Chapter 4 Molecular Sizes and Association Forces of Humic Substances in Solution Alessandro Piccolo, Pellegrino Conte, Annunziata Cozzolino, and Riccardo Spaccini, Dipartimento di Scienze Chimico-Agrarie, Universita di Napoli “Federico IT”, Portici, Italy Abstract .... 89 4-1 Introduction . 90 4-2 Experimental Approach 4 See 91 4-2.1 Humic Substances Extraction and Characterization OL 4.2.2 High-Pressure Size Exclusion Chromatography . 92 4-2.3 First Experiment: Additions of Mineral and Organic Acids . oe 42.4 Second Experiment: Variation of Eluent Composition .. ae 42.5 Reproducibility of Chromatograms and Column Performance . 94 4-3 Results and Discussion ..............+ 94 43.1 Molecular Structure and Hydrophobicity of Humic Substances 94 43.2 Humic Conformational Stabilities in Acids ..........--..+ ‘ 97 4-3.3 Changes of Molecular-Size Distributions with Mobile Phases . 102 43.4 Ultraviolet Spectra of Humic Acids in Modified Solutions. . 14 44 Conclusions .... aoe eee 116 References . 7 ABSTRACT Molecular sizes of humic (HA) and fulvic acids (FA) of different origin were evalu- ated by high performance size exclusion chromatography (HPSEC) in two different exper- iments. In the first experiment, the molecular sizes of the humic materials were significantly decreased when the pH of the medium was adjusted to 3.5 using monocarboxylic acids be- fore injection into the HPSEC system. In the second experiment, molecular dimensions of humic substances (HS) were followed by HPSEC after dissolution in different mobile phases of constant ionic strength. Additions of CH,OH, HCI, and ethanoic acid to the con- trol mobile phase gave rise to a progressive decrease in molecular size. Bulk size reductions of the humic fractions, as observed by means of a refractive index detector, were accompa- nied by substantial decreases in absorbance peaks detected by use of an ultraviolet light (UV)- visible light (Vis) detector. Changes in molecular absorptivity with solution composition were Copyright © 2001. Soil Science Society of America, 677 S. Segoe Rd., Madison, WI 53711, USA. Humic Substances and Chemical Contaminants 8990 POCCGLO ET AL. also observed in the UV spectra of the whole HA. The size reduction of the humic fractions in both HPSEC experiments was attributed to disruptions of supramolecular associations of small heterogeneous molecules held together at pH 7 by weak hydrophobic forces (van der Waals, x1, and CH—1 bondings) rather than to a shrinking of the random coil structures of polymeric species. The apparent high molecular-sizes of the supramolecular associations var- ied with humic matter, thereby indicating the relationship between humic chemical compo- sition and conformational structure. In the first experiment, the decrease in humic molecu- lar size was a function of the affinities between the humic components and different monocarboxylic acids. This behavior was confirmed by the second experiment that indicated that the higher the hydrophobicity of the humic material, the larger was the size reduction by CHjOH in comparison to the control, and by ethanoic acid compared with HCl, These results may contribute to explain the role of dissolved HS in the geochemical C cycle as well as in the environmental transport of pollutants. 4-1 INTRODUCTION Humic substances (HS) are the natural organic compounds present in all ter- restrial and aquatic systems. The processes of their accumulation and decomposi- tion in the different environmental domains (Piccolo, 1996; Hedges et al., 1997) are still largely unclear. Nevertheless, the characteristics and quantity of HS greatly affect the environmental fate of organic pollutants in soils and natural waters (Carter & Suffet, 1982; Chin et al., 1990). The molecular properties of dissolved HS have recently been recognized to influence the removal of synthetic organic com- pounds from municipal and industrial wastewaters (Karanfil et al., 1996a,b). The molecular properties of HS give rise to the binding and environmental transport of both nonpolar (Chiou et al., 1986; Chin & Weber, 1989) and slightly polar (pesti- cides) organic contaminants (Piccolo et al., 1996a, 1998). Despite their prominent environmental role, the chemical structures of HS are still far from clear mainly because of their chemical heterogeneities and spatial vari- abilities. As a consequence the conformational structures of HS are also ill-defined. There is a general acceptance of the concept that HS are polydisperse, long-chain, randomly coiled molecules that may have a slight degree of crosslinking (Steven- son, 1994). Negative charges arising from the ionization of carboxyl groups are re- sponsible for the mutual repulsion and expansion of the coils (Cameron et al., 1972). The random coil model (Ghosh & Schnitzer, 1980) depicts HS as most densely coiled at high concentrations, low pH, and high ionic strength, and as flexible lin- ear colloids at neutral pH, low ionic strength, and low concentrations. This model has helped several investigators to explain results obtained by gel permeation chro- matography (De Haan, 1987; Ceccanti, 1989), or by diffusion through ultrafiltra- tion membranes (Cornel et al., 1986). A common observation was that elution at the larger permeation volumes was retarded considerably when the ionic strength of the mobile phase was increased. The changes were attributed to coiling of the humic molecules with consequent decrease of the hydrodynamic radius and en- hanced diffusion through the smaller gel pores. The same phenomenon was also observed by varying the ionic strength of the mobile phase in HPSEC experiments with organic colloids from natural waters (Becher et al., 1985; Chin & Gschwend, 1991; Chin et al., 1994). Another conformational model still represents humic ma-MOLECULAR SIZES AND ASSOCIATION FORCES ” terials as polymers but aggregated in micellelike or membranelike structures (Wer- shaw, 1986, 1993). In this description, the humic molecules would have inner hy- drophobic domains with structural voids that could entrap hydrophobic organic com- pounds (Schlautman & Morgan, 1993), and quench their fluorescence activity (Engebretson & von Wandruszka, 1994, 1997). Recent results, mainly from low-pressure size-exclusion chromatography (Piccolo et al., 1996b,c, 2000) experiments, have indicated that humic aggregates are composed of relatively small subunits weakly held together by hydrophobic forces. The apparent aggregation of humic material in high molecular dimensions is considered not to be permanent, but found to be reversibly disrupted by the pres- ence of an organic acid in a rapid change from acidic to alkaline pH. These find- ings have suggested that HS in solution would appear to consist of randomly self- assembling supramolecular associations rather than polymeric coils, as in biological macromolecules (Cantor & Schimmel, 1980, p. 144). This innovative explanation of the conformational behavior of HS had to be verified in other experimental con- ditions and HPSEC was utilized for further investigations. For a number of reasons, HPSEC isa more valid chromatographic system than low-pressure gel permeation (Conte & Piccolo, 1999a,b). The major advantages of HPSEC are: (i) high repro- ducibility of chromatograms (5%); (ii) relatively higher rapidity of analyses (about 60 min per chromatogram); (iii) longer column life (more than 1000 injections/HPSEC column); and (iv) higher sensitivity to chemical changes in the injected samples because of much lower loading masses. Two main experiments were conducted by HPSEC: (i) modifications of mol- ecular dimensions of humic materials were followed when these were treated with different mineral and organic acids prior to HPSEC analyses (Piccolo et al., 1999a); and (ii) elution through HPSEC by mobile phases of different solution composi- tions, but of constant ionic strength (Conte & Piccolo, 1999b). In the latter exper- iment a Refractive Index detector was used in series with a UV detector in order to obtain the actual mass distribution in the various molecular-size intervals, instead of the sole distribution of chromophores as revealed by the UV detector. 4-2 EXPERIMENTAL APPROACH 42.1 Humic Substances Extraction and Characterization Humic acids were obtained from: a volcanic soil (Typic Xerofluvent, Vico, Italy) near Rome (HA1); an oxidized coal provided by Eniricerche, Italy, SpA (HA2); and a North Dakota Leonardite from the Mammoth Chemical Co., Hous- ton, TX (HA3). Fulvic acids (FA) were obtained from: a German agricultural soil (Eutric Luvisol, Munich, Germany) (FA1); and an Italian soil (Eutric Regosol, Caserta, IT) (FA2). The HS were extracted by standard procedures (Stevenson, 1994). Original materials were shaken overnight in a 0.5 M NaOH and 0.1 M Na,P20; solution under an atmosphere of No. The HAs were precipitated from al- kaline extracts by lowering the pH to 1 with 6 M HCI; extensively purified by three cycles of dissolution in 0.1 M NaOH; and subsequently precipitated in 6 M HCI. The HAs were then treated with a 0.5% (v/v) HCI-HF solution for 36 h, dialyzed