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92 “PICCOLO ET AL. [Spectrapore 3 dialysis tubes, 3500 molecular weight (MW) cut-off] against dis- tilled water until CF--free, and freeze dried. The FAs, the humic material left in so- lution after precipitation of HAS at pH 1, were purified by adsorbing on an Am- berlite XAD-8 resin, eluting with a 1 MNaOH solution, and, after adjusting the pH to 5, dialyzing in Spectrapore 3 tubes against distilled water until Cl--free, and freeze drying. The HAs and FAs were then redissolved in 0.5 M NaOH and passed through a strong cation exchange resin (Dowex 50) to further eliminate divalent and poly- valent metals, and again freeze dried. All humic samples were characterized for their elemental content, using a Fisons EA 1108 Elemental Analyzer (Fisons Scientific Equipment, Italy). The ash content was less than 5% in all humic samples. In preparation for HPSEC analysis, both HA and FA samples (50 mg) were subsequently suspended in distilled water (50 mL) and titrated for 3h to pH 7 using a CO;-free solution of 0.5 M NaOH in an atmosphere of N3. An automatic titrator (VIT 90 Videotitrator, Radiometer, Copenhagen) was used and constant stirring was maintained using a magnetic follower. The resulting sodium-humates were then fil- tered through a Millipore 0.45-1m membrane, and freeze dried. 4-2.2 High-Pressure Size Exclusion Chromatography The HPSEC system used consists of a Perkin Elmer LC-200 solvent pump (Perkin Elmer, Norwalk, CT), and two detectors in series, a UV-Vis variable wave- length detector (Perkin Elmer LC-295) set at 280 nm and a refractive index (RI) detector (Fisons Instruments, Refractomonitor IV). A Rheodyne rotary injector equipped with a 100-L sample loop was used to charge the humic solutions. The size exclusion separation used two types of high-pressure columns (Conte & Pic- colo, 1999a). In the first experiment a Biosep $2000 column (Phenomenex, Tor- rance, CA) (600 by 7.5 mm i.d.), preceded by a 7.5-cm Biosep Guard column was used, while a TSK G3000SW (600 by 7.5 mm i.d.) by Toso Haas (Japan), preceded by a7.5-cm TSK Guard column, was adopted in the second experiment. In both experiments, the columns were preceded by a 0,22-m stainless-steel inlet filter. A water bath was used to keep the temperature of analyses at 25°C, Column calibration to obtain MWs of HS is complicated by the lack of awareness of their chemical structures. Any calibration standard that might be used would be likely to have a different hydrodynamic radius and interaction with the stationary phase than would apply for HS (Cameron et al., 1972). Polysaccharides have been successfully used to evaluate the molecular size distributions of dissolved HS from different sources when eluted with dilute salts such as a 0.05 M NaNO; at pH 7 and constant ionic strength (Rausa et al., 1991; Conte & Piccolo, 1999a,b). This neutral mobile phase was suitable for the rapid and efficient HPSEC of HS, and the phenomena of solute-stationary phase interactions were avoided (Berden & Berggren, 1990; Rausa et al., 1991; Conte & Piccolo, 1999a,b). Polysaccharides (Polymer Sciences Laboratories, UK) of known MW values (186 100, 100 000, 48 000, 23 700, 12 200, 5800 Da) were used as the calibration standards in the studies reported here, and water was used to determine the total volume of the permeation system. The calibration curves were semilog linear over the range defined by our standards, and these were used to determine the MW ofMOLECULAR SIZES AND ASSOCIATION FORCES 93 an analyte, Mj, at some elution volume i. Elution was carried out at the flow rate of 0.6 mL min"! as suggested elsewhere (Rausa et al., 1991; Conte & Piccolo, 1999a). Size exclusion chromatograms for both the UV-Vis and RI detectors were evaluated by using the Perkin-Elmer-Nelson Turbochrom 4.0-SEC peak integra- tion and MW software. Calculations of the weight (M,,), and number-average (M,) MW values, and polydispersity (M,,/M,) were carried out by the method of Yau et al. (1979) using the following equations: _ 2 iM) Ww Zh hy 0 and 7 Ei (i/M) (2) where /; is the height of the size exclusion chromatogram of each sample eluted at volume i. Based on the described method, the system dispersion was found to be less than the error (<5%), and the chromatograms were evaluated without additional correction factors. 42.3 First Experiment: Additions of Mineral and Organic Acids Humic solutions for HPSEC analysis were prepared by dissolving 2.0 mg of each HA sample in 10 mL of the HPSEC eluent. Prior to the injection in the HPSEC system, each control solution was titrated to pH of 3.5 with HCI or with a monocarboxylic acid (methanoic, ethanoic, propanoic, and butanoic acids). The treatment with HCI was done to evaluate the sole effect of pH on the conformational structure of the HA and to distinguish it from the additional effect provided by the hydrophobic components of carboxylic acids. Calculations showed that addition of HCI and monocarboxylic acids did not significantly affect the ionic strength of humic solutions that remained constant at 0.05 M. 4-2.4 Second Experiment: Variation of Eluent Composition Four different mobile phases were used for sample dissolution and HPSEC elution: (i) a solution at pH 7.01 of 0.05 MNaNO; and 4.0 x 107 M NaN; (the lat- ter to suppress microbial activity); (ii) the same solution as in (i), but 4.6 x 10-7 M in CHOH (the final pH was 6.97); (iii) the same solution as in (i) but adjusted to pH 5.54 with concentrated HCI; (iv) the same solution as in (i), but 4.6 x 10-7 M in ethanoic acid (the final pH was 5.69). The low ionic changes introduced in so- lutions 3 and 4 (<2.0 x 10-6 M) did not significantly change the / value, and so all mobile phases were considered to have the same ionic strength (J = 0.0504 M) as the control solution (i). This ionic strength was reported not to affect exclusion chro- matography in the specific stationary phase used here because it prevents ionic un-94 PICCOLO ET AL. coiling of HS and minimizes hydrophobic adsorption to the stationary phase (Barth, 1980; Becher et al., 1985; Berden & Berggren, 1990; Conte & Piccolo, 1999a,b). All mobile phases were made with MilliQ water (Waters, UK) and HPLC-grade reagents, and were filtered through Millipore 0.45-ym membrane (Millipore, Bed- ford, MA) filters. Freeze-dried sodium salts of the humic samples were dissolved in the same mobile phase solution, then immediately passed through a 0.2-pm fil- ter (polyvinyldifluoroamide [PVDF], Millipore), and injected into the HPSEC sys- tem operating with one of the four selected mobile phases. In this experiment, a con- centration of 0.5 g L~' was used for each humic solution. The molecular compositions of the HA and FA were evaluated using CPMAS. '3C-nuclear magnetic resonance (NMR) spectroscopy employing common proce- dures and using variable contact time (VCT) pulse sequence (Conte et al., 1997a; Piccolo & Conte, 1998). ‘The same HA samples were also analyzed by UV-Vis spectroscopy. The HA, previously titrated to pH 7, were redissolved in 0.05 M'NaCl to reach a concentra- tion of 0.5 g L~! and added with the same amounts of CH,OH, HCl, and ethanoic acid as those used for the HPSEC experiments. These HA solutions were diluted 100 times in 0.05 M NaCl and scanned for the light absorbance from 250 to 450 nm in a Perkin-Elmer Lamba 3B UV/Vis spectrophotometer using quartz cuvettes of I-cm pathlength. 4-2.5 Reproducibility of Chromatograms and Column Performance Humic solutions were prepared anew before each injection. Three replicates for each humic sample were run to obtain a chromatogram. The relative standard deviation of M, values in subsequent injections of the sample never exceeded 5%, and this confirmed the reproducibility of the HPSEC analyses of HS as reported in other studies (Berden & Berggren, 1990; Rausa et al., 1991; Conte & Piccolo, 1999a). Such precision should also be interpreted as evidence for the absence of reversible and irreversible adsorption of solute on the stationary phase (Rausa et al.. 1991). The column calibration by the polysaccharides standards was checked under the conditions of the two experiments. No significant differences in the calibration were found either with additions of mineral and organic acids or with changes of mobile phase. This was considered to be evidence that indicated that neither the acids present in the 10-mL humic solution injected into the HPSEC system (first exper- iment), nor the changes in the composition of the eluent (second experiment) af- fected the size-exclusion behavior of the chromatographic column. 4-3 RESULTS AND DISCUSSION 4-3.1 Molecular Structure and Hydrophobicity of Humic Substances The structural features shown by the NMR spectra of HA and FA (Fig. 4-1) are not different from those reported for terrestrial HS by other investigators (Pre- ston. 1996: Zech & Guggenberger, 1996: Piccolo & Conte. 1998). In general, HAsMOLECULAR SIZES AND ASSOCIATION FORCES 95 showed sharper resonances for aromatic C atoms than FAs, and the FAs in turn had more intense signals for peptides and carbohydrates (at about 50 and 66 ppm, re- spectively). In particular, the spectra for the HA1, from a volcanic soil, contained more carbohydrate (71- and 101-ppm peaks) and peptide (53-ppm peak) C atoms than the HA2 and HA3 samples that appeared instead to be richer in aromatic and in carboxylic C atoms. Such significant differences in chemical structure may well have influences on the molecular conformations of the three HAs in solution. The elemental compositions and C/H and C/N ratios (Table 4—1) also indicated differ- ences in chemical nature between these HS. In general, quantitative evaluations of the NMR signals (Table 4-2) confirmed these observations. The HA3 showed the highest content of aliphatic and aromatic C atoms, whereas HA1 was richest in polar C atoms and poorest in aliphatic and aromatic C atoms (Table 4-2). The amount of polar C atoms, as measured by 32 nn) 250 200 160 100 80 0 -5D Fig 41 CPMAS "C-NMR spectra of humic ppm ‘materials.96 PICCOLO ET AL. Table 4-1. Elemental analyses of the humic samples. Samplet c H N CH CIN %6— HAL 41.11 3.33 2.22 123 18.5 HAZ 49.06 3.06 1.02 16.0 48.1 HA3 45.90 371 0.96 123 47.8 FAL 38.60 4.42 412 87 92 FA2 21.96 4.39 3.29 5.0 67 + Ashefree and moisture-free basis. See Section 4-2.1 for identification. CPMAS |3C-NMR, was used to indicate the hydrophilicity (HI) of the humic mat- ter, whereas the content of apolar C atoms were related to the hydrophobicity (HB) of the humic samples. Table 4-2 gives the HI/HB ratio for different humic materi- als, The ratio varied in the order: HA1 > HA2 > HA3, indicating that HA3 was the most hydrophobic material, whereas HA1 had the highest hydrophilicity. As ex- pected, FAs were generally more hydrophilic than HAs. Data from CPMAS !3C-NMR can indicate the humic structural differences that may affect their reactivities in aqueous solution (Conte et al., 1997b). It may be expected that, as in the case of proteins [that decrease the free-energy of solva- tion by assuming the conformation that best excludes hydrophobic components from water (Tanford, 1991)], HS in solution should arrange their conformation to orient most of their hydrophobic components away from water (Wershaw, 1986, 1993). As suggested previously (Piccolo et al., 1996a,b; Piccolo, 1996), humic matter may be composed of self-associating subunits in conformational arrangements in which hydrophobic domains, held together by weak forces such as van der Waals, m1 and CH-m bondings, are contiguous to hydrophilic domains that are mainly stabilized by H bonds. The sizes and numbers of both domains are dependent on the molec- ular nature of the humic components that ultimately control the reactivites of the humic materials. Hence, a simple hydrophobic index, defined as the ratio between the quantity of hydrophilic and hydrophobic C atoms in a humic material as found by NMR spectroscopy, may be an indication of the capacities of HS to interact with organic compounds of different polarities. The greater the hydrophobicity of a humic material, the larger might its potential be to interact with compounds of low polarity and the lesser to interact with those of higher polarity. ‘Table 4-2. Distribution of C intensity in different regions of !3C-NMR spectra of humic samples. 0-45 ppm — 45-60 ppm 60-110 ppm 110-160 ppm 160-190 ppm Samplet aliphatic C-O,C-N_anomeric aromatic Carboxyl —_ HI/HBt HAI 278 20.2 25.6 10.9 93 1.42 HA2 20.4 8.39 20.4 24.8 25.0 119 HA3 26.6 4.72 18.9 25.6 15.8 0.92 FAL 34.6 42.0 09 28 149 1.34 FA2 40.2 36.6 2.4 24 18.3 134 + See Section 4-2.1 for identification. } HI/HB = [45-60) + (60-110) + (160-190)}[(0-45) + (110-160)],MOLECULAR SIZES AND ASSOCIATION FORCES 97 Table 4-3. Weight-average molecular weight values, and percentage variation with respect to control solutions (+) of humic acids treated with HCI and different monocarboxylic acids. HAt Control HCI Methanoic Ethanoie Propanoic Butanoic Mat HAL 32961 37462+13.7 23483-20823 140-29.8 9 31065-5.75 33. 716+2.29 HA2 10024 -9647-3.76 —9272-7.50 6 RO6-32.1. 6 128-38.9 = 7526-24.9 HA3 12162 19 015455.1__ 11 088-9.57 7 878-35.8 7200-413 5 9R6-S1.2 + See “Humic Substances Extraction and Characterization” for identification. + Daltons, The weight-average MW (M,) values of the humic samples, and their per- centage changes under the different conditions are reported in Table 4-3. These data show that HA] had a higher M,, (32 961 Da) than HA2 (10 024 Da) and HA3 (12 162 Da). The high HI/HB ratio of HA1 may explain its large molecular size. The high hydrophilicity of HAI gives rise to the most hydrated conformation because H-bonds can readily form between the hydrophilic components and the water mol- ecules of the medium. In contrast, HA2 and HA3, which showed very similar My values, have also lower HI/HB ratios, and thus a more hydrophobic nature than HAI. This implies a relative tendency to repel water molecules and a consequent decrease in hydration water in the HA2 and HA3 conformations in solution, resulting in smaller molecular sizes than for HAI. 4-3.2 Humic Conformational Stabilities in Acids Figures 4-2, 4-3, and 44 show that all chromatograms recorded after treat- ing HAs with either HCI or monocarboxylic acids showed lower peak intensities than the control. Such change in the UV absorption capacity of HAs may be ex- plained by the phenomenon of hypochromism (Cantor & Schimmel, 1980, p. 144; Freifelder, 1982, p. 500-503). The alteration of reciprocal orientation between the transition dipole moment of absorbing chromophores and the induced dipoles of the neighboring chromophores, when chromophore groups are separated from each other, causes changes in molecular absorptivity that may result in a decrease of absorption intensity in the chromatograms. Hence, the observed hypochromic effect in the chromatograms may be interpreted as an indication that HS have a supramolecular rather than a polymeric nature. In fact, notwithstanding the constant ionic strength, if HAs were covalently bound polymers, their conformations would have been coiled down by the pH decrease (from pH 7-3.5), and the molecular ab- sorptivity would have increased, thereby showing a hyperchromic UV reading with respect to the control solution. Conversely, the decrease in absorbance is evidence that the total molecular absorptivity of the eluting material was lower (bypochromism) than in the control humic solution. This may be attributed to a sep- aration of molecules (or chromophores) rather than to their compaction into a coil. A logical conclusion is that the addition of the acids to the humic solution disrupts the molecular associations of humic samples giving rise to much smaller compo- nents. Moreover, chromatograms of humic solutions treated with HCI showed a vari- ation in retention times when compared to control solutions. Figures 4-2, 4-3, and98 PICCOLO ET AL. 4-4 show that although a peak was still present at the void volume for all HAs, the diffuse second peak was not only considerably lowered in intensity, but was also shifted to longer retention times. This also indicates that the number of small size- fractions increased with the treatment of the HAs with HC1. Hypochromism of chro- matographic peaks, combined with an increase of their retention times suggest that HCI caused the apparent high molecular sizes of the HAs to be dispersed into sub- units of smaller molecular sizes. The M,, values reported in Table 4-3 do not account for the real conforma- tional changes of the HA induced by the addition of HCl. In fact, some of the data (for HA1 and HA3) show increases of M, values upon HCl addition, and that con- trasts with the changes observed in the chromatograms. This is due to the impor- 70: Intensity (V3 Elution Volume (mL) Fig. 4-2. Chromatograms of HA 1. A, control solution; B, as for A but with HCI added; C, as for A but ‘with methanoie acid added; D, as for A but with ethanoic acid added; E, as for A but with propanoic acid added; F, as for A but with butanoic acid added.MOLECULAR SIZES AND ASSOCIATION FORCES 99 tance that the calculation method attributes to the relative heights of chromatographic peaks. The positive differences shown for HA1 and HA3 (Table 4-3) were due to the overevaluation of the peak at the void volume in comparison to the second peak that represents smaller molecules diffusing through the gel pores. While the first peaks at the V, only slightly decreased, with respect to the control, the second peak was more significantly decreased. The result is that the average M,, values reflect the high-molecular size fractions more than the low-molecular size fractions, and thereby fail to provide a real quantitative comparison between the chromatograms. The conformational disruption of humic matter following the addition of HCI can be explained by the thermodynamic changes induced by the formation of H 605 mrensrty (mv) Elution Volume (mL) Fig. 4-3. Chromatograms of HA2. A, control solution; B, as for A but with HCI added; C, as for A but with methanoic acid added; D, as for A but with ethanoic acid added; E, as for A but with propanoic acid added; F, as for A but with butanoic acid added.100 * PICCOLO ET AL. bonds between protonated acidic functions of humic molecules and their comple- mentary O-containing functions. Hydrogen bonds are relatively strong linkages in comparison to van der Waals bonds, since these involve a gain in energy from 10 to 20 kJ mol"! (Schwarzenbach et al., 1993). The increase in energy content is the reason for the considerable thermodynamic drive to pass from a less stable con- formation, mainly held together by hydrophobic interactions, to a more rigid con- formation predominantly stabilized by H bonding. Hence, these results indicate that the original conformation of the humic material in solution must be predominantly stabilized by weak intermolecular hydrophobic forces (van der Waals, n—m, CH—1 intensity (mV) Elution Volume (mL) Fig, 4-4. Chromatograms of HA3. A, control solution; B, as for A but with HCl added; C, as for A but with methanoic acid added; D, as for A but with ethanoic acid added; B, as for A but with propanoic acid added; F, as for A but with butanoic acid added.MOLECULAR SIZES AND ASSOCIATION FORCES 101 bondings) that hold together smaller humic subunits. Such molecular aggregation can be disrupted when more energetic intermolecular interactions, such as H bond- ing, are favored by acid addition and when the formation of stronger H bonds pro- mote the spatial associations to rearrange that were previously governed by weaker hydrophobic forces. These results are somewhat different from those previously ob- tained by means of the low-pressure chromatography of humic materials after ad- dition of HCl (Piccolo et al., 1996b,c). The discrepancies must be attributed not only to different humic materials but also, and mainly to the properties of HPSEC that is a much more sensitive technique for evaluating the effects of small amounts of acids on the size-distributions of HS (Conte & Piccolo, 1999a). Figures 4-2 through 44 show that additions of monocarboxylic acids to humic solutions causes similar but more extensive conformational changes than those for HCI. The chromatograms obtained with methanoic, ethanoic, propanoic, and butanoic acids were significantly decreased in intensities and shifted to longer retention times in comparison to the control and to those for the HCI treatment. The important result is that, while the conformational disruptions of HAs should be driven by H bond formation upon the addition of any acid, changes in molecular size distributions of HAs varied with the structures of the monocarboxylic acids. Methanoic acid, the most hydrated organic acid, significantly decreased the chromatographic peaks of the highly hydrophilic HAI (Fig. 4-2), whereas its cf- fect on the more hydrophobic HA2 and HA3 (Fig. 4-3 and 4~4) samples was less substantial. Ethanoic acid was also very effective in decreasing the apparent size of HAI, but it had a more significant effect than that brought about by methanoic acid on the size-distributions of HA2 and HA3. In general, the increase in the num- ber of C atoms or methylene groups in the organic acids structures, as in the cases of propanoic and butanoic acids, resulted in a progressive decrease of conforma- tional disruption for the hydrophilic HA1, and an increase in the disruption for the more hydrophobic HA2 and HA3. These variations are explained by the interactions of organic acids with the hydrophobic components of HA. Ethanoic acid is relatively small and hydrated and can still enter the hydrophilic arrangement of HA. It has, however, sufficient hy- drophobic character (in the methyl group) to also disrupt hydrophobic associations in HA2 and HA3. The conformation of HA3, with the highest hydrophobicity and predominant aliphatic composition (Fig. 4-1 and Table 4-2), can be increasingly altered by the progressively higher aliphaticities of propanoic and butanoic acids (Fig. 4-4). Conversely, HA2, in which the hydrophobicity is intermediate and mainly due to aromatic moieties (Fig. 4-1 and Table 4-2), is less disturbed by the longer aliphatic chain of butanoic acid. This acid may not easily interact with the presumably tighter hydrophobic associations of HA2, which are likely to be stabi- lized by mainly x bonds among the aromatic functionalities (Fig. 4-3). The results from this first experiment indicate that humic conformations in solution can be disrupted by breaking the weak hydrophobic associations of the small heterogeneous molecules of HA while concomitantly forming more energy-rich in- termolecular H bondings. The consequent conformational rearrangements lead to decreased molecular sizes of HA and decreases in molecular absorptivity (hypochromism). However, the extent of the conformational changes is related to the hydrophobic character of the organic acids. The higher the number of C atoms102 * PICCOLO ET AL, in the acids, the larger are their affinities for hydrophobic components of HA, and the greater their capacities to alter the humic hydrophobic associations. 4-3.3 Changes of Molecular-Size Distributions with Mobile Phases Molecular-size distributions of humic materials in four different HPSEC mobile phases were recorded concomitantly by both UV-Vis and RI detectors. The UV detector reveals only the molecular absorptivity of groups absorbing at a cho- sen UV wavelength, whereas the RI detector follows the overall mass distribution of humic matter. The RI detector is thus very useful since it shows not only the dis- MILLIVOLTS ELUTION VOLUME (mL) Fig. 4-5, Size exclusion chromatograms of HAI recorded with the UV-Vis detector. A = control mo- bile phase (0.05 M NaNOs, pH = 7, |= 0.05); B= same as A but 4.6 « 107 Mf in CH,OH (final pH 6.97); C= same as A but to pH 5.54 with HCI; D= same as A but 4.6 10-7 M in ethanoic acid (final pH 5.69).MOLECULAR SIZES AND ASSOCIATION FORCES 103 tribution of certain groups (the chromophores) in HAs, but also the conformational changes of the bulk humic mass that take place with changes in the mobile phase. The M,, values, their percentage changes in different mobile phases, and the poly- dispersity (M,,/M,) of humic samples are reported in Table 4-4 for both the UV and RI detectors. The chromatograms of HAI, HA2, HA3, FAI and FA2 recorded by UV and RI detectors, are shown in Fig. 4-5 and 4-6; 4-7 and 4-8; and 4-9 and 4-10; 4-11 and 4-12; and 4-13 and 4-14, respectively. In the control mobile phase A, M,, values for both UV- and RI-detectors showed that molecular sizes of HA were higher than for FA and so was the poly- dispersity (Table 44). The HA2 was found to have the lowest M,, of the three HAs MILLIVOLTS 2 B 24 30 3B ELUTION VOLUME (mL) Fig. 4-6. Size exclusion chromatograms of HA | recorded with the RI detector. A = control mobile phase (0.05 M'NaNOs, pH = 7, I = 0.05); B = same as A but 4.6 « 10-7 M in CH;OH (final pH = 6.97); C = same as A but to pH = 5.54 with HCI; D = same as A but 4.6 * 10-7 M in ethanoic acid (final pH 5.69),104 *PICCOLO ET AL. with good agreement between UV and RI values. Moreover, its lower polydisper- sity implies that HA2 is more homogeneous in chemical composition than HA1 and HA3. Differences in molecular property are readily inferred by comparing the chro- matograms of HA and FA in both UV and RI modes (Fig. 4-5 through 4-14). In fact, UV chromatograms (Fig. 4-5, 4-7, and 4-9) obtained in mobile phase A (con- trol) show that a small first peak (high-molecular-size material eluting at the void volume) was increasingly higher passing from HA2 to HA3 and to HA1, whereas a large second peak (low molecular-size material diffusing through the smaller col- umn pores) was present, though with varying intensity, in all three HAs. These dif- ferences in the molecular size distributions among the HAs was also shown in RI chromatograms (Fig. 4-6, 4-8, and 4-10). Similar My, values and polydispersities MILLIVOLTS 6 2 8 a nan J ELUTION VOLUME (mi) Fig, 4-7. Size exclusion chromatograms of HA2 recorded with the UV-Vis detector. A = control mo- bile phase (0.05 M NaNOs, pH = 7,1= 0.05); 6.97); C= same as A but to pH 5.54 with HCI; pH 5.69). ame as A but 4.6 « 10-7 M in CH;OH (final pH yme as A but 4.6 « 10-7 M in ethanoic acid (finalMOLECULAR SIZES AND ASSOCIATION FORCES 105 were found for all FAs in mobile phase A (Table 4-3). This was shown by both UV (Fig. 4-1 and 4-13) and RI (Fig. 4-12 and 4-14) chromatograms. The significantly lower molecular size of FA, as compared to HA, was indicated both by higher clu- tion volumes of their diffused peak in UV chromatograms and by the considerable material eluting at total volumes in RI chromatograms of both FAI and FA2, No material eluting at the total volumes was in fact observed in the RI chromatograms of HA (Fig. 4-6, 4-8 and 4-10). The use of mobile phase B (solution A, made only 4.6 x 107 M in CH,OH and with very similar pH) modified substantially the chromatograms of the HA. The calculated M,, values decreased by more than 50% for HA2 and HA3, while these 5.0: A 8 Cc ------ 4.0: : MILLIVOLTS a ° 2.0: 1.0; ELUTION VOLUME (mL) Fig. 4-8. Size exclusion chromatograms of HA2 recorded with the RI detector. A = control mobile phase (0.05 M NaNO;, pH = 7, 1 = 0.05); B= same as A but 4.6 x 10-7 M in CHOH (final pH 6.97); C same as A but to pH 5.54 with HCI; D= same as A but 4.6 x 10-7 M in ethanoic acid (final pH 5.69).106 ‘PICCOLO ET AL. values increased by 26% for HA1 (Table 4-4). Similar results were obtained for the RI detector, except in the case of HA2 that showed a decrease in My, values as the other two HAs. The UV chromatograms of both HA2 and HA3 (Fig. 4-7 and 4-9) showed the disappearance of the first peak and a significant reduction of the second (diffused) peak that was more than halved in intensity, while the elution of its maximum was retarded at a larger volume than in solution A. Conversely, soil humic material (HA 1) showed an increase of the first peak intensity and a decrease of that of the second peak (Fig. 4-5), thereby accounting for the increase of M,, val- ues observed (Table 4-4). The RI chromatograms confirmed these changes, and also vo ELUTION VOLUME (mL) Fig. 4-9, Size exclusion chromatograms of HA3 recorded with the UV-Vis detector. A = control mo- bile phase (0.05 NaNO, pH = 7, I = 0.05); B = same as A but 4.6 10-7 M in CH,OH (final pH 6.97); C= same as A but to pH 5.54 with HCl; D = same as A but 4.6 10-7 M in ethanoic acid (final pH 5.69).MOLECULAR SIZES AND ASSOCIATION FORCES 107 showed the appearance of a low-molecular size material eluting at the total volume (Fig. 4-6, 4-8, and 4-10). Methanol slightly shifted the main diffused peak to higher elution volumes for both FAs, but a significant reduction in peak intensity was more noticeable in the UV chromatogram of FA2 than for FA1 (Fig. 4-11 and 4-13). The RI chromatograms (Fig. 4-12 and 4-14) also showed a different mass distributions for the FA. Mobile phase C (solution A brought to pH 5.54 with HCl) was generally very effective in reducing the My values of all humic samples (Table 4-4). However, the MILLIVOLTS Cietlca. B 2 30 3B ELUTION VOLUME (mL) Fig. 4-10. Size exclusion chromatograms of HA3 recorded with the RI detector. A = control mobile phase (0.05 M NaNOs, pH = 7, I= 0.05); C= same as A but to pH 5.54 with HCl; 5.69). = same as A but 4.6 x 1077 M in CH;OH (final pH 6.97); = same as A but 4.6 x 10-7 M in ethanoic acid (final pH108 PICCOLO ET AL. UV chromatograms showed that reduction of peak intensities was much larger for HA2 and HAI than for HA3 (Fig. 4-5, 4-7, and 4-9). The chromatogram of the latter material was similar to that obtained in solution B, and even showed a small peak still eluting at the void volume. The RI chromatograms confirmed the behavior observed with the UV detector. In fact, most of the humic materials of HAI and HA3 (Fig. 4-6 and 4-10) were eluted at the total volume when in mobile phase C, thereby confirming the substantial size reduction that is suggested by the UV chro- ELUTION VOLUME (mi) Fig. 4-11. Size exclusion chromatograms of FA recorded with the UV-Vis detector. A = control mo- bile phase (0.05 M NaNOs, pH = 7, I = 0.05); B = same as A but 4.6 « 10-7 M in CH3OH (final pH 6.97); C= same as A but to pH 5.54 with HCl; D = same as A but 4.6 x 10-7 in ethanoic acid (final pH 5.69).MOLECUEAR SIZES AND ASSOCIATION FORCES 109 matograms because of the large decreases in peaks intensites. The RI chromatogram of HA3 (Fig. 4-10) substantiated UV results by showing that the elution curve is similar to that in mobile phase B and that only part of HA3 was shifted beyond the total volume. This indicates that solutions B and C had similar effects on the size distribution of HA3. Mobile phase C shifted the main peak of both FAs to higher elution volumes in the UV chromatograms (Fig. 4-11 and 4-13). However, peak intensities were slightly increased in FAI, but reduced substantially in FA2. The RI chromatograms confirmed the general molecular size reduction of both FAs (Fig. 4-12 and 4-14) in mobile phase C. Mobile phase D (solution A made 4.6 x 10-7 M in ethanoic acid with a pH of 5.69) was the most effective in reducing M,, values of all humic samples with respect to the control solution (Table 4-4). The UV chromatograms of all the HAs (Fig. 4-5, 4-7, and 4-9) showed, not only a reduction of peak intensity of about one order of magnitude in comparison to the control solution A, but also a shift of the remaining peak at higher elution volumes than for solution C. The RI chro- matograms confirmed these large changes by showing that the bulk of humic sam- ples in each case was shifted to the total volume (Fig. 4-6, 4-8, and 4-10), thereby indicating the extreme reduction in size caused by the presence of ethanoic acid in the eluent. Similarly, the large peak of both FAs was shifted to higher elution vol- 4.0. MILLIVOLTS ELUTION VOLUME (mL) Fig. 4-12. Size exclusion chromatograms of FAI recorded with the RI detector. A = control mobile phase (0.05 M NaNO3, pH = 7, 1 = 0.05); B = same as A but 4.6 « 10-7 M in CH;OH (final pH 6.97); C = same as A but to pH 5.54 with HCI; D =same as A but 4.6 « 10°? M in ethanoic acid (final pH 5.69).110 PICCOLO ET AL. umes, and were substantially reduced in intensity (Fig. 4-11 and 4-13). The RI chro- matograms confirmed the UV findings in that most of the FAs were eluted at the total volume (Fig. 4-12 and 4-14). These results cannot be explained by changes in ionic strength and the ran- dom coil model (Ghosh & Schnitzer, 1980), but would appear to substantiate the description of HS as supramolecular associations of relatively small molecules (Pic- colo et al., 1996b,c; Piccolo, 1997; Conte & Piccolo, 1999b) that are weakly held together by dispersive forces (van der Waals, nm, CH-1 bondings) and H bond- 1504 MILLIVOLTS 0 20 30 40 50 60 RETENTION TIME (min) Fig, 4-13. Size exclusion chromatograms of FA2 recorded with the UV-Vis detector, A = control mo- bile phase (0.05 M NaNO, pH = 7, I= 0.05); B = same as A but 4.6 10-7 M in CH,OH (final pH 6.97); C= same as A but to pH 5.54 with HCI; D~ same as A but 4.6 * 10-7 M in ethanoic acid (final pH 5.69).MOLECULAR SIZES AND ASSOCIATION FORCES 1 ings. The size of the HS is only apparently macromolecular, and the weak inter- molecular forces would appear to be easily overcome and the humic conformational structure disrupted. However, the possible conformational rearrangement of the humic superstructures appeared dependent not only on the composition of the mo- bile phase, but also on the particular molecular structures of the humic components. When the control mobile phase is only 4.6 x 10-7 M in CH;OH (solution B), no new ions nor pH changes are introduced. The observed alteration in molecular- size distribution of the humic samples may be attributed to the capacity of CH;OH to form both van der Waals bonds with the apolar, hydrophobic, components of the humic aggregate and H bonding with the O-containing functionalities in HS. These interactions seemed sufficient to modify the appearances of the UV chromatograms of HA2 and HA3 by disrupting the weak bondings that temporarily stabilize the humic components into apparently large aggregates. The result is the dispersion of these materials into smaller humic associations, and their diffusion through smaller gel pores with a reduction in average molecular sizes. In the case of HA1, addi- tion of CH;OH to the mobile phase gave rise to an increase of the first chromato- graphic peak (Fig. 4-5), thereby indicating that the molecular composition and con- formational association this HA must be different from those of HA2 and HA3. MALLIVOLTS ELUTION VOLUME (mi) Fig. 4-14. Size exclusion chromatograms of FA2 recorded with the RI detector. A = control mobile phase (0.05 M NaNO,, pH = 7, I= 0.05); B = same as A but 4.6 x 10-7 M in CH;OH (final pH 6.97); C = same as A but to pH 5.54 with HCI; D = same as A but 4.6 x 10°? M in ethanoic acid (final pH 5.69).112 “PICCOLO ET AL. Table 4-4. Weight-average molecular weight and polydispersity of humic samples in different mobile phases as determined by UV and RI detectors. My changes are reported as compared to the control mobile phase (A). At Bt ct Dt Samplet M,§ P€ M, P % My, P % My P % HAL UV 23000 28 29000 3.5 +26.1 8200 1.9 -64.3 4000 1.2 -82.6 RI 22700 2.9 9200 2.9 -68.8 3400 1.3 -R5.2 2250 1.1 90.2 HA2 UV 9700 1.7 4200 1.7 -S6.7 3600 10 -62.9 2900 12 -70.1 RI 9500 1.6 2930 16 -69.1 2100 10 -779 2200 1.0 -76.8 HAS UV 17000 2.0 7900 2.0 -53.5 9000 1.8 -47.0 3500 11 -79.4 RI 16650 3.5 7790 3.5 ~-53.2 7990 15 -52.0 2200 1.0 -86,7 FAL Uv 12000 1.8 11000 18 = -83 8600 1.7 -28.3 6700 14 44.2 RI 6550 2.1 6230 2.1 48 $170 16 -21.0 1490) 1.1) -77.2 FA2 UV 11000 1.7 9600 1.7) -12,7 7000 1.5 -36.4 5100 1.3 -53.6 RI 6000 1.9 4910 2.1 -18.2 4280 1.5 -28.7 3480 1.7) -42.0 + See Section 4-2.1 for identification t Mobile phase: A = NaNO; (pH 7); B = CH,OH (pH 6.97); C = HCI (pH 5.54); D = ethanoic acid (pH 5.69). § Daltons. {| P= polydispersion (M,/M,). This reasoning was confirmed by the RI chromatograms that showed a mass shift for all HAs towards elution volumes typical of lower MW material (eluted in the total volume). The RI variation was less for HAI (Fig. 4-6) that suggested that HOH changed the reciprocal arrangement of the chromophores more than in- creasing the size of this hydrophilic humic material, as one would have concluded by only evaluating UV results (Fig. 4-5). However, the results from the RI detector data would not rule out the possi- bility that humic conformational changes, rather than a disaggregation of weakly associated small molecules, could result from a coiling down of large macromol- ecules that, by assuming a reduced hydrodynamic radius (radius of gyration, R,), would elute at higher elution volumes. This possibility appears to contrast with the large reduction in the intensities of the peaks observed for mobile phase B in the UV chromatograms of HA2 and HA3. This reduction of peak intensities may be again explained with the phenomenon of hypochromism described in “Humic Conformational Stabilities in Acids”. Reduction in peak absorbance implies that total molecular absorptivity of the eluting humic matter is lower (hypochromism) than in the mobile phase of the control. This may be attributed to a separation of molecules (or chromophores) rather than to their compaction into a coil. A logical conclusion is that the addition of CH;OH to the mobile phase disrupted the humic supramolecular associations and that conformations of smaller dimension were formed,MOLECULAR SIZES AND ASSOCIATION FORCES 113 The greater changes produced by decreasing the pH of the control solution to 5.54 (mobile phase C) may be attributed to a greater disruption of the humic supramolecular structures than had taken place in CH,OH. The additional H atoms present in this mobile phase would protonate the weaker carboxylic functionalities that were in the dissociated form at pH 7 in the case of the control mobile phase. A number of negative charges were then neutralized, and more H bonding (than in the control solution) could be concomitantly formed among the complementary functionalities of the humic molecules. Hence, the humic conformational structure, which originally was stable in the mobile phase at pH 7, was largely altered. Be- cause of the parallel observation that a reduction in peak absorbance was shown in the chromatograms (hypochromism), and that a shift to high elution volumes of the humic mass was evident in RI chromatograms, this change cannot simply be due to the reduction of the hydrodynamic radius of the humic random coil, as previously explained (Swift & Posner, 1971; Ghosh & Schnitzer, 1980; Berden & Berggren, 1990). A more plausible explanation of this experiment is that the humic confor- mation had collapsed into molecular associations of smaller dimension but of greater thermodynamic stability than in the control solution. As stated earlier, the formation of a H bond represents a gain in energy from 10 to 20 kJ mol over that for a van der Waals bond (Schwarzenbach et al., 1993). In this new conformation, the humic molecules in mobile phase C are prone to aban- don the loose conformation assumed at pH 7 in the control solution because they now form relatively strong intermolecular H bondings. Similarly, as in the first ex- periment, it can be concluded that the weaker conformation of apparently high mol- ecular size shown by the HA in the control solution must have arisen predominantly from weak intermolecular hydrophobic forces holding together smaller molecules. The even greater changes in the molecular-size distribution of HAs caused by ethanoic acid in mobile phase D at a pH similar to that of solution C may be ex- plained by an additional effect due to the methyl group of ethanoic acid. The weak acidity of CH;COOH allows both undissociated and dissociated forms of ethanoic acid to be present at pH 5.69, and mixed H bonding can be formed with humic mol- ecules (Piccolo & Celano, 1994), thereby stabilizing their conformation. As for mo- bile phase C, such energy-driven rearrangement overrides the humic associations that are only weakly stabilized by hydrophobic forces at pH 7. However, the shift to higher elution volumes, and the general reduction of molecular absorptivity of the peaks in the UV chromatograms (hypochromism), as well as the almost com- plete mass shift to the total volume in the RI chromatograms for all HAs, indicates that the ethanoic acid methyl! group must play an additional role in further enhancing the disaggregation of humic associations. As shown also in the first experiment with monocarboxylic acids of different chain lengths, it is likely that the methyl group in mobile phase D interfered with the residual hydrophobic forces that still con- tributed to the stabilization of the humic conformation in solution C, thereby break- ing humic molecular aggregation into even smaller molecular associations. The much smaller variation in molecular-size distribution shown by the FAs suggests that these had profoundly different molecular compositions than the HAs (Table 4-2). The absorbing chromophores of the FAs must be too few and spread out for their additive absorptivity to be affected by conformational changes, and to give the large hypocromic effect shown by the HAs. The small changes in the UV14 PICCOLO ET AL. chromatograms of the FAs (Fig. 4-11 and 4—13) can be related to the low content of highly absorbing electronic systems such as aromatic or olefinic structures (Table 4~2). The more sensitive UV changes observed for FA3 must be ascribed to its higher aromaticity content. Nevertheless, the RI chromatograms were self- evident in showing a progressive shift to higher elution volumes with changes in the mobile phase, thereby indicating some decrease in molecular size also for the FAs. The use of poorly UV-absorbing fulvic-like DOM, and the lack of RI detec- tors prevented earlier studies to appreciate size reduction in mobile phases of vary- ing pH values (Berden & Berggren, 1990). 4-3.4 Ultraviolet Spectra of Humic Acids in Modified Solutions In order to verify that variations in absorbance intensity (hypochromism) were not limited to one wavelength, UV spectra of the HA solutions were recorded over arange of wavelengths. Figure 4-15 shows that three HAs produced different vari- ations in absorbance values upon modification of the solution. Whereas the addi- tion of CH;OH caused a reduction in molecular absorptivity at all wavelengths in HA2 and only for some wavelengths in HAI, it did not have any effect with respect to the control in the case of HA3. In contrast, addition of HCI produced lower ab- sorbance values (than for the control solution) for all HAs, except for the 290- to 330-nm range in HA1. Addition of ethanoic acid gave a significant decrease in ab- sorbance for all three HA solutions over all wavelengths. These results confirm that molecular absorptivity of the bulk HA varies with the compositions of the solutions, and that the absorptivity is reduced by adding chemicals that disrupt their loose mol- ecular associations and produce a hypochromic effect. It is noteworthy that the different light absorption properties of HAs in mod- ified solutions can be related to their specific molecular compositions. The major discrepancy observed among UV spectra of HAs was the different molecular ab- sorptivity shown by HAI in the CH;OH-amended solution (as compared to HA2 and HA3). This may be explained by the relatively larger aromaticity and carboxylic acidity (Table 4-2) of HA2. The slight amount of CH;OH in solution B was capa- ble of a larger conformational disruption of HA2 due to formation of H bonds be- tween CH;OH and humic carboxylate groups. These were sufficient to disaggre- gate a humic association that was weakly stabilized, mainly by 1 interactions among the aromatic structures, and give rise to hypochromism when compared with the control solution (Table 44, Fig. 4-15). Conversely, the lower acidic-C and larger aliphatic-C content of HAI (Table 4-2) suggests that a smaller number of inter- molecular H bonds were formed with CH3OH, and that these, instead of disrupt- ing, gave rise to humic aggregates that were already stabilized by hydrophobic forces acting through the aliphatic moieties. The overall large hydrophobicity of HA3 pos- sibly balances the two opposite CH;OH effects towards a new humic conforma- tion showing an absorptivity similar to that of control solution. While the RI chromatograms showed that the order of humic resistance to dis- ruption by CH,OH was: HA! > HA3 > HA2, the UV chromatogram of HAI re- vealed an increase of the first peak intensity with respect to the control solution (Fig. 4.5, Table 4-4). As noted before, this enhancement with CH;OH must be attrib- uted to an association with the first peak of a chromophore that was previously in-MOLECULAR'SIZES AND ASSOCIATION FORCES 115 250 300 360 400 460 Wavelength (nm) Fig. 4-15. UV-Vis spectra (250-450 nm) of the whole HAs. A = control solution (0.05 M NaNO, pH same as A but 4.6 x 10-7 M in CH3OH (final pH 6.97); C = same as A but to pH same as A but 4.6 x 10-7 M in ethanoic acid (final pH 5.69).116 PICTOLO ET AL. corporated into the second diffuse peak. The latter, in fact, showed a decrease in absorbance with respect to the control solution. Again, the highest hydrophobicity of HA3 (Table 4--2), and its consequent stable conformation can explain the lim- ited disruption noted in both the UV and RI chromatograms (Fig. 4-9 and 4-10) of this material when treated with HCI. Conversely, HA] and HA2, of higher HI/HB ratios, and hence of less hydrophobic character, showed a more extensive disruption into smaller size-fractions as indicated by both the UV and RI chro- matograms. Finally, ethanoic acid, containing both a methyl group and a carboxyl group, was able to overcome even the strong hydrophobic association in HA3 and to reduce its average molecular size to the same extent as for HA1 and HA2. 4-4 CONCLUSIONS Our findings indicate that the changes in molecular associations of HS can be followed by HPSEC. This technique provides molecular size distributions that are a combination of two factors: (i) the elution volume; and (ii) the molecular ab- sorptivity of the chromatographic peaks. This was shown in the cases of both ex- periments reported here. In particular, the first experiment has confirmed that the weak molecular forces holding together relatively smal] humic components can eas- ily be overcome by the action of monocarboxylic acids. The consequent reduction in molecular size depends on the nature of the humic materials and on the number of C atoms in the organic acid. The second experiment, in which HAs of different molecular structures were compared in HPSEC mobile phases of different com- positions, but of constant ionic strength, showed that the self-assembling of humic components in supramolecular associations of apparent high molecular sizes is de- pendent on solution composition. Previous studies have failed to indicate the combination of the above factors because either: (i) HAs that were closely similar in nature were detécted only by UV in leS$ Sensitive low-pressure size-exclusion systems (Swift & Posner, 1971); or (ii) poorly absorbing FAs or aqueous DOM samples were studied in HPSEC sys- tems without the support of a RI detector (Becher et al., 1985; Berden & Berggren, 1990; Chin & Gschwend, 1991; Chin et al., 1994, 1997). Our results showed that when the mobile phase of the chromatographic sys- tem was varied in composition but without changing significantly the ionic strength, or when humic solutions were treated with monocarboxylic acids, the molecular size distributions of the HS was dramatically reduced. The size re ion, which was proven by peak shifts to higher elution volumes, as detected tation, was accompanied by a significant decrease in molecular abs termined by the UV detector. This reduction in peak absorbance could only be at- tributed to a disaggregation into smaller associations of the humic conformational arrangement. The constant ionic strength of the different mobile phases excluded the possibility that such an effect was caused by the compaction | conformation. Furthermore, differences in humic molecular struct different hydrophobicities that appeared to be related to conformational stabilities and thus to the extents of changes in a8s0ciations. These results are in line with previous findings that have suggested that HS in solution form loosely bound supramolecular conformations of self-associatingMOLECULAI SIZES AND ASSOCIATION FORCES 7 molecules. The predominant binding forces appear to be hydrophobic, probably be- cause of the entropy-driven fendency to exclude water molecules out of the humic associations and thus stabilize molecular conformations (Israelachvili, 1991; Tan- ford, 1991; Schwarzenbach et al. » 1993). Such a model of dissolved HS based on the self-association of small molecules rather than on the macromolecular random coil model appears to be promising both from the viewpoint of advancing knowl- edge on the ultimate chemical structures of HS and in explaining their role in the processes of organic matter accumulation and decomposition in environmental com- partments (Piccolo et al., 1999b), REFERENCES Barth, HL 1200. A practical approach to steric exclusion chromatography of water-soluble polymers. 1 Chromatogr. Sci. 18:409-429, Becher, G.. GE. Carlberg, E.T. Gjessing, J.K. Hongslo, and S. Monarca. 1985. High-performance size Gxclusion chromatography of chiorinated natural humic water and mutagenicity studies using the microscale fluctuation essay. Environ. Sci. Technol, 19:422 436, Berden. M.,and D, Berggren. 1990, Gel filtration chromatography of humic substances in sol solutions using HPLC-determination of the molecular weight distribution. J Soil Sci. 41-61-72, Cameron, R'S. 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