Natural organic matter (NOM) is a key component in the physical, chemical, and biological processes that occur in all ecosystems. In terrestrial environments, NOM is known to play an important role in metal speciation and transport, nutrient retention, organic and inorganic contaminant mobility, mineral weathering, and acid buffering [1,2]. Much of the chemical activity of these molecules is the result of a high concentration of oxygen containing functional groups, particularly carboxyl groups. In many respects, carboxyl groups are the most important functional groups in natural organics, accounting for the majority of their charge behavior, acidity, and metal / surface binding characteristics.
Though carboxyl group contents in NOM fractions may be determined easily, detailed mechanistic models of many of the above processes are difficult to construct. This is because the chemical behavior of carboxyl groups is also dependent on their "structural environment;" the types and positions of other functional groups neighboring the carboxyl. For example, acidity and metal-complex stabilities vary for different carboxylic acids based on local molecular structure [3,4,5]. Molecular configuration also governs the degree to which additional functional groups such as ketones and hydroxyls may participate with a carboxyl group in metal chelation, which accounts for the strong metal affinity of many natural organic acids. The chemical structures of complex, highly degraded molecules such as humic substances have not been completely determined, and our understanding of carboxyl group chemistry in NOM is therefore limited. The goal of this work is to gain insight on the types of carboxyl structural environments in NOM using infrared spectroscopy.
IR Spectroscopy of Simple Organic Acids
A carboxyl group will give rise to two main absorption features in the infrared, with energies that depend on the protonation state of the carboxyl (Figure 1). The protonated carboxylic acid yields absorption bands corresponding to a carbonyl stretch (νC=O) between 1690 and 1750 cm-1, and C-OH vibrations (νC-OH) between 1200 and 1300 cm-1 (comprised of a mixture of C-O stretch and C-O-H bend that often yields a single, broad absorption band). On deprotonation, νC=O shifts to lower energy as its vibrational mode becomes coupled to that of the other oxygen, giving rise to an asymmetric feature (νas) between 1540 and 1650 cm-1. Similarly, the C-OH band shifts to higher energy on deprotonation, yielding a symmetric COO- mode (νs) between 1300 and 1420 cm-1.
The precise energies of these bands are dependent on a number of factors, two of which include a) the electron density on the carboxyl, as affected by the presence of electron donating / withdrawing functional groups on the molecule, and b) inter / intramolecular H-bonding involving a carboxylic oxygen or the proton in the carboxylic acid. The effects of these two factors are typically more consistent and predictable in the νas mode relative to the other modes, making νas a useful probe for carboxyl group structural environment. Specifically, α-substitution of electron withdrawing groups on aliphatic carboxylic acids causes an increase in νas. The degree of shift depends on the type and quantity of substitutions, but in general ranges from 30 cm-1 for oxygen substitution, to over 100 cm-1for halogen substitution (Figure 2). Aromatic acid (benzoic acid type) νas values occur above those of unsubstituted aromatics, but substitution on the aromatic ring has less of an effect on νas.
Carboxylate Bands in Humic Substances
We collected ATR-FTIR spectra of several humic substance isolates from the International Humic Substances Society (IHSS), as well as leachate water from the organic / mulch layer of a forest soil in the Pine Barrens, NJ (visibly colored due to high organic matter content). Representative spectra are shown in Figure 3. It is clear from the figures that, despite the complexity of natural organic molecules, IR spectroscopy is useful in selectively studying carboxyl groups due to their high polarity (i.e., high infrared activity) and pH behavior.
After a 10 cm-1 correction for air-drying effects (see Air Drying Procedure below), the νas values for the 6 IHSS isolates, as well as for the Pine Barrens leaf leachate, fell within a range of 1574-1582 cm-1 (Figure 4). Since we expect the same effects mentioned above to govern the νas position in the NOM samples, comparison with νas values for the simple organic acids should give insight on the carboxyl group types present in NOM. Based on the overlap shown in Figure 4, we conclude that the dominant fraction of carboxyl groups in all NOM fractions studied are aliphatic, with some form of α-substitution. From a simple elemental abundance perspective, we suspect that the majority of these substitutions are in the form of oxygen functional groups, primarily hydroxyls and neighboring carboxyls.
Though we believe these structural types to account for the majority based on the peak position, other types (including unsubstituted aliphatic acids and aromatic acids) may also be present in significant proportions, which may give rise to spectral features that do not contribute to the main peak. Heterogeneity in structural types is also expected to produce a broadening of the main νas peak (that is, the single observed peak represents a sum of individual peaks that do not coincide precisely). The observed full width at half maximum (FWHM or "halfwidth") for the NOM samples deviates from the halfwidth for simple carboxylic acids by approximately 10 cm-1, represented in Figure 4 with the lighter "error" bars.
These and other results will be discussed in further detail in an upcoming publication:
- “Probing the structural environment of carboxyl groups in natural organic molecules using infrared spectroscopy” (to be submitted to Geochimica et Cosmochimica Acta).
Air Drying Procedure for Studying Dilute Aqueous Solutions
For these experiments, the natural organic samples were dried onto the attenuated total reflection (ATR) crystal, rather than collecting spectra of the compounds in the aqueous phase. The samples were dried under a stream of dry, CO2-free air (obtained from an FTIR Purge Gas Generator unit) until the samples took on a very thin, shiny, oily appearance and no bulk water was visibly present. This method was convenient because pH adjustments could be made easily and accurately with minimal amounts of the NOM in solution by using a large, dilute sample volume. More importantly, the drying procedure proved to be a simple means of collecting clear, intense infrared spectra on the Pine Barrens DOM sample, without the need for potentially harmful extraction or concentration methods.
Spectra were collected on the thin films after the samples became visibly dry, but retained a shiny, oily appearance. In successive scans, the second set collected showed a slight decrease in intensity in the 1620-1680 cm-1 and 2900-3600 cm-1 regions, corresponding to the H-O-H bending and stretching modes of the water molecule, respectively. These slight decreases in signal intensity demonstrate that a small amount of residual water was present in the samples, and was lost on further desiccation within the dry air environment of the sample chamber. The hydrated state of the samples may suggest that the infrared spectra are more representative of molecules in a "pseudo-aqueous" environment, rather than in solid form, or in a KBr matrix. However, when compared with a highly concentrated aqueous NOM sample, a shift in νas of 10 cm–1 was observed. This deviation is minimal but consistent with shifts observed between aqueous and solid spectra observed previously .
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