shimadzu UV-2550 and a HORIBA FluoroMax-4 spectrofluorometer, respectively. The fluorescence excitation wavelength (λ_exc) was increased every 5 nm over the 300–550 nm range, and the emission spectra were collected from 10 nm greater than λ_exc to 650 nm. The spectra were corrected for the instrument response using the manufacturer's factor prior to each test.

Fluorescence excitation–emission matrices of ESHA and PLFA (100 mg L⁻¹) were prepared using a HORIBA Aqualog fluorometer. The excitation wavelength varied from 240–600 nm in 3 nm steps, while the emission wavelength was also recorded from 260–620 nm in 3 nm steps.

Infrared spectrometry was performed on a Bruker VERTEX 70 infrared spectrometer using the ATR method from 4000 to 400 cm⁻¹ at room temperature. Raman spectrometry was performed on a LabRAM HR800 confocal laser Raman spectrometer at a laser wavelength of 514 nm.

NMR spectroscopy

Cross-polarization and magic angle spinning (CP/MAS) ¹³C NMR was performed on a 400 MHz Bruker AVANCE III spectrometer with 4 mm sample rotors in a double resonance probe head. All the NMR experiments were performed at 298 K with a 2 s delay time and scanned 10 000 times. Primary quantitative analysis of functional groups was calculated from the corresponding peak areas.

Other Measurements

Dynamic light scattering (DLS) was performed on a Delsa™ Nano Submicron Particle Size and Zeta Potential Analyzer (Beckman Coulter, USA). The data were recorded 70 times and obtained from the average value of three experiments.

The concentration of Cu²⁺ in Aldrich HA was determined by the flame method using an atomic absorption spectrometer (PinAAcle 900T, PerkinElmer, USA).

Results and discussion

Fluorescence increase

Fluorescence originates from the lowest energy excited state with few exceptions. Therefore, both the maximum wavelength of emission and the fluorescence quantum yield are independent of the excitation wavelength for an individual chromophore. Here, the fluorescence emission of Aldrich HA, presenting a unique envelope spectra over the λ_exc 300–650 nm range (Fig. 1a), had similar features to those of other aquatic HS such as Suwannee River humic (SRHA), Suwannee River fulvic acids (SRFA), alkali-extracted and carboxylated lignin (LAC), and extracted environmental marine samples.⁶ In each fluorescence spectrum, the emission spectrum line excited by a longer wavelength light was enveloped below the emission line excited by a shorter wavelength light. This phenomenon indicated that the moieties excited by a longer wavelength light account for a lower energy subset of those that can be excited by a shorter wavelength light. Compared with the original sample (original), the sample after reduction with NaBH₄ (reduced) exhibited substantially enhanced (100% to more than 200%) fluorescence emission intensity (Fig. 1a and b). Moreover, the maximum emission wavelength shifted to a short wavelength, especially at shorter excitation wavelengths of 300–420 nm (Fig. 1d). The same trend was observed for the fluorescence peak shape, which became much sharper after reduction and was more obvious at shorter excitation wavelengths. In addition, with significantly enhanced fluorescence emission compared with the original sample, the reduced sample had a notable loss in absorption across the ultraviolet and visible regions (Fig. 1c). Therefore, the fluorescence quantum yield of the reduced Aldrich HA was much higher than that of the original compared with the increasing folds in the emission intensity.

Fig. 1 Optical changes of Aldrich HA following NaBH₄ reduction: fluorescence emission spectra of the samples before and after reduction are shown in (a) and (b), respectively; a comparison of the absorption spectra is set in (c), whereas the fluorescence emission maxima are compared in (d).

As a supplement, the excitation–emission matrix of PLFA and ESHA were also examined (Fig. 2). A similar fluorescence enhancement (around 2 folds) with blue-shifted emission maximum were also observed for the two samples after NaBH₄ reduction. Furthermore, the results for the abovementioned three HS used in this study were consistent with those of a previous report on aquatic HS samples.⁶ Thus, to some extent the results indicate that the fluorescence properties of natural HS, which is derived from terrestrially or microbially dominated sources, possess similarity and universality on a structural basis for the reduction induced enhanced and blue-shifted fluorescence emission regardless of the origin of the samples and whether the structure of HS was either dramatically or trivially changed after reduction. Thus, the following experiments investigated and analyzed the structural puzzle at the molecular level using multiple techniques.

Fig. 2 Excitation–emission matrix of PLFA before and after reduction are shown in (a) and (b), respectively, and those corresponding to ESHA are shown in (c) and (d).

It is worth noting that the induction of excess NaBH₄ resulted in a large increase in pH due to the reduction of H⁺ to H₂. Therefore, the pH of the solution was observed to increase to 10.2 during reduction. Diluted hydrochloric acid was added to adjust the pH back to the original (around 7.0), and it was discovered that the fluorescence spectrum of the reduced solution (pH∼10 and ∼7.0) was completely unchanged, which excluded the influence of pH on the increase in fluorescence emission of HS after reduction. In addition, the fluorescence of HS may be influenced by the binding of metal ions such as Cu²⁺, which possibly quenches fluorescence. Thus, the original Aldrich HA was examined by atomic absorption, and Cu²⁺ was not detected, confirming that the observed changes in fluorescence were not due to changes in the degree of Cu–organic complexation.

Structural characterization

IR and Raman analysis. After reduction, it could be observed from IR that the characteristically wide peak at 3500–2500 cm⁻¹ due to strong hydrogen-bond interactions became sharper, and a significant peak at 3550 cm⁻¹ corresponding to “free” hydroxyl groups appeared (Fig. 3a). Clearly, some amount of the original “associated” –OH groups were replaced by a considerable sum of “free” –OH groups, indicating that the H-bond interactions were considerably reduced (see discussions below). Moreover, a substantial reduction of the C=O stretching vibration at 1700–1500 cm⁻¹ was observed. Meanwhile, more peaks at 1000–700 cm⁻¹ (C–H bending vibration) appeared and exhibited stronger intensity than the original state. All of the abovementioned data indicate the reduction of carbonyl groups of aliphatic and aromatic ketones as well as quinones to the corresponding alcohols and hydroquinones/phenols, respectively. Complementarily, Raman spectroscopy was also applied to probe the structural changes in Aldrich HA (Fig. 3b). Despite the poor baseline due to the fluorescence property of HS, the carbonyl peak of the sample before reduction was still clearly observed around 1600 cm⁻¹, which peak completely disappeared after reduction. The Raman result was consistent with the IR results, confirming that the carbonyl groups of both the ketones and quinones of the sample were reduced to alcohol groups, and this structural change should be responsible for the very distinct increase in emission.

Fig. 3 ATR-IR (a) and Raman spectra (b) of Aldrich HA before and after reduction.

The dynamic light scattering (DLS) data were in good agreement with the abovementioned results (Fig. 4). The size of Aldrich HA after reduction was significantly larger than that of the original sample, changing from 75–180 nm to 150–280 nm. Consistent with the IR results, because the significant “free” –OH peak (3550 cm⁻¹) could be clearly observed only when relatively weak H-bond interactions existed, increasing molecular diameter of HS resulted from the weakened H-bond interactions (especially for intra/inter-molecular association), referring to the original wide peak at 3500–2500 cm⁻¹. Moreover, the intensity of H-bonds generated from two –OH groups (strong H-bond donors and weak acceptors) was much weaker than that of the H-bonds between C=O (strong H-bond acceptors) and –OH groups. Thus, in the original state when H-bonds were generated between C=O and –OH groups, the HS molecules were arranged more compactly; however, after C=O groups were reduced into C–OH, the corresponding extent of interaction of H-bonds between two –OH groups was considerably weakened, leading to a more dispersed distribution of molecules than before. On the other hand, after carbonyls were converted to alcohols, the probability of H-bond formation may increase. Because the number of newly formed –OH groups was consistent with the number of reduced C=O groups, the possibility of new H-bond formation should increase very restrictedly but the H-bond intensity was decreased largely. Thus, the size of HS after reduction was still overall larger.

Fig. 4 The results of DLS for Aldrich HA before (HSori) and after (HSred) NaBH₄ reduction.

Solid-state ¹³C NMR analysis. Because of the structural and compositional complexity of HS, the ability of conventional 1D ¹H NMR to identify the tiny changes in the chemical environment from very complex crowding signals is limited. Thus, solid-state ¹³C NMR was further employed for more credible deduction.^25–27 During reduction, Aldrich HA was analyzed with different reduction times (Fig. 5a). Compared with the original sample, the relative abundance of carbon at 160–200 ppm following reduction (30 min) was reduced from 19.60% to 12.12%, which was assigned to the carbonyl groups (ketones as well as quinones). Moreover, the relative abundance of carbon at 60–100 ppm following the reduction (30 min) was increased from 0.98% to 7.07%, which was assigned to alcohol-like C or O-alkyls.^8,20,23 Furthermore, this proportion increased to 9.90% when the sample was reduced for a longer duration (60 min). These changes in the NMR spectra clearly demonstrate that the C=O groups were transformed to C–OH groups. In contrast, the relative abundance of carbon at 0–60 ppm and 100–160 ppm remained substantially unchanged during reduction, indicating that the optical changes could not be related to aliphatic series or aromatics (Fig. 5b). Thus, the amount of carbonyl moieties (160–200 ppm) decreased, and thus, the electron acceptors of both ketones and quinones decreased. Consequently, intramolecular charge transfer transitions between acceptors and donors were reduced. Therefore, the excited-state chromophores can return back to ground state more easily and successfully by emitting fluorescence due to fewer obstacles for charge transfer transitions.

Fig. 5 CP/MAS solid-state ¹³C NMR spectra (a) and relative abundance integration (b) of Aldrich HA with different reduction times of 0, 20, and 30 min. The total area integration from 0–200 ppm was set to 100%.

Conclusions

This study targeted the relationship between the unique optical properties of HS and the relevant structural changes. Together with the enhanced emission, all the results of IR spectroscopy, Raman spectroscopy, NMR spectroscopy and DLS uniformly indicated the transformation of carbonyl-containing moieties to alcohol-like structures during NaBH₄ reduction, which led to a decrease in charge transfer interactions and then reduced the fluorescence quenching in HS molecules. Thus, this transformation should be responsible for the increase in fluorescence emission. Based on the abovementioned results and discussions, it is striking that HS exhibited substantially enhanced emission along with decreased UV absorption after reduction. Therefore, these results highlight the role that the charge transfer transitions play in the fluorescence quenching process. This study not only supports the charge transfer model, which is a relatively new concept framework for understanding HS, but also benefits the study of the complete HS structure and more broad physical and chemical properties of NOM.

Acknowledgements

This work was supported by the NSFC (nos. 21007089 and 21377126) and the SRF for ROCS, SEM of China.

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