Optical spectroscopy to gain further insights on the chemical structure of humic substances (HS) and chromophoric dissolved organic matter (CDOM). Rossana Del Vecchio1,2,*,Tara M Schendorf1, Kevin Koech 1, Marla Bianca1 and Neil V Blough1 (1) Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20740, USA (2) Earth System Science Interdisciplinary Center, University of Maryland, College Park, MD 20740, USA (*correspondence : rossdv@umd.edu)

Why CDOM…… It is found ubiquitously in all aquatic environments It is a complex mixture of unknown structure It absorbs light across UV and visible wavelengths It contributes to the optical properties of most surface waters It can alter the Global Carbon Cycle Goals…… Employ optical spectroscopy coupled with chemical reduction/reoxidation and pH titrations to characterize and monitor HS/CDOM in natural waters 08/07/2017 NEMC Meeting

HS/CDOM: optical properties Longer wavelength excitation emitting species - subset of shorter wavelength excitation Individual chromophores cannot be resolved within our 5 nm resolution Not a simple linear superposition of the spectra of numerous independent chromophores Near-continuum of emitting species or states. Short wavelength (<350 nm) absorption: local donor/acceptor states fluorescence: local donor states Long wavelength (>350 nm) absorption/fluorescence tail: optical charge-transfer transitions Optical charge-transfer transitions can arise via a ground-state association between electron donors (D) (phenols and carboxylics) and electron acceptors (A) (ketones/aldehydes and/or quinones) to form a D-A charge-transfer complex. These D-A complexes exhibit new broad absorption bands that are not shown by either the D or A molecules alone. Hernes and Benner (2003), JGR, 108(C9), 3291 Del Vecchio and Blough (2004), EST, 38, 3885-3891. Boyle et al. (2009), EST, 43, 2262-2268. Furman and Lonsky (1986). JWC. 1-16. 08/07/2017 NEMC Meeting

Optical properties and structure Donors Acceptors CT complexes Hernes and Benner (2003), JGR, 108(C9), 3291 Del Vecchio and Blough (2004), EST, 38, 3885-3891. Boyle et al. (2009), EST, 43, 2262-2268. Furman and Lonsky (1986). JWC. 1-16. 08/07/2017 NEMC Meeting

Functional groups in HS/CDOM Aromatic ketones have been proposed to comprise the majority of these carbonyl-groups content (Leenheer, 1987). Quinones from the degradation and biological reworking of plant tissue are also believed to be significant constituents of HS (Thorn EST 1992). Phenolic & carboxyl moieties (Ritchie and Perdue, GCA, 2003) ~5-10 and ~10-20% of C, respectively; ratio of 21:79 of total acidity, respectively; FA~HA (Phenolic);TFA>AFA>NOM>AHA>THA (carboxyl); However, the relative contribution of each group to the HS optical properties is still ill-defined 08/07/2017 NEMC Meeting

Selective manipulation of specific functional groups Chemical reduction pH titration NaBH4 Na2S2O4 08/07/2017 NEMC Meeting

Schendorf et al., L&O Methods, 2016. Chemical reduction of HS/CDOM (NaBH4) Schendorf et al., L&O Methods, 2016. 08/07/2017 NEMC Meeting

Schendorf et al., L&O Methods, 2016. Chemical reduction of HS /CDOM (NaBH4) Schendorf et al., L&O Methods, 2016. 08/07/2017 NEMC Meeting

NaBH4 reduction preferential loss of long-wavelength absorption Del Vecchio et al., submitted Schendorf et al., L&O Methods, 2016. Ma et al., EST, 44, 5395-5402, 2010. preferential loss of long-wavelength absorption 3 components mostly irreversible minor reversibility enhanced, blue-shifted emission primarily due to aromatic ketones/aldehydes minor contribution from quinones 08/07/2017 NEMC Meeting

(Del Vecchio et al., submitted) Na2S2O4 reduction (Del Vecchio et al., submitted) minor loss of VIS absorption minor enhanced, blue-shifted emission consistent with previous work (Maurer et al EST 2010; Fimmen et al EST 2007; Aeschbacher et al EST 2010) reversible quinones 08/07/2017 NEMC Meeting

Na2S2O4 vs NaBH4 Del Vecchio et al., submitted Schendorf et al., L&O Methods, 2016. Ma et al., EST, 44, 5395-5402, 2010. 08/07/2017 NEMC Meeting

Selective manipulation of specific functional groups Chemical reduction pH titration NaBH4 Na2S2O4 08/07/2017 NEMC Meeting

pH dependence of optical properties Dryer, D. J.; Korshin, G. V.; Fabbricino, M., Environ. Sci. Technol. 2008. Korshin, G. V.; Benjamin, M. M.; Li, C.-W., Water Sci. Technol. 1999. absorbance increased with increasing pH pH dependence of absorption changes indicates occurrence of specific functional groups carboxylic acids at low pH phenols at high pH 08/07/2017 NEMC Meeting

pH dependence of optical properties Increasing pH 08/07/2017 NEMC Meeting

pH dependence of optical properties ApH(x)/ApH(2) 08/07/2017 NEMC Meeting

pH dependence of asborption difference Dryer, D. J.; Korshin, G. V.; Fabbricino, M., Environ. Sci. Technol. 2008. ΔA = A[pH (x)] – A[pH (2)] pH<7: peak at 280 nm pH>7: broad peak at 300-400 nm 08/07/2017 NEMC Meeting

pH dependence of absorption difference: carboxylic acids (pKa=3-5) vs phenols (pKa >8) Dryer, D. J.; Korshin, G. V.; Fabbricino, M., Environ. Sci. Technol. 2008. 𝐀(𝐩𝐇 𝐗 )−𝐀(𝐩𝐇 𝟐 ) 𝐀(𝐩𝐇 𝐗 )−𝐀(𝐩𝐇 𝟖 ) Low pH (carboxylic acid) A peak at 280 nm Extends into the long wavelengths Consistent among HS High pH (phenol groups) A broad band at 300-400 nm Extends into the long wavelengths Wide variation among HS 08/07/2017 NEMC Meeting

pH dependence of optical properties Untreated vs NaBH4 reduced Increasing pH 08/07/2017 NEMC Meeting

pH dependence: untreated vs reduced HS Peak at 280 is unaffected Peak at 330 nm is significantly affected Peak at ~375 nm is substantially decreased Peak ratio is inverted A at >400 nm is diminished Trend varies among HS ΔA = A(pH (x)) – A(pH (2)) 08/07/2017 NEMC Meeting

pH dependence of absorption for reduced HS Summary 𝐀(𝐩𝐇 𝐗 )−𝐀(𝐩𝐇 𝟐 ) 𝐀(𝐩𝐇 𝐗 )−𝐀(𝐩𝐇 𝟖 ) Peak at 280 is unaffected A at >330 nm is eliminated Trend ~ consistent among HS A at 280 is unaffected A at ~330/400 nm is lowered A at >450 nm is lowered Trend varies among HS 08/07/2017 NEMC Meeting

Conclusions Selective manipulation of functional groups has allowed us getting further insights on HS/CDOM chemical structure in relation to their optical/photochemical properties NaBH4/Na2S2O4 reduction of HS allowed to conclude that aromatic ketones/aldehydes are significant contributors to absorption quinones are minor contributors to absorption pH titrations……… absorption both carboxylic and phenolic groups contribute to the HS absorption with the latter contributing more 08/07/2017 NEMC Meeting

Conclusions …… continued NaBH4 reduction + pH titrations both carboxylic and phenolic groups are coupled with carbonyls to form long-wavelength CT transitions ……………consistent with the CT model absorption changes are more variable among different HS ……………a new method for discriminating among HS 08/07/2017 NEMC Meeting

Thank you for your attention! 08/07/2017 NEMC Meeting