Secondary Structures in a Freeze-Dried Lignite Humic Acid Fraction

Article pubs.acs.org/est

Secondary Structures in a Freeze-Dried Lignite Humic Acid Fraction Caused by Hydrogen-Bonding of Acidic Protons with Aromatic Rings Xiaoyan Cao,† Marios Drosos,‡ Jerry A. Leenheer,§ and Jingdong Mao*,† †

Department of Chemistry and Biochemistry, Old Dominion University, 4541 Hampton Blvd, Norfolk, Virginia 23529, United States Centro Interdipartimentale di Ricerca sulla Risonanza Magnetica Nucleare per l′ Ambiente, l′ Agroalimentare ed i Nuovi Materiali (CERMANU), Università di Napoli “Federico II”, Via Università 100, 80055 Portici, Italy § 4024 Douglas Mountain Drive, Golden, Colorado 80403, United States ‡

S Supporting Information *

ABSTRACT: A lignite humic acid (HA) was separated from inorganic and non-HA impurities (i.e., aluminosilicates, metals) and fractionated by a combination of dialysis and XAD-8 resin. Fractionation revealed a more homogeneous structure of lignite HA. New and more specific structural information on the main lignite HA fraction is obtained by solid-state nuclear magnetic resonance (NMR) spectroscopy. Quantitative 13 C multiple cross-polarization (multiCP) NMR indicated oxidized phenyl propane structures derived from lignin. MultiCP experiments, conducted on potassium HA salts titrated to pH 10 and pH 12, revealed shifts consistent with carboxylate and phenolate formation, but structural changes associated with enolate formation from aromatic beta keto acids were not detected. Two-dimensional 1H−13C heteronuclear correlation (2D HETCOR) NMR indicated aryl-aliphatic ketones, aliphatic and aromatic carboxyl groups, phenol, and methoxy phenyl ethers. Acidic protons from carboxyl groups in both the lignite HA fraction and a synthetic HA-like polycondensate were found to be hydrogen-bonded with electron-rich aromatic rings. Our results coupled with published infrared spectra provide evidence for the preferential hydrogen bonding of acidic hydrogens with electron-rich aromatic rings rather than adjacent carbonyl groups. These hydrogen-bonding interactions likely result from stereochemical arrangements in primary structures and folding.

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INTRODUCTION Humic substances (HS) consist of complex associations of molecules that originate from degraded organic matter (especially plant vegetation), and play key roles in many environmental processes.1−6 Lignin is a major component of plant vegetation7 and is a major precursor of humic substances.1 Humic acid isolated from lignite coal and defined by the standard alkali extraction/acid precipitation method8 is being increasingly used to promote plant growth and yields, and to remediate contaminated soils, aquifers, and sediments.3,9 The biochemical mechanisms of these beneficial effects of humic acids are presently unknown. A major limitation to understanding these mechanisms is the lack of knowledge of primary and secondary structures of lignite humic acid. Secondary structures resulting from hydrogen bonding between acids and bases in nucleic acids and proteins are essential to the functions of life.10,11 Hydrogen bonding in lignite humic acid is limited to carboxylic acids and phenols as proton donors and carbonyl groups (carboxylic acids and ketones) and π-electrons in aromatic rings as proton acceptors. Infrared spectroscopy has long been used to detect hydrogen bonding in HS by shifts in hydroxyl and carbonyl group stretching frequencies,1 but little is known about aromatic ring π-electrons as proton accepting groups in HS. Hydrogen bonding between water12−14 and various acids with various aromatic compounds (benzene, phenol, furan, etc.) has been well described in the chemical literature.10,15,16 Solid-state © XXXX American Chemical Society

nuclear magnetic resonance (NMR) spectroscopy has proven to be one of the most powerful methods for characterizing complex organic matter such as HS.17−20 Two-dimensional correlation of 13C-chemical shifts with 1H chemical shifts, termed 2D 13C−1H heteronuclear correlation (HETCOR) NMR, can identify the chemical environment of a given peak (functional group) and provide structural information that is not available with 13C or 1H NMR alone or other spectroscopic methods.21,22 An additional advantage of 2D HETCOR NMR is that it probes carbon proximity to both nonexchangeable and exchangeable (alcohols, carboxyls, and phenols) protons, revealing H-bonding properties of humic acids including aromatic rings as proton acceptors. The objectives of the present study were to better understand the primary and secondary structures of a freezedried lignite HA acid fraction derived primarily from lignin after removal of inorganic and non-HA impurities. Primary structures in freeze-dried H-form and K-salt form (titrated to pH 10 and 12) HA were examined by solid-state 13C multiple cross-polarization (multiCP) spectroscopy. The K-salt HA studies were performed to determine possible structural changes by disruption of H-bonding at high pH and structural Received: June 11, 2015 Revised: January 13, 2016 Accepted: January 18, 2016

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DOI: 10.1021/acs.est.5b02859 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

Article

Environmental Science & Technology

small (10 ms) CP from 1H without significant magnetization losses due to relaxation and with a moderate duty cycle of the radio frequency irradiation, by multiple CP periods alternating with 1H spin−lattice relaxation periods that repolarize the protons. The spectra were measured at a spinning speed of 14 kHz, where spinning sidebands are fairly

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RESULTS AND DISCUSSION Characterization of Lignite-HA Fraction and Humic Acid-Like Polycondensate by 1D NMR. Figure 1 shows the multiCP and CP/TOSS spectra of the lignite-HA fraction B


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of syringyl structures with methoxy, or aryl ether substituents at the 3-, 4-, and 5-positions, which would result in characteristic peaks (C-4 near 135 ppm, C-2 and C-6 near 105 ppm),32 and these phenols/phenyl ether carbons would account for 50% of the aromatic carbon structures. The aliphatic C−O accounted for 6% of all C. The COO/NCO comprised ∼13% of all C. The contribution of NCO was negligible because the infrared spectra of LC2 showed no evidence of amide peaks26 and LC2 had relatively high C/N atomic ratio (26.2). The relative abundances of carboxyl increased while those of aromatic C−O, OCH3, and alkyl C decreased relative to lignin (SI Table S1). These trends are consistent with diagenetic processes such as demethylation and oxidation of the propane side chains.26 The CP/TOSS spectra (Figure 1(d)) of HALP were different from those of LC2 (Figure 1(b)). The signals from aliphatic structures were evident but small in the spectra of HALP. The signals in the aromatic region also had different features. The spectra of HALP mainly showed aromatic C−O (143 ppm, 149 ppm), unsubstituted aromatic C ortho to aromatic C−O structures (∼118 ppm) as well as signals from carboxyl groups (161 ppm, 170 ppm), which are abundant in gallic acid and protocatechuic acid precursors27,28 (Figure 1(e)). The 13C multiCP NMR spectra of the potassium salts of the lignite humic acid at pH 10 and pH 12 were obtained to examine possible phenol, phenolic ethers, and enol structures in LC2 (Figure 2). It is of our interest to test the hypothesis of the

Figure 1. (a) 13C NMR spectra obtained with multiple crosspolarization (multiCP) of LC2. 13C cross-polarization total sideband suppression (CP/TOSS) spectra of (b) LC2, (c) lignin, and (d) HALP. Thin lines: spectra of all C; bold lines: corresponding spectra of nonprotonated and mobile C, obtained after dipolar dephasing (68 μs for multiCP and 48 μs for CP/TOSS). (e) Structures of gallic acid (GA) and protocatechuic acid (PA).

(LC2, Figure 1(a) and (b), thin lines), CP/TOSS spectra of lignin (Figure 1(c), thin line), humic-acid-like polycondensate (HALP, Figure 1(d), thin line), and their corresponding dipolar-dephased spectra (bold lines). The CP/TOSS spectra of LC2 (Figure 1(b)) matched its quantitative multiCP spectra (Figure 1(a), Supporting Information (SI) Figure S1) surprisingly well, except that the CP/TOSS spectra showed slightly enhanced ketone signals. The good matching of CP/ TOSS to multiCP spectra with enhancement of sp2-hybridized C can be explained by excitation effects (carrier frequency for 13 C: 150 ppm for CP/TOSS experiments, and 90 ppm for multiCP experiments). A comparison between these spectra and that of lignin (Figure 1(c)) confirmed that the carbons in the fractionated lignite humic acid (LC2) were primarily derived from lignin residues. For instance, methoxy groups (OCH3) produced a sharp peak near 56 ppm, and aromatic C− O structures in lignin yielded shoulders near 150 ppm. The aliphatic C−O signals likely arose from phenyl propane side chains. Integration of the multiCP spectrum of LC2 (SI Table S1) revealed that phenols and phenyl ether substituents (aromatic C−O) constituted 27% of the aromatic carbon structures. This is reasonable given that there was no indication

Figure 2. 13C multiCP spectra of potassium salts of LC2 at pH 10 (a) and pH 12 (c). Corresponding spectra of nonprotonated and mobile C, obtained after 68-μs of dipolar dephasing of LC2 at pH 10 (b) and pH 12 (d). The corresponding spectra of free humic acid LC2 are shown in dashed lines for comparisons.

presence of aromatic beta-keto acids in humic acid. Beta-keto acid structures were hypothesized to exist based upon oxidative degradation of the phenyl propane side chains in lignin, hydrogen-bonding shifts in the infrared spectra of aromatic ketone carbonyl groups in various HAs,1 decarboxylation upon heating, and the formation of conjugated double bond structures in enols which lead to an increase in color intensity C


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spectral changes in the aliphatic region, indicating no structural changes occurring in the phenyl propane side chain as the pH was increased. Titrating the humic acid to pH 12 further yielded some spectral changes: a progressive decrease in the aromatic C−O peak near 150 ppm and the appearance of a peak at ∼170 ppm (Figure 2(c, d)). This could be explained by the conversion of phenols to phenolates. However, the new peak at ∼170 ppm can also be due to the presence of carbonate, which was confirmed by additional chemical-shift-anisotropy filtering and long-range C−H dipolar dephasing experiments (SI Figure S2). In summary, the spectra of the potassium salts formed at pH 10 and pH 12, revealed shifts consistent with carboxylate and phenolate formation, but structural changes associated with enolate formation from aromatic beta keto acids were not detected, disproving the aromatic beta-keto acid hypothesis. Functional Group Proximities of LC2 and HALP by 2D HETCOR NMR. Information on the chemical environments of functional groups in LC2 and HALP is obtained with 2D HETCOR NMR (Figures 3 and 4) which provides throughspace 1H−13C correlations via the dipolar interactions. 1H slices at different 13C chemical shifts were extracted to observe the correlations more clearly. Proton chemical shifts were assigned as follows: 0.8−3 ppm, nonpolar alkyl protons; 3−5.5 ppm, protons associated with O/N-alkyl groups; 6−8.5 ppm, aromatic/amide NH/phenolic OH protons; and above 9.5 ppm, aldehyde and COOH protons, and some hydrogenbonded OH/NH protons.21,33 The structural information on LC2 and HALP from the 2D NMR spectra is presented below. Lignite Humic Acid Fraction (LC2). Alkyl CHn carbons resonating at 30 and 41 ppm (SI Figure S3(a, b)) were predominantly associated with their directly bonded alkyl protons at tm,e = 0.01 ms, with their proton spectra showing peak maximums at ∼2 ppm and ∼3 ppm, respectively. With increasing mixing times, the correlations of these alkyl carbons with aromatic protons and OCH protons became more

at high pH. If present, H-bonding in beta-keto acids would likely be preferred to H-bonding with other carboxyl groups or aromatic ring π-electrons because of entropic and energetic considerations. Titrating the humic acid to pH 10 would change the aromatic beta-keto acid to its enolized form (Scheme 1). The following 13C NMR spectral changes would Scheme 1. Keto-enol tautomerization

be expected accordingly: a decrease in intensity of the protonated beta carbon on the propane side chain (35−50 ppm and/or 75−85 ppm depending on variations in lignin structure); a change in the aromatic carbon profile resulting from the formation of double bonds in enolates; and a decrease in intensity of aromatic ketones from 185 to 215 ppm. Titrating the humic acid to pH 12 would quantitatively convert all free phenol groups to phenolate groups which are shifted downfield in the 13C NMR spectra from phenyl ether groups. Figure 2 (a, b) shows that the carboxyl peak shifted from 171 ppm in the spectra of LC2 to 176 ppm in the spectra of LC2 at pH10, and the aromatic peak decreased in intensity at pH 10. The former observation likely resulted from the conversion of the acid form (COOH) to the salt form (COO− K+). The decrease in aromatic carbon intensity could be associated with the loss of protons (carboxyl and phenol) available for crosspolarization of aromatic carbons. There were no evident

Figure 3. 2D HETCOR NMR spectra of LC2 with (a) 1 ms LG-CP and (b) 1 ms HH−CP. (c-i) 1H spectra extracted at different 13C chemical shifts at different mixing times. Please see text for further details. D


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Figure 4. 2D HETCOR NMR spectra of HALP with (a) 1 ms LG-CP and (b) 1 ms HH−CP. (c-d) 1H spectra extracted at different 13C chemical shifts at different mixing times. Please see text for further details.

ms, the proton spectra associated with alkyl C, OCH, and aromatic C showed contributions predominantly from their directly attached protons, that is, alkyl protons (∼4 ppm), OCH protons (∼5 ppm), and aromatic protons (∼7 ppm), respectively (Figure 4(c)). The unusually high chemical shift of alkyl protons (∼4 ppm) suggested that the alkyl carbons were likely bonded to two sp2-hybridized carbons. The aromatic C, aromatic C−O, and COO carbons were predominantly associated with aromatic protons, consistent with the structures of gallic and protocatechuic acid precursors (Figure 1(e)). Ketones seemed to be mainly associated with aliphatic protons. As the mixing time increased to 0.3 ms, the proton spectrum of alkyl carbons showed greater contributions from OCH, and aromatic protons, whereas that of OCH carbons showed increasing intensities of alkyl and aromatic protons. The proton spectra associated with aromatic and COO carbons showed relatively greater intensities from alkyl protons. The ketones showed correlations with all types of protons. The HALP spectra showed different proximities of functional groups from those of the lignite humic acid. Hydrogen Bonding of Acidic Protons with Aromatic Rings. The proton spectra of aromatic carbons in LC2 (Figure 3(c−f)) showed contributions from COOH and hydrogenbonded OH protons (∼12 ppm, indicated by arrow) even at the shortest mixing time of 0.01 ms, suggesting the strong interaction of acidic protons from carboxyl and possibly phenolic groups with aromatic ring carbons from 110 to 160 ppm. Such interaction of acidic protons from carboxyl and phenol groups with aromatic carbons was more apparent with the HETCOR spectra of HALP than with the LC2 spectra because HALP contained less aliphatic carbons and protons (Figure 4(c,d)). Similarly, the 2D HETCOR spectra of an Amherst HA (Figure 4 in Mao et al.21) showed even more pronounced correlations of carboxyl protons with aromatic C groups. Phenol and methoxy groups strongly donate electrons to aromatic π-electron clouds, which makes this hydrogenbonding interaction more favorable. The peak at 1640 cm−1 in the infrared spectra likely includes aromatic ketone groups hydrogen-bonded to phenols (SI Figure S4). Furthermore, when acidic protons were replaced with potassium ions at pH

prominent (SI Figure S3(a, b)). The absence of alkyl proton peak maxima near 1.2 ppm indicated that almost all alkyl carbons were adjacent to CO or aromatic rings which is consistent with lignin structure, and that they were not attached to paraffinic structures. Such paraffinic components as found in suberin, while likely present in lignite, were removed by the fractionation procedure. In the case of OCH3 groups, strong cross peak was observed between OCH3 carbons and their own protons (∼3.7 ppm) in 2D spectrum at tm,e = 0.01 ms (Figure 3(a)), whereas that of OCH3 carbons and aromatic protons (7 ppm) was weaker (SI Figure S3(c)). The corresponding 1H spectra (SI Figure S3(c)) showed growing contributions from aromatic protons with increasing mixing time, suggesting ligninlike structures: CaromH−Carom−O−CH3. The OC carbons near 76 ppm (SI Figure S3(d)) showed correlations with their directly attached protons, and with aromatic protons at longer mixing times. This is consistent with the phenyl propane structures. Aromatic C and aromatic C−O groups (Figure 3(c− f)) were found near the aromatic protons as expected, but also showed proximity to alkyl and OCH protons. Compared to the proton spectra associated with aromatic C, those of aromatic C−O groups (Figure 3(e,f)) showed relatively greater contributions from OCH protons, consistent with lignin-like phenolic ether structures. Both aromatic and aliphatic COO groups were present in LC2, supporting the conclusion that aromatic carboxyls may not be the dominant carboxyl structures in all humics.35 The COO groups resonating at relatively lower chemical shifts (168 ppm, Figure 3(g)) were found primarily near aromatic protons, whereas the COO groups near 174 ppm (Figure 3(h)) were closer to alkyl protons. The ketones showed relatively weaker cross peaks (Figure 3(a,b)), but their correlations to both aromatic and alkyl protons were evident (Figure 3(i)). Overall, the 2D HETCOR data of LC2 indicated aliphatic structures in close proximity with aromatics, aliphatic carboxyls, and aromatic structures substituted with phenol, phenolic ether, carboxylic acid, and aromatic ketone, consistent with oxidized lignin structures. Humic-Acid-Like Polycondensate. The 2D HETCOR spectra of HALP are shown in Figure 4(a, b). At tm,e = 0.01 E


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our view, occluded hydronium ions are not simply floating inside the hydrophobic humic matrix, but they may represent a structural part, shaping the supramolecular association of small organic molecules, adjusting their conformational ability, therefore imparting characteristic properties to humic substances.2,40 For example, changes in pH are directly related to changes in fluorescence,42 verifying the disruption of the novel π−π bonds that are also introduced in the present study. Our findings hence add new and more specific information that can be investigated by studies of spectral changes on natural humic substances and model compounds such as HALP. Aromatic ring hydrogen bonding with acidic hydrogens in lignite humic acid may also bind hydrophilic carboxylic acids such as citric acid,25 and coordinate metallic cations. The lignite humic acid in the present study could have been modified from its form in the environment by the extraction and purification procedures, the freeze-drying procedure in the acid form that maximizes hydrogen bonding interactions, and by the titration to pH 10 and 12 that disrupts secondary structures resulting from hydrogen bonding. These structural modifications were a necessary part of the experimental procedures to interpret secondary structures, but the results should be regarded as an “end member starting point” for more realistic studies of humic acid structure in environments where rehydration with water, pH changes, interactions with different components of natural organic matter, various metal ions, and aluminosilicates cause changes in secondary structures. One of the most important findings of the study is the importance of phenolic aromatic rings as a binding site for acids and possibly other cationic species.

10, the intensity of aromatic carbon signals decreased (Figure 2) partially because of the loss of acidic exchangeable protons which further lowers the cross-polarization efficiency of aromatic carbons, providing additional evidence for H-bonding of carboxylic acids with aromatic rings. In addition, the IR spectra of LC2 (SI Figure S4) contained a broad peak near 2500 cm−1 indicating H-bonded acid groups, but the carbonyl oxygen of carboxyl groups was not H-bonded as indicated by its unshifted frequency of 1710 cm−1. Thus, three independent lines of spectral evidence point to the preferential hydrogen bonding of carboxylic acid hydrogens with electron-rich aromatic rings rather than adjacent carboxyl groups. These hydrogen-bonding interactions likely result from the stereochemical arrangements in primary structures and folding, and such interactions of acidic protons with aromatic rings involving phenols, water, and hydronium ions are also likely. Intramolecular interactions such as folding and ring formation are entropically favored, but intermolecular interactions may also occur as illustrated by HALP where folding is less favored because of the lack of the aliphatic linkage to a carboxyl group. Tests with ball and stick models indicated that at least one hydronium ion hydrogen-bonded to a carboxyl and aromatic πelectrons was necessary for folding to occur without bond strains. In addition, aliphatic structures found in LC2 do not seem to be a necessary condition for folding to allow hydrogenbinding of acidic hydrogens with aromatic rings, because HALP contained only aromatic carboxyl groups, which are also hydrogen bonded to aromatic rings. Nonconventional π-hydrogen bonds involving X-H donors (X may be a N, O, or C atom) and the π-electrons of aromatic rings have been well documented in structural organic chemistry.10−16,34 Though π-hydrogen bonds are typically several kcal mol−1 weaker than conventional hydrogen bonds,34 they are generally of significance in structural biology. For instance, N−H···π and O−H···π interactions regularly occur in proteins.15 π-hydrogen bonds are formed between liquid water and benzene, and between water and other aromatic solutes including phenylalanine, implying their common occurrence at hydrated biological interfaces.14 To the best of our knowledge, aromatic ring interactions with acidic hydrogens have not been reported in the humic literature. Hydrogen bonding of acidic hydrogens to aromatic rings may be a common phenomenon occurring in humic and fulvic acids from various environments. For instance, the 2D HETCOR spectra of an Amherst HA that is enriched in ligninderived segments (Figure 4 in Mao et al.21) also demonstrated strong correlations of carboxyl protons with aromatic C groups. Similar observations have also been made in the 2D HETCOR spectrum of a fulvic acid isolated from Laramie-Fox Hills aquifer (Figure 34 in Cao35). These interactions may explain the visible absorption tail of humic substances, which was attributed to intramolecular charge-transfer interactions between hydroxyl-substituted aromatic donors and quinoid acceptors formed by the partial oxidation of lignin precursors.36 However, our 13C NMR data and reported NMR data of humic acids24,37−39 show that quinones or aromatic ketone structures are present in such low abundances (∼3% of all C) to adequately explain charge-transfer interactions between donor (hydroxy−benzene) and acceptor (quinoid) groups. Schaumann et al. introduced the hypothesis that water molecules may bridge soil organic matter molecular segments40 and later Nebbioso et al. found that occluded hydration water to a soil HA accounted for up to 40% of the HA weight.41 According to

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.5b02859. Methods and obtained spectra of high spinning speed multiple cross-polarization (multiCP) with 13C chemical shift anisotropy filtering, and multiCP with recoupled long-range dipolar dephasing. Table on functional group compositions of LC2 and lignin. FT-IR spectrum of LC2 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone: 757-683-6874; fax: 757-683-4628; e-mail: jmao@odu. edu. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Partial support by the Sedimentary Geology and Paleobiology Program of the National Science Foundation under the award EAR-1226323 (Old Dominion University) is acknowledged. The lignite humic acid fractionation was performed at the U.S. Geological Survey National Water Quality Laboratory, Denver, CO. We thank Professor Klaus Schmidt-Rohr (Brandeis University) for providing availability to the NMR spectrometer required for this study, for his assitance with collecting and discussing the NMR spectra, and providing the 13C spectrum of lignin. F


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Secondary Structures in a Freeze-Dried Lignite Humic Acid Fraction

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