The Science of the Total Environment, 81/82 (1989) 489-499 489 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

EVIDENCE OF CONFORMATIONAL CHANGES IN FULVIC ACIDS FROM DIALYSIS

M.T.S.D.VASCONCELOS, A.P.L.M.G.SANTOS and A.A.S.C.MACHADO

CIQ(LrP),Chemistry Department, Faculty of Science,P4000 Porto (Portugal)

SUMMARY

A dialysis study was conducted to evaluate the influence of the solution composition

on the permeability of a membrane to fulvic adds (FA). It has been found how the

permeability depends on the concentrations of the electrolytes in solution and pH, and that

dialysis is not suitable for complexation studies on solutions with high ionic strength.

Additional support for the present view of the dependence of the aqueous conformation of

FA molecules on their solution environment has been found.

INTRODUCTION

The conformational state of molecules of humic substances is important because it

conditions the extent of their interaction with other molecules in natural systems. In

consequence, the study of the aggregation state and conformation in solution of molecules

of humic materials, specially of fulvic adds (FA), has aroused much attention in recent years.

Particular interest has been focussed on the influence of the solution characteristics (FA

concentration, ionic strength, pH, presence of compexing cations,etc.) on the FA molecular

shape and aggregation state, which have been studied by a variety of experimental tech-

niques (refs.l-7). However, the results of these studies not always have been consistent and

the use of different values for fixed experimental parameters make comparisons difficult or

without meaning (ref.1).

In the course of a comparative study (ref. 8) of three analytical methods for determina-

tion of complexation parameters of copper(H) ion with FA extracted from soil (ion-selective

electrodes, fluorescence and dialysis), we have confirmed that dialysis (ref. 9) is the most

suitable technique for studies of metal-FA interactions in natural waters but we have found

that it is not adequate for studies at higher concentrations of FA and of background

electrolyte. Previous works on the use of dialysis for studying FA solutions have been

0048-9697/89/$03.50 © 1989 Elsevier Science Publishers B.V.

490

directed only to the determination of complexing capacities at low concentrations of FA and

low ionic strength (refs. 9-12). In our studies, we have found that if dialysis experiments are

made at different values of the variables (FA concentration, ionic strength, pH, etc.), they

provide interesting results about FA conformational changes, which we considered to be

worth reporting in the present paper.

EXPERIMENTAL

Reagents and materials

Analytical grade reagents were used to prepare all solutions. The water was de-ionised

and double-destilled in a quartz still.

Copper(U) ion solutions were prepared from a Cu(NOs)2standard solution (Radiome-

ter S-3546). KNO 3 was used as background electrolyte. For pH adjustments, 0.1 M HNO 3 and

0.1 M KOH were used.

The sample of FA used in the study was isolated from a Portuguese forest soil collected

at Famalic-~fo (at 30 km from Oporto), by the standard procedure recommended by the IHSS

(ref.13). In a few experiments concerning the influence of the age of the FA solutions on the

permeability, a sample extracted from another forest soil from the same region was used.

The dialysis cells consisted of bags made of 15 cm pieces of MW 1000 cutoff Spectra/

Pot6 tubing tied with double knots at both ends.

Determinations and sample storage were carried out in polyethylene vessels. All

material, glassware and polyethylene vessels were cleaned by soaking in 10% HNO s followed

by rinsing with water. Upon cleaning, dialysis bags were stored in water until used.

Equipment

Free and total copper(II) ion concentrations (M t and M t respectively) in dialysis

solutions were determined with an IL551 atomic absorption spectrophotometer and an IL655

graphite furnace. An IL63041 hollow-cathode lamp was used. Measurements were made at

324.7 nm.

pH was measured with a Crison Digilab 517 pHmeter and Ingold U102.023088 glass

and Orion 90-02-00 (double junction) reference electrodes. Solutions were stirred with a

Metrohm E649 magnetic stirrer.

491

FA UV spectrophotometry determinations in the internal dialysis solutions were made

with a Cary 17D UV spectrophotometer at 260 nm.

Procedures

FA solutions were prepared by dissolving aliquots of the solid sample in water or in the

background electrolyte solution. Solutions were prepared dally (except for the experiments

to study the influence of the age of the FA solutions on the permeability).

Dialysis experiments were carried out at room temperature (ca. 20°C) following a

procedure similar to that described in the literature (ref. 9). A 200 mL batch of solution

containig FA (Lt), with or without CufNO3) 2 and KNOy after adjustement of the pH to the

required value, was dialysed against 10 mL of water or KNO 3 solution with concentration

fixed to provide ionic strength as similar as possible to that of the external solution. If

copper(H) ion was present, its concentrations in the external (M~ and internal (Ms) solutions

after osmotic equilibrium had been established, were measured as required. FA concentra-

tion in the internal solution was measured by UV spectrophotometry, after calibration with

FA solutions (range: 5-40 mg L -1) at the same pH as the sample. Dialysis experiments were

made at pH=4 and pH=6.

Several types of experiments were carried out to characterize the permeability of the

dialysis membrane to copper(U) ion and FA. For measurement of the influence of the time

on the permeability, several experiments with systems of identical composition were started

in parallel and the dialysis process was interrupted after different lapses of time (experiments

I-W in Table 1).

To determine the influence of the different parameters (L e M e backgrond electrolyte

and pH) on the permeability to FA, several systems were used in which only one of those

parameters was varied, the dialysis time being kept constant (16 hours or more) in each series

of experiments (experiments V-VII in Table 1).

To determine the influence of the age of solutions on the permeability to FA, large

volumes (1000 mL) of original solutions of different concentrations were prepared. During

one week, aliquots were taken from these solutions every day and dialysed against 0.001 M

KNO s during a constant period of time (experiments VIII in Table 1).

The composition of the solutions used in the experiments is presented in Table 1.

402

TABLE 1 Details on the permeability experiments"

Exp. Mtxl0 s L t I s pH Time Results c (rag L -~) (M_.M_) (h)

IA 1.5 0 0.1 6 1-18 100% Cu after 16 h IB 1.5 0 0.001 6 1-18 100% Cu after 16 h

IIA 1.0 51 0.001 6 4-20 % Cu constant after 16 h IIB 8.0 30 0.001 6 4-20 % Cu constant after 16 h

UI 1.5 30 0.1 6 1-35 100% FA after 35 h

IVA 0 107 NA 4 21/93 9%/30% FA IVB 0 48 NA 4 21/93 4 % / 9 % FA IVC 0.5 32 0.003 4 20/48 11%/22% FA

V 0 32 0.001 2-6 19 Fig.1

VIA 0 30-800 0.001 6 16 Fig. 2 VIB 0 30-1000 0.1 6 16 Fig. 2 VIB 0 30-100 0.1 6 19 Table 2 VIC 0 30-800 NA 6 19 Table 2 VIB' 0 40-100 0.1 4 21 Table 2 VIC' 0 40-800 NA 4 21 Table 2

VIIA 0.3-7 30 0.001 4 16 Table 3 VIIB 0.3-7 30 0.001 6 16 Table 3

VIII d 0 30-1000 0.001 6 19 No age effect on % FA

a) At 20 °C; "-" : range; "/": fixed values. b) Background electrolyte concentration:

0.1 M: Internal and external solution 0.001 M: Internal solution only NA, Background electrolyte not added: Internal and external solution.

c) Expressed as permeability %. d) Series of identical experiments made daily, during a week, on the original solution (see text).

493

RESULTS AND DISCUSSION

The literature (refs.l-7) describes experimental evidence about the influence of the

concentrations of FA and metal ions, the ionic strentgh and the pH, on the FA conformation

and aggregation in solution. Therefore, it was decided to investigate the influence of these

variables on the dialysis of this type of macromolecules. Before this, some experiments were

made to evaluate the establishment of the dialysis equilibrium.

Dialysis equilibrium

Experiments to determine the time required for the establishment of the copper(U) ion

osmotic equilibrium, which is an essencial parameter for dialysis complexation studies, were

performed in the absence and in the presence of FA, at different background electrolyte

concentrations (Table 1, Experiments I and ]I). It was found that 16 h were sufficient to reach

equilibrium in all situations.

Experiments to evaluate the influence of the dialysis time on the permeability to FA

were performed in the absence and in the presence of copper(ID ion, at different background

electrolyte concentrations and at different pH values (Table 1, Experiments IT[ and IV). It was

found that the permeability to FA increased with time. Therefore, in all experiments to

determine the permeability to FA, dialysis t~nes were kept constant. In the determination of

the complexation parameters, the FA permeability should be kept at values as low as

possible. In consequence, the time used in experiments for this purpose (ref. 8) was whenever

possible close to the 16 hours minimum.

Membrane permeability to FA

To investigate the influence of FA and metal ion concentrations, ionic strentgh and pH,

on the permability to FA, solutions with different FA concentrations, with or without

coppeffII) nitrate and potassium nitrate, were dJalysed against pure water or potassium

nitrate at different concentrations (Table 1, Experiments V to VII). The more relevant results

are detailed in Figs. I and 2 and Tables 2 and 3.

Influence of ionic stren2h and p H

Considering the nature of FA, it is difficult to calculate accurately their contribuition to

494

TABLE 2 Influence of pH, FA concentration and ionic strength on the membrane permeabi l i ty to FA

Exp. External solution Internal solution Permeabil i ty

L, (mg L "~) P (M__) P (M__) (%)

p H = 6, Dia lys i s Time 19 h

VIB1 30 0.1 0.1 98 VIC1 30 N A N A 6 VIB2 100 0.1 0.1 76 VIC2 100 N A N A 4 VIC3 800 N A N A 18

pH = 4, Dialys is Time 21 h

VIBI' 40 0.1 0.1 85 VICI' 40 N A N A 0 VIB2' 107 0.1 0.1 95 VIC2' 110 N A N A 9 VIC3' 800 N A N A 35

a) See note a) on Table 1.

~FA

7O. 4[

5 0

30.

pH

Fig. 1. Influence of p H on the permeabil i ty to FA (%AF dialysed). External solution: Lt= 31.8 mg L 1. Internal solution: I= 0.001 M. Dialysis time: 19 h.

495

TABLE 3 Typical results of batch titration of FA with copper(U) ion for determinat ion of complexation parameters by dialysis experiments"

Exp. M t (rag L 1) L t (mg L "1) Permeabil i ty

External Internal External b Internal ¢ (% FA)

p H = 4 1 4.17 3.24 30 8.70 29 2 2.72 1.98 30 8.30 28 3 1.86 1.12 30 4.80 16 4 0.755 0.337 30 4.46 15 5 0.392 0.170 30 4.23 14 6 0.301 0.124 30 4.12 14 7 0.182 0.0510 30 3.39 13

p H = 6 1' 4.08 2.25 31 7.20 23 2' 2.59 1.06 31 4.98 16 3' 1.81 0.544 31 2.88 9 4' 0.689 0.350 31 1.34 4.3 5' 0.373 0.015 31 1.16 3.7

a) At 20°C, with 0.001 M background electolyte concentration (internal solution), 16 h dialysis time. b) Analytical concentration. c) Determined by UV sprectrophotometry.

~ F A

~ . *

40] *

2 *

~n~n~ ~- ~- ,, |

,,;o ' ' 5 , ; o . . . . . T o o ,oo Lt(n~ I: I)

Fig. 2. Influence of the concentration of FA on their permeabi l i ty (%AF dialysed) at p H = 6 and ( ~ ) I= 0.001 M and ( ~ ) I= 0.1 M. Dialysis time: 16 h.

496

the ionic strength of the solution. The literature refers that to a 10 mg L -1 FA solution

corresponds a value of ionic strength of 0.001 M (ref. 12). In the present work, this value was

used as reference and an attempt was made to use ionic strengths as similar as possible in the

internal and external solutions. Due to the difficulty of fixing ionic strengths at low values

in series of experiments to evaluate ionic strength effects, their levels were made markedly

different to obtain meaningfull comparisons.

The study showed that the fraction of FA dialysed increases markedly with the ionic

strength (Table 1, Experiment VI). As shown in Fig. 2 and Tables 2 and 3, it is ca. 100% at 0.1

M ionic strength, and very low in very diluted solutions (low concentrations of FA, copper(U)

nitrate and potassium nitrate, at pH--4 or 6). This means that under the experimental

conditions used, a decrease in the ionic strength provokes an increase of the volume of the

FA partides.

At low ionic strength, an increase of pH in the range 2 to 6 provoked also a decrease of

the FA fraction which was dialysed (Table 1, Experiment V, and Fig. 1). These results are in

agreement with results obtained by other techniques, namely gel filtration (refs. 6,7), ~-ae-

dependent fluorescence depolarization (re£ 1) and laser light scattering (ref. 4), from which

it was concluded that the size of the FA molecules increases with pH at low ionic strength.

The volume increase was attributed (ref.1) to the expansion of the monomers due to negative

charge accumulation in consequence of the deprotonation of carboxylic groups and not to

their aggregation. Such an aggregation (by hydrogen bonding) occurs only at low pH

(protonated molecules) and high concentration of background electrolyte which screens the

charge on dissociated groups, and these two conditions will have to occur simultaneously to

originate aggregation (ref.1). Viscosity studies (ref. 7) showed that at low ionic strength and

neutral pH, the FA molecules are expanded disaggregated monomers but that they take

ellipsoidal configuration at high ionic strengths. In conclusion, the observed decrease of the

dialysis fraction with increase of pH and decrease of ionic strength can be explained as the

result of a conformational alteration in which the flexible linear AF monomers change from

ellipsoidal shape to an expanded linear elongated shape with increased volume.

Influence of FA concentration

It was found (Table 1, Experiments VI, Fig. 2 and Table 2) that the d i a l ~ d fraction of

497

FA depends on its concentration but that the ionic strength influences the direction and

extent of such dependence. At a background electrolyte level of 0.1 M_M_, the dialysed fraction

decreases when the concentration increases (Fig. 2 and Table 2). In dilluted solutions, the

inverse is observed, although the variation is less pronounced.

The results at 0.1 M ionic strength are consistent with results reported by Lochmuller

and Saavedra (ref.1) from ~ne-dependent fluorescence depolarization studies. These au-

thors explained the same type of variation, observed at macroscopic level, by the formation

of aggregates of monomers when the FA concentration increases at high ionic strength. The

aggregation is probably due not only to hydrogen bonding and charge screening on the

ionised groups (see above) but also to interactions of the hydrophobic regions of the FA

molecules (ref. 1).

At low ionic strength and high pH, aggregation is not expected to occur, in consequence

of the repulsions between the charged groups of the FA polyanions. These are not neutralized

by cations of the background electrolyte whose concentration is too low for achieving such

a neutralization. The observed increse of the dialysed fraction of FA with concentration at

low ionic strength may be only an effect of concentration gradient at the surface of the dialysis

membrane.

Influence of cop_per(U) ion concentration

The results of the studies of the complexation (ref.8) of copper(n) ion by FA (Table 1,

Experiment VII) show (Table 3) that, for the experimental conditions used (low background

electrolyte and FA concentrations, pH = 4 or 6), the FA dialysed fraction increases with the

concentration of copper(U) ion (Mr). This result suggests that aggregation of FA molecules

due to complexation of copper(U) ion by two carboxylic groups in separate ligand molecules

(ref.14) does not occur in the present case, which may be a consequence of the low level of

FA concentration used in this study. The same conclusion is also supported by other results

obtained in the complexation studies (ref. 8). It was found that a metal-ligand model

consisting only of the 1:1 species describes well the experimental situation in the complete

range of the titration curves, is., no evidence was found of the occurrence in significative

amount of 1:2 or species with larger ligand to metal ratios. The increase of the FA diaiysed

fraction with the concentration of copper(u) ion may be explained as an effect of the excess

498

free ions from copper(U) nitrate on the charged groups of the FA polyanions, similar to the

effect of the background electrolyte which was discussed above.

Influence of the age of solution on the permeability

Experiments to evaluate the influence of the age of the solution on the permeability to

FA (Table 1, Experiments VIII) were made in a large range of FA concentration (up to 1000

mg L -~) but at low ionic strength and at pH = 6. The results show no change of dialysed fraction

with the age of the solution, which suggests that aggregation does not occur along the time

in these experimental conditions, as expected at low ionic strength.

CONCLUSIONS

The results obtained in the present work show that the permeability to FA depends on

FA concentration, level of other electrolytes in solution and pH, for instance, it was found that

the permeability increases when the ionic strength increases and pH decreases from 6 to 2.

It was concluded that dialysis, although an excellent technique for complexation

studies on natural waters (refs. 8,9), is not suitable when high FA and background electrolyte

concentrations are present.

On the other hand, this work provides additional support for the present view on the

dependence of the aqueous conformation of FA molecules on their solution environment,

which was formulated from sudies where different experimental techniques were used (refs.

1,3,7).

ACKNOWLEDGEMENTS

INIC, Lisbon, provided financial support for this work through Research Line 4A of

CIQ(UP). We thank Ant6nio J.T.Sousa for routine work.

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