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Molecular weight of aquatic fulvic acids by vapor pressure osmometry
00167037/8763.00
+ .xl
Molecular weight of aquatic fulvic acids by vapor pressure osmometry GEORGE R. AKEN and RONALDL. MALCOLM U.S. Geological Survey, Water Resources Division, Box 25046, Denver Federal Center, MS 407, Denver, Co 80225, U.S.A. (Received October 22, 1986; accepted in revised form May 19, 1987) Abstract--The molecular weightsof aquatic fulvic acids extracted from five riverswere determined by vapor pressure osmometry with water and tetrahydrofuran as solvents. The values obtained ranged from 500 to 950 daltons, indicating that the molecular tights of aquatic fulvic acids are not as great as has been m in some other molecular weight studies. The samples were show-nto be relatively monodisperse from radii of gyration measurements determined by small angle x-ray scattering. THF affords greater precision and accuracy than Hz0 in VP0 measurements, and was found to be a suitable solvent for the determination of molecular weight of aquatic fulvic acid because it obviates the dissociation problem. An inverse correlation was observed with these samples between the concentration of Ca++ and Mg++ in the river water and the radii of gyration and molecular weights of the corresponding fulvic acid samples. solvents with low dielecvic constants. Dielectric con-
INTRODUCI’ION
stant is a measure of a solvent’s ability to solvate ions. MOLECULARWEIGHT is an integral property in understanding the physical and chemical characteristics of a compound. Determining the molecular weights of humic substances is a task complicated by the nature of these materials. Humic substances are complex mixtures of organic, polyelectrolytic acids that do not form ideal solutions. The problems associated with molecular weight determinations on mixtures have been recognized (LANSINGand KRAEMER, 1935). The usual methods for determining molecular weight yield molecular weight averages; depending on which of the methods is used, the results are not directly comparable. Vapor pressure osmometry (VPO) is a colliitive property method that has been used to determine molecular weights of humic substances (HANSEN and SCHNITZER,1969; WIMN and WEBER, 1977; REUTER and PERDUE, 198 1; GILLAMand RILEY, 198 1). By definition, a colligative property is a thermodynamic property that depends only on the number of particles in solution, and not on the nature of these particles. A “number-average” molecular weight, ?i;i, where every species present, regardless of weight, gives the same response, can be determined from colligative property measurements (MOORE, 1972). A significant problem, that can lead to incorrect m,, values for humic substances, is the dissociation of the organic acids in -aqueous solution. A molecular-weight value lower than the actual molecular weight is obtained for a dissociated organic acid (HANSENand SCHMTZER, 1969), because the dissociated protons are “counted” as solute molecules, increasing the value for the number of solute molecules present. The most common method for dealing with this problem has been to carry out the measurements in water, and to correct the data mathematically for dissociation (GILLAM and RILEY, 1981; REUTER and PERDUE, 198 1; AIKEN and GILLAM, 1987). Dissociation of protons from humic substances can be suppressed by making measurements in organic
In solvents with dielectric constants lower than lO- 15, ionization of an organic acid essentially is non-existent
and the solvent is class&d as non-polar (CHARLOT and TREMILLON,1969). It is important that the solute be sufficiently soluble in the solvent; that no side reand that the solvent actions, such as association, OCCUT; have a vapor pressure of 50 to 400 torr at the operating temperature to maintain a well-saturated vapor atmosphere around the thermistors (BURGE, 1977). In this sttidy, tetrahydrofimm (THF) is employed as a solvent for the vapor pressure osmometry (VPO) determination of molecular weights of aquatic fulvic acids. In THF, with a dielectric constant of 7.58, organic acids, such as fulvic acid, are present in the undissociated form, and VP0 data need not be corrected for the presence of dissociated protons. 2” data for six aquatic fulvic acids, representing five different environments, were obtained with THF as solvent. These values were compared with %” values for each of the samples obtained in water, and corrected for dissociation by the method of GILLAM and RILEY (198 1). EXPERIMENTAL PROCEDURE Reagents. The THF used in this study was HPLC grade from Burdick and Jackson. Naphthoic acid, toluic acid, hexanoic acid, tartaric acid, and ascorbic acid were high purity reagents (99 percent +) obtained from Aldrich Chemical Co. aqclodextrin and &cyclodextrin standards were also obtained from Aldrich Chemical Co. a tocopherol acetate and dls-tocopherol were obtained from ICN pharmaceuticals,Inc. All other reagents were analytical reagent grade. Samples. Samples of riverinefulvic acid were collected from 5 different sites in the United States (Table 1). The predominant vegetation types of the watershed, upstream of each sample site, are listed in Table 1;they representa wide diversity of source material for each fulvic acid sample. Each sample was extracted according to the method of THURMAN and MALCOLM (1981). in brief, river water was filtered through 0.45 pm silver-membrane filters, acidified to pH 2, isolated and concentrated by sorption onto XAD-8 resin, and eluted with 0.1 N NaOH. Humic acid was precig itated at pH 1 and separatedfrom fulvic acid by centrifiqation.
2177
G. R. Aiken and R. L. Malcolm
2178 Table 1.
Site descriptions and sampling dates for riverine fulvic acid samples.
Sample Coal Creek Ohio River
Sampling date
Site description SlaallstreamdrainingtheFlattopsWlldernessArea,Colorado. Vegetation type: Spruce-Fir Forest (Picea. Abies).
6182
Major river draining east central United States. Sampled at Cincinnati, Ohio. Vegetationtypes: Appalachianoak forest (Quercus), MixedNesophyticForest(Acer.Acsculus.Fagus,Liriodendron, Quercus. Tilla) and Oak Hickory Forest (Quercus. Carya).
10/81 4/02
Missouri River
8/81
Major river draining north central United States. Sampled at Sioux City. Iowa. Vegetation types: Northern Floodplain Forest (Populus. Salix, Ulmus) and Wheatgrass, Needlegrass,and Bluestem grassland (Agropyron. Stipa. Andropogon).
Ogeechee River
5182
Small river draining Piedmont. Sampled at Grange, Georgia. Vegetation types: Oak-Hickory-PineForest (Quercus. Carya. Pinus).
Suwannee River
12/17/02
River in southeasternGeorgia draining the Okefenokee Swamp. Sampled at Fargo, Georgia. Vegetation types: Southern Floodplain Forest (Quercus. Nyassa. Taxodium).
The fulvic acid then was desalted on XAD-8 resin, H-saturated by passing through AC-MP 50 cation-exchange resin and lyophiliaed. Elemental analysis was performed on each sample; these analyses are presented in Table 2. A review of the methods used for the determination of each element has been publi&edbyHUFFlvUN and STUBER(1985). Moltdar size and weight measurement. The radius of gyration of each of the samples was obtained by small angle xray scattering measurements, according to the method of W~HAW and PINCKNEY (1973). These measurements were made on 1 percent aqueous solutions of isolated aquatic htlvic acid at pH 10, using a Kratky’ small angle x-ray scattering goniometer. A Knauer vapor pressureosmometer was used to determine vapor pressures of the fulvic acid solutions in both water and THF. The THF was mdistihed immediately before dissolving the htlvic acid to eliminate peroxides and water from the solvent. Precise pH measurements were made on the aqueous solutions, using an Orion Model No. 810200 combination
’ Use of trade names in this report is for identification purposes only and does not constitute endorsement by the U.S. Geological Survey.
pH electrode. In all cases, the fulvic acid was dried at 60°C over P20J to a constant weight to eliminate moisture before dissolving in the appropriate solvent. A number of standards were used in each solvent system to determine the depet&nce of the apparatus constant, K_, with molecular weight. VPG analyses were carried out by HutTman Laboratories, Lakewood, colorado. Data analysis. A number of publications, most notably BONNAR et al. ( 1958) and CLOVER( 1975), have been devoted to discussing the methods used to make colhgative property measurements, and the analysis and treatment of data. The general expression for relating measured changes in colhgative properties to molecular weight (CLOVER,1975), is:
-*
e=e,+aW+bW2+cWJ.
where, for vapor pressureosmometry, 9 is the change in temperature of the thermistor Wis the weight ratio of solute to solvent, 0, is the value of 0 when W = 0, and a, b, and c are virial coefficients. The molecular weight of the solute, M, is related to the first virial coefficient, a, and the calibration constant, K,,,,,, by the following equation: z, = K&a.
(2)
Km is determined for a given solvent and osmometer system
Table2. Elementalcompositions,radiiofgyration and molecularweightsofaquaticfulvicacids. Radiusof gyrttion'
Elementalcomposition(percent) Sample
xn
C
H
0
N
S
Cl
Ash
(A)
THF
HissouriRiver
55.4
5.3
35.0
1.3
0.78
----
0.1
4.5
::: (540)
585 (640) 696
OMo River(fall)
55.5
5.4
35.9
1.5
1.28
1.15
0.57
6.2
556
594
55.0
5.4
35.9
1.5
1.28
0.25
0.38
5.7
554
607 (618) 629
Coal Creek
52.6
4.5
3G.4
0.95
0.7
0.78
1.23
8.3
790 (751) 713
743
Ogeechee Rfver
54
4.02
30.5
0.93
1.27
0.29
0.39
7.1
592 (592) 593
714
Suwannee River
54.2
3.92
38.0
0.72
0.35
1.01
0.19
a.8
840 (840) 841
959 (829) 689
(spring)
(1)
1. Average valuesofduplicateanalysesarein parentheses.
"20
Fulvic acid moiecuiar weights by determining the first virial coefficients for standards of
known molecular weight. The appropriate solution to Eqn. (1) wasobtained arithmetically by subjecting the data to three of the model equation procedures outlined by GLOVER (1975). Ihe three model pnxxxiures used were the first virial model appropriate for near ideal solutions (E~ONNAR et al., 1958); the second virial model appropriate for non-ideal solutions; and the first point zero method (BONNAR et al., 1958) appropriate for non-ideal behavior caused by zero-point error. The model that exhibited the best fit of the data was used to calculate the molecular weight. Account has not been taken in the previous discussion for the dissociation of acidic poiyelectrolytes, such as fulvic acid, in aqueous solution. AIICENand GILLAM (1987) reviewed several procedures that have been described in the literature to correct colligative property data for the dissociation of humic substances. The method of Giw and RILEY( 1981) which utilizes the solution pH and &, to correct for dissociation has been used in this work. RExJL-rs
Molecular size measurements Small-angle x-my scattering measurements were made on each sample to determine radii of gyration data (Table 2), and to estimate tbe degme of polydispersity of each sample. The radius of gyration of a molecule, which is defined as the root-mean-square distance of tbe electrons in tbe molecule from tbe center of charge, is a usefm general characteristic of comparative molecular size (WERSHAWand AKEN, 1985) and is related to molecular weight (TANFORD, 1961). The values for the radii of gyration for the aquatic fulvic acids reported in this paper range from of 8.8 A for the Suwannee River FA, to 4.5 A for the Missouri River FA. These values fall within the range of radii of gyration for aquatic humic substances reported by THURMAN et al. ( 1982). In the small-angle x-ray experiment, the radius of gyration, R, is given by the following expression:
--#R=
In I(h) = 3
+ constant
(3)
where I(h) is the intensity of scattered radiation, and h = 27r sin26/X, where 20 is the scattering angle, and A is the wavelength of the impinging x-my. The radius is calculated from the slope of tbe plot of In I(h) versus h* which is the so-called Guinier plot. For a monodisperse system, in which all of the scattering particles are of tbe same molecular size, the Guinier plot is a straight line; for a polydisperse system, the plot is concave upward. Each of the Guinier plots for the fulvic acid samples in this study is linear. Within the limits of the x-my experiment, the samples appear to be monodisperse; however, this term is misl~ng as fulvie acid is recognized as a complex mixture of organic acids. These samples are therefore said to be monodisperse, meaning that the distribution of particle sizes in the sample is narrow, rt 0.5 A (R. L. WERSHAW, pets. commun.). This information is relevant because am, which is the arithmetic mean of the weights of all molecules in the sample, is of little value for polydis-
2179
perse systems. Because each sample can be shown to be relatively monodisperse, the a,, values obtained by VPG are of significance. Further analysis is required to quanti~ tbe range of particle sixes present in each of the fulvic acid samples.
Vaporpressure osmometry results Because H20 has a high dielectric constant (78.54), a correction for dissociation must be applied for this solvent. The data obtained for tbe molecular weights of tbe organic acid standards determined in Hz0 are given in Table 3. The corrected values obtained for these standards are within a few percent of the actual values of tbe standards. The molecular weight values determined by VPG for tbe suite of aquatic fulvic acids are given in Table 2. The molecular weight values obtained in water range from a bigh value of 829 daltons for the Suwannee River FA to a low value of 594 dahons for the Ohio River FA. The problems of reproducibility for the weight values obtained in water are caused by a combination of the low K,, for water, and the ~fficulty of obtaining accurate pH measurements on low conductivity solutions. However, these values are in good agreement with molecular weights determined by other workers on aquatic fulvic acid utilizing colligative property measurements (WILSON and WEBER, 1977; GILLAMand RILEY, 198 1; REUTER and PERDUE, 198 1). The literature values range from 600 to 950 daltons and represent fulvic acids extracted from a variety of environments. With the exception of the Coal Creek FA, the s,, values obtained in THF are lower than those obtained in water, ranging from 536 daltons for the Missouri River FA to 840 daltons for the Suwannee FA. THF has a higher apparatus constant, Km, (Table 4) than water and is a more suitable solvent for VP0 than water is. A high apparatus constant is advantageous because of tbe greater change in instrument response (6) per molecule of solute. Reproducibility of meaTable 3. Molecular weight values of standards
determined by VPOin THFand
Solvent THF
X20
Standanf
H20.
Molfcularweight Percent calcuated actual difference
Hexanoic acid
120
116.16
+3.8
Toluicacid
136
139.26
+2.2
a-Naphthoic acid
173
172.12
+0.8
Menad+one
177
172.2
+3.2
Tacopherolacetatc
447
472.7
-5.3
Ascorbic acid
174 186
175
* 0.1 + 6.3
Tartar-kacid
149 154
150
- 0.2 t3.0
cr-cyclodextrln
1010
973
+3.8
,+cyclodextrin
1151
1135
+1.4
G. R. A.&enand R. L. Malcolm
2180
Table 4. Vapor pressureosmometry data for Suwannee Fulvic Acidfn THFand %$I.
Flnt virlal Response, coefflcent Concentrepon a"' Solvent (mglm L) pH (mv) Kanob
THF
8.07 10.24 12.83 16.01
---------
49.8 64.6 79.9 99.5
5266
x20
5.24 7.83 9.34 11.55
2.28 2.14 2.06 1.99
8.5 11.9 15.2 18.2
752
NW
6.27
840
0.78d
959
a Strictly speaking.theuse of tagfulvicacid/mL of solventfor thesoluteconcentrations is unconventional.A morn appropriate set of concentrationunits would be mg fulvicacid/gsolvent. The two are relatedby the factorgsolvent/mLsolution, which IS essentially constantover the range offulvlcacidconcentrations used in thfsstudy. The ewor. therefore,disappearsinto the apparatusconstant, K
librium ultracentrifugation. In addition, the data presented in this paper provides independent substantiation of the general dissociation correction methods of Gu_LAMand RIJ.EY( 1981)and REUTERand PERDUE (198 1). These authors illustrated the validity of their correction methods utilizing standards such as tartaric acid and benxenepentacarboxylic acid. Unfortunately, no good model compounds for aquatic fulvic acid exist, and it is reassuring to obtain similar results using two exeunt solvent systems. KESCUSSiON Efects ofisolation procedure on molecular weight of aquatic fulvic acid
Disagreement exists between the values obtained by VPG and the molecular weights reported in the literature determined by using ultral%ration and gel filtmtion chromatography (THURMANet al., 1982). TheoaW bK app determinedusingbenrilfor TR F and dextrose for H20. retically, these methods separate solutes based on molecular size; however, data are almost exchrsively ' Obtainedby quadraticregression. reported as molecular weight fractionations. It can be d Correctedfor dissociation. argued that, because these methods of analysis are subject to different constraints, the data they provide are not directly comparable. However, it is useful in the study of humic substances to evaluate the conclusions surements in THF was very good. In the case of the drawn from one type of analysis with those arrived at Suwannee FA, the solution appeared cloudy, indicating that the solubility of that particular FA in THF was by a different method. As pointed out by MJZYER( 1986) in a recently published report of ultraf%ration data for exceeded. In the VP0 experiment, a higher apparent the Ggeechee River, the molecular weights obtained molecular weight value will be obtained for a sample are subject to the chemical pro@%ties which does not completely dissolve in the solvent. It by ~~~tmtion of the substances being filtered. Meyer reported that, is sign&ant that despite some solubility probiems in for the Ogeechee River, approximately 60 percent of THF, the molecular weight values obtained are lower the dissolved organic carbon (DOC) was in the IOOflthan those obtained in water. Small amounts of water 10000 MW fraction and 20 percent of the DOC was in the fblvic acid will contribute significantly to the in the < 1000 MW &action. The an value obtained by overall colhgative effect in THF because each water VP0 for the fulvic acid sample isolated from the Ggeemolecule, with a molecular weight of 18, is counted as a solute molecule. The same water has only a minor thee River, which accounted for 40 percent of the DOC effect when the measurements are made in water, be- in this system, was 7 14 daltons. Based on the VP0 cause it only contributes to an error in actual sample data for the isolated f%vic acid, it would be expected weight. To minimize this problem, samples were dried that the
Fulvic acid molecular weights inorganic and organic constituents to form larger species that are retained by the ultrafihration membrane. Adjustment of the sample to pH 2 results in the disruption of these interactions, and upon passing the sample through the column of XAD-8 resin, the fulvic acid is retained while the other constituents pass through the column. The resulting fidvic acid sample, therefore, has been effectively isolated from other chemical species present in the original water sample. The low ash contents for the firlvic acids reported in Table 2 am evidence for this occurring. In the case of the Ogeechee sample, MEYER (1986) reports that the Ogeechee River has high concentrations of iron, and notes that interactions of the iron with the DOC may be a factor in the fm~oM~on data. The isolated fulvic acid from the Ogeechee River with an ash content of 0.39 percent is free of iron and has a lower molecular weight. A potentially more serious possibility for the study of aquatic fulvic acid is that the isolation procedure may result in chemical and structural alteration of the fulvic acid. Of particular significance is the possibility of ester hydrolysis in the presence of 0.1 N NaOH during the elution of the fulvic acid from the XAD resin. One result of ester hydrolysis would be the lowering of the molecular weight of the sample. Ester hydrolysis in the presence of base has been reported for soil humic substances by SCHNITZERand NEYROUD (1975), and for aquatic humic substances by L~AOet al. ( 1982). In the isolation of fulvic acid from water, contact time with 0.1 N NaOH is minimized to avoid ester hydrolysis. Data have been presented that suggest that ester hydrolysis is not a significant problem as far as the extraction of fulvic acid from water is concerned. MCKNIGHT et al. ( 1983) demonstrated that the Cu(I1) binding capacity of fulvic acid extracted from the Shawsheen River was not altered by the isolation procedure. In other studies utilizing colligative property measurements to determine molecular weights of aquatic firlvic acids, the isolated fuIvic acids were adjusted to pH 7 (REUTER and F%RDUE, 1981), to pH
2181
10 (GILLAM and RILEY, 198 1), and to pH 13 (WILSC)N and WEBER, 1977) during sample preparation. The general good agreement between molecular weight data presented by these authors and data presented in this study is further evidence that ester hydrolysis resulting from the isolation of &lvic acid from water is not a significant problem. Effects of divalent cations on molecular weightsof fulvic acids General water chemistry data for each of the sites sampled are contained in Table 5. The systems fall into two groups-those with high specific conductance and those with low specific conductance. Compa~ng the ;i?, values obtained by VP0 and the radii of gyration data with the water chemistry data, it is observed that the radii of gyration and the ;i?, values for the high specific conductance systems are noticeably lower than the values for the low conductance systems. Of particular significance are the values for Ca+’ and Mg+* in each of these groups. The sites with low specilic conductance values have con~ntmtions of Ca++ and Mg++ that are below the mean values for world rivers of 15 mg/L for Ca++ and 4.1 mg./L for Mgf+ (WETZEI., 1983) while the sites with high specific conductance values have Ca++ and Mg++ concentrations that are significantly higher than the mean values. Ca++ and Mg++ are the predominant cations neutralizing the charges on humic substances in the soil environment resulting in decreased ~lubi~~tion (HAYES and SWIM, 1978), and it appears that in the systems reported on in this paper the presence of these divalent cations suppresses the solubility of the higher molecular weight humic substances. A similar phenomenon has been noted in estuaries where, as salinity increases, high molecular weight fractions of humic substances ffocculate (SHOLKOVITZ,1978). The effect of the presence of divalent cations on the molecular weight of aquatic fulvic acid is manifested in many different systems. Recently, ERTELet al. ( 1986)
Table 5. General water chcllistry data for each site, and molecularweights detemined tn water for the cornspondlng fulvic acids.
Sample
MiSSOWl
pH
Specific conductance Cat* ( mhos) !JlqiL
7.9
Ohio (fall) 7.2
Ug*+ agfL
Re+ WL
K' WL
Alkalinity
DOC W./L
Suspended w sediment (da,~on~) sg/c
975
b3a
26"
81a
5.4a
n.a
3.4
313a
640
600
38b
Ilb
30b
2.6b
59b
3.4
9b
594
(spring)
7.5
330
38b
12b
18b
21b
79b
2.3
13b
618
Coal Creek
7.6
75
9
2.7
1.3
0.6
35
5.4
n.a.
743
Oqeechee
6.8
53
8’
1.5’
5.2’
1.1
23’
5.7
7’
714
Sunannee
4.2
47
0.58’
0.4ld
2.6d
0.15d
1.0
37
n.a.
829
n/a
- not available
a. Oata from U.S. GeologicalSurvey Rater Data Report IA-81-1,
Missouri River at Sioux City, Iowa.
b. Data from U.S. GeoloqicalSurvey Water Data Report KY-82-1.Ohfo River at Markland Dam. c. Data from U.S. GeologicalSurvey Yater Data Report GA-82-1, Oqeechee River at Eden, Georgia. d. Oata from Rykiel (1984).Suwannee River at Fargo. Georgia.
2182
G. R. Aiken and R. L. Malcolm
found that dissolved humic acids of the Rio Negro, which is low in suspended sediments, are removed from solution by adsorption on fine sediments below the confluence of the Rio Negro and the Rio Solinoes, which has a high sediment load. In the case of the present study, no correlation was observed between molecular weight of dissolved fulvic acid and the amount of suspended sediment. The Amazon system is characterized by waters that have low specific conductances and low concentrations of divalent cations. LEENHEER and SANTOS(1980) reported Ca+’ values of 0.17 mg/L and 10 mg/L and Mg++ values of 0.06 mg/L and 1.48 mg/L, respectively, for the Rio Negro and Rio Solinoes. The concentrations of Ca++ and Mg++ are an order of magnitude higher in the Rio Solinoes than in the Rio Negro. By suppressing the charge on the humic substances, Ca++ and Mg++ may play a significant role in precipitating humic substances from the river and by enhancing the sorption of humic substances by suspended sediment.
The data presented in this paper represent fulvic acid samples from a wide variety of aquatic environments-from dark water systems of low conductivities and high dissolved organic carbon inanitions, to river systems with high conductivity values and low dissolved organic carbon concentrations. The narrow range of molecular weights obtained for aquatic fulvic acids, 500-950 daltons, is controilcd by the effects of polarity and, most irn~~~y, with regard to this work, molecular size on soiubility. Molecules of high molecular weight that are fairly soluble in water are limited to moiecules that are very polar, such as polyacrylic acid and po1yethylene glycol. Large, polar biochemicals and secondary metabolitcs, such as carbohydrates, proteins and nucleic acids, do not occur in appreciable amounts in natural waters because they are labile. In the case of solutes such as aquatic fulvic acid, molecules have been selectively solubilized and separated from the potential pool of available organic compounds based on polarity and size considerations. In retrospect, it is not surprising to find that the upper limit for aquatic fulvic acid is less than 1000 daltons. The fact that the molecular weights of aquatic fulvic acids are low is a major constraint on structural modeling, and influences our understanding of the chemistry of these species. The chemical implications of low molecular weight can be observed by taking as an example Suwannee FA, which has been extensively studied. From the elemental analysis data and the molecular weight of 829 daltons (as determined in water) it can be shown that this sample is a heterogenous mixture. Only one out of every 2.6 molecules can contain a nitrogen atom, for instance. Similarly, not all molecuies can possess the same chemical characteristics or trace constituents. M&NIGHT et al. (1983) report the presence of two types of Cu(II) binding sites in aquatic fulvic acids. For the case of the Suwannee
sample, the concentrations of types I and II sites is 1.2 X 10T6and 2.7 X lo-’ moles/mg C, respectively. Only one out of every 2 molecules of this sample, therefore, can have a type I site, and only one out of every 8 molecules can have a type II site. Other structural information can be obtained by considering the distribution of functional groups with this sample. THURMAN and MAJXOLFA (1983) report that the carboxyl content of Suwannee FA is 6.0 mmol/gram. With a molecular weight of 829 daltons, this sample contains fewer than 5 carboxyl groups per molecule. These molecules, therefore, arc best described as oligoelectrolytes and should not be thought of as polyelectrolytes in the sense that polyacrylic acid is a polyelectrolyte. The low molecular weights of aquatic fulvic acids have important geochemical and biochemical implications. STEWARTand WETZEL (1981), for instance, have suggested that lower molecular weight fulvic acids are more inhibitory to calcite precipitation than higher molecular weight humic acids. In other work, CHIOU etal. (1986) have shown that significant solubility enhancements of hydrophobic organic pollutants and pesticides by humic substances can be described in terms of a partition-like interaction. The effectiveness of humic substances in enhancing solute solubility is largely controlled by the molecular size and polarity of the humic material, with larger soil-derived humic substances being more effective than low molecular weight aquatic fulvic acid. A relationship between the molecular weights of humic substances and their biological activity has also been noted (STEINBERG and MUENSTER, 1985). STEWART and WE-EEL (1982) have reported that humic substances of low apparent molecular weight are more stimulatory to both 14Cassimilation and alkaline phosphatase activity by algal-bacterial assemblages than humic substances of high apparent molecular weight. VEER (1985) has also noted the increased effect of lower molecular weight humic substances on the physiological activity of microbial cells. Others have noted that the presence of soil humic substances enhanced the uptake of iron by diatoms in laboratory studies (PROVASOLI, 1963; PRAKASH er al., 1973). While the biochemical mechanisms of this effect have not been elucidated, it is interesting to note that the molecular weights of aquatic fulvic acids arc similar to those of siderophores (approximately 400-650 daltons), and aquatic fulvic acid may be capable of passing cellular membranes. SUMMARY Small angle x-ray scattering measurements were made on aquatic fulvic acid samples to obtain radii of gyration data and to estimate the degree of polydisper&y of each sample. The samples were found to be relatively monodisperse, indicting that ;i?, values, as obtained by VPO, wouid be useful data in understanding the chemical nature of aquatic fulvic acid. an values were then obtained in water and in THF as solvents.
Fulvic acid molecular wei8hu
2183
metric number-average wei8hts of humic substances for dissociation. Chem. GeOt.33,355-366. GLOVPRC. A. (1975) Absolute colligative property measurements. In PolvmerMoltxxdar W&&s. Pt. 1 ted. P. E. SLADE JR.), pp. 79-159. Marcel Dekke< New York. HANSEN E. H. and SCHNITZRM. (1969) Molecular wei&t measurements of polycarboxylic acids in water by vapor pressure osmometry. Anal. Chim. Acta 46,247-254. HAYES M. H. B. and Swa R. S. (1978) The chemistry of soil colloids. In The Chemistry of Soil Co~t~t~~s feds. D. J. GREEMANDand M. H. B. Hnves), pp. 179-203. ~ey-~n~en~, New York. HUFF%%AN E. W. D. and STLIBERH. A. (1985) Analytical methodology for elemental analysis of humic substances. In Humic Substances in Soil, Sediment and Water (eds. G. R. AIKEN,D. M. MCKNIGHT,R. L. WERSHAWand P. M&CAR-THY),pp. 433-456. John Wiley and Sons, New York. LANSING W. D. and RRAEMER E. 0. (1935) Molecular wei@ analysis of mixtures by sedimentation equilibrium on the Svedbeq ultracentrifuge. f. Amer. Chem. Sot. 57, 13691377. LEENHIZER J. A. and Smos U. M. (1980) Consideracoes sobre os pmcessos de ~rnen~~o no aqua preta acida do rio Negro (Amaxonia Central). Acta Amazonica IO, 343355. LIAOw., cHtu!XMANR. F., JOHNSON J. D. and MILL~NGTON D. S. (1982) Structural characterization of aquatic humic material. Environ. Sri. Technol. 16,403-4 10. MCKNIGHT D. M., FEDERG. L., THURMAN EM., WERSHAW R. L. and W~~TALL J. L. ( 1983)Complexion of copper by aquatic humic substances horn different environments. Sci. Tot. Environ. 28,65-76. MEYERJ. L. (1986) Dissolved organic carbon dynamics in two subttopical blackwater rivers. Arch. H~robio~. 108, 119-134. MOORE W. J. (1972) Ph~ic~ Chemists, 4th ed. PrenticeHall, Eqlewood Cliffs, N.J. 977p. PRAKASH A., RASHIDM. A. and RAOD. V. S. (1973)Influence Acknowledgements-The authors wish to express their 8ratof humic substances on the growth of marine phytoplankitude to D. M. McKninht. E. M. Perdue. R. A. Thorn. and ton: Diatoms. Limnoi. Oceanogr.18,s 16-524. several anonymous r&w& for their c4&tmctive criticisms f’ROVASOLIL. (1963) Organic regulation of phytoplankton on the manuscript. fmty. In TheSea. Vol.2, pp. 165-219.Wiley-Interscience, New York. REUTERJ. H. and PERDUEE. M. ( 1981)Calculation of moEditoriul handling:J. I. Hedges lecular weights of humic substances from colligative data: Application to aquatic humus and its molecular size Rattions. Geochim. Cosm~him. Acta 45,2017-2022. RYKIELE. J. ( 1984)General hydrology and mineral budgets AXEN G. R. (1984) Evaluation of ultrafiltration for deterfor Okefenokee Swamp: Ecologicalsignificance in the Okemining molecular weight of fidvic acid. Environ. Sci. Techfenokee Swamp (eds. A. D. COHEN,D. J. CASAGRANDE, nol. 18,978-98 1. M. J. ANDWKO and G. R. Bzs~), pp. 212-228. Wetland AIKEN G. R. and GILLAMA. H. (1987) Determination of Surveys, Los Alamos, New Mexico. molecular weights of humic substances by colliknuiveprop SCHNITZERM. and NEYROUD J. A. (1975) Alkanes and fatty erty measurements. In Humic Substances II. StructuralAsacids in humic substances. Fuel 54, 17- 19. pects (als. M. HAYES,R. MA~ARTHY, R MAuxlLM and SHOLKOV~Z: E. R., BOYLEE. A. and PRKEN. B. ( 1978)The R. SWIFT).J. Wiley and Sons. Chichester (in mess). removal of dissolved humic acids and iron during estuarine BONNAR R.V., Du.&ATM. and SrItiXs F. H:(1958)~r&er mixing. Earth Planet. Sci. L&t. 40, 130-136. AverageMoiec&r Weights.Wiley-In&science, New York, STEIF~ERG C. and MUENSTER U. (1985) Geochemistry and 31Op. ecolqical role of humic substances in lakewater. In Humic BURGE D. E. (1977) Mo~~ weight Davison by osSubstances in Soil, Sedim~t and Water feds. G. R. AIKEN. mometty. Amer. tib. 9,4 l-5 1. D. M. MCRN~GHT, R. L. WERSHAW and P. MCCARTHY), CH~OUC. T., M,uco~~ R. L., BRINIQNT. I. and RILED. E. pp. 10.5-145.John Wiley and Sons, New York. ( 1986)Water solubihty enhancement of some organic polA. J. and WETZELR. G. (1981) Dissolved humic lutants and pesticides by dissolved humic aod fulvic acids. STEWART matem& Photcdegradation, sediment effects,and reactivity Environ. Sci. Techtwl. X&502-508. with phosphate and calciumprecipitation. Arch. Hydrobiof. CHARLOT G. and TREMILLION B. ( 1969)Chemical Reactions 92,265-286. in Solvents and Melts. Per8amon Press, Oxford, 528~. ERTEL J. R., HEDGESJ. I., ~EVOL A. H., RKHEYJ. E. and STEWART A. J. and WETZELR. G. (1982) Influence of disRmztno M. (1986) Dissolved humic substances of the solved humic material on carbon assimilation and alkaline phosphatase activity in natural algalbacterial assemblages. Amazon River system. Linmol. Oceanogr.37,739-754. Freshwater Bioi. 12, 369-380. GILLAMA. H. and BLEY J. P. (1981) Cormction of ammo-
Because THF has a low dielectric constant, the acidic protons on fulvic acid do not dissociate, and the VP0 data do not need correction for Mation. The molecular weight values obtained in THF compared favorably with the data obtained in water, and THF was found to be a useful solvent for determining molecular weights of aquatic fulvic acids. in water, %,, values for aquatic hlvic acid ranged from 500 to 950 dahons; these values are not as large as has been suggesmd in other molecular weight studies utilizing ultiltration or gel filtration; however, a number of factors may contribute to this difference. While ultrafiltration and gel filtration are subject to interactions which can lead to higher molecular weight fractionation values, it is a strong possibility that the pH adjustments used in the isolation of aquatic fulvic acid disrupt interactions between the fulvic acid and other chemical constituents in the water, such as silica and iron, resulting in lower molecular weights for the isolated materials. A correlation was observed between the concxmtrations of Ca++ and Mg++ in the river water and the radii of gyration and mohxular weights of the corresponding fulvic acid samples. It appears that the presence of these divalent cations in the soils of the watershed and in the river water deemases the solubility of higher molecular weight components of the humic substances by suppressing charge on the organic polyanions. The lower molecular weight values and the small range of values obtained for aquatic fulvic acid are probably the result of sohtbility constraints for this class of compounds in water.
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TANFORD C. (1961) Physical Chemistry of Macromolecules.
John Wiley and Sons, New York. THURMAN E. M. and MALCOLMR. L. (1981) Preparative isolation of aquatic humic substances. Environ. Sci. Technol. 15,463-466.
THURMANE. M. and MALCOLMR. L. (1983) Structural study of humic substances: New approaches and methods. In Aquatic and Terrestrial Humic Materials (eds. R. F. CHRISTMANand E. T. GJESING), pp. l-24. Ann Arbor Science, Ann Arbor, Michigan. THURMAN E. M., WERHSAW R. L., MALCOLMR. L. and PINCKNEY D. J. (1982) Molecular size of aquatic humic substances. Org. Geochem. 4,27-35. VISSERS. A. (1985) Physiological action of humic substances on microbial cells. Soil Bil. B&hem. 17,457-462.
WERSHAWR. L. and AIKENG. R. (1985) Molecular size and weight measurements of humic substances. In Humic Substances in Soil. Sediment and Water (e&. G. R. AIKEN, D. M. MIXNIGHT, R. L. WERHSAWand P. MAcCA~I’I.N), pp. 477-492. John Wiley and Sons, Inc., New York. WERSHAWR. L. and PINCXNEYD. J. (1973) Determination of the association and dissociation of humic acid fractions by small angle x-ray scattering. J. Res. U.S. Geol. Surv. 1, 701-707. WETZELR. G. (I 983) Limnology. Saunders College Publishing, Philadelphia, 767~. WILSONS. A. and WEBERJ. H. (1977) A comparative study of number-average dissociation corrected molecular weights of fulvic acids isolated from water and soil. Chem. Geol. 19,285-293.
+ .xl
Molecular weight of aquatic fulvic acids by vapor pressure osmometry GEORGE R. AKEN and RONALDL. MALCOLM U.S. Geological Survey, Water Resources Division, Box 25046, Denver Federal Center, MS 407, Denver, Co 80225, U.S.A. (Received October 22, 1986; accepted in revised form May 19, 1987) Abstract--The molecular weightsof aquatic fulvic acids extracted from five riverswere determined by vapor pressure osmometry with water and tetrahydrofuran as solvents. The values obtained ranged from 500 to 950 daltons, indicating that the molecular tights of aquatic fulvic acids are not as great as has been m in some other molecular weight studies. The samples were show-nto be relatively monodisperse from radii of gyration measurements determined by small angle x-ray scattering. THF affords greater precision and accuracy than Hz0 in VP0 measurements, and was found to be a suitable solvent for the determination of molecular weight of aquatic fulvic acid because it obviates the dissociation problem. An inverse correlation was observed with these samples between the concentration of Ca++ and Mg++ in the river water and the radii of gyration and molecular weights of the corresponding fulvic acid samples. solvents with low dielecvic constants. Dielectric con-
INTRODUCI’ION
stant is a measure of a solvent’s ability to solvate ions. MOLECULARWEIGHT is an integral property in understanding the physical and chemical characteristics of a compound. Determining the molecular weights of humic substances is a task complicated by the nature of these materials. Humic substances are complex mixtures of organic, polyelectrolytic acids that do not form ideal solutions. The problems associated with molecular weight determinations on mixtures have been recognized (LANSINGand KRAEMER, 1935). The usual methods for determining molecular weight yield molecular weight averages; depending on which of the methods is used, the results are not directly comparable. Vapor pressure osmometry (VPO) is a colliitive property method that has been used to determine molecular weights of humic substances (HANSEN and SCHNITZER,1969; WIMN and WEBER, 1977; REUTER and PERDUE, 198 1; GILLAMand RILEY, 198 1). By definition, a colligative property is a thermodynamic property that depends only on the number of particles in solution, and not on the nature of these particles. A “number-average” molecular weight, ?i;i, where every species present, regardless of weight, gives the same response, can be determined from colligative property measurements (MOORE, 1972). A significant problem, that can lead to incorrect m,, values for humic substances, is the dissociation of the organic acids in -aqueous solution. A molecular-weight value lower than the actual molecular weight is obtained for a dissociated organic acid (HANSENand SCHMTZER, 1969), because the dissociated protons are “counted” as solute molecules, increasing the value for the number of solute molecules present. The most common method for dealing with this problem has been to carry out the measurements in water, and to correct the data mathematically for dissociation (GILLAM and RILEY, 1981; REUTER and PERDUE, 198 1; AIKEN and GILLAM, 1987). Dissociation of protons from humic substances can be suppressed by making measurements in organic
In solvents with dielectric constants lower than lO- 15, ionization of an organic acid essentially is non-existent
and the solvent is class&d as non-polar (CHARLOT and TREMILLON,1969). It is important that the solute be sufficiently soluble in the solvent; that no side reand that the solvent actions, such as association, OCCUT; have a vapor pressure of 50 to 400 torr at the operating temperature to maintain a well-saturated vapor atmosphere around the thermistors (BURGE, 1977). In this sttidy, tetrahydrofimm (THF) is employed as a solvent for the vapor pressure osmometry (VPO) determination of molecular weights of aquatic fulvic acids. In THF, with a dielectric constant of 7.58, organic acids, such as fulvic acid, are present in the undissociated form, and VP0 data need not be corrected for the presence of dissociated protons. 2” data for six aquatic fulvic acids, representing five different environments, were obtained with THF as solvent. These values were compared with %” values for each of the samples obtained in water, and corrected for dissociation by the method of GILLAM and RILEY (198 1). EXPERIMENTAL PROCEDURE Reagents. The THF used in this study was HPLC grade from Burdick and Jackson. Naphthoic acid, toluic acid, hexanoic acid, tartaric acid, and ascorbic acid were high purity reagents (99 percent +) obtained from Aldrich Chemical Co. aqclodextrin and &cyclodextrin standards were also obtained from Aldrich Chemical Co. a tocopherol acetate and dls-tocopherol were obtained from ICN pharmaceuticals,Inc. All other reagents were analytical reagent grade. Samples. Samples of riverinefulvic acid were collected from 5 different sites in the United States (Table 1). The predominant vegetation types of the watershed, upstream of each sample site, are listed in Table 1;they representa wide diversity of source material for each fulvic acid sample. Each sample was extracted according to the method of THURMAN and MALCOLM (1981). in brief, river water was filtered through 0.45 pm silver-membrane filters, acidified to pH 2, isolated and concentrated by sorption onto XAD-8 resin, and eluted with 0.1 N NaOH. Humic acid was precig itated at pH 1 and separatedfrom fulvic acid by centrifiqation.
2177
G. R. Aiken and R. L. Malcolm
2178 Table 1.
Site descriptions and sampling dates for riverine fulvic acid samples.
Sample Coal Creek Ohio River
Sampling date
Site description SlaallstreamdrainingtheFlattopsWlldernessArea,Colorado. Vegetation type: Spruce-Fir Forest (Picea. Abies).
6182
Major river draining east central United States. Sampled at Cincinnati, Ohio. Vegetationtypes: Appalachianoak forest (Quercus), MixedNesophyticForest(Acer.Acsculus.Fagus,Liriodendron, Quercus. Tilla) and Oak Hickory Forest (Quercus. Carya).
10/81 4/02
Missouri River
8/81
Major river draining north central United States. Sampled at Sioux City. Iowa. Vegetation types: Northern Floodplain Forest (Populus. Salix, Ulmus) and Wheatgrass, Needlegrass,and Bluestem grassland (Agropyron. Stipa. Andropogon).
Ogeechee River
5182
Small river draining Piedmont. Sampled at Grange, Georgia. Vegetation types: Oak-Hickory-PineForest (Quercus. Carya. Pinus).
Suwannee River
12/17/02
River in southeasternGeorgia draining the Okefenokee Swamp. Sampled at Fargo, Georgia. Vegetation types: Southern Floodplain Forest (Quercus. Nyassa. Taxodium).
The fulvic acid then was desalted on XAD-8 resin, H-saturated by passing through AC-MP 50 cation-exchange resin and lyophiliaed. Elemental analysis was performed on each sample; these analyses are presented in Table 2. A review of the methods used for the determination of each element has been publi&edbyHUFFlvUN and STUBER(1985). Moltdar size and weight measurement. The radius of gyration of each of the samples was obtained by small angle xray scattering measurements, according to the method of W~HAW and PINCKNEY (1973). These measurements were made on 1 percent aqueous solutions of isolated aquatic htlvic acid at pH 10, using a Kratky’ small angle x-ray scattering goniometer. A Knauer vapor pressureosmometer was used to determine vapor pressures of the fulvic acid solutions in both water and THF. The THF was mdistihed immediately before dissolving the htlvic acid to eliminate peroxides and water from the solvent. Precise pH measurements were made on the aqueous solutions, using an Orion Model No. 810200 combination
’ Use of trade names in this report is for identification purposes only and does not constitute endorsement by the U.S. Geological Survey.
pH electrode. In all cases, the fulvic acid was dried at 60°C over P20J to a constant weight to eliminate moisture before dissolving in the appropriate solvent. A number of standards were used in each solvent system to determine the depet&nce of the apparatus constant, K_, with molecular weight. VPG analyses were carried out by HutTman Laboratories, Lakewood, colorado. Data analysis. A number of publications, most notably BONNAR et al. ( 1958) and CLOVER( 1975), have been devoted to discussing the methods used to make colhgative property measurements, and the analysis and treatment of data. The general expression for relating measured changes in colhgative properties to molecular weight (CLOVER,1975), is:
-*
e=e,+aW+bW2+cWJ.
where, for vapor pressureosmometry, 9 is the change in temperature of the thermistor Wis the weight ratio of solute to solvent, 0, is the value of 0 when W = 0, and a, b, and c are virial coefficients. The molecular weight of the solute, M, is related to the first virial coefficient, a, and the calibration constant, K,,,,,, by the following equation: z, = K&a.
(2)
Km is determined for a given solvent and osmometer system
Table2. Elementalcompositions,radiiofgyration and molecularweightsofaquaticfulvicacids. Radiusof gyrttion'
Elementalcomposition(percent) Sample
xn
C
H
0
N
S
Cl
Ash
(A)
THF
HissouriRiver
55.4
5.3
35.0
1.3
0.78
----
0.1
4.5
::: (540)
585 (640) 696
OMo River(fall)
55.5
5.4
35.9
1.5
1.28
1.15
0.57
6.2
556
594
55.0
5.4
35.9
1.5
1.28
0.25
0.38
5.7
554
607 (618) 629
Coal Creek
52.6
4.5
3G.4
0.95
0.7
0.78
1.23
8.3
790 (751) 713
743
Ogeechee Rfver
54
4.02
30.5
0.93
1.27
0.29
0.39
7.1
592 (592) 593
714
Suwannee River
54.2
3.92
38.0
0.72
0.35
1.01
0.19
a.8
840 (840) 841
959 (829) 689
(spring)
(1)
1. Average valuesofduplicateanalysesarein parentheses.
"20
Fulvic acid moiecuiar weights by determining the first virial coefficients for standards of
known molecular weight. The appropriate solution to Eqn. (1) wasobtained arithmetically by subjecting the data to three of the model equation procedures outlined by GLOVER (1975). Ihe three model pnxxxiures used were the first virial model appropriate for near ideal solutions (E~ONNAR et al., 1958); the second virial model appropriate for non-ideal solutions; and the first point zero method (BONNAR et al., 1958) appropriate for non-ideal behavior caused by zero-point error. The model that exhibited the best fit of the data was used to calculate the molecular weight. Account has not been taken in the previous discussion for the dissociation of acidic poiyelectrolytes, such as fulvic acid, in aqueous solution. AIICENand GILLAM (1987) reviewed several procedures that have been described in the literature to correct colligative property data for the dissociation of humic substances. The method of Giw and RILEY( 1981) which utilizes the solution pH and &, to correct for dissociation has been used in this work. RExJL-rs
Molecular size measurements Small-angle x-my scattering measurements were made on each sample to determine radii of gyration data (Table 2), and to estimate tbe degme of polydispersity of each sample. The radius of gyration of a molecule, which is defined as the root-mean-square distance of tbe electrons in tbe molecule from tbe center of charge, is a usefm general characteristic of comparative molecular size (WERSHAWand AKEN, 1985) and is related to molecular weight (TANFORD, 1961). The values for the radii of gyration for the aquatic fulvic acids reported in this paper range from of 8.8 A for the Suwannee River FA, to 4.5 A for the Missouri River FA. These values fall within the range of radii of gyration for aquatic humic substances reported by THURMAN et al. ( 1982). In the small-angle x-ray experiment, the radius of gyration, R, is given by the following expression:
--#R=
In I(h) = 3
+ constant
(3)
where I(h) is the intensity of scattered radiation, and h = 27r sin26/X, where 20 is the scattering angle, and A is the wavelength of the impinging x-my. The radius is calculated from the slope of tbe plot of In I(h) versus h* which is the so-called Guinier plot. For a monodisperse system, in which all of the scattering particles are of tbe same molecular size, the Guinier plot is a straight line; for a polydisperse system, the plot is concave upward. Each of the Guinier plots for the fulvic acid samples in this study is linear. Within the limits of the x-my experiment, the samples appear to be monodisperse; however, this term is misl~ng as fulvie acid is recognized as a complex mixture of organic acids. These samples are therefore said to be monodisperse, meaning that the distribution of particle sizes in the sample is narrow, rt 0.5 A (R. L. WERSHAW, pets. commun.). This information is relevant because am, which is the arithmetic mean of the weights of all molecules in the sample, is of little value for polydis-
2179
perse systems. Because each sample can be shown to be relatively monodisperse, the a,, values obtained by VPG are of significance. Further analysis is required to quanti~ tbe range of particle sixes present in each of the fulvic acid samples.
Vaporpressure osmometry results Because H20 has a high dielectric constant (78.54), a correction for dissociation must be applied for this solvent. The data obtained for tbe molecular weights of tbe organic acid standards determined in Hz0 are given in Table 3. The corrected values obtained for these standards are within a few percent of the actual values of tbe standards. The molecular weight values determined by VPG for tbe suite of aquatic fulvic acids are given in Table 2. The molecular weight values obtained in water range from a bigh value of 829 daltons for the Suwannee River FA to a low value of 594 dahons for the Ohio River FA. The problems of reproducibility for the weight values obtained in water are caused by a combination of the low K,, for water, and the ~fficulty of obtaining accurate pH measurements on low conductivity solutions. However, these values are in good agreement with molecular weights determined by other workers on aquatic fulvic acid utilizing colligative property measurements (WILSON and WEBER, 1977; GILLAMand RILEY, 198 1; REUTER and PERDUE, 198 1). The literature values range from 600 to 950 daltons and represent fulvic acids extracted from a variety of environments. With the exception of the Coal Creek FA, the s,, values obtained in THF are lower than those obtained in water, ranging from 536 daltons for the Missouri River FA to 840 daltons for the Suwannee FA. THF has a higher apparatus constant, Km, (Table 4) than water and is a more suitable solvent for VP0 than water is. A high apparatus constant is advantageous because of tbe greater change in instrument response (6) per molecule of solute. Reproducibility of meaTable 3. Molecular weight values of standards
determined by VPOin THFand
Solvent THF
X20
Standanf
H20.
Molfcularweight Percent calcuated actual difference
Hexanoic acid
120
116.16
+3.8
Toluicacid
136
139.26
+2.2
a-Naphthoic acid
173
172.12
+0.8
Menad+one
177
172.2
+3.2
Tacopherolacetatc
447
472.7
-5.3
Ascorbic acid
174 186
175
* 0.1 + 6.3
Tartar-kacid
149 154
150
- 0.2 t3.0
cr-cyclodextrln
1010
973
+3.8
,+cyclodextrin
1151
1135
+1.4
G. R. A.&enand R. L. Malcolm
2180
Table 4. Vapor pressureosmometry data for Suwannee Fulvic Acidfn THFand %$I.
Flnt virlal Response, coefflcent Concentrepon a"' Solvent (mglm L) pH (mv) Kanob
THF
8.07 10.24 12.83 16.01
---------
49.8 64.6 79.9 99.5
5266
x20
5.24 7.83 9.34 11.55
2.28 2.14 2.06 1.99
8.5 11.9 15.2 18.2
752
NW
6.27
840
0.78d
959
a Strictly speaking.theuse of tagfulvicacid/mL of solventfor thesoluteconcentrations is unconventional.A morn appropriate set of concentrationunits would be mg fulvicacid/gsolvent. The two are relatedby the factorgsolvent/mLsolution, which IS essentially constantover the range offulvlcacidconcentrations used in thfsstudy. The ewor. therefore,disappearsinto the apparatusconstant, K
librium ultracentrifugation. In addition, the data presented in this paper provides independent substantiation of the general dissociation correction methods of Gu_LAMand RIJ.EY( 1981)and REUTERand PERDUE (198 1). These authors illustrated the validity of their correction methods utilizing standards such as tartaric acid and benxenepentacarboxylic acid. Unfortunately, no good model compounds for aquatic fulvic acid exist, and it is reassuring to obtain similar results using two exeunt solvent systems. KESCUSSiON Efects ofisolation procedure on molecular weight of aquatic fulvic acid
Disagreement exists between the values obtained by VPG and the molecular weights reported in the literature determined by using ultral%ration and gel filtmtion chromatography (THURMANet al., 1982). TheoaW bK app determinedusingbenrilfor TR F and dextrose for H20. retically, these methods separate solutes based on molecular size; however, data are almost exchrsively ' Obtainedby quadraticregression. reported as molecular weight fractionations. It can be d Correctedfor dissociation. argued that, because these methods of analysis are subject to different constraints, the data they provide are not directly comparable. However, it is useful in the study of humic substances to evaluate the conclusions surements in THF was very good. In the case of the drawn from one type of analysis with those arrived at Suwannee FA, the solution appeared cloudy, indicating that the solubility of that particular FA in THF was by a different method. As pointed out by MJZYER( 1986) in a recently published report of ultraf%ration data for exceeded. In the VP0 experiment, a higher apparent the Ggeechee River, the molecular weights obtained molecular weight value will be obtained for a sample are subject to the chemical pro@%ties which does not completely dissolve in the solvent. It by ~~~tmtion of the substances being filtered. Meyer reported that, is sign&ant that despite some solubility probiems in for the Ogeechee River, approximately 60 percent of THF, the molecular weight values obtained are lower the dissolved organic carbon (DOC) was in the IOOflthan those obtained in water. Small amounts of water 10000 MW fraction and 20 percent of the DOC was in the fblvic acid will contribute significantly to the in the < 1000 MW &action. The an value obtained by overall colhgative effect in THF because each water VP0 for the fulvic acid sample isolated from the Ggeemolecule, with a molecular weight of 18, is counted as a solute molecule. The same water has only a minor thee River, which accounted for 40 percent of the DOC effect when the measurements are made in water, be- in this system, was 7 14 daltons. Based on the VP0 cause it only contributes to an error in actual sample data for the isolated f%vic acid, it would be expected weight. To minimize this problem, samples were dried that the
Fulvic acid molecular weights inorganic and organic constituents to form larger species that are retained by the ultrafihration membrane. Adjustment of the sample to pH 2 results in the disruption of these interactions, and upon passing the sample through the column of XAD-8 resin, the fulvic acid is retained while the other constituents pass through the column. The resulting fidvic acid sample, therefore, has been effectively isolated from other chemical species present in the original water sample. The low ash contents for the firlvic acids reported in Table 2 am evidence for this occurring. In the case of the Ogeechee sample, MEYER (1986) reports that the Ogeechee River has high concentrations of iron, and notes that interactions of the iron with the DOC may be a factor in the fm~oM~on data. The isolated fulvic acid from the Ogeechee River with an ash content of 0.39 percent is free of iron and has a lower molecular weight. A potentially more serious possibility for the study of aquatic fulvic acid is that the isolation procedure may result in chemical and structural alteration of the fulvic acid. Of particular significance is the possibility of ester hydrolysis in the presence of 0.1 N NaOH during the elution of the fulvic acid from the XAD resin. One result of ester hydrolysis would be the lowering of the molecular weight of the sample. Ester hydrolysis in the presence of base has been reported for soil humic substances by SCHNITZERand NEYROUD (1975), and for aquatic humic substances by L~AOet al. ( 1982). In the isolation of fulvic acid from water, contact time with 0.1 N NaOH is minimized to avoid ester hydrolysis. Data have been presented that suggest that ester hydrolysis is not a significant problem as far as the extraction of fulvic acid from water is concerned. MCKNIGHT et al. ( 1983) demonstrated that the Cu(I1) binding capacity of fulvic acid extracted from the Shawsheen River was not altered by the isolation procedure. In other studies utilizing colligative property measurements to determine molecular weights of aquatic firlvic acids, the isolated fuIvic acids were adjusted to pH 7 (REUTER and F%RDUE, 1981), to pH
2181
10 (GILLAM and RILEY, 198 1), and to pH 13 (WILSC)N and WEBER, 1977) during sample preparation. The general good agreement between molecular weight data presented by these authors and data presented in this study is further evidence that ester hydrolysis resulting from the isolation of &lvic acid from water is not a significant problem. Effects of divalent cations on molecular weightsof fulvic acids General water chemistry data for each of the sites sampled are contained in Table 5. The systems fall into two groups-those with high specific conductance and those with low specific conductance. Compa~ng the ;i?, values obtained by VP0 and the radii of gyration data with the water chemistry data, it is observed that the radii of gyration and the ;i?, values for the high specific conductance systems are noticeably lower than the values for the low conductance systems. Of particular significance are the values for Ca+’ and Mg+* in each of these groups. The sites with low specilic conductance values have con~ntmtions of Ca++ and Mg++ that are below the mean values for world rivers of 15 mg/L for Ca++ and 4.1 mg./L for Mgf+ (WETZEI., 1983) while the sites with high specific conductance values have Ca++ and Mg++ concentrations that are significantly higher than the mean values. Ca++ and Mg++ are the predominant cations neutralizing the charges on humic substances in the soil environment resulting in decreased ~lubi~~tion (HAYES and SWIM, 1978), and it appears that in the systems reported on in this paper the presence of these divalent cations suppresses the solubility of the higher molecular weight humic substances. A similar phenomenon has been noted in estuaries where, as salinity increases, high molecular weight fractions of humic substances ffocculate (SHOLKOVITZ,1978). The effect of the presence of divalent cations on the molecular weight of aquatic fulvic acid is manifested in many different systems. Recently, ERTELet al. ( 1986)
Table 5. General water chcllistry data for each site, and molecularweights detemined tn water for the cornspondlng fulvic acids.
Sample
MiSSOWl
pH
Specific conductance Cat* ( mhos) !JlqiL
7.9
Ohio (fall) 7.2
Ug*+ agfL
Re+ WL
K' WL
Alkalinity
DOC W./L
Suspended w sediment (da,~on~) sg/c
975
b3a
26"
81a
5.4a
n.a
3.4
313a
640
600
38b
Ilb
30b
2.6b
59b
3.4
9b
594
(spring)
7.5
330
38b
12b
18b
21b
79b
2.3
13b
618
Coal Creek
7.6
75
9
2.7
1.3
0.6
35
5.4
n.a.
743
Oqeechee
6.8
53
8’
1.5’
5.2’
1.1
23’
5.7
7’
714
Sunannee
4.2
47
0.58’
0.4ld
2.6d
0.15d
1.0
37
n.a.
829
n/a
- not available
a. Oata from U.S. GeologicalSurvey Rater Data Report IA-81-1,
Missouri River at Sioux City, Iowa.
b. Data from U.S. GeoloqicalSurvey Water Data Report KY-82-1.Ohfo River at Markland Dam. c. Data from U.S. GeologicalSurvey Yater Data Report GA-82-1, Oqeechee River at Eden, Georgia. d. Oata from Rykiel (1984).Suwannee River at Fargo. Georgia.
2182
G. R. Aiken and R. L. Malcolm
found that dissolved humic acids of the Rio Negro, which is low in suspended sediments, are removed from solution by adsorption on fine sediments below the confluence of the Rio Negro and the Rio Solinoes, which has a high sediment load. In the case of the present study, no correlation was observed between molecular weight of dissolved fulvic acid and the amount of suspended sediment. The Amazon system is characterized by waters that have low specific conductances and low concentrations of divalent cations. LEENHEER and SANTOS(1980) reported Ca+’ values of 0.17 mg/L and 10 mg/L and Mg++ values of 0.06 mg/L and 1.48 mg/L, respectively, for the Rio Negro and Rio Solinoes. The concentrations of Ca++ and Mg++ are an order of magnitude higher in the Rio Solinoes than in the Rio Negro. By suppressing the charge on the humic substances, Ca++ and Mg++ may play a significant role in precipitating humic substances from the river and by enhancing the sorption of humic substances by suspended sediment.
The data presented in this paper represent fulvic acid samples from a wide variety of aquatic environments-from dark water systems of low conductivities and high dissolved organic carbon inanitions, to river systems with high conductivity values and low dissolved organic carbon concentrations. The narrow range of molecular weights obtained for aquatic fulvic acids, 500-950 daltons, is controilcd by the effects of polarity and, most irn~~~y, with regard to this work, molecular size on soiubility. Molecules of high molecular weight that are fairly soluble in water are limited to moiecules that are very polar, such as polyacrylic acid and po1yethylene glycol. Large, polar biochemicals and secondary metabolitcs, such as carbohydrates, proteins and nucleic acids, do not occur in appreciable amounts in natural waters because they are labile. In the case of solutes such as aquatic fulvic acid, molecules have been selectively solubilized and separated from the potential pool of available organic compounds based on polarity and size considerations. In retrospect, it is not surprising to find that the upper limit for aquatic fulvic acid is less than 1000 daltons. The fact that the molecular weights of aquatic fulvic acids are low is a major constraint on structural modeling, and influences our understanding of the chemistry of these species. The chemical implications of low molecular weight can be observed by taking as an example Suwannee FA, which has been extensively studied. From the elemental analysis data and the molecular weight of 829 daltons (as determined in water) it can be shown that this sample is a heterogenous mixture. Only one out of every 2.6 molecules can contain a nitrogen atom, for instance. Similarly, not all molecuies can possess the same chemical characteristics or trace constituents. M&NIGHT et al. (1983) report the presence of two types of Cu(II) binding sites in aquatic fulvic acids. For the case of the Suwannee
sample, the concentrations of types I and II sites is 1.2 X 10T6and 2.7 X lo-’ moles/mg C, respectively. Only one out of every 2 molecules of this sample, therefore, can have a type I site, and only one out of every 8 molecules can have a type II site. Other structural information can be obtained by considering the distribution of functional groups with this sample. THURMAN and MAJXOLFA (1983) report that the carboxyl content of Suwannee FA is 6.0 mmol/gram. With a molecular weight of 829 daltons, this sample contains fewer than 5 carboxyl groups per molecule. These molecules, therefore, arc best described as oligoelectrolytes and should not be thought of as polyelectrolytes in the sense that polyacrylic acid is a polyelectrolyte. The low molecular weights of aquatic fulvic acids have important geochemical and biochemical implications. STEWARTand WETZEL (1981), for instance, have suggested that lower molecular weight fulvic acids are more inhibitory to calcite precipitation than higher molecular weight humic acids. In other work, CHIOU etal. (1986) have shown that significant solubility enhancements of hydrophobic organic pollutants and pesticides by humic substances can be described in terms of a partition-like interaction. The effectiveness of humic substances in enhancing solute solubility is largely controlled by the molecular size and polarity of the humic material, with larger soil-derived humic substances being more effective than low molecular weight aquatic fulvic acid. A relationship between the molecular weights of humic substances and their biological activity has also been noted (STEINBERG and MUENSTER, 1985). STEWART and WE-EEL (1982) have reported that humic substances of low apparent molecular weight are more stimulatory to both 14Cassimilation and alkaline phosphatase activity by algal-bacterial assemblages than humic substances of high apparent molecular weight. VEER (1985) has also noted the increased effect of lower molecular weight humic substances on the physiological activity of microbial cells. Others have noted that the presence of soil humic substances enhanced the uptake of iron by diatoms in laboratory studies (PROVASOLI, 1963; PRAKASH er al., 1973). While the biochemical mechanisms of this effect have not been elucidated, it is interesting to note that the molecular weights of aquatic fulvic acids arc similar to those of siderophores (approximately 400-650 daltons), and aquatic fulvic acid may be capable of passing cellular membranes. SUMMARY Small angle x-ray scattering measurements were made on aquatic fulvic acid samples to obtain radii of gyration data and to estimate the degree of polydisper&y of each sample. The samples were found to be relatively monodisperse, indicting that ;i?, values, as obtained by VPO, wouid be useful data in understanding the chemical nature of aquatic fulvic acid. an values were then obtained in water and in THF as solvents.
Fulvic acid molecular wei8hu
2183
metric number-average wei8hts of humic substances for dissociation. Chem. GeOt.33,355-366. GLOVPRC. A. (1975) Absolute colligative property measurements. In PolvmerMoltxxdar W&&s. Pt. 1 ted. P. E. SLADE JR.), pp. 79-159. Marcel Dekke< New York. HANSEN E. H. and SCHNITZRM. (1969) Molecular wei&t measurements of polycarboxylic acids in water by vapor pressure osmometry. Anal. Chim. Acta 46,247-254. HAYES M. H. B. and Swa R. S. (1978) The chemistry of soil colloids. In The Chemistry of Soil Co~t~t~~s feds. D. J. GREEMANDand M. H. B. Hnves), pp. 179-203. ~ey-~n~en~, New York. HUFF%%AN E. W. D. and STLIBERH. A. (1985) Analytical methodology for elemental analysis of humic substances. In Humic Substances in Soil, Sediment and Water (eds. G. R. AIKEN,D. M. MCKNIGHT,R. L. WERSHAWand P. M&CAR-THY),pp. 433-456. John Wiley and Sons, New York. LANSING W. D. and RRAEMER E. 0. (1935) Molecular wei@ analysis of mixtures by sedimentation equilibrium on the Svedbeq ultracentrifuge. f. Amer. Chem. Sot. 57, 13691377. LEENHIZER J. A. and Smos U. M. (1980) Consideracoes sobre os pmcessos de ~rnen~~o no aqua preta acida do rio Negro (Amaxonia Central). Acta Amazonica IO, 343355. LIAOw., cHtu!XMANR. F., JOHNSON J. D. and MILL~NGTON D. S. (1982) Structural characterization of aquatic humic material. Environ. Sri. Technol. 16,403-4 10. MCKNIGHT D. M., FEDERG. L., THURMAN EM., WERSHAW R. L. and W~~TALL J. L. ( 1983)Complexion of copper by aquatic humic substances horn different environments. Sci. Tot. Environ. 28,65-76. MEYERJ. L. (1986) Dissolved organic carbon dynamics in two subttopical blackwater rivers. Arch. H~robio~. 108, 119-134. MOORE W. J. (1972) Ph~ic~ Chemists, 4th ed. PrenticeHall, Eqlewood Cliffs, N.J. 977p. PRAKASH A., RASHIDM. A. and RAOD. V. S. (1973)Influence Acknowledgements-The authors wish to express their 8ratof humic substances on the growth of marine phytoplankitude to D. M. McKninht. E. M. Perdue. R. A. Thorn. and ton: Diatoms. Limnoi. Oceanogr.18,s 16-524. several anonymous r&w& for their c4&tmctive criticisms f’ROVASOLIL. (1963) Organic regulation of phytoplankton on the manuscript. fmty. In TheSea. Vol.2, pp. 165-219.Wiley-Interscience, New York. REUTERJ. H. and PERDUEE. M. ( 1981)Calculation of moEditoriul handling:J. I. Hedges lecular weights of humic substances from colligative data: Application to aquatic humus and its molecular size Rattions. Geochim. Cosm~him. Acta 45,2017-2022. RYKIELE. J. ( 1984)General hydrology and mineral budgets AXEN G. R. (1984) Evaluation of ultrafiltration for deterfor Okefenokee Swamp: Ecologicalsignificance in the Okemining molecular weight of fidvic acid. Environ. Sci. Techfenokee Swamp (eds. A. D. COHEN,D. J. CASAGRANDE, nol. 18,978-98 1. M. J. ANDWKO and G. R. Bzs~), pp. 212-228. Wetland AIKEN G. R. and GILLAMA. H. (1987) Determination of Surveys, Los Alamos, New Mexico. molecular weights of humic substances by colliknuiveprop SCHNITZERM. and NEYROUD J. A. (1975) Alkanes and fatty erty measurements. In Humic Substances II. StructuralAsacids in humic substances. Fuel 54, 17- 19. pects (als. M. HAYES,R. MA~ARTHY, R MAuxlLM and SHOLKOV~Z: E. R., BOYLEE. A. and PRKEN. B. ( 1978)The R. SWIFT).J. Wiley and Sons. Chichester (in mess). removal of dissolved humic acids and iron during estuarine BONNAR R.V., Du.&ATM. and SrItiXs F. H:(1958)~r&er mixing. Earth Planet. Sci. L&t. 40, 130-136. AverageMoiec&r Weights.Wiley-In&science, New York, STEIF~ERG C. and MUENSTER U. (1985) Geochemistry and 31Op. ecolqical role of humic substances in lakewater. In Humic BURGE D. E. (1977) Mo~~ weight Davison by osSubstances in Soil, Sedim~t and Water feds. G. R. AIKEN. mometty. Amer. tib. 9,4 l-5 1. D. M. MCRN~GHT, R. L. WERSHAW and P. MCCARTHY), CH~OUC. T., M,uco~~ R. L., BRINIQNT. I. and RILED. E. pp. 10.5-145.John Wiley and Sons, New York. ( 1986)Water solubihty enhancement of some organic polA. J. and WETZELR. G. (1981) Dissolved humic lutants and pesticides by dissolved humic aod fulvic acids. STEWART matem& Photcdegradation, sediment effects,and reactivity Environ. Sci. Techtwl. X&502-508. with phosphate and calciumprecipitation. Arch. Hydrobiof. CHARLOT G. and TREMILLION B. ( 1969)Chemical Reactions 92,265-286. in Solvents and Melts. Per8amon Press, Oxford, 528~. ERTEL J. R., HEDGESJ. I., ~EVOL A. H., RKHEYJ. E. and STEWART A. J. and WETZELR. G. (1982) Influence of disRmztno M. (1986) Dissolved humic substances of the solved humic material on carbon assimilation and alkaline phosphatase activity in natural algalbacterial assemblages. Amazon River system. Linmol. Oceanogr.37,739-754. Freshwater Bioi. 12, 369-380. GILLAMA. H. and BLEY J. P. (1981) Cormction of ammo-
Because THF has a low dielectric constant, the acidic protons on fulvic acid do not dissociate, and the VP0 data do not need correction for Mation. The molecular weight values obtained in THF compared favorably with the data obtained in water, and THF was found to be a useful solvent for determining molecular weights of aquatic fulvic acids. in water, %,, values for aquatic hlvic acid ranged from 500 to 950 dahons; these values are not as large as has been suggesmd in other molecular weight studies utilizing ultiltration or gel filtration; however, a number of factors may contribute to this difference. While ultrafiltration and gel filtration are subject to interactions which can lead to higher molecular weight fractionation values, it is a strong possibility that the pH adjustments used in the isolation of aquatic fulvic acid disrupt interactions between the fulvic acid and other chemical constituents in the water, such as silica and iron, resulting in lower molecular weights for the isolated materials. A correlation was observed between the concxmtrations of Ca++ and Mg++ in the river water and the radii of gyration and mohxular weights of the corresponding fulvic acid samples. It appears that the presence of these divalent cations in the soils of the watershed and in the river water deemases the solubility of higher molecular weight components of the humic substances by suppressing charge on the organic polyanions. The lower molecular weight values and the small range of values obtained for aquatic fulvic acid are probably the result of sohtbility constraints for this class of compounds in water.
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WERSHAWR. L. and AIKENG. R. (1985) Molecular size and weight measurements of humic substances. In Humic Substances in Soil. Sediment and Water (e&. G. R. AIKEN, D. M. MIXNIGHT, R. L. WERHSAWand P. MAcCA~I’I.N), pp. 477-492. John Wiley and Sons, Inc., New York. WERSHAWR. L. and PINCXNEYD. J. (1973) Determination of the association and dissociation of humic acid fractions by small angle x-ray scattering. J. Res. U.S. Geol. Surv. 1, 701-707. WETZELR. G. (I 983) Limnology. Saunders College Publishing, Philadelphia, 767~. WILSONS. A. and WEBERJ. H. (1977) A comparative study of number-average dissociation corrected molecular weights of fulvic acids isolated from water and soil. Chem. Geol. 19,285-293.