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Correction of osmometric number-average molecular weights of humic substances for dissociation
Chemical Geology, 33 ( 1981 ) 355--366
355
Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
CORRECTION OF OSMOMETRIC NUMBER-AVERAGE MOLECULAR WEIGHTS OF HUMIC SUBSTANCES FOR DISSOCIATION
A.H. GILLAM *''*: and J.P. RILEY Department o f Oceanography, The University of Liverpool, Liverpool L69 3BX (Great Britain)
(Received July 18, 1980; revised and accepted May 7, 1981 )
ABSTRACT
Gillam, A.H. and Riley, J.P., 1981. Correction of osmometric number-average molecular weights of humic substances for dissociation. Chem. Geol., 33: 355--366. It is necessary to correct the number-average molecular-weight (Mn) values of organic compounds obtained by colligative methods such as vapour-pressure osmometry (VPO) for dissociation of any acidic functional groups present. A novel correction method for the determination of Mncorz-values is described which does not require prior knowledge of the concentrations or dissociation constants of the acidic functional groups present. The procedure has been applied to the evaluation of 1VIncorr-valuesof a range of freshwater and marine humic substances and gave results in the range 500--1000. A correlation between salinity and Mncorr has been found for a range of marine fulvic acids, providing further evidence for the removal of the higher-molecular-weight fractions of dissolved organic matter (DOM) with increasing salinity.
INTRODUCTION
A l t h o u g h the h u m i c c o m p o u n d s o f soils and sediments have been studied for over t w o h u n d r e d years, it is o n l y in r e c e n t years t h a t progress has been m a d e in unravelling their c o m p l e x c h e m i s t r y (see, e.g., K o n o n o v a , 1 9 6 6 ; Schnitzer and Khan, 1 9 7 2 ; J a c k s o n , 1 9 7 5 ; Schnitzer, 1978). In c o n t r a s t , o u r k n o w l e d g e o f the h u m i c material present in a q u a t i c s y s t e m s is m u c h less detailed, p a r t l y because o f t h e d i f f i c u l t y o f recovering w o r t h w h i l e a m o u n t s of a q u a t i c h u m i c substances. Until r e c e n t l y , a l m o s t all t h e w o r k o n h u m i c substances has c o n c e n t r a t e d on b u l k p r o p e r t i e s (see review b y Williams, 19'75; Gjessing, 1 9 7 6 ; Kerr and Q u i n n , 1 9 8 0 ) and on f u n c t i o n a l ~ o u p d e t e r m i n a tions ( S c h n i t z e r and Khan, 1 9 7 2 ; Weber and Wilson, 1 9 7 5 ) . However, r e c e n t d e v e l o p m e n t s ( M a c C a r t h y et al., 1 9 7 9 ; Saito and H a y a n o , 1 9 7 9 ) in t h e sepa* ' Present address: Department of Oceanography, The University of British Columbia,
6270 University Boulevard, Vancouver, B.C. V6T 1 W5, Canada. ,2 Author to whom correspondence should be addressed.
0009-2541/81/0000--0000/$02.50 © 1981 Elsevier Scientific Publishing Company
356 ration of the components of aquatic and sedimentary humic substances may enable more meaningful investigations of their structures ar_d origins to be made. The measurement of the molecular weights of humic substances remains a controversial topic in the study of these compounds. Values ranging from a few hundred to several million have been reported for humic materials from various sources and utilising different analytical techniques (Schnitzer and Khan, 1972}. This spread of values may, in part, reflect differences in the nature of the sample, arising from the use of a variety of extraction and purification techniques. However, it seems likely that to a large extent they result from the difficulty of making meaningful measurements of the molecular weights of such substances. In c o m m o n with all polymeric materials, humic substances are composed of a complex mixture of species of various molecular weights and dimensions. If the method used is based on a colligative property, then the result obtained is the number-average molecular weight, MN, whereas the weight-average molecular-weight value, MW, is calculated from measurements of the weights of the individual polymeric species. Both gel filtration and ultrafiltration have been widely used for the determination of Mw-values of humic substances (Gjessing, 1965, 1976; Gjessing anti Lee, 1967; Shapiro, 1967; Ghassemi and Christman, 1968; Rashid and King, 1969; Swift and Posner, 1971; Cameron et al., 1972; de Haan, 1972; Wershavv and Pinckney, 1973; Stuermer, 1975; Alberts et al., 1976; Mantoura, 1976; Starikova et al., 1976; Kwak et al., 1977; Buffle et al., 1978; MacFarlane, 1978) although they both have considerable limitations and data obtained by such means must be treated with caution. Vapour-pressure o s m o m e t r y (Schnitzer and Desjardins, 1962; Hansen and Schnitzer, 1969; Martin and Reuter, 1973; Buffle et al., 1977; Linehan, 1977! and cryoscopic methods (De Borger and De Backer, 1968; Wilson and Weber. 1977) have been used to measure Mn-values of a wide range of humic substances. Hansen and Schnitzer (1969) and, more recently, Wilson and Weber (1977) have produced detailed theoretical treatments of the calculation of M---ncorr-values (Mn-values corrected for the dissociation of acidic functional groups) of naturally-occurring humic substances, including some isolated from river waters. However, both of these correction systems have limitations, whiL.h will be discussed in the following section. This paper describes a novel system for the correction of Mn-values of hum~c substances which only requires a knowledge of the pH of the solution. The procedure has been used to derive Mncorr-values for a range of freshwater and marine humic substances. EXPERIMENTAL AND CALCULATIONS
Isolation of aquatic hurnic substances The isolation, purification and chemical analysis of the humic samples used for the vapour-pressure o s m o m e t r y measurements have been described in de
357 tail (Gillam, 1979). Briefly, large volumes (100--200 l) of filtered (WhatmanC-~ GF/C) water were acidified to pH 2.0 and passed through columns (30 cm X 2 cm 2) of acetone-extracted Amberlite® XAD-2 resin. The adsorbed organic matter was eluted from the resin with aqueous ammonia (2 M, five bed volumes). The resultant solution was rotary-evaporated to a small volume and the organic matter was separated into its humic and fulvic acid fractions by acidification (HCI, pH 1.0). After removal of the precipitated humic acids, the fulvic acid remaining in the filtrate was purified by ultrafiltration, using a MW 500 cut-off filter. The protonated fulvic acid was recovered by rotary evaporation. Both materials were ground to a fine powder and thoroughly dried, in vacuo, over magnesium perchlorate.
Preparation of the solutions for vapour-pressure osmometry Weighed amounts of fulvic substances and standards (8--20 mg) were carefully dissolved in doubly distilled water to give a total volume of 2 ml. The solutions were thoroughly stirred to ensure complete mixing.
Instrumentation. The pH-values of the solutions of humic substances were measured with a Radiometer ® PHM research pH meter fitted with a GK ® 2301 C combined glass--calomel electrode. The linear response of the pH electrode was checked with 0.05 m solutions of potassium tetroxalate and potassium hydrogen phthalate (pH-values = 1.68 and 4.01, respectively, at 25°C) and it was standardised prior to each set of measurements with respect to the phthalate buffer. Vapour-pressure osmometric measurements were made at 37°C, using a Mecrolab ® 301 osmometer, following the manufacturer's instructions and general guidelines for Mn determinations given by Dr. D. Dare and the staff of the Polymer Characterisation Unit (P.C.U.), Department of Inorganic, Physical and Industrial Chemistry, University of Liverpool. The apparatus constant was determined using glucose solutions and was calculated to be 69.36 kg ~2 g-'. The accuracy of the vapour-pressure osmometer in the range 500--1000 D was verified by measurement of the Mn-value of a Waters Associates Ltd. GPC standard (polyethylene glycol), having an Mn-value of 790. Using a first virial model, an Mn-value of 730 was found. As the result from a linear leastsquares solution was in satisfactory agreement with the certified value, no corrections were applied.
Calculations o f Mn in vapour-pressure osmometry Bonnar et al. (1958) have reviewed the principal methods of measuring Mn and have also provided an excellent source of computational methods for the evaluation and interpretation of vapour-pressure-osmometry data. Techniques
358 for the d e t e r m i n a t i o n of Mn have been reviewed more recently by Glover (1975) who has also r e c o m m e n d e d c o m p u t a t i o n a l m et hods similar to those suggested by Bonnar et al. (1958). Meeks and Goldfarb (1967) have shown that the solution theories of Bonnar et al. are applicable to vapour-pressure o s m o m e t r y and that an expression of the type: :: = ~
+ aW
+ bW 2 +
is applicable. For this case, ,~,, often called the instrument readout, is expressed as a change in resistance of the thermistor network used to measure the temperature difference between the solution of the test material and the solvent. is the zero-point error, a and b are constants for a given instrument and so"JO lutions used, and W is the weight ratio of solute to solvent. The term a is directly related to Mn. As b and other higher terms can generally be neglected for all but th e most c om pl ex systems, it is normally then possible to write: Mn
=
Kapp/a
where Kapp is the apparatus constant Glover (1975) has discussed the principal types of model used to predict the behaviour of p o l y m e r solutions. For low molecular weight, ideal compounds. the P.C.U. r e c o m m e n d a simple, first-virial model of the t ype: ~:~= :)0 + a W This model accurately describes the majority of the data obtained for the a q u a t ic humic substances used in the present study. As there was no apparent deviation f r o m linearity for these data, corrections for non-ideality, using secondor third-virial models (Glover, 1975), were not made. The zero-point m e t h o d of Bonnar et al. was used to correct for the non-zero value of () in the absence of a solute. In the theoretical t r e a t m e n t of vapour-pressure o s m o m e t r y discussed above, no allowance has been made for the dissociation of organic c o m p o u n d s in aqueous solutions. Both De Borger and De Backer (1968) and Hansen and Schnitzel (1969) corrected their vapour-pressure o s m o m e t r y data by assuming that the dissociation of a p o l y f u n c t i o n a l acid (HrTA) could be described by the following equation: H n A ~ nH÷ + A n-
~1
At equilibrium, for a dibasic acid HnA, the total n u m o e r of particles was considered to be 1 + ha, rather than 1 + a~ + 2a2, where the a terms refer to the consecutive dissociation steps of the acid. Wilson and Weber (1977) have described a more complex correction system, based on measurements of the concentrations and "dissociation c o n s t a n t s " of the individual acidic functional groups. Although this latter correction is satisfactory for pure di- and polycarboxylic acids, difficulties arise when a t t e mpt s are made to apply it ~o naturally-occurring humic substances (see the Section "Results and Discussion"). The proposed correction method is based on the identity that, for the dis-
359 sociation of a polyacidic compound, the total concentration of particles at equilibrium is given both by the expression Ca+[H ÷] and by W/Mnexp., where Ca, [H+], W and Mnex are, respectively, the total acid and hydrogen 1on concentrations, the weigh~ ratio of solute to solvent, and Mnex p is the experimentally determined value of Mn. Thus: Ca +
[H ÷] = W/Mnex p
and
Mnex p = K a p p / a (*)
Then: Ca + [ H ÷] = W a / K a p p and so: C a = W a / K a p p - [H ÷]
(2)
Since [H ÷] can be measured and C a calculated from eq. 2, the value of ~ corrected for dissociation, (0corr), can be calculated from: ecorr = 8/(1 + [H ÷]/C a)
(3)
From a set of data for G vs. W, the most suitable model was selected for the experimental data. A value for the constant a was then calculated for the complete determination. A value for Ca for each data point was calculated from eq • 2, using the known values of K a p , pH and W and this value of a • 0 c o r r was then evaluated for each data point ~ o m eq. 3 and the most suitable model for the corrected data was again selected• The corrected value for a, acorr, was calculated and the corrected value for the molecular weight (Mncorr) of the compound under investigation was then determined. RESULTS AND DISCUSSION m
The values of Mncorr for several known organic compounds were calculated, using the novel correction system described above (Table I). The compounds were chosen because of their partial chemical similarity to fulvic acids. Experience with the vapour-pressure osmometer suggested that a mean deviation between the actual and the experimental values of Mn of 5% was to be expected. The results in Table I indicate, therefore, that the correction system does compensate adequately for the dissociation of these low-molecular-weight organic compounds. Further evidence for the suitability of the system was provided by re-evaluation of the data of Wilson and Weber (1977) for tartaric acid and tartaric acid--succinic acid mixtures. Using a second-virial model with a zero-point correction, it was shown (Table II) that it was possible to obtain accurate Mneorr-values w i t h o u t prior knowledge of the dissociation constants or of the initial concentrations of each t y p e of acidic functional group. The major difficulties with the application of the correction m~thod of t-hnsen and Schnitzer (1969) to aquatic humic substances are that: (1) an * A similar approach was used by Glover (1975) and Wilson and Weber (1977).
360 TABLE I
Mn ~ o r r -values for known organic c o m p o u n d s (values given are the mean of three determinations) (_k)mpound
Mnactua!
Mncorr
Per cent deviation
Tartaric acid Ascorbic acid Oxalic acid
150.1 176.1 90.0
154.2 180.6 93.8
+2.8 +2.6 +4.2
] ' A B L E II Comparison of correction methods Compound
Mnactual
Mn Otl: (Wilson and Weber, 1977)
Mn COl'[ (proposed m e t h o d ,
Tartaric acid Tartaric acid--succinic acid
l 50.1 1 38
152..I 142.4
15[.2 139.1
i n a c c u r a t e a p p r o x i m a h o n for the dissociation of a p o l y f u n c t i o n a l acid is made, and (2) relatively large c o n c e n t r a t i o n s of h u m i c substances are required to ensure that linearity o f t h e ,,--W relationship is o b t a i n e d (Hansen and Schnitzer, 1969). Curvature of the lines at low c o n c e n t r a t i o n s , s h o w n in Fig. 1, makes it d i f f i c u l t to e x t r a p o l a t e to t h e p o i n t at which W = 0, and this means that the I
.
•
.g
,~',.,.
i W .']:
,
•
i O-
i -[ .
" r~,
.
.
.
.
!~00
.
.
.
.
.
.
":)'2 ~
.
.
.
.
.
"~O,,C..
.
.
.
.
.
2'J)O(~
.
.
J
2~S)C ,
Fig. 1. A p p l i c a t i o n o f tile correction system o f Hansen and Schnitzer ( 1 9 6 9 ) to VPO data o f water fulvic acids.
361
method is unsuitable for use with either our own fulvic acid data or with those published by Wilson and Weber (1977). The differences in Mn-values obtained by Wilson and Weber for their water fulvic acid and the value obtained using the correction method of Hansen and Schnitzer (see Fig. 1) may be partly explained by the approximations of these methods. As Wilson and Weber {1977) have noted, both De Borger and De Backer (1968) and Hansen and Schnitzer (1969) assumed that the dissociation of a polyfunctional acid could be described by a one-step process. Wilson and Weber (1977) described a more complex system, based on the approximation that their fulvic acid samples behaved as a mixture of two monobasic acids. It has been suggested that the presence of two distinct groups of carboxylic acids may not accurately represent the polyacidic nature of humic substances (J.H. Reuter, pers. commun., 1979). In order to calculate values for the individual dissociation constants, Wilson and Weber (1977) utilised data for soil fulvic acids previously reported by Gamble {1970, 1972). Furthermore, they utilised the potentiometric approach devised by Borggaard (1974) to calculate the concentrations of each of the acidic functional groups. Stuermer (1975) and Gillam (1979) have both provided evidence to suggest t h a t no secondary maxima are present in derivative plots of potentiometric titrations of aquatic fulvic acids. Perdue et al. (1980) have recently discussed the difficulties of obtaining representative values for the concentrations and dissociation constants of carboxylic functional groups in humic substances. Thus, the correction methods of Hansen and Schnitzer and Wilson and Weber are both of limited value for the fulvic acid samples used in this study. The proposed correction m e t h o d requires neither the use of an extrapolation procedure nor any previous knowledge of the dissociation constants of the compound. Although pH is not strictly defined as - log [ H÷], all previous methods for the determination of Mn c orr-values of aquatic__ humic substances have made this assumption. Despite this, accurate Mn¢orr-values for several known organic compounds have been obtained. The accurate calculation of [H ÷] requires knowledge of the E °- and/~-values of the electrode system. On the basis of previously reported methods, it was decided that, although the assumption that the concentration and activity terms are identical is incorrect, the differences in the Mncorr-values would be within the bounds of experimental error. Data for the calculation of Mncorr-values for several types of sample are given in Table III. The relatively small range of concentrations for the aquatic humic substances was determined by the limited amounts (< 100 mg) of organic material. However, despite the limited number of data points, accurate M--n-values were obtained for tartaric and ascorbic acids. In preparing samples of humic substances for vapour-pressure osmometry, ultrafiltratiQn was used to ensure that t h e y were free from inorganic salts and low-molecular-weight organic compounds, as these would cause the molecular weights to appear to be anomalously low.
362 TABLE III
Sample data for vapour-pressure-osmometry measurements Compound
W (g/kg)
pH
:
Tartaric acid
3.900 5.880 7.796 10.352
2.19 2.08 2.00 1.93
1.030 2.165 3.098 4.428
4.045 5.946 7.988 10.020
2.46 2.39 2.32 2.28
3.892 6.111 7.999 10.689 4.177 5.983 7.826 9.421
Ascorbic acid
SK-8
HM-I
~
i," C O L T
acorr
Mncorr
0.523
0.804 1.759 2.571 3.762
0.455
152.3
1.160 2.070 2.788 3.750
0.425
0.997 1.839 2.514 3.429
0.399
173.8
2.38 2.19 2.10 2.01
0.785 1.238 1.603 2.112
0. t95
0.486 0.773 1.037 1.425
0.138
503
2.32 2.21 2.05 2.03
0.628 0.860 1.105 1.358
0.138
0.266 0.415 0.472 0.681
0.074
943
T h e Mn-values o f a series o f fulvic acid s a m p l e s f r o m a variety of soils and n a t u r a l w a t e r s are s h o w n in Fig. 2. As a general t r e n d , it a p p e a r s t h a t :
soil fulvies
lake,'river fulvics
marine fulvics
F r o m t h e results o b t a i n e d using the p r o p o s e d c o r r e c t i o n s y s t e m , it w o u l d appear t h a t it is possible to d i f f e r e n t i a t e b e t w e e n fulvic acids f r o m w i d e l y differing origins. When this s y s t e m is a p p l i e d t o t h e soil a n d w a t e r fulvic acid d a t a o f Wilson and W e b e r ( 1 9 7 7 ) , Mncorr-values o b t a i n e d are 1103 and 888, r e s p e c t i v e l y . W h e t h e r this is indicative o f t h e e x t e n t to w h i c h t h e h u m i c materials in t h e t w o s y s t e m s have b e c o m e d e g r a d e d c a n n o t be stated f r o m this o n e set o f values. H o w e v e r , t h e d e c r e a s e in Mncorr-values o n passing f r o m a lacustrine to a m a r i n e e n v i r o n m e n t m a y be a t t r i b u t a b l e to d i f f e r e n c e s in the solubility o f the various s u b f r a c t i o n s o f the h u m i c substances. As the salinity increases, the h i g h e r - m o l e c u l a r - w e i g h t f r a c t i o n s o f t h e h u m i c c o m p o u n d s will t e n d to p r e c i p i t a t e selectively. This will lead to an overall l o w e r i n g o f t h e ~Invalues (see, e.g., B r o w n , 1975, 1 9 7 7 ; S h o l k o v i t z , 1 9 7 6 ; S h o l k o v i t z et al., 1 9 7 8 ~ In general, t h e c o r r e c t e d m o l e c u l a r weights are several t i m e s l o w e r t h a n t h o s e o b t a i n e d b y gel p e r m e a t i o n c h r o m a t o g r a p h y and u l t r a f i l t r a t i o n , which d e p e n d m a i n l y on m o l e c u l a r size and s h a p e ( C a m e r o n et al., 1972; Buffle et al., 1978).
363
2000 i u
h
[: O~
I000i
~O
Vo W
~7~2V
400 [--
So,:
I Lak@/Riv(2r
I
iVlarln~
Fig. 2. Mncorr-values for a range of fulvic acid samples. v = this study; ~ = Buffle et al. (1977); o = Wilson and Weber (1977)* ; a = De Borger and De Backer (1968)* ; • = Hansen and Schnitzer (1969)* ; • = Martin and Reuter (1973); Footnote: * values obtained using the novel correction method. TABLE IV Mncorr-values for the aquatic fulvic acids of this study Sample
Salinity (°/oo)
Mncorr
Sample
Salinity (°/oo)
Mncorr
SK-I SK-2 SK-4 SK-5 SK-6 SK-7
31.37
591 755 626 650 720 792
SK-8 HM-I CN-2 DE-/ IS-2
33.20
501 943 846 761 623
31.30 31.10 28.28 27.80
32.59
SK = samples collected during R.S.S. "Shackelton" cruise, June 1977, Skaggerak; HM = sample from peaty stream feeding Hatchmere, Delamere Forest, Cheshire; CN = sample from Lake Celyn, north Wales; DE = sample from River Dee, north Wales; IS = sample collected during R.V. "Edward Forbes" cruise, October, 1977, Irish Sea.
T h e M--ncorr d a t a f o r t h e m a r i n e f u l v i c a c i d s o f t h i s s t u d y w e r e p l o t t e d against the salinity of the water samples from which they were concentrated. T h e r e g r e s s i o n b e t w e e n t h e s e t w o p a r a m e t e r s is s h o w n in Fig. 3 a n d y i e l d e d a c o r r e l a t i o n c o e f f i c i e n t o f - 0 . 8 6 , w h i c h is s i g n i f i c a n t a t t h e 9 5 % c o n f i d e n c e limit. This provides further evidence that one of the major effects of increasing s a l i n i t y o n t h e D O M p r e s e n t in n a t u r a l w a t e r s is t h e r e m o v a l o f t h e h i g h e r molecular-weight fractions of the DOM.
364 350r
i. 3OO~-
i 270 I 4OO
1 .... M
Fig. 3. Correlation of salimty with Mncorr-values of marine fulvic acid samples of this s t u d y
Although water has been widely used as a solvent for vapour-pressure osm o m e t r y of fulvic acid samples, this medium cannot be employed as a solvent for humic acids. Although sulpholane has been used for this purpose, with the humic acid fractions of various soils (Schnitzer and Desjardins, 1962), it requires extensive purification. Trichloromethane and 2-butanone have also been used for Mn determinations, and values in the range 410--1000 were obtained for a series of subfractions of a sedimentary humic acid sample (Ishiwatari, 1973). CONCLUSIONS
The Mn-values obtained for the fulvic acids in the present study are within the range of those obtained by other workers using the VPO technique. Accurate values for the molecular weights of naturally-occurring aquatic humic substances are essential for studies of the metal complexation ability of these compounds. Few studies have been made to determine the nature of the humic fractions which are involved in metal complexation. Previous reports (Mantoura and Riley, 1975; Buffle et al., 1977) indicate, however, that it is the low er-molecular-weight compounds which possess the greater complexing ability. It appears that vapour-pressure o s m o m e t r y will remain one of the more important techniques for the determination of the molecular weights of aquatic humic substances. ACKNOWLEDGEMENTS
The authors would like to thank the staff of the Polymer Characterisation Unit, University of Liverpool, for their guidance with the vapour-pressure osmometer used in this study. One of the authors (A.G.) is indebted to the Natural Environment Research Council for the award of a research studentship.
365 REFERENCES Alberts, J.J., Schindler, J.E., Nutter, D.E. and Davis, E., 1976. Elemental, infra-red spectrophotometric and electron spin resonance investigations of non-chemically isolated humic material. Geochim. Cosmochim. Acta, 40: 369--372. Bonnar, R.U., Dimbat, M. and Stross, F.H., 1958. Number Average Molecular Weights -Fundamentals and Determination. Interscience, New York, N.Y., 310 pp. Borggaard, O.K., 1974. Titrimetric determination of acidity and pK values of humic acid. Acta Chem. Seand. (A), 28: 121--122. Brown, M., 1975. High molecular weight material in Baltic seawater. Mar. Chem., 3: 253-258. Brown, M., 1977. Transmission spectroscopy of natural waters, C. Ultraviolet spectral characteristics of the transition from terrestrial humus to marine yellow substance. Estuarine Coast. Mar. Sci., 5: 309--3i7. Buffle, J., Greter, F.-L. and Haerdi, W., 1977. Measurement of complexation properties of humic and fulvic acids in natural waters with lead and copper ion-selective electrodes. Anal. Chem., 49: 216--222. Buffle, J., Deladoey, P. and Haerdi, W., 1978. The use of ultrafiltration for the separation and fractionation of organic ligands in fresh waters. Anal. Chim. Acta, 101: 339--357. Cameron, R.S., Swift, R.S., Thornton, B.K. and Posner, A.M., 1972. Calibration of gel permeation chromatography materials for use with humic acid. J. Soil. Sci., 23: 342--349. De Borger, R. and De Backer, H., 1968. D4termination du poids mol6culaire moyen des acides fulviques par cryoscopie en milieu aqueux. C.R. Acad. Sci. (Paris), Ser. D, 266: 2052--2055. de Haan, H., 1972. Molecular size distribution of soluble humic compounds from different natural waters. Freshwater Biol., 2: 235--241. Gamble, D.S., 1970. Titration curves of fulvic acid: the analytical chemistry of a weak acid polyeleetrolyte. Can. J. Chem., 48: 2662--2669. Gamble, D.S., 1972. Potentiometric titration of fulvic acid: equivalence point calculations and acidic functional groups. Can. J. Chem., 50: 2680--2690. Ghassemi, M. and Christman, R.F., 1968. Properties of the yellow organic acids of natural waters. Lirrmol. Oeeanogr., 13: 583--597. Gillam, A.H., 1979. The chemical and physical characteristics of aquatic humic substances. Ph. D. Thesis, University of Liverpool, Liverpool. Gjessing, E.T., 1965. Use of Sephadex gel for the estimation of molecular weight of humic substances in natural water. Nature (London), 208: 1091--1092. Gjes~ing, E.T., 1976. Physical and chemical characteristics of aquatic humus. Ann Arbor Science, Publishers, Ann Arbor, Mich. Gjessing, E.T. and Lee, G.F., 1967. Fractionation of organic matter in natural waters on Sephadex columns. Environ. Sci. Technol., 1: 631--638. Glover, C.A., 1975. Absolute colligative property methods. In: P.E. Slade, Jr. (Editor), Polymer Molecular Weights, Part 1, Marcel Dekker, New York, N.Y., Ch. 4. Hansen, E.H. and Schnitzer, M., 1969. Molecular weight measurements of polycarboxylic acids in water by aqueous vapour pressure osmometry. Anal. Chim. Acta., 46: 247--254. Ishiwatari, R., 1973. Chemical characterization of fractionated humic acids from lake and marine sediments. Chem. Geol., 12: 113--126. Jackson, T.A., 1975. Humic matter in natural waters and sediments. Soil Sci., 119: 56--64. Kerr, R.A. and Quinn, J.G., 1980. Chemical comparison of dissolved organic matter isolated from different oceanic environments. Mar. Chem., 8: 217--229. Kononova, M., 1966. Soil Organic Matter. Pergamon, Oxford, 544 pp. Kwak, J.C.T., Nelson, R.W.P. and Gamble, D.S., 1977. Uitrafiltration of fulvtc and humic acids, a comparison of stirred cell and hollow fiber techniques. Geochim. Cosmochim. Acta, 41 : 993--996.
366
Linehan, D.J., 1977. A comparison of the polycarboxylic acids extracted by water from an agricultural top soil with those extracted by alkali. J. Soil Sci., 28: 369--378. MacCarthy, P., Peterson, M. J., Malcolm, R.L. and Thurman, E.M., 1979. Separation of humic substances by pH gradient desorption from a hydrophobic resin. Anal. Chem., 51: 2041'--2043. MacFarlane, R.B., 1978. Molecular weight distribution of humic and fulvic acids of sediments from a north Florida estuary. Geochim. Cosmochim. Acta, 42: 1579--1582. Mantoura, R.F.C., 1976. Humic compounds in natural waters and their complexation witt~ metals. Ph.D. Thesis, University of Liverpool, Liverpool. Mantoura, R.F.C. and Riley, J.P., 1975. The use of gel filtration in the study of metal binding by humic acids and related compounds. Anal. Chim. Acta, 7 8 : 1 9 3 - - 2 0 0 Martin, S.J. and Reuter, J.H., 1973. Chemistry of river water organic matter. Geol. Soc. Am. Boulder, Colo., Abstr. Progr., 5: 727. Meeks, A.C. and Goldfarb, l.J., 1967. Time dependence and drop size effects in determination of number average molecular weight by vapour pressure osmometry. Anal. Chem.. 39: 908--911. Perdue, E.M., Reuter, J.H. and Ghosal, M., 1980. The operational nature of acidic functional group analyses and its impact on mathematical descriptions of acid--base equilibria in humic substances. Geochim. Cosmochim. Acta, 44: 1841--1851. Rashid, M.A. and King, L.H., 1969. Molecular weight distribution measurements on humlc and fulvic acid fractions from marine clays on the Scotian Shelf. Geochim. Cosmochim Acta, 33: 147--151. Saito, Y. and Hayano, S., 1979. Application of high performance aqueous gel permeation chromatography to humic substances from marine sediment. J. Chromatogr.. 177 : 390392. Schnitzer, M., 1978. Humic substances chemistry and reactions. In: M. Schnitzer and S.U. Khan (Editors), Soil Organic Matter. Elsevier, New York, N.Y., Ch. ]. Schnitzer, M. and Desjardins, J.G., 1962. Molecular and equivalent weights of the organic matter of a podzol. Soil Sci. Soc. Am. Proc., 26: 362--365. Schnitzer, M. and Khan, S.U., 1972. Humic Substances in the Environment. Marcel Dekker, New York, N.Y., 327 pp. Shapiro, J., 1967. In: Yellow organic acids of lake water: differences in their composition and behaviour. H.L. Golterman and R.S. Clymo (Editors), Chemical Environment in the Aquatic Habitat. K. Ned. Akad. Wet., pp. 202--216. Sholkovitz, E.R., 197 6. Flocculation of dissolved organic and inorganic matter during the mixing of river water and sea water. Geochim. Cosmochim. Acta, 40: 831--845. Sholkovitz, E.R., Boyle, E.A. and Price, N.B., 1978. The removal of dissolved humic acids and iron during estuarine mixing. Earth Planet. Sci. Lett., 40: 130--136. Starikova, N.D., Yablokova, O.G. and Korzhikova, L.I., 1976. Determining the molecular composition of dissolved organic matter by gel filtration. Oceanology, 16:571--575. Stuermer, D.H., 1975. The characterization of humic substances in seawater. Ph.D. Thesis, Woods Hole Oceanogr. Inst.--Mass. Inst. Technol., Woods Hole, Mass.--Cambridge, Mass. Swift, R.S. and Posner, A.M., 1971. Gel chromatography of humic acid. J. Soil Sci., 23: 237--249. Weber, J.H. and Wilson, S.A., 1975. The isolation and characterization of fulvic acid and humic acid from river water. Water Res., 9: 1079--1084. Wershaw, R.L. and Pinckney, D.J., 1973. The fractionation of humic acids from natural water systems. J. Res. U.S. Geol. Surv., 1: 361--366. Williams, P.J. le B., 1975. Biological and chemical aspects of dissolved organic matter in seawater. In: J.P. Riley and G. Skirrow (Editors), Chemical Oceanography, Vol. 2. Aca dernic Press, London, 2nd ed., pp. 301--363. Wilson, S.A. and Weber, J.H., 1977. A comparative study of number-average dissocationcorrected molecular weights of fulvic acids isolated from water and soil. Chem. Geol.. 19: 285--293.
355
Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
CORRECTION OF OSMOMETRIC NUMBER-AVERAGE MOLECULAR WEIGHTS OF HUMIC SUBSTANCES FOR DISSOCIATION
A.H. GILLAM *''*: and J.P. RILEY Department o f Oceanography, The University of Liverpool, Liverpool L69 3BX (Great Britain)
(Received July 18, 1980; revised and accepted May 7, 1981 )
ABSTRACT
Gillam, A.H. and Riley, J.P., 1981. Correction of osmometric number-average molecular weights of humic substances for dissociation. Chem. Geol., 33: 355--366. It is necessary to correct the number-average molecular-weight (Mn) values of organic compounds obtained by colligative methods such as vapour-pressure osmometry (VPO) for dissociation of any acidic functional groups present. A novel correction method for the determination of Mncorz-values is described which does not require prior knowledge of the concentrations or dissociation constants of the acidic functional groups present. The procedure has been applied to the evaluation of 1VIncorr-valuesof a range of freshwater and marine humic substances and gave results in the range 500--1000. A correlation between salinity and Mncorr has been found for a range of marine fulvic acids, providing further evidence for the removal of the higher-molecular-weight fractions of dissolved organic matter (DOM) with increasing salinity.
INTRODUCTION
A l t h o u g h the h u m i c c o m p o u n d s o f soils and sediments have been studied for over t w o h u n d r e d years, it is o n l y in r e c e n t years t h a t progress has been m a d e in unravelling their c o m p l e x c h e m i s t r y (see, e.g., K o n o n o v a , 1 9 6 6 ; Schnitzer and Khan, 1 9 7 2 ; J a c k s o n , 1 9 7 5 ; Schnitzer, 1978). In c o n t r a s t , o u r k n o w l e d g e o f the h u m i c material present in a q u a t i c s y s t e m s is m u c h less detailed, p a r t l y because o f t h e d i f f i c u l t y o f recovering w o r t h w h i l e a m o u n t s of a q u a t i c h u m i c substances. Until r e c e n t l y , a l m o s t all t h e w o r k o n h u m i c substances has c o n c e n t r a t e d on b u l k p r o p e r t i e s (see review b y Williams, 19'75; Gjessing, 1 9 7 6 ; Kerr and Q u i n n , 1 9 8 0 ) and on f u n c t i o n a l ~ o u p d e t e r m i n a tions ( S c h n i t z e r and Khan, 1 9 7 2 ; Weber and Wilson, 1 9 7 5 ) . However, r e c e n t d e v e l o p m e n t s ( M a c C a r t h y et al., 1 9 7 9 ; Saito and H a y a n o , 1 9 7 9 ) in t h e sepa* ' Present address: Department of Oceanography, The University of British Columbia,
6270 University Boulevard, Vancouver, B.C. V6T 1 W5, Canada. ,2 Author to whom correspondence should be addressed.
0009-2541/81/0000--0000/$02.50 © 1981 Elsevier Scientific Publishing Company
356 ration of the components of aquatic and sedimentary humic substances may enable more meaningful investigations of their structures ar_d origins to be made. The measurement of the molecular weights of humic substances remains a controversial topic in the study of these compounds. Values ranging from a few hundred to several million have been reported for humic materials from various sources and utilising different analytical techniques (Schnitzer and Khan, 1972}. This spread of values may, in part, reflect differences in the nature of the sample, arising from the use of a variety of extraction and purification techniques. However, it seems likely that to a large extent they result from the difficulty of making meaningful measurements of the molecular weights of such substances. In c o m m o n with all polymeric materials, humic substances are composed of a complex mixture of species of various molecular weights and dimensions. If the method used is based on a colligative property, then the result obtained is the number-average molecular weight, MN, whereas the weight-average molecular-weight value, MW, is calculated from measurements of the weights of the individual polymeric species. Both gel filtration and ultrafiltration have been widely used for the determination of Mw-values of humic substances (Gjessing, 1965, 1976; Gjessing anti Lee, 1967; Shapiro, 1967; Ghassemi and Christman, 1968; Rashid and King, 1969; Swift and Posner, 1971; Cameron et al., 1972; de Haan, 1972; Wershavv and Pinckney, 1973; Stuermer, 1975; Alberts et al., 1976; Mantoura, 1976; Starikova et al., 1976; Kwak et al., 1977; Buffle et al., 1978; MacFarlane, 1978) although they both have considerable limitations and data obtained by such means must be treated with caution. Vapour-pressure o s m o m e t r y (Schnitzer and Desjardins, 1962; Hansen and Schnitzer, 1969; Martin and Reuter, 1973; Buffle et al., 1977; Linehan, 1977! and cryoscopic methods (De Borger and De Backer, 1968; Wilson and Weber. 1977) have been used to measure Mn-values of a wide range of humic substances. Hansen and Schnitzer (1969) and, more recently, Wilson and Weber (1977) have produced detailed theoretical treatments of the calculation of M---ncorr-values (Mn-values corrected for the dissociation of acidic functional groups) of naturally-occurring humic substances, including some isolated from river waters. However, both of these correction systems have limitations, whiL.h will be discussed in the following section. This paper describes a novel system for the correction of Mn-values of hum~c substances which only requires a knowledge of the pH of the solution. The procedure has been used to derive Mncorr-values for a range of freshwater and marine humic substances. EXPERIMENTAL AND CALCULATIONS
Isolation of aquatic hurnic substances The isolation, purification and chemical analysis of the humic samples used for the vapour-pressure o s m o m e t r y measurements have been described in de
357 tail (Gillam, 1979). Briefly, large volumes (100--200 l) of filtered (WhatmanC-~ GF/C) water were acidified to pH 2.0 and passed through columns (30 cm X 2 cm 2) of acetone-extracted Amberlite® XAD-2 resin. The adsorbed organic matter was eluted from the resin with aqueous ammonia (2 M, five bed volumes). The resultant solution was rotary-evaporated to a small volume and the organic matter was separated into its humic and fulvic acid fractions by acidification (HCI, pH 1.0). After removal of the precipitated humic acids, the fulvic acid remaining in the filtrate was purified by ultrafiltration, using a MW 500 cut-off filter. The protonated fulvic acid was recovered by rotary evaporation. Both materials were ground to a fine powder and thoroughly dried, in vacuo, over magnesium perchlorate.
Preparation of the solutions for vapour-pressure osmometry Weighed amounts of fulvic substances and standards (8--20 mg) were carefully dissolved in doubly distilled water to give a total volume of 2 ml. The solutions were thoroughly stirred to ensure complete mixing.
Instrumentation. The pH-values of the solutions of humic substances were measured with a Radiometer ® PHM research pH meter fitted with a GK ® 2301 C combined glass--calomel electrode. The linear response of the pH electrode was checked with 0.05 m solutions of potassium tetroxalate and potassium hydrogen phthalate (pH-values = 1.68 and 4.01, respectively, at 25°C) and it was standardised prior to each set of measurements with respect to the phthalate buffer. Vapour-pressure osmometric measurements were made at 37°C, using a Mecrolab ® 301 osmometer, following the manufacturer's instructions and general guidelines for Mn determinations given by Dr. D. Dare and the staff of the Polymer Characterisation Unit (P.C.U.), Department of Inorganic, Physical and Industrial Chemistry, University of Liverpool. The apparatus constant was determined using glucose solutions and was calculated to be 69.36 kg ~2 g-'. The accuracy of the vapour-pressure osmometer in the range 500--1000 D was verified by measurement of the Mn-value of a Waters Associates Ltd. GPC standard (polyethylene glycol), having an Mn-value of 790. Using a first virial model, an Mn-value of 730 was found. As the result from a linear leastsquares solution was in satisfactory agreement with the certified value, no corrections were applied.
Calculations o f Mn in vapour-pressure osmometry Bonnar et al. (1958) have reviewed the principal methods of measuring Mn and have also provided an excellent source of computational methods for the evaluation and interpretation of vapour-pressure-osmometry data. Techniques
358 for the d e t e r m i n a t i o n of Mn have been reviewed more recently by Glover (1975) who has also r e c o m m e n d e d c o m p u t a t i o n a l m et hods similar to those suggested by Bonnar et al. (1958). Meeks and Goldfarb (1967) have shown that the solution theories of Bonnar et al. are applicable to vapour-pressure o s m o m e t r y and that an expression of the type: :: = ~
+ aW
+ bW 2 +
is applicable. For this case, ,~,, often called the instrument readout, is expressed as a change in resistance of the thermistor network used to measure the temperature difference between the solution of the test material and the solvent. is the zero-point error, a and b are constants for a given instrument and so"JO lutions used, and W is the weight ratio of solute to solvent. The term a is directly related to Mn. As b and other higher terms can generally be neglected for all but th e most c om pl ex systems, it is normally then possible to write: Mn
=
Kapp/a
where Kapp is the apparatus constant Glover (1975) has discussed the principal types of model used to predict the behaviour of p o l y m e r solutions. For low molecular weight, ideal compounds. the P.C.U. r e c o m m e n d a simple, first-virial model of the t ype: ~:~= :)0 + a W This model accurately describes the majority of the data obtained for the a q u a t ic humic substances used in the present study. As there was no apparent deviation f r o m linearity for these data, corrections for non-ideality, using secondor third-virial models (Glover, 1975), were not made. The zero-point m e t h o d of Bonnar et al. was used to correct for the non-zero value of () in the absence of a solute. In the theoretical t r e a t m e n t of vapour-pressure o s m o m e t r y discussed above, no allowance has been made for the dissociation of organic c o m p o u n d s in aqueous solutions. Both De Borger and De Backer (1968) and Hansen and Schnitzel (1969) corrected their vapour-pressure o s m o m e t r y data by assuming that the dissociation of a p o l y f u n c t i o n a l acid (HrTA) could be described by the following equation: H n A ~ nH÷ + A n-
~1
At equilibrium, for a dibasic acid HnA, the total n u m o e r of particles was considered to be 1 + ha, rather than 1 + a~ + 2a2, where the a terms refer to the consecutive dissociation steps of the acid. Wilson and Weber (1977) have described a more complex correction system, based on measurements of the concentrations and "dissociation c o n s t a n t s " of the individual acidic functional groups. Although this latter correction is satisfactory for pure di- and polycarboxylic acids, difficulties arise when a t t e mpt s are made to apply it ~o naturally-occurring humic substances (see the Section "Results and Discussion"). The proposed correction method is based on the identity that, for the dis-
359 sociation of a polyacidic compound, the total concentration of particles at equilibrium is given both by the expression Ca+[H ÷] and by W/Mnexp., where Ca, [H+], W and Mnex are, respectively, the total acid and hydrogen 1on concentrations, the weigh~ ratio of solute to solvent, and Mnex p is the experimentally determined value of Mn. Thus: Ca +
[H ÷] = W/Mnex p
and
Mnex p = K a p p / a (*)
Then: Ca + [ H ÷] = W a / K a p p and so: C a = W a / K a p p - [H ÷]
(2)
Since [H ÷] can be measured and C a calculated from eq. 2, the value of ~ corrected for dissociation, (0corr), can be calculated from: ecorr = 8/(1 + [H ÷]/C a)
(3)
From a set of data for G vs. W, the most suitable model was selected for the experimental data. A value for the constant a was then calculated for the complete determination. A value for Ca for each data point was calculated from eq • 2, using the known values of K a p , pH and W and this value of a • 0 c o r r was then evaluated for each data point ~ o m eq. 3 and the most suitable model for the corrected data was again selected• The corrected value for a, acorr, was calculated and the corrected value for the molecular weight (Mncorr) of the compound under investigation was then determined. RESULTS AND DISCUSSION m
The values of Mncorr for several known organic compounds were calculated, using the novel correction system described above (Table I). The compounds were chosen because of their partial chemical similarity to fulvic acids. Experience with the vapour-pressure osmometer suggested that a mean deviation between the actual and the experimental values of Mn of 5% was to be expected. The results in Table I indicate, therefore, that the correction system does compensate adequately for the dissociation of these low-molecular-weight organic compounds. Further evidence for the suitability of the system was provided by re-evaluation of the data of Wilson and Weber (1977) for tartaric acid and tartaric acid--succinic acid mixtures. Using a second-virial model with a zero-point correction, it was shown (Table II) that it was possible to obtain accurate Mneorr-values w i t h o u t prior knowledge of the dissociation constants or of the initial concentrations of each t y p e of acidic functional group. The major difficulties with the application of the correction m~thod of t-hnsen and Schnitzer (1969) to aquatic humic substances are that: (1) an * A similar approach was used by Glover (1975) and Wilson and Weber (1977).
360 TABLE I
Mn ~ o r r -values for known organic c o m p o u n d s (values given are the mean of three determinations) (_k)mpound
Mnactua!
Mncorr
Per cent deviation
Tartaric acid Ascorbic acid Oxalic acid
150.1 176.1 90.0
154.2 180.6 93.8
+2.8 +2.6 +4.2
] ' A B L E II Comparison of correction methods Compound
Mnactual
Mn Otl: (Wilson and Weber, 1977)
Mn COl'[ (proposed m e t h o d ,
Tartaric acid Tartaric acid--succinic acid
l 50.1 1 38
152..I 142.4
15[.2 139.1
i n a c c u r a t e a p p r o x i m a h o n for the dissociation of a p o l y f u n c t i o n a l acid is made, and (2) relatively large c o n c e n t r a t i o n s of h u m i c substances are required to ensure that linearity o f t h e ,,--W relationship is o b t a i n e d (Hansen and Schnitzer, 1969). Curvature of the lines at low c o n c e n t r a t i o n s , s h o w n in Fig. 1, makes it d i f f i c u l t to e x t r a p o l a t e to t h e p o i n t at which W = 0, and this means that the I
.
•
.g
,~',.,.
i W .']:
,
•
i O-
i -[ .
" r~,
.
.
.
.
!~00
.
.
.
.
.
.
":)'2 ~
.
.
.
.
.
"~O,,C..
.
.
.
.
.
2'J)O(~
.
.
J
2~S)C ,
Fig. 1. A p p l i c a t i o n o f tile correction system o f Hansen and Schnitzer ( 1 9 6 9 ) to VPO data o f water fulvic acids.
361
method is unsuitable for use with either our own fulvic acid data or with those published by Wilson and Weber (1977). The differences in Mn-values obtained by Wilson and Weber for their water fulvic acid and the value obtained using the correction method of Hansen and Schnitzer (see Fig. 1) may be partly explained by the approximations of these methods. As Wilson and Weber {1977) have noted, both De Borger and De Backer (1968) and Hansen and Schnitzer (1969) assumed that the dissociation of a polyfunctional acid could be described by a one-step process. Wilson and Weber (1977) described a more complex system, based on the approximation that their fulvic acid samples behaved as a mixture of two monobasic acids. It has been suggested that the presence of two distinct groups of carboxylic acids may not accurately represent the polyacidic nature of humic substances (J.H. Reuter, pers. commun., 1979). In order to calculate values for the individual dissociation constants, Wilson and Weber (1977) utilised data for soil fulvic acids previously reported by Gamble {1970, 1972). Furthermore, they utilised the potentiometric approach devised by Borggaard (1974) to calculate the concentrations of each of the acidic functional groups. Stuermer (1975) and Gillam (1979) have both provided evidence to suggest t h a t no secondary maxima are present in derivative plots of potentiometric titrations of aquatic fulvic acids. Perdue et al. (1980) have recently discussed the difficulties of obtaining representative values for the concentrations and dissociation constants of carboxylic functional groups in humic substances. Thus, the correction methods of Hansen and Schnitzer and Wilson and Weber are both of limited value for the fulvic acid samples used in this study. The proposed correction m e t h o d requires neither the use of an extrapolation procedure nor any previous knowledge of the dissociation constants of the compound. Although pH is not strictly defined as - log [ H÷], all previous methods for the determination of Mn c orr-values of aquatic__ humic substances have made this assumption. Despite this, accurate Mn¢orr-values for several known organic compounds have been obtained. The accurate calculation of [H ÷] requires knowledge of the E °- and/~-values of the electrode system. On the basis of previously reported methods, it was decided that, although the assumption that the concentration and activity terms are identical is incorrect, the differences in the Mncorr-values would be within the bounds of experimental error. Data for the calculation of Mncorr-values for several types of sample are given in Table III. The relatively small range of concentrations for the aquatic humic substances was determined by the limited amounts (< 100 mg) of organic material. However, despite the limited number of data points, accurate M--n-values were obtained for tartaric and ascorbic acids. In preparing samples of humic substances for vapour-pressure osmometry, ultrafiltratiQn was used to ensure that t h e y were free from inorganic salts and low-molecular-weight organic compounds, as these would cause the molecular weights to appear to be anomalously low.
362 TABLE III
Sample data for vapour-pressure-osmometry measurements Compound
W (g/kg)
pH
:
Tartaric acid
3.900 5.880 7.796 10.352
2.19 2.08 2.00 1.93
1.030 2.165 3.098 4.428
4.045 5.946 7.988 10.020
2.46 2.39 2.32 2.28
3.892 6.111 7.999 10.689 4.177 5.983 7.826 9.421
Ascorbic acid
SK-8
HM-I
~
i," C O L T
acorr
Mncorr
0.523
0.804 1.759 2.571 3.762
0.455
152.3
1.160 2.070 2.788 3.750
0.425
0.997 1.839 2.514 3.429
0.399
173.8
2.38 2.19 2.10 2.01
0.785 1.238 1.603 2.112
0. t95
0.486 0.773 1.037 1.425
0.138
503
2.32 2.21 2.05 2.03
0.628 0.860 1.105 1.358
0.138
0.266 0.415 0.472 0.681
0.074
943
T h e Mn-values o f a series o f fulvic acid s a m p l e s f r o m a variety of soils and n a t u r a l w a t e r s are s h o w n in Fig. 2. As a general t r e n d , it a p p e a r s t h a t :
soil fulvies
lake,'river fulvics
marine fulvics
F r o m t h e results o b t a i n e d using the p r o p o s e d c o r r e c t i o n s y s t e m , it w o u l d appear t h a t it is possible to d i f f e r e n t i a t e b e t w e e n fulvic acids f r o m w i d e l y differing origins. When this s y s t e m is a p p l i e d t o t h e soil a n d w a t e r fulvic acid d a t a o f Wilson and W e b e r ( 1 9 7 7 ) , Mncorr-values o b t a i n e d are 1103 and 888, r e s p e c t i v e l y . W h e t h e r this is indicative o f t h e e x t e n t to w h i c h t h e h u m i c materials in t h e t w o s y s t e m s have b e c o m e d e g r a d e d c a n n o t be stated f r o m this o n e set o f values. H o w e v e r , t h e d e c r e a s e in Mncorr-values o n passing f r o m a lacustrine to a m a r i n e e n v i r o n m e n t m a y be a t t r i b u t a b l e to d i f f e r e n c e s in the solubility o f the various s u b f r a c t i o n s o f the h u m i c substances. As the salinity increases, the h i g h e r - m o l e c u l a r - w e i g h t f r a c t i o n s o f t h e h u m i c c o m p o u n d s will t e n d to p r e c i p i t a t e selectively. This will lead to an overall l o w e r i n g o f t h e ~Invalues (see, e.g., B r o w n , 1975, 1 9 7 7 ; S h o l k o v i t z , 1 9 7 6 ; S h o l k o v i t z et al., 1 9 7 8 ~ In general, t h e c o r r e c t e d m o l e c u l a r weights are several t i m e s l o w e r t h a n t h o s e o b t a i n e d b y gel p e r m e a t i o n c h r o m a t o g r a p h y and u l t r a f i l t r a t i o n , which d e p e n d m a i n l y on m o l e c u l a r size and s h a p e ( C a m e r o n et al., 1972; Buffle et al., 1978).
363
2000 i u
h
[: O~
I000i
~O
Vo W
~7~2V
400 [--
So,:
I Lak@/Riv(2r
I
iVlarln~
Fig. 2. Mncorr-values for a range of fulvic acid samples. v = this study; ~ = Buffle et al. (1977); o = Wilson and Weber (1977)* ; a = De Borger and De Backer (1968)* ; • = Hansen and Schnitzer (1969)* ; • = Martin and Reuter (1973); Footnote: * values obtained using the novel correction method. TABLE IV Mncorr-values for the aquatic fulvic acids of this study Sample
Salinity (°/oo)
Mncorr
Sample
Salinity (°/oo)
Mncorr
SK-I SK-2 SK-4 SK-5 SK-6 SK-7
31.37
591 755 626 650 720 792
SK-8 HM-I CN-2 DE-/ IS-2
33.20
501 943 846 761 623
31.30 31.10 28.28 27.80
32.59
SK = samples collected during R.S.S. "Shackelton" cruise, June 1977, Skaggerak; HM = sample from peaty stream feeding Hatchmere, Delamere Forest, Cheshire; CN = sample from Lake Celyn, north Wales; DE = sample from River Dee, north Wales; IS = sample collected during R.V. "Edward Forbes" cruise, October, 1977, Irish Sea.
T h e M--ncorr d a t a f o r t h e m a r i n e f u l v i c a c i d s o f t h i s s t u d y w e r e p l o t t e d against the salinity of the water samples from which they were concentrated. T h e r e g r e s s i o n b e t w e e n t h e s e t w o p a r a m e t e r s is s h o w n in Fig. 3 a n d y i e l d e d a c o r r e l a t i o n c o e f f i c i e n t o f - 0 . 8 6 , w h i c h is s i g n i f i c a n t a t t h e 9 5 % c o n f i d e n c e limit. This provides further evidence that one of the major effects of increasing s a l i n i t y o n t h e D O M p r e s e n t in n a t u r a l w a t e r s is t h e r e m o v a l o f t h e h i g h e r molecular-weight fractions of the DOM.
364 350r
i. 3OO~-
i 270 I 4OO
1 .... M
Fig. 3. Correlation of salimty with Mncorr-values of marine fulvic acid samples of this s t u d y
Although water has been widely used as a solvent for vapour-pressure osm o m e t r y of fulvic acid samples, this medium cannot be employed as a solvent for humic acids. Although sulpholane has been used for this purpose, with the humic acid fractions of various soils (Schnitzer and Desjardins, 1962), it requires extensive purification. Trichloromethane and 2-butanone have also been used for Mn determinations, and values in the range 410--1000 were obtained for a series of subfractions of a sedimentary humic acid sample (Ishiwatari, 1973). CONCLUSIONS
The Mn-values obtained for the fulvic acids in the present study are within the range of those obtained by other workers using the VPO technique. Accurate values for the molecular weights of naturally-occurring aquatic humic substances are essential for studies of the metal complexation ability of these compounds. Few studies have been made to determine the nature of the humic fractions which are involved in metal complexation. Previous reports (Mantoura and Riley, 1975; Buffle et al., 1977) indicate, however, that it is the low er-molecular-weight compounds which possess the greater complexing ability. It appears that vapour-pressure o s m o m e t r y will remain one of the more important techniques for the determination of the molecular weights of aquatic humic substances. ACKNOWLEDGEMENTS
The authors would like to thank the staff of the Polymer Characterisation Unit, University of Liverpool, for their guidance with the vapour-pressure osmometer used in this study. One of the authors (A.G.) is indebted to the Natural Environment Research Council for the award of a research studentship.
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