Implications of Mixture Characteristics on Humic ... - ACS Publications

2 Implications of Mixture Characteristics on Humic-Substance Chemistry J. A. Leenheer, P. A. Brown, and T. I. Noyes

Downloaded by UNIV OF SYDNEY on September 4, 2014 | http://pubs.acs.org Publication Date: December 15, 1988 | doi: 10.1021/ba-1988-0219.ch002

U.S. Geological Survey, Denver Federal Center, Denver, CO 80225

The chemistry of complex mixtures usually cannot be described as the sum of the characteristics of the individual components because of intermolecular interactions that modify component characteristics. A study of both inter- and intramolecular interactions in the Suwannee River fulvic acid, by measurement of the dependence of density on concentration in various solvents, indicated that polar functional groups (carboxyl, phenolic, hydroxyl, and ketone) were responsible for most of the molecular interactions in water. A two -stage fractionation of Suwannee Riverfulvic acid on silica gel resulted in 31 fractions that had variable molecular weight distributions and acid-group contents. Intramolecular interactions were determined to predominate over intermolecular interactions for the Suwannee River fulvic acid in all solvents at weight-volume concentrations less than 1%.

HUMIC SUBSTANCES ARE H E T E R O G E N E O U S with respect to the diversity of molecules that compose the mixture, and with respect to the diverse functional groups, structural units, and configurations that compose individual molecules. Intermolecular interactions between molecular components in the mixture, or intramolecular interactions between organic functional groups within a molecule, may change chemical and physical properties of humic substances among various environments. Molecular interactions of humic substances are dependent on temperature, pH, ionic strength, type of solvation, degree of hydration (on drying), type of countercations, and concentration of the humic substance in solution. Environmentally significant phenomena that may be affected by molecular interactions of humic substances include organic-contaminant partitioning, trace-element inter0065-2393/89/0219-0025$06.00/0 © 1989 American Chemical Society

In Aquatic Humic Substances; Suffet, I., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1988.


26

AQUATIC HUMIC SUBSTANCES

actions, and rates of decomposition of humic substances in various environ ments. Humin and humic acid are reactive substances that form amorphous organic phases in soil, sediment, and water; types of interactions and ordering of these interactions into a membrane model are discussed by Wershaw and Marinsky (I). Fulvic acid exists as a solute in natural waters more commonly than does humic acid, and molecular interactions are controlled by the chemistry of water. A question frequently asked concerning fulvic acid is whether fulvic acid exists in water as a solute dissolved in an ideal solution, or does it interact to form nonideal molecular aggregates? If fulvic acid exists as a molecular aggregate in water, the chemistry of fulvic acid cannot be described as the sum of the characteristics of the individual components because of intermolecular interactions that modify component characteris tics. This chapter will investigate the mechanisms of molecular interactions in a stream fulvic acid isolated from the origin of the Suwannee River at the Okefenokee Swamp in southern Georgia. Variations in solution densities and molecular-size distributions with solvents of differing polarity and concen trations were used to distinguish intermolecular from intramolecular inter actions. A two-stage fractionation of the fulvic acid on silica gel was designed to disrupt heterogeneous molecular aggregates (if they exist) and obtain smaller-sized particles in solutions of the fractions that are more homoge neous than fulvic acid. Both polar and nonpolar interactions undoubtedly are responsible for the often unique chemical and physical properties of extremely complex mixtures that are called humic substances. Polar interactions such as hy drogen bonding will be emphasized in this chapter over nonpolar interactions because of the predominantly polar character of the Suwannee River fulvic acid. Molecular aggregates of fulvic acid caused by nonpolar interactions are not likely to form in natural waters, because concentrations of fulvic acid at natural concentrations are less than critical micelle concentrations of known detergents and because of the lack of significant nonpolar moieties in fulvic acid structure. However, polar interactions, both intramolecular and inter molecular, will increase the nonpolar character of fulvic acid, and they may increase partitioning of nonpolar contaminants into molecular structures of fulvic acid. The findings of polar-interaction mechanisms in fulvic acid also can be related to interactions between polar moieties in humin and humic acid structures. However, nonpolar interactions also should be recognized as relevant to humin and humic acid.

Experimental Methods and Materials The Suwannee River was sampled at its origin at the outlet of the Okefenokee Swamp near Fargo, Georgia, during November 1983. Onsite, 8104 Lof water were processed through a filtration and column-adsorption system that fractionated and isolated In Aquatic Humic Substances; Suffet, I., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1988.

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Implications of Mixture Characteristics

organic solutes into hydrophobic-neutral, strong hydrophobic fulvic acid, weak hy drophobic fulvic acid, hydrophilic acid, and hydrophilic-neutral fractions (2). The theory and chemical significance underlying these operational fractionation proce dures are given in previous reports (3-7). Most (94.6%) of the fulvic acid was contained in the strong hydrophobic fulvic acid fraction; 382.4 g of this fraction was isolated from water and used in the study described in this chapter. The strong hydro phobic fulvic acid fraction was 66% of the dissolved organic-carbon concentration, 38.4 mg/L of the Suwannee River water. Molecular-weight distributions of the Suwannee River fulvic acid were estimated by the degree of polydispersity determined by the ratio of M (weight-average mo lecular weight) to M (number-average molecular weight). M and M were deter mined by equilibrium centrifugation. Equilibrium-ultracentrifugation experiments were conducted on an ultracentrifuge (Beckman L8-70 M, equipped with a Beckman Prep UV scanner) that assayed solute concentrations at 280 nm throughout the ultracentrifuge cells during the run. A four-phase titanium rotor (AnF, 5.7-cm radius) was used at speeds of 20,000 to 40,000 rpm. Equilibrium between sedimentation and molecular diffusion of the solutes was virtually complete between 24 and 48 h of centrifugation. Analytical ultracentrifugation cells were constructed of aluminum bodies with centerpieces of Kel-F (for aqueous 0.2 M KC1 solvent) or aluminum (for organic solvents). Each cell had a sample and reference compartment with quartz windows in the centerpiece that contained 300 μL· of solution. The immisciblefluorocarbonoil (FC-43), used to determine the bottom of the solution in the cell, was used only for aqueous solvents and not for organic solvents that partially solubilized the oil. The M and M values were determinedfromthe equilibrium-ultracentrifugation curves by the method of Lansing and Kraemer (8). Solution densities of the Suwannee River fulvic acid at 20 °C were determined by pycnometry (9) in the solvent used for equilibrium ultracentrifugation. Pycnometers of 2-mL capacity gave results accurate to 0.02 g/mL for the samples at 1-2% w/v solute concentrations; 10-mL pycometers were used at the 0.4-1% solute con centrations. Partial molar volume or apparent molar volumes (for nonideal solutions) were calculated as the reciprocal of the solution density. All concentrations of humic substances in this chapter are expressed on a weight-per-solution-volume basis. Solvents used for density determinations included distilled water, aqueous 0.2 M KCl, tetrahydrofuran (freshly distilled), dioxane (freshly distilled), N,Ndimethylformamide (dried over calcium hydride and distilled), acetonitrile, and gla cial acetic acid. Equilibrium ultracentrifugation was conducted with aqueous 0.2 M KCl, tetrahydrofuran, and glacial acetic acid. Both tetrahydrofuran and glacial acetic acid have small dielectric constants, so that the carboxyl group does not ionize to produce negative charges on fulvic acid solutes. These negative charges interfere with solute sedimentation during centrifugation. Tetrahydrofuran was preferred for use as a solvent for centrifugation because of its minimal density and viscosity; however, glacial acetic acid was used when samples were not soluble in tetrahydro furan. Fulvic acid ionization was suppressed by 0.2 M KCl for centrifugation of samples dissolved in water. Fractionation of fulvic acid on silica gel was conducted with 100-200-mesh activated silica (ICN Biochemicals). Thefirst-stagesilica fractionation used a 2-L bed volume of silica contained in a 50-mm-i.d. X 950-m m glass column. The column was conditioned by passing 0.1 M tetrabutylammonium acetate in chloroform through until solution equilibrium of the tetrabutylammonium acetate with the silica was obtained. Six grams of the Suwannee River fulvic acid was titrated in water with tetrabutylammonium hydroxide to pH 8; then the sample was freeze-dried. The tetrabutylammonium salt of fulvic acid was dissolved in chloroform, and a total volume of 22 mL was added to the silica column. Fraction 1 eluted with w


n

w

n

w


n


28


3100 mL of chloroform;fraction2 eluted with 970 mL of methyl ethyl ketone; fraction 3 eluted as a distinct band at the interface of methyl ethyl ketone and acetonitrile in 400 mL of solvent;fraction4 eluted in 2000 mL of acetonitrile;fraction5 eluted in 1425 mL of 75% acetonitrile and 25% 2-propanol;fraction6 eluted in 1310 mL of 75% acetonitrile and 25% water; andfraction7 eluted in 1350 mL of 75% ace tonitrile and 25% water, with 0.25 M oxalic acid dissolved in the mixed solvent. The volumes of solvent used during the elution sequence were determined by observing the cessation of color elutedfromthe column by each solvent. After elution, the solvents were removed by vacuum-rotary evaporation; the fractions were redissolved in 50:50 acetonitrile-water; then the samples were con verted back tofreeacids by passing them through columns of cation-exchange resin in the hydrogen form (MSC-1H, Dow Chemical). Thefractionswere (1) vacuumevaporated to about 20% of their original volume to remove acetonitrile, (2) adjusted to pH 2 with HC1, (3) applied to a 500-mL bed volume resin column (XAD-8), (4) washed with 1 L of 0.01 M HC1 to remove acetic acid and oxalic acid, and (5) eluted with 300 mL of acetonitrile. The acetonitrile was removed by vacuum evaporation, and the water was removed byfreeze-drying.The driedfractionswere weighed to calculate yield. The second-stage fractionation separatedfree-acidfulvic acidfractions2, 4, 5, and 6 on silica gel. Silica gel was packed in glass columns, with bed volumes cor responding to a loading of 2.5 mg of sample per 1 mL of bed volume. Silica was packed with chloroform as the initial mobile phase and thefractionswere applied and then dissolved in tetrahydrofuran at a volume of 5% of the bed volume. The elution sequence was (1) chloroform, (2) diethyl ether, (3) 75% diethyl ether and 25% methyl ethyl ketone, (4) methyl ethyl ketone, (5) acetonitrile, (6) 75% acetonitrile and 25% isopropyl alcohol, (7) 75% acetonitrile and 25% water, and (8) 75% aceto nitrile and 25% acetonitrile with 0.25 M oxalic acid. Varying quantities of solvents were used for each fraction; cessation of eluted color was the sign for the beginning of the next solvent. Each solvent was removed by vacuum-rotary evaporation; the oxalic acid was removedfromfraction8 through readsorption on resin (XAD-8) by the procedure discussed in the previous paragraph. Unfractionated fulvic acid was methylated with diazomethane. Nitrogen gas was slowly bubbled in a polytetrafluoroethylene (Teflon) tube through two 25-mm i.d. X 150-mm test tubes, which were sealed with rubber stoppers. Thefirsttest tube was two-thirds filled with diethyl ether; the second test tube, kept in an ice bath, contained 8 mL of diethyl ether, 8 mL of 2-(2-ethoxyethoxy)ethanol, 8 mL of 30% KO H solution; and 3 g of 99% N-methyl-N-nitroso-p-toluenesulfonamide. Diazo methane gas (CH N ), generated by the reagents, was bubbled with nitrogen through 100 mg of fulvic acid sample and then suspended in methylene chloride in an ice bath while being dispersed by ultrasonic vibration. The fulvic acid sample was dis solved in methylene chloride during methylation; the reaction was stopped after complete dissolution, and methylene chloride and excess diazomethane were re moved by vacuum evaporation. 2

2

Results and Discussion Density-concentration curves for Suwannee River fulvic acid in water, te trahydrofuran, dioxane, and Ν,Ν-dimethylformamide are shown in Figure 1. Pimentel and McClellan (10) state that intramolecular interactions are concentration-independent in their effects on density, whereas intermolec ular interactions result in increases in density with concentration. Che-


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29

1.8

1.7 Φ Ξ 1.6


ε

ο • Δ A •

Free acid in water Free acid in tetrahydrofuran Free acid in dioxane Free acid in N,N-dimethylforamide Methylated fulvic acid in Ν ,N -dimethylforamide • Methylated fulvic acid in acetonitrile

'55 1.4 c Φ Û 1.3

02

0.4

0.6 0.8 1.0 1.2 1.4 1.6 1.8 Fulvic-Acid Concentration (percent weight/volume )

2.0

2.2

Figure 1. Density-concentration curves for Suwannee River fulvic acid in various solvents. baevskii et al. (II) reported that soil fulvic acid aggregates in water near 1% concentrations, as determined by density measurements. Both the water and Ν,Ν-dimethylformamide density-concentration curves in Figure 1 in dicate similar density-concentration dependence, with little or no density increase beyond 1% concentration. However, the density indicated by the Ν,Ν-dimethylformamide curve is 0.1-0.2 g / m L less than that indicated by the water curve. The tetrahydrofuran and dioxane curves are concentrationindependent from 0.4-1.0% concentration (within limits of error); the larger value for density in tetrahydrofuran at 2.0% may be due to lack of complete solubility, because a slight precipitate was observed at this concentration. The range of density values in different solvents of different polarity indicates that some type of molecular interaction is occurring. The concentration independence of the density data for tetrahydrofuran and dioxane indicates that one type of interaction is intramolecular. A conclusion for intramolecular interactions compared with intermo lecular interactions cannot be made from the data of Figure 1 alone because of the lack of data from 0-0.4% concentration, where the analytical error for pycnometric measurements of density becomes too large to detect sig nificant differences. However, a minimum density for the sample can be calculated from Traube's rule (12), which states that the molar volume of an organic liquid with no interactions is equal to the summation of atomic volumes, plus a correction term for double bonds and aromatic rings, plus a correction term for end groups. Brown and Leenheer (13) applied Traube's rule and used elemental analysis, molecular weight data, C nuclear mag netic resonance (NMR) data, *H N M R data, and titrimetric data for the 1 3


30


Suwannee River fulvic acid. They calculated a minimum solution-state den sity of 1.45 g / m L . Methylation is known to decrease hydrogen-bonding interactions (9), and is likely to produce a decrease in density compared to free-acid samples. However, a methyl ester group is less dense (d = 1.26 g/mL) than a free carboxyl group (d = 1.90 g/mL) (12), and methylation will decrease the minimum calculated density of the sample. The degree of methylation of the sample was measured by H N M R spectroscopy (14). The calculated minimum density of the sample, using Traube's rule (12), decreased to 1.37 g / m L . The solution-state density values for the methylated sample in iV,N-dimethylformamide (Figure 1) are identical (within limits of error) to the calculated minimum density; the density values for the methylated sam ples in acetonitrile are about 0.1 g / m L larger. Ν,Ν-Dimethylformamide is a good hydrogen-bonding solvent, whereas acetonitrile cannot hydrogenbond. Perhaps unmethylated hydroxyl groups in the fulvic acid dissolved in acetonitrile are interacting with carbonyl and ether groups of fulvic acid, but not in Ν,Ν-dimethylformamide, which hydrogen-bonds to these groups. Conversion of a free carboxyl group to a carboxylate anion by an increase in p H in water increases hydrogen-bonding interactions with un-ionized weak-acid groups such as phenols (10, 15) because of the greater dipole of the carboxylate anion. However, the creation of negative charges with the p H increase tends to disrupt molecular interactions because of electrostatic repulsion (16). Density-concentration curves of the potassium salt of fulvic acid at p H 8 in water and in 0.2 M K C l are shown in Figure 2. Densities of potassium salts of fulvic acid in water are much greater than density of


l

2.2|

j

J

1

1

1

1

1

1

1

1

ο Potassium salt, fulvic acid in water (pH 8) • Potassium salt, fulvic acid in 0.2 Ν KCl (pH 8) 0.4

JL _1_ J0.6 0.8 1.0 1.2 1.4 1.6 1.8 Fulvic-Acid Concentration (percent weight/volume )

2.0

2.2

Figure 2. Density-concentration curves for the potassium salt of Suwannee River fulvic acid at pH 8 in water and in 0.2 M KCl.



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31

hydrogen-saturated fulvic acid in water (Figure 1). The initial interpretation of these densities is that molecular interactions are enhanced markedly in the salt-anion forms of fulvic acid. However, electrolyte salts are known to increase the density of water by closer packing of the water molecules around the ions in a process known as électrostriction (17). To determine the density and molecular weight effects of électrostriction on a potassium-carboxylate ion pair, solution-density determinations in dis tilled water were performed on a series of 16 acids in free-acid and potassiumsalt forms. The resulting data are presented in Table I. Carboxyl-group contribution to compound density, calculated from Traube's rule (12, 23), generally increased by more than a factor of 2 between the free-acid and potassium-salt forms. The magnitude of the increase from acetic through benzoic acids i n Table I can be accounted for by adding four to six water molecules per potassium-carboxylate ion pair. This water is the bound water in the electrical double layer. Larger densities of potassium-carboxylate groups in phthalic through salicylic acids in Table I are probably caused by inter- and intramolecular interactions in these acids. The carboxylate group seems to form an intramolecular chelate with the free carboxyl group in monopotassium phthalate, with aromatic tr electrons in phenylacetic acid, and with the phenolic hydroxyl group in salicylic acid. Carboxylate-phenol interactions apparently cause a dimer with p-hydroxybenzoic acid. The large density values of the potassium salts of the Suwannee River fulvic acid presented in Figure 2 can be explained best by eleetroTable I. Solution Densities for Carboxylic Acids and Their Salts Acid Form Potassium Salt Forms Carboxyki Carboxyl Compound Group Compound Group Density Density Density Density Carboxylic Acid (g/mL) (g/mL) (g/mL) (g/mL) Acetic 3.73 1.12 2.02 1.92 Propionic 4.62 1.04 1.91 1.99 Succinic 4.25 2.54 1.43 2.04 Adipic 4.59 1.28 2.07 2.21 Pimelic 4.51 1.22 2.17 1.88 Azelaic 4.74 1.14 1.70 2.23 Sebacic 4.84 1.11 1.63 2.25 Tartaric 4.05 1.85 2.90 1.93 Citric 4.88 1.85 2.37 3.10 Benzoic 4.81 1.20 1.61 2.08 PhthaliC' 5.12 1.47 2.10 1.83 ρ- H y droxy benzoic 5.52 1.39 1.89 2.16 Phenylacetic 6.07 1.19 1.59 2.85 — — Salicylic 7.99 2.25 0

b

0

"Densities determined in Ν,Ν-dimethylformamide at 20 °C at 1% (w/v). ''Densities determined in water at 20 °C at 1% (w/v). 'Calculated as carboxyl group contribution to compound density using Traube's rule. ''Monopotassium salt.


0


32


lyte-electrostriction effects on water, rather than by molecular interactions within and among fulvic acid solutes, although the density-concentration dependence of the 0.2 M KC1 curve may indicate intermolecular interac tions. The smaller solution-density values of 0.2 M KC1 curve i n Figure 2 compared to the water curve illustrates the loss of the bound water layer at greater ionic strength. Not as many water molecules are bound to potassium carboxylate ion pairs in fulvic acid as are bound with dissociated potassium and carboxylate ions at lesser ionic strength. E q u i l i b r i u m ultracentrifugation was used to determine molecular weight distribution data i n different solvents at small concentrations (0.002-0.004%). Changes i n molecular weight distribution of fulvic acid dissolved in solvents of differing polarity may indicate changes in molecular conformation and aggregate states at concentrations approaching infinite dilution, where accurate density measurements were not available. Data for M , M , and degree of polydispersity of the Suwannee River fulvic acid i n water, tetahydrofuran, Ν,Ν-dimethylformamide, and acetonitrile are given in Table II. The data of Table II has only semiquantitative significance because a U V detector measures only compounds with aromatic nuclei that vary in their molar absorptivity. Furthermore, computation of M by the method of Lansing and Kraemer (8) provides only a semiquantitative estimate. Table II clearly indicates that the molecular weights of the potassium salt of the fulvic acid are much greater. However, the degree of polydispersity of the potassium salt data is much less than the data for fulvic acid i n organic solvents. Larger molecular weights were expected for the potassium salt of the fulvic acid because of the additional weight added by the potassium ions and bound water; however, the reason for the large decrease i n the degree of polydispersity was unknown. The lesser degree of polydispersity for potas sium salt in water, coupled with the large density, indicated that the presence of stable aggregates at a small concentration should be considered a possin

w

n

Table Π. Molecular-Weight Distribution Data for Suwannee River Fulvie Acid in Various Solvents

Sample Form" Potassium salt (density =1.8 g/mL) Free acid (density =1.55 g/mL) Methylated (density = 1.37 g/mL) Methylated (density = 1.43 g/mL)

Solvent 0.2 Ν KC1, pH 8 in water tetrahydrofiiran

Number Weight Average Average Degree of Polydispersity (M ) (MJ B

1060

1340

1.27

470

1110

2.36

530

1250

2.36

530

1190

2.25

Ν,Ν-dimethylformamide acetonitrile

"Density values were derived from 0.4% weight-volume values of fulvic acid concentrations in Figures 1 and 2.



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LEENHEER ET AL.

33

bility. Intermolecular aggregation is not likely to increase the degree of polydispersity in organic solvents because molecular weights were lower. Two-stage silica-gel fractionation was designed as a chemical fraction ation on the basis of polarity differences within the fulvic acid sample. If stable aggregates of fulvic acid were held together by polar hydrogen-bond ing forces, two-stage silica-gel fractionation would have a large probability of disaggregating the sample because of competitive polar-adsorption effects of the silica gel. The fractionation and the yields of the silica-gel fractionation are shown in Figure 3, and the yields are shown in Table III. Losses (10.9% for the first stage, 12.2% for the second stage) for the two-stage fractionation were within expectations for normal handling and processing losses, and the yield for each fraction can be regarded as representative of the whole sample. Molecular-weight distributions of 15 of the major fractions from silicagel fractionation are presented in Table IV. Molecular-weight distributions were determined in tetrahydrofuran, rather than in water, to avoid élec trostriction effects, which result in variable densities and large uncertainties in molecular weight determinations. The 15 fractions in Table IV accounted for nearly 88% of the weight of the sample. Large variations in molecular weight distributions were determined among the 15 fractions, but the sum mations of M and M adjusted for the yield of each fraction (Table IV) gave virtually the same M and M as the whole sample. Correspondence of the Af and M values of the fractions with the M and M values of the whole sample provide definitive evidence that Suwannee River fulvic acid is not aggregated in tetrahydrofuran. The 15 fractions also were titrated with 0.1 Ν N a O H to p H 8.5 to determine acid-group content. Variable acid-group contents occurred, as shown in Table IV, and will produce variable électrostriction effects on den sity for the potassium-salt form of the fulvic acid. These effects are probably the reason that the potassium salt of fulvic acid is less polydisperse in water than in tetrahydrofuran (Table II). Some indication exists that fractions of large acid-group content (such as fraction 6-2 in Table IV) have lesser M and degrees of polydispersity than fractions with small acid-group contents. Fractions with large acid-group contents would indicate the largest increase in density and apparent molecular weight on conversion to potassium salt. Fulvic acid molecular weight distribution, skewed to low-molecular-weight solutes in tetrahydrofuran, would appear to be less polydisperse in aqueous 0.2 M KC1. The silica-gel fractionation data coupled with molecular weight distri bution data do not support the existence of stable aggregate structures for fulvic acid at small concentrations (0.003%) in either tetrahydrofuran or aqueous 0.2 M KC1. Therefore, density increases of 0.1-0.2 g / m L (Figure 2) from the minimum calculated values for fulvic acid can be ascribed to intramolecular interactions for the underivatized sample in tetrahydrofuran and dioxane, and for the methylated sample in acetonitrile. The more polar solvents with good hydrogen-bonding characteristics (water and N , N - d i n

w

n

n

w

w

n

w

w


34


Silica r- Bu N*salt-i fractionation 4

1

1

Silica — Free acid — fractionation

ι

Si-2. 1&2

1

Si-2. 3

1 Si-2, * 1 Si-2, 5


Si-2, 6 Si-2. 7 1

1

Figure 3. Two-stage fractionation of the Suwannee River fulvic acid on silica gel. Bar sizes are proportional to yields.


2.

LEENHEER ET AL.


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Table III. Yields of Silica-Gel Fractionation of Suwannee River Fulvic Acid Weight Percent Fraction Number Yield (mg) (from Figure 3) Elution Solvent


Si-1 Si-2 Si-3 Si-4 Si-5 Si-6 Si-7 Si-2,1 and 2 Si-2,3 Si-2,4 Si-2,5 Si-2,6 Si-2,7 Si-4,2 and 3 Si-4,4 Si-4,5 Si-4,6 Si-4,7 Si-4,8 Si-5,1 Si-5,2 Si-5,3 Si-5,4 Si-5,5 Si-5,6 Si-5,7 Si-5,8 Si-6,1 Si-6,2 Si-6,3 Si-6,4 Si-6,5 Si-6,6 Si-6,7 Si-6,8

72 chloroform 658 methyl ethyl ketone 50% methyl ethyl ketone, 50% acetonitrile 66 559 acetonitrile 2420 75% acetonitrile, 25% 2-propanol 1500 75% acetonitrile, 25% water 75% acetonitrile, 70 25% water, 0.25 M oxalic acid 57 chloroform and diethyl ether 23 75% diethyl ether, 25% methyl ethyl ketone methyl ethyl ketone 105 49 acetonitrile 187 75% acetonitrile, 25% 2-propanol 58 75% acetonitrile, 25% water diethyl ether and 75% diethylether, 25% 6.8 methyl ethyl ketone methyl ethyl ketone 127 acetonitrile 54 244 75% acetonitrile, 25% 2-propanol 62 75% acetonitrile, 25% water 75% acetonitrile, 25% water, 0.25 M oxalic 3.6 acid 25 chloroform 360 diethyl ether 42 75% diethyl ether, 25% methyl ethyl ketone 913 methyl ethyl ketone 162 acetonitrile 391 75% acetonitrile, 25% 2-propanol 362 75% acetonitrile, 25% water 75% acetonitrile, 25% water, 0.25 M oxalic 67 acid 15 chloroform 548 diethyl ether 200 75% diethyl ether, 25% methyl ethyl ketone 336 methyl ethyl ketone acetonitrile 37 136 75% acetonitrile, 25% 2-propanol 88 75% acetonitrile, 25% water 75% acetonitrile, 25% water, 0.25 M oxalic 31 acid

1.35 12.32 1.23 10.45 45.21 28.15 1.30 1.21 0.49 2.23 1.04 4.00 1.23 0.14 2.72 1.15 5.21 1.31 0.08 0.54 7.68 0.89 19.47 3.45 8.33 7.71 1.26 0.33 11.69 4.27 7.18 0.80 2.89 1.88 0.65

methylformamide) are more effective at disrupting intramolecular hydrogen bonds in fulvic acid. Extrapolation of the density-concentration curves for these solvents in Figure 1 to zero concentration would result in values reasonably similar to the minimum calculated density of 1.45 g/mL. The occurrence of intramolecular interactions in the potassium-salt form of the Suwannee River fulvic acid cannot be deduced from the density-



36

Table IV. Molecular-Weight Distributions of Suwannee River Fulvic Acid Fractions from Silica-Gel Fractionation Degree of Polydispersity Acid-Group Weight Number Content* Percent Average * Average * (meq/g) (MJ Weight (MJ Fraction" 3.85 5.27 1060 200 2-4 2.23 3.65 4.26 1700 400 2-5 1.04 3.46 5.11 2920 570 2-6 4.00 2.96 3.07 1270 410 2-7 1.23 5.29 2.56 1170 460 4-4 2.72 4.30 1.09 820 750 5.21 4- 6 6.97 1.45 1060 730 7.68 5- 2 5.63 3.06 1160 380 5-4 19.47 5.70 3.73 1050 280 5-5 3.45 4.59 5.17 1500 290 5-6 6.50 4.74 2.30 1200 510 8.34 5- 7 7.16 1.17 520 440 11.69 6- 2 6.57 1.14 730 640 4.27 6-3 5.29 1.26 1110 880 7.18 6-4 4.40 1.09 1670 1540 2.89 6-6 1


1

Weight yield

87.9

χ %w = 476 476 M = = 542 0.879 542 M fractions = 1.15 M whole sample 470 n

n

=

n

χ %W = 1008 1008 = 1147 M = 0.879 1147 M fractions = 1.03 M whole sample 1110 w

w

w

"First stage-second stage. M and M are determined by equilibrium ultracentrifugation in tetrahydrofuran. Measured by the base titer to pH 8.5. b

n

w

c

concentration data of Figure 2 because of the large and variable effects of électrostriction of water on density.

Implications of Findings Intermolecular association is indicated by the density-concentration depen dence of Suwannee River fulvic acid at concentrations of about 1% in water and Ν,Ν-dimethylformamide. This indication, together with findings of in tramolecular association i n organic solvents with lesser polarity than water, has important implications regarding partitioning of nonpolar organic con taminants into humic substances. Both intermolecular and intramolecular hydrogen bonding (illustrated in reactions 1 and 2) dehydrate a polar portion of a polar molecule. Dehydration renders it more nonpolar, such that nonpolar contaminants can more readily partition into the hydrogen-bonded structure if this structure is sufficiently large to accommodate the contami nant. In Aquatic Humic Substances; Suffet, I., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1988.

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LEENHEER ET AL.

H l

37

Ο

0

I H

H

g 0

0

C - O H m O

C-OHimO

II

4 H 0


2

Benzoic-acid monomer: polar; low density; predominates at small concentrations

Benzoic-acid dimer: nonpolar; high density; predominates at large concentrations

Reaction 1. Intermolecular hydrogen tending. H I Ο

Ο C-OHn«iO H H O H «a Ο 2H 0 2

Salicyclic acid: low density; polar

Η-bonded chelate: high density; less polar

Reaction 2. Intramolecular hydrogen bonding. If polar humic substances like Suwannee River fulvic acid exist at large concentrations, such as on moist soil or sediment surfaces, intermolecular interactions can decrease the polarity and enhance contaminant partitioning. If polar humic substances are dried in soils or sediments, increased inter molecular and intramolecular interactions will decrease polarity and enhance partitioning of nonpolar contaminants. The lack of molecular interactions for the Suwannee River fulvic acid in water at small concentrations is significant. This lack of interactions substantiates the findings by Chiou et al. (18) that the Suwannee River fulvic acid at small concentrations in water has little affinity for various nonpolar contaminants. However, Chiou et al. (18) and Carter and Suffet (19) determined that other aquatic fulvic acids and humic acids from different environments had significant affinities for nonpolar con taminants. In Aquatic Humic Substances; Suffet, I., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1988.


38


Polar interactions of inorganic substances with humic substances are likely to occur in many environments. Hydrogen bonding with silica gel was used to fractionate the Suwannee River fulvic acid. The last fraction desorbed interacted so strongly with silica that oxalic acid had to be added to the water to break up the complex. In addition to weakly acidic silica, boric acid, acids of sulfides and polysulfides, acids of sulfites and polythionates, phosphoric acid, selenious acid, arsenous acid, and a number of other weak inorganic acids may hydrogen-bond to electron-donating groups in humic substances. Inorganic electron-donating groups that may hydrogen-bond to weak acid sites in humic substances include the oxides of iron, manganese, uranium, and other metals. The successful chromatographic fractionation of fulvic acid on silica gel has relevant implications for chromatography of humic substances to gain structural information. The variable acid-group content of the Suwannee River fulvic acid was unknown before silica-gel fractionation. Additional chromatography using basic adsorbents, such as magnesium oxide or alu mina, may resolve humic substances into more homogeneous fractions that will provide more useful structural information.

Summary This study illustrates some of the difficulties encountered because of the complex mixture characteristics of humic substances. When chemical prop erties of constituent fractions were additive, such as the molecular weight distributions and acid-group contents of Table IV, the skewed nature of the molecular weight distribution and the differential effects of électrostriction of water on acid-group density required the use of silica-gel fractionation to interpret the density-concentration curves of Figure 2. Because molecular interactions within fulvic acid change the properties of polar functional groups, the concept of minimum density calculation by Traubes rule (12) had to be introduced to detect the effect. Intermolecular interactions could not be distinguished from intramolecular interactions without silica-gel frac tionation data. This study, as well as a number of other reviews (20, 21), continues to identify the need to separate the complex mixture of humic substances into more homogeneous fractions, in order to extend our knowl edge of their molecular nature and properties.

References 1. Wershaw, R. L.; Marinsky, J. A. Presented at the 193rd National Meeting of the American Chemical Society, Denver, CO, April 1987. 2. Leenheer, J. Α.; Noyes, T. I. U.S. Geological Survey Water Supply Paper 2230 1984, 16 p. 3. Leenheer, J. A. Environ. Sci. Technol. 1981, 15, 578-587.



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LEENHEER ET AL.


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4. Leenheer, J. Α.; Huffman, E. W. D., Jr. J. Res. U.S. Geol. Surv. 1976, 4, 737-751. 5. Leenheer, J. Α.; Huffman, E. W. D., Jr. Water Resour. Invest. (U.S. Geol. Surv.) 1979, 79-4, 16 pp. 6. Thurman, E. M.; Malcolm, R. L.; Aiken, G. R. Anal. Chem. 1978, 50, 775-779. 7. Aiken, G. R. In Organic Pollutants in Water: Sampling, Analysis, and Toxicity Testing; Suffet, I. H.; Malaiyandi, M., Eds.; Advances in Chemistry 214; Amer ican Chemical Society: Washington, DC, 1987, p 295-307. 8. Lansing, W. D.; Kraemer, E. O. J. Am. Chem. Soc. 1935, 57, 1369-1377. 9. American Society for Testing and Materials. Petroleum Products and Lubricants: Method D-941-55; American Society for Testing and Materials: Philadelphia, 1966; pp 17, 310-315. 10. Pimentel, G.; McClellan, A. The Hydrogen Bond; W. H. Freeman: San Fran cisco, 1960; Chapter 5, pp 167-192. 11. Chebaevskii, A. I.; Tuev, Ν. Α.; Stepanova, N. P. Pochvovedenie 1971, 7, 31-37. 12. Traube, J. Ber. Dtsch. Chem. Ges. 1895, 28, 2722. 13. Brown, P. Α.; Leenheer, J. A. In Humic Substances in the Suwannee River, Florida and Georgia: Interactions, Properties, and Proposed Structures; Averett, R. C., Ed.; U.S. Geological Survey Water Supply Paper, in press. 14. Noyes, T. I.; Leenheer, J. A. In Humic Substances in the Suwannee River, Florida and Georgia: Interactions, Properties, and Proposed Structures; Averett, R. C., Ed.; U.S. Geological Survey Water Supply Paper, in press. 15. Davis, M. M. Acid-Base Behavior in Aprotic Organic Solvents; National Bureau of Standards Monograph 105; U.S. Government Printing Office: Washington, DC, 1968; 151 pp. 16. Hayes, M. H. B. In Humic Substances in Soil, Sediment, and Water: Geochem istry, Isolation, and Characterization; Aiken, G. R.; McKnight, D. M.; Wer shaw, R. L.; MacCarthy, P., Eds.; John Wiley and Sons: New York, 1985; pp 329-362. 17. Gurney, R. W. Ionic Processes in Solution; Constable and Company: London, 1953; pp 190-195. 18. Chiou, C. T.; Kile, D. E.; Brinton, T. I.; Malcolm, R. L.; Leenheer, J. A. Environ. Sci. Technol. 1987, 21(12), 1231-1234. 19. Carter, C. W.; Suffet, I. H. Environ. Sci. Technol. 1982, 16, 735-740. 20. Swift, R. S. In Humic Substances in Soil, Sediment, and Water: Geochemistry, Isolation, and Characterization; Aiken, G. R.; McKnight, D. M.; Wershaw, R. L.; MacCarthy, P., Eds.; John Wiley and Sons: New York, 1985; 387-408. 21. Leenheer, J. A. In Humic Substances in Soil, Sediment, and Water: Geochem istry, Isolation, and Characterization; Aiken, G. R.; McKnight, D. M.; Wer shaw, R. L.; MacCarthy, P., Eds.; John Wiley and Sons: New York, 1985; 409-429. RECEIVED for review October 21, 1987. ACCEPTED for publication December 21, 1987.


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