Volume and structure of humic acids studied by viscometry

Colloids and Surfaces A: Physicochemical and Engineering Aspects 151 (1999) 213–224

Volume and structure of humic acids studied by viscometry pH and electrolyte concentration effects M.J. Avena 1, *, A.W.P. Vermeer, L.K. Koopal Laboratory for Physical Chemistry and Colloid Science, Wageningen Agricultural University, Dreijenplein 6, 6703 HB Wageningen, The Netherlands Received 4 November 1997; accepted 29 April 1998

Abstract Viscometry was used to evaluate the effects of pH and supporting electrolyte concentration on the intrinsic viscosities of eight humic acids and one fulvic acid. Two synthetic poly(acrylic acid ) (PAA) samples of different molecular weight were also studied for comparison. Humic and fulvic acid molecules behave as flexible entities that can swell or shrink in response to changes in pH and ionic strength. An increase in the solution pH leads to the development of negative charges in the molecules with the consequent electrostatic repulsion between ionized groups and molecular swelling. Increasing the ionic strength increases the screening of charges and leads to molecular shrinkage. The pH dependence decreases with increasing electrolyte concentration and at 10−1 M electrolyte the intrinsic viscosity is almost pH independent. The general behavior of PAAs is similar to that of the humics, though the effects of pH and electrolyte concentration are much larger for the PAAs. The degree of hydration of the humics differs for different samples. There are compact samples with low water content and swelling properties whereas other humics are more hydrated and flexible. All the studied humics have an internal structure that limits the expansion of the molecules when the electrolyte concentration is decreased. The latter is in accordance with the low values of the Mark–Houwink coefficient, a, of humics. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Branching; Degree of hydration; Flexibility; Hydrated fulvic acid structure; Hydrated humic acid structure; Internal structure; Intrinsic viscosity; Mark Houwink coefficient

1. Introduction Information on the structure and size of humic molecules is relevant for the understanding of their physical–chemical properties and the geochemical role they play in soils, aquifers and sediments. The molecular structure of humics is not known in * Corresponding author. Fax: +31 317 483777; e-mail: [email protected] or [email protected] 1 On leave of absence from INFIQC, Departamento de Fisicoquı´mica, Facultad de Ciencias Quimicas, Universidad Nacional de Co´rdota, Co´rdota, Argentina.

detail yet and several models have been proposed. Chen and Schnitzer [1] mentioned that fulvic acids ( FA) and humic acids (HA) behave like flexible, linear, synthetic polyelectrolytes and concluded that there must be numerous linkages about which relatively free rotation occurs. Ghosh and Schnitzer [2] concluded that HA and FA behave as rigid sphero-colloids at high sample concentration, low pH or in the presence of sufficient amounts of neutral electrolytes, but they are flexible linear colloids at low sample concentrations, relatively high pH and low electrolyte concentration. Cameron et al. [3], on the other hand,

0927-7757/99/$ – see front matter © 1999 Elsevier Science B.V. All rights reserved. PII S0 9 2 7- 7 7 5 7 ( 9 8 ) 0 05 0 4 - 4

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visualized the HA molecules in solution as a series of charged, occasionally branched strands. They concluded that branching results in an increased coil density within the molecule giving rise to more compact spheres compared with a linear molecule of equivalent weight. They also described the HA molecules as a structure that is perfused with solvent molecules which are able to exchange with bulk solvent molecules. More recently, Schulten [4] and Schulten and Schnitzer [5] proposed a structure that resulted from a comprehensive investigation combining different experimental techniques with molecular mechanics and dynamic calculations. The optimized HA structure turned out to be a crosslinked network with voids of various dimensions that can trap and bind other organic components such as carbohydrates or proteinaceous materials as well as inorganics and water. The branched structure was shown to be dynamic, with mobility of the side chains and occluded molecules, changes in hydrogen bonding, and voids formation. This picture of flexible and permeable molecules has led to the use of the Donnan model to theoretically represent the electrostatic properties of humics [6,7]. In this model a HA molecule is seen as a gel type domain with a homogenous electrostatic potential inside it. Outside the gel the potential drops to zero. Together with the NICA model that accounts for the site binding of ions to the HA molecules [8], the so-called NICA–Donnan model has been used successfully to describe H+, Cd, Ca, Cu and Pb binding to humics at different pH and electrolyte concentrations [9,10]. The only parameter the Donnan model requires is the hydration volume, i.e. the volume of solution inside the solvated particles per unit mass of the dry particles. This volume has been used as an adjustable parameter in previous articles and it was assumed to be ionic strength dependent and pH independent. However, experimental evidence supporting these assumptions is lacking. Viscometry offers a way to obtain the hydration volumes of humics in aqueous solutions. According to the Einstein equation [11], the intrinsic viscosity is related to the specific volume, V , HA of the HA molecules by: [g]=nV HA

(1)

where n is a shape factor whose value is 2.5 for spheres and larger than 2.5 for ellipsoids [12], and: N V V = A h HA M

(2)

where V is the hydrodynamic volume of the HA h molecule, M its molecular weight and N A Avogadro’s number. The specific volume V in HA an aqueous solution can also be expressed as a function of the partial specific volume of the dry HA, V , and the specific hydration volume, dry V : hydr V =V +V (3) HA dry hydr Thus, the intrinsic viscosity can also be written as: [g]=n(V +V ) (4) dry hydr which allows us to estimate the hydration of the humics provided the other quantities are known [11,13]. If the shape factor is constant, the hydration volume is directly proportional to [g]. In this article we report the viscometric properties of a series of humic acids, a fulvic acid and two poly(acrylic acid) samples. We mainly address the effects of varying pH and electrolyte concentration on the conformational properties and hydration volumes of the studied materials.

2. Materials and methods 2.1. Samples Eight different humic acids and a fulvic acid were studied. The elementary composition, atomic ratios and origins of the samples are listed in Table 1. Higashiyama L (HLHA), Kinshozan P ( KPHA), Kinshozan F ( KFHA), Kinshozan OH ( KOHHA) and Shitara Black (SBHA) humic acids were obtained from Y.-H. Yang [14]. They are soil humics. The origins and methods of extraction and purification of these samples together with their physical and chemical properties were reported by Kuwatzuka et al. [15] and by Tsutsuky and Kuwatsuka [16 ].

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M.J. Avena et al. / Colloids Surfaces A: Physicochem. Eng. Aspects 151 (1999) 213–224 Table 1 Elementary composition (% weight on an ash-free basis) and atomic ratios of the studied samples Sample

PPHA HLHA FSHA KPHA KFHA PAHA KOHHA SBHA LFA

Elementary composition

Atomic ratio

Origin

C

H

N

O

H/C

N/C

O/C

52.1 59.1 52.9 57.3 57.1 55.8 55.2 58.7 45.1

5.1 5.7 5.4 3.8 4.5 4.6 5.4 3.4 4.1

2.4 3.3 2.4 2.6 3.9 0.6 3.3 3.4 1.1

39.9 32.0 39.3 36.3 34.6 38.9 36.0 34.5 49.7

1.17 1.16 1.22 0.79 0.94 0.99 1.17 0.70 1.09

0.039 0.048 0.039 0.039 0.059 0.009 0.051 0.050 0.021

0.57 0.41 0.56 0.48 0.45 0.52 0.49 0.44 0.83

Purified peat humic acid (PPHA) was obtained from D.K. Kinniburgh [6 ]. This is a well-studied sample prepared from a commercial Irish horticultural peat. Its chemical composition, solid state 13C NMR and UV–vis analyses as well as proton and cation (Cd, Cu, Pb, and Ca) binding properties were reported previously [6,10]. Equilibrium UV scanning ultracentrifugation showed that the sample is polydisperse with a weight average molecular weight of approximately 23 000 D. Purified Aldrich humic acid (PAHA) is the result of a purification treatment applied to Aldrich humic acid (Aldrich Chemie, code: H1,675-2). The purification procedure of PAHA was essentially the same as that applied by Vermeer et al. [17]. Extensive characterization of this sample was performed by Vermeer [9] using fluorescence, UV–vis and FT-IR spectroscopies, liquid and solid state 1H and 13C NMR, viscometry, gel permeation chromatography and ellipsometry. Although Aldrich HA is believed to be coal derived, the results showed that, by comparing the PAHA properties with those of humics derived from fresh water, marine water, soil or peat, PAHA is most similar to soil HA. However, its nitrogen content is lower and it is also somewhat more hydrophobic than most soil humic acids. The average molecular weight obtained by size exclusion chromatography using proteins as standards is 21 000 D. This value is similar to that reported for Aldrich HA in general [18–20]. Forest soil humic acid ( FSHA) was obtained from E.J.M. Temminghoff [21]. It was extracted

Peat Soil Soil Soil Soil Coal? Soil Soil Soil

from the forest floor material, taken from the Tongbergen forest (Oisterwijk, The Netherlands). Some physical–chemical properties and its proton and copper binding capacity were described by Temminghoff et al. [21]. Laurentian fulvic acid (LFA) was obtained from C.H. Langford [22]. It derives from a sample of a podsol collected in the Laurentian Forest Preserve of Laval University, Quebec, Canada. Physicochemical characterization of the sample has been reported by Wang et al. [22]. Besides humic and fulvic acids, two poly(acrylic acid ) samples (M=150 000 and M=500 000) were also studied for comparison. The samples were purchased from Polysciences Inc. ( Warrington, USA) and were used without further purification. 2.2. Viscosimetric measurements All measurements were performed at 23.0±0.1°C. The ionic strength, I, was adjusted to nominal 10−3 M, 10−2 M and 10−1 M solutions by adding the necessary amount of either solid KNO or a 1 M KNO solution. 3 3 Before use the samples were dissolved in a KOH solution at pH around 10 and equilibrated overnight. Results of potentiometric titrations have shown the necessity of this procedure to avoid hysteresis in the charging behavior of humic acids [6,9]. After the equilibration, the electrolyte concentration and pH were adjusted by adding KNO and HNO /KOH solutions respectively. 3 3 The viscosities were measured with an automatic

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dilution Ubbelohlde viscometer ( Viscometric MS type 53000). Special care was taken in measuring the flow times for the different KNO blank solu3 tions. The values were 293.7 s, 293.5 s and 291.4 s for 10−3 M, 10−2 M and 10−1 M solutions respectively. This variation is in agreement with the corresponding variation in the viscosity of KNO 3 solutions as a function of salt concentration [23]. In a typical sample run, 8 ml of a 2 g/l solution of given pH and salt concentration was introduced into the viscometer and a series of readings of the flow time was made. After that, 5 ml of a KNO 3 solution of the same concentration and pH was added and a new series of readings performed. This procedure was repeated until the concentration of the HA solution was around 0.5 g/l. The pH was checked after the measurements. In all cases, the difference between the pH of the initial solution and the diluted one was less than 0.5 units. The presented viscosities are averages of a sequence of at least 10 stable readings. From the measured viscosities and those of the blank solutions, the reduced viscosities, g , were red calculated. The intrinsic viscosity was found by extrapolating g to zero concentration [11]. red 3. Results A typical result is shown in Fig. 1, where the g versus c curves for PPHA at pH 7 and at red HA four nominal KNO concentrations are plotted. 3

Fig. 1. Reduced viscosity, g , as a function of the concenred tration of PPHA, c , at pH 7 and four nominal KNO concenHA 3 trations. Dilutions were performed with salt of the same nominal concentration except for the ‘‘water’’ curve where 10−3 M KNO solution was used. 3

At a given HA concentration, the reduced viscosity decreases as the electrolyte concentration increases. The curves at 10−1 M and 10−2 M are straight lines parallel to the c axis, indicating very weak HA effect of dilution on g . In these cases, the intrinsic red viscosity of the samples can be accurately evaluated by extrapolating the curves to c =0. Although HA the curve at 10−3 M is also a straight line, it has a negative slope that corresponds to a decrease in the ionic strength as the dilution with 10−3 M KNO progresses. Conductivity measurements 3 confirm that this is the case: the conductivity of the initial solution (c =3.7 g/l ) was around HA 0.5 mmho/cm, which is the conductivity that corresponds to a KNO solution between 3 3×10−3 M and 4×10−3 M. This high ionic strength is probably due to the presence of additional potassium and nitrate ions provided by the acid and base added to set the pH to 7. Upon dilution with 10−3 M solution, the initially high ionic strength decreased, with a consequent decrease in the intrinsic viscosity. Support for this explanation is given by the ‘‘water’’ curve, which was obtained with PPHA initially dissolved in pure water. The only source of K+ and NO− at the 3 beginning of the measurements was the acid and/or base additions to set the pH to 7. The conductivity of this initial solution (c =2 g/l ) corresponded HA to a KNO concentration around 6×10−4 M. By 3 diluting with 10−3 M KNO the ionic strength 3 increased and the resulting curve had a positive slope as can be seen in Fig. 1. In spite of the fact that the electrolyte concentration changed in nominal 0 M and 10−3 M experiments, the good linearity of the curves allows us to evaluate [g] at 10−3 M because this is the resulting electrolyte concentration at infinite dilution. Fig. 2 shows the [g] versus pH curves for the humic and fulvic acids studied at three KNO 3 concentrations. By analyzing the curves for PPHA, HLHA and PAHA it is possible to exemplify the behavior of all samples. PPHA has the largest [g] values. The intrinsic viscosity decreases with increasing electrolyte concentration. In the pH range 4–10 the pH effects are not important at high ionic strengths, however, for 10−3 M and 10−2 M KNO [g] increases with pH. For all salt 3 concentrations there is an increase in the intrinsic

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Fig. 2. Effects of pH and KNO concentration on the intrinsic viscosity of a series of humic acids and a fulvic acid. $ 10−3 M; c 3 10−2 M; , 10−1 M KNO . 3

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viscosity at pH lower than 4. This is due to aggregation and subsequent precipitation of the material. The HLHA sample shows intermediate [g] values. The behavior is similar to that of PPHA except that no increase in [g] at low pH is observed. Similar viscosities and behavior are found for FSHA, KPHA and KFHA. PAHA, SBHA and LFA have low intrinsic viscosities. The high relative uncertainties do not allow us to evaluate [g] very accurately. Also included in this group is KOHHA, which has a somewhat higher [g]. The general trends observed for these samples are similar to those observed for PPHA and HLHA, i.e. decreasing [g] with increasing electrolyte concentration or decreasing pH. However, the effects of the KNO concentration 3 and the pH are relatively small as compared with those of the HLHA group. Fig. 3 shows the [g] versus pH curves for PAA500 and PAA150 samples. The behavior is typical for linear polyelectrolytes; the intrinsic viscosity decreases strongly as the electrolyte concentration increases. [g] depends on the pH in acidic media and is constant at pH>7. Similar behavior for different PAA samples in aqueous NaBr solu-

Fig. 3. Effects of pH and KNO concentration on the intrinsic 3 viscosity of two PAA samples. $ 10−3 M; c 10−2 M; , 10−1 M KNO . 3

tions was found by Noda et al. [24] and Takahashi and Nagasawa [25]. Although the general trends are similar, all the effects observed for the synthetic polyelectrolytes are much larger than for the humics.

4. Discussion 4.1. Effects of HA concentration and ionic strength on the viscosity The plots of g versus c are straight lines at red HA all the conditions studied. Their slopes are close to zero in 10−2 M and 10−1 M solutions, which indicates that in the concentration range between 0.5 and 4 g/l the reduced viscosity of humic samples accurately approximates the intrinsic viscosity. This also means that the effects of intermolecular interactions are negligible and that the volume and size of the hydrated molecule remain unchanged in the considered range of humic acid concentration. As stated above, the non-zero slope at low salt concentration can be explained as an effect of changing ionic strength upon dilution. A great variety of g versus c behaviors has red HA been forwarded in the literature. It is well known that g of humics increases upon dilution with red pure solvent as in the case of polyelectrolytes [26– 30]. As the concentration of molecules gradually decreases, the concentration of counterions and the ionic strength also decrease, lowering the screening of charges and swelling the molecules. An increase in g with decreasing c has also red HA been noted for HA and FA samples, even in the presence of a constant nominal supporting electrolyte concentration. Ghosh and Schnitzer [2], for example, reported bending of curves towards high g at low HA concentrations for samples prered pared in 10−3 M, 5×10−3 M and 10−2 M NaCl solutions at pH 6.5. The less concentrated the diluting solution, the more pronounced the bending of the curves. Highly concentrated solutions (4 g/l4 g/l they postulated that the moleHA

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cules behaved like uncharged polymers or spherocolloids, due to the lack of available space to expand the structure. However, the overall behavior can also be explained by invoking a decrease in the ionic strength as dilution progresses. If it is assumed that the studied samples had a charge of 3 mmol/g at pH 6.5 [7,9], an 8 g/l solution would have a total counterion concentration of about 2.4×10−2 M. This high initial value indicates that the ionic strength of the HA solution should decrease upon dilution with either 10−3 M, 5×10−3 M or 10−2 M NaCl solutions, leading to a bending up in the g versus c curves. The red HA less concentrated the diluting solution, the more pronounced the bending. Besides the behaviors indicated above, other kinds of g versus c curves have been reported. red HA Visser [31], for example, found linear variations in the 2.5–5 g/l concentration range for different humic and fulvic acids of different molecular weight, even when dilutions were performed with water. Recently, Rey et al. [32], by studying a soil FA, reported a minimum in the curves at a fulvic concentration value of around 0.16 g/l at pH 4 at ionic strengths varying from 5×10−3 M to 10−1 M. A maximum in the curves has also been observed [28]. These different behaviors reported in the literature indicate that the dependence of g on concentration is sometimes rather complired cated to explain for HA and FA, and is not yet fully understood. The shape of the curves seems to be highly dependent on the particular material studied and the experimental conditions. As in the present measurements no obscure effects have been observed, it follows that a good control of the ionic strength appears to be critical in order to obtain flat or straight g versus c curves that red HA allow us to accurately evaluate [g]. 4.2. Effect of ionic strength and pH on the intrinsic viscosity The effect of the ionic strength, I, on the intrinsic viscosity of the HA samples is similar to the effects of I on polyelectrolytes [29,30]. As the salt concentration decreases, the concentration of counterions also decreases, lowering the screening of charges of the polymer. This results in increased repulsive

219

forces between charged groups which cause the molecule to expand, assuming an extended configuration. As a consequence of the swelling process, [g] increases, as indicated by Eq. (1). At a constant ionic strength, the decrease in [g] by decreasing pH shown in Fig. 2 can also be explained in terms of molecular volume variations. It is well known that carboxylic and phenolic groups in HA and FA deprotonate to generate negative charges in the molecules. The charge is very low at low pH and monotonously increases as the pH increases. Normal charge densities for humics are around 1 mmol/g at pH 3 and 4–5 mmol/g at pH 10 [6,33]. Therefore, alkaline media lead to molecular expansion because of the relatively high repulsive forces among charged groups. As the pH decreases, the charge decays and the molecule shrinks. Since the pH effect decreases with increasing ionic strength, [g] versus pH curves at different salt concentrations approach each other as the pH decreases. They should meet at the point of zero charge of the sample where electrostatic repulsion between groups should be zero. For most of the samples studied, the curves at different electrolyte concentrations tend to merge around pH 3, indicating that the point of zero charge is approached. If molecules were able to develop positive charges, [g] would increase again and the curves should separate on further decrease in pH. This behavior is not observed, except for PPHA ( Fig. 3). However, it is unlikely that PPHA carries positive charges at pH<4. As pointed out above, the increase in [g] at pH lower than 4 for PPHA is due to molecular aggregation and/or precipitation. Also, the increase of [g] at 10−1 M KNO for 3 KPHA must be due to aggregation. Otherwise, the effects should have occurred more strongly at 10−2 M and 10−3 M. Not too many reports can be found in the literature about the [g] versus pH behavior of humic and fulvic acids at different electrolyte concentrations to compare with the present results. Kumada and Kawamura [34,35] measured the reduced viscosity of several humics at a constant concentration of 5 g/l and reported a diminution in g as the pH was decreased from alkaline red media. The curves had a minimum at pH around

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4–5, and the reduced viscosity increased again by further lowering the pH, as in the case of PPHA or KPHA at 10−1 M electrolyte. Chen and Schnitzer’s [1] data for FA show that both the intrinsic and the reduced viscosity of the materials had also a minimum at pH around 3, in the absence of added electrolyte. Later, Ghosh and Schnitzer [2] found minima in the curves of HA and FA at pH around 6.5. Recently, Rey et al. [32] found a minimum at pH around 6 for the [g] versus pH curves of FA at different KNO concen3 trations. The effects of pH were interpreted as a consequence of varying molecular associations [2,32,34,35], electrostatic repulsion among charged groups [1,34,35], and clustering of water molecules around ionized groups [1]. Ghosh and Schnitzer [2] also proposed that HA and FA molecules are coiled at low pH and/or high electrolyte concentration, and uncoiled and more or less linear at low salt concentration and high pH values. Although several explanations have been postulated, the present results can easily be understood if the molecules are thought to be flexible entities that can swell or shrink in response to ionization of groups and screening of charges. Low pH and high ionic strengths decrease the repulsive forces between charged groups, leading to molecular shrinkage. At very low pH and high electrolyte concentration, intermolecular repulsive forces are also decreased so that molecular association and aggregation may take place. PAA samples have also intrinsic viscosities that are a function of the charge and the screening by supporting electrolyte ions. Protonation of carboxylic groups at pH lower than 7 diminishes the net negative charge of the molecules, decreasing [g]. Above pH 7 the net charge of these linear polyelectrolytes hardly increases due to counterion condensation effects [36,37]. As in the case of our HA and FA results, the pH effects are diminished by increasing the ionic strength because of the screening of charges. 4.3. Hydration Eq. (4) shows that [g] depends on the shape factor n and the degree of hydration. These two

parameters cannot be independently resolved considering viscosity measurements alone. Following Tanford [13], and in order to obtain some information about the water content of the studied samples, maximum values for V (using n=2.5) hydr and a compromise value for n (using V =1.5 ml/g) were calculated. For V an hydr dry average value of 0.6 ml/g was used [38]. The intrinsic viscosities at pH 10 and 10−1 M or 10−3 M electrolyte were used in the calculations, and the results are shown in Table 2. KPHA, SBHA, PAHA and LFA are neither highly hydrated nor highly asymmetric in 10−1 M electrolyte. Their respective V and n are comparable hydr to those of globular proteins like serum albumin, ribonuclease and hemoglobin [13]. The molecules appear to be somewhat compact entities. The other considered humic acids are more hydrated and/or more asymmetric, especially PPHA, which would have more than 90% water in its structure if the molecule is considered to be spherical. By decreasing the electrolyte concentration to 10−3 M, the volume and hydration of the molecules increase considerably. Flexible molecules such as HLHA, FSHA, KPHA and KFHA, increase their specific volume by a factor of two to three, whereas more structured humics increase it by a factor of 1.5 to 1.8. Fig. 2 indicates that the specific volume is both ionic strength and pH dependent. A decrease in the pH from 10 to 3 is as effective as an increase in the electrolyte concentration from 10−3 M to 10−1 M in order to shrink the molecules. Thus, by varying the pH from 3 to 10 at 10−3 M electrolyte, the volume changes two to three times for flexible humics whereas it changes 1.5 to 1.8 times for more rigid ones. 4.4. Structure An interesting way to gain further insight into the structure of organic polyelectrolytes is by analyzing the effects of supporting electrolyte concentration on the intrinsic viscosity, similarly to what was done by Mays [39], who compared the expansion of different linear and star-branched poly(styrene sulphonate) (PSS) samples. Mays compared the measured hydrodynamic radius at a given ionic

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Table 2 Specific volumes, V , specific hydration volumes, V , and the shape factor n of a series of humic samples at pH 10 and HA hydr 10−1 M KNO ( less hydrated state) and 10−3 M KNO (most hydrated state) 3 3 Sample

n=2.5

V

V (ml/g) HA

V

=1.5 hydr

Flexibility

n

hydr

10−1 M

10−3 M

10−1 M

10−3 M

10−1 M

PPHA

10.0

17.2

9.4

16.6

11.9

HLHA FSHA KPHA KFHA

3.6 3.4 1.8 2.4

9.2 7.2 5.2 6.4

3.0 2.8 1.2 1.8

8.6 6.6 4.6 5.8

4.3 4.0 2.1 2.9

f f/s f f

PAHA KOHHA SBHA LFA

1.4 3.4 1.4 2.2

2.6 5.6 2.2 3.4

0.8 2.8 0.8 1.6

2.0 5.0 1.6 2.8

1.7 4.0 1.7 2.6

s s s s

s

f=flexible, s=structured.

strength to that measured for the same sample in another electrolyte solution. He found that the hydrodynamic radius of a branched PSS increased by about 30% (this means an increase in the intrinsic viscosity by a factor of 2.2) on decreasing the ionic strength from 5×10−1 M to 5×10−3 M. Much larger expansions, up to around 80% (increase in [g] by a factor of 5.8), were observed for the corresponding linear material. This clearly shows that branching limits the expansion and contraction of the molecule. In order to do a similar analysis, a viscosity ratio is defined as:

Fig. 4. The viscosity ratio, R (=[g]/[g] ) as a function of g0.1 0.1 I −1/2 (using I=0.1 M as standard ) for linear and branched synthetic polyelectrolytes and the studied humics (pH 10).

[g] R = x (5) g0.1 [g] 0.1 where [g] and [g] are the intrinsic viscosities x 0.1 measured in x M and 0.1 M electrolyte concentration respectively. Fig. 4 presents the viscosity ratio of the studied HA samples and compares them with the values for linear and branched PSS samples as measured by Mays and with the values for several linear PAA samples obtained from Fig. 3 and from Takahashi and Nagasawa [25]. Although not shown here, R values for several g0.1 other linear PSS samples [40] (M from 2.6×106 D to 3.5×105 D) lie very close to the results of Mays. It can be observed that the

viscosity ratio of all HA and the FA are in the range of the branched molecules; they are remarkably lower than those of linear polyelectrolytes. Several factors can be responsible for this behavior: (i) differences in molecular weight of HAs compared with those of the synthetic polyelectrolytes, which could modify the response of [g] to changes in the ionic strength; (ii) differences in charge density, since a completely dissociated PAA molecule should have a charge density of 14 mmol/g, which is around three times higher than that of the HA; and (iii) an internal structure similar to branching that limits expansion in HAs and FAs,

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as in the case of branched PSSs studied by Mays [39]. For the well-studied linear PAA and PSS, the intrinsic viscosity and the ionic strength are related according to Eq. (6) [41]: [g]=[g] +kI −1/2 (6) 2 where k is a constant and [g] , the minimum value 2 for [g], is referred to as the intrinsic viscosity at infinite salt concentration. The value of k is strongly dependent on the molecular weight [41]. However, by using R the following expression g0.1 can be obtained: I −1/2 [g] +kI −1/2 2 # (7) R = g0.1 [g] +k(0.1)−1/2 (0.1)−1/2 2 which indicates that R should be much less g0.1 dependent on k and therefore on M. Moreover, if [g] %[g] the viscosity ratio becomes indepen2 0.1 dent of k and M. The low dependence of the slope of R versus I −1/2 curves as a function of M can g0.1 be observed in Fig. 4. In fact, the slope changes by a factor of 1.7 times in the M range 15 000 to 500 000 for PAA, while the corresponding change in k is around 25 as can be estimated from literature data [25]. Similarly, changes in M for linear PSSs [40] from 2.6×106 D to 3.5×105 D modify the slope of the lines just by a factor of 1.2. The low dependence of R on molecular g0.1 weight also holds for humic acids. Visser [31] measured viscosities of different humics that had been separated in several molecular weight fractions (range 750–200 000) by ultrafiltration. The data of Visser obtained in water and 10−1 M NaCl were used here to calculate the viscosity ratios. For a given type of humic acid the R values g0.1 differ at most by a factor of 2, depending on the molecular weight, but there is no clear relation between M and R . Therefore, it may be cong0.1 cluded that variations in molecular weight cannot be responsible for the low R values as compared g0.1 with those of the linear synthetic polyelectrolytes. Also, variations in the charge density do not change appreciably the viscosity ratio, at least for PAA. Data reported by Noda et al. [24] for PAA at different ionic strengths and degree of dissociation were used to estimate R at 10−2 M electrog0.1

lyte. For M=500 000 PAA the viscosity ratio was 3.5, 3.13 and 3.23 for the respective degrees of ionization 0.2 (charge 2.8 mmol/g), 0.6 (8.4 mmol/g) and 1 (14 mmol/g). For M=15 000 PAA the corresponding values were 1.86, 1.99 and 1.88. The analysis shows that neither variations in molecular weights nor variations in charge density can account for the low R of the studied HAs g0.1 and FA. Thus, some kind of internal structure comparable to branching should be invoked in order to explain the low salt sensitivity of the HAs and FA. A closer view of the R values of the humics g0.1 is presented in Fig. 5, where R is plotted versus g0.1 I −1/2 at pH 10. Note the I −1/2 is proportional to the Debye length, a measure of the electrostatic screening. It follows that the response to changes in the Debye length is different for different humics. A strong response is observed for KPHA, KFHA and HLHA. FSHA holds an intermediate position and the PAHA, PPHA, KOHHA, SBHA and LFA group are least sensitive to the ionic strength. These results indicate that the first group is relatively flexible with a low internal structure, whereas the second group has a relatively high internal structure. In Table 2, the degree of flexibility is summarized in the last column. The flexibility of the PPHA molecules is very low. Combining this information with the rather high value of [g] may lead to the conclusion that PPHA is probably nonspherical, rather than extremely strongly hydrated. Additional support for branching can be obtained by analyzing the coefficient a in the

Fig. 5. Viscosity ratios as a function of I −1/2 (~Debye length) of the studied humics (pH 10).

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Mark–Houwink equation: [g]=KMa

(8)

The coefficient a can take values between 2 and −2, depending on the properties of the macromolecules and on the solvent quality [30]. For rigid macromolecules a=0 because the hydrodynamic volume is directly proportional to M, a=2 for a rigid rod with constant diameter, whose height is proportional to M, a=1 for unbranched free draining coils with no excluded volume and between 0.5 (theta solvents) and 0.9 (good solvents) for unbranched, no-draining coils with excluded volume [30]. It is generally accepted that the presence of branches reduces the hydrodynamic volume relative to the mass of the molecule [13,30]. Therefore, the intrinsic viscosity of a branched polymer is lower than that of an unbranched one [42–44]. Several cases of branched polymers with a lower than 0.5 [43–45], a decreasing with M [44], or even negative a [45] can be found in the literature. Visser [31] reports K and a values for FAs and HAs that were fractioned by ultrafiltration. The estimated a values were 0.34 (Aldrich HA), 0.45 (soil HA), 0.43 (aquatic HA), 0.44 (aquatic FA) and 0.47 (microbial FA). All a values are lower than the corresponding value for an unbranched coil in a theta solvent. These low a values are good evidence for branching and/or crosslinking.

changing pH are lower at higher electrolyte concentration. Although the degree of hydration differs for different samples, the hydration is, in general, dependent on both the pH and ionic strength. Assuming that the shape factor is constant, an approximately linear relationship between hydrodynamic volume and pH is found. The proportionality factor decreases with increasing electrolyte concentration, and at 10−1 M the hydrodynamic volume is about constant. All the samples studied are internally structured. This conclusion is reached by comparison of the behavior of humics with that of branched synthetic polyelectrolytes. The low values of the Mark– Houwink coefficient a of humics are also indicative of internal structure. As compared with the linear flexible polyelectrolytes, the internal structure of the humics limits the expansion of the molecules when the electrolyte concentration is decreased or the pH is increased. The present data and conclusions are in agreement with the pictures proposed by Cameron et al. [3], Schulten [4] and Schulten and Schnitzer [5] for humics. HA appears to be a branched and crosslinked network with room for solvent molecules, electrolyte ions and perhaps larger organic molecules. The internal structure allows them to keep a somewhat compact structure, whereas the dynamism of the molecules allows them to swell or shrink when the charge or the screening of charge are changed by changing the pH or the electrolyte concentration.

5. Conclusions Humic and fulvic acid molecules undergo conformational changes when they are exposed to changes in either pH or ionic strength. The general behavior is similar for all the samples studied, i.e. the hydrodynamic volume decreases with both increasing electrolyte concentration and decreasing pH. This is mainly the result of a combined effect of electrostatic repulsion between negatively charged groups that tends to expand the molecule, screening of electrical charges by supporting electrolyte ions that decreases the repulsion effect, and flexibility of the molecules to respond to expansion–contraction processes. The effects of

Acknowledgment MJA thanks fellowship.

WAU

for

a

postdoctoral

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