Effects of extraction procedures on fulvic acid properties

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the Total Environment, Publishers

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62 (1987) 3-12

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EFFECTS OF EXTRACTION PROCEDURES ON FULVIC ACID PROPERTIES J.E. GREGOR and H.K.J. POWELL Chemistry Department, University of Canterbury, Christchurch, New Zealand

SUMMARY Acid pyrophosphate (pH 2-4) is proposed as an extractant for soil fulvic acids. After XAD treatment of the extractant solution low-ash fulvic acids are obtained in high yield. The fulvic acids were subjected to the high pH and low pH conditions normally encountered in fulvic acid extraction. The observed changes induced in molecular weight distribution, equivalent weight, acid dissociation constants (pKt) and metal binding strength are reported.

INTRODUCTION The mOSt convenient source of terrestrial humic and fulvic acids is the Bn horizon of podzolised soils. The traditional method of extraction involves treatment of the soil with alkali (0.1-0.5 M), followed by acidification (pH l-2) to precipitate humic acids, then ion exchange to obtain salt-free fulvic acids which are finally isolated by freeze drying (ref. 1). In recent years use of hydrophobic XAD resins for desalting of fulvic acids has been used to advantage (ref. 2): the non-ionic acids are adsorbed at pH < 2, the resins are washed free of salts, then the fulvic acids released by deprotonation in neutral or alkaline solution. For studies on fulvic and humic acids to be meaningful it is essential that the acids are not modified in the extraction process; this is most unlikely if the traditional method of extraction is used. The propensity of phenolic materials to undergo oxidation in alkaline solution is well known (ref. 3); complete removal of 02 from the soil matrix prior to alkali treatment requires careful procedures. Oxygen uptake by fulvic acids in alkali has been reported (ref. 4). Further degradation of the organic matter is possible in the acidification step. In the presence of iron(lll) salts at pH c 6 polyphenols are rapidly and irreversibly oxidised to quinones (ref. 5) themselves reactive species, while hydroxy carboxylic acids undergo photochemical oxidation (ref. 6). Waite and Morel (ref. 7) have reported the photochemical oxidation of fulvic acid by colloidal iron(lll) hydroxide in acid solution.

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If the isolated humic and fulvic acids are to be used in metal binding studies it is also essential that the organic material has a low ash content. Specifically, it must be low in silicon, Als+, Fes+, Ca2+ and divalent transition metal ions, all of which will bind preferentially with the strongest donor sites. Fulvic acids isolated by the alkali - ion exchange method are variously reported to have 2-10% ash, whereas those treated on XAD resins are likely to have l-2% (ref. 8). OBJECTIVES The objectives in the present study were as follows. (i) To develop a method for extraction of fulvic acids which avoided the use of alkaline conditions, and masked any possible redox reactions by iron(lll) in acid solution. [The use of acid pyrophosphate (pH 2-4) as an extractant and masking agent is outlined: details of this method have been published (ref. 9).] (ii) To study the changes in physical and chemical properties of fulvic acids induced by treatment with aqueous alkali (pH 13) or acid (pH 1.7). [Molecular weight distributions, equivalent weights and pK, values, and g.1.c analysis of hydrolysis fragments are reported.] (iii) To compare the metal binding properties of fulvic acids extracted by the pyrophosphate method and by the alkali method. [Metal binding curves are reported, as determined by copper ion selective electrode.] EXTRACTION PROCEDURE The extraction procedure is summarized in Scheme 1, and is detailed elsewhere (ref. 9). Salient points are as follows: (i) Leaching is more efficient than is batch extraction. The pH of the leachate, initially 2.0, is quickly buffered to pH 3 by the soil or 4-4.5 by peat samples. Complete leaching of 300 g soil (200 g peat) required 800 ml (1800 ml) of 0.1 M pyrophosphate (pH 2). Dissolution of humic acids was minimal. (ii) Pyrophosphate masks iron(lll) and prevents any redox reaction with polyphenols; this masking has been demonstrated by sampled dc polarographic measurements on iron(lIl)/pyrophosphate solutions in the presence of catechol. (iii) On acidification, prolonged standing at pH 1.7 can lead to polymerization and precipitation of fulvic acids. When this polymerization occurs on the XAD resin complete retrieval of fulvic acids from the resin is not possible (ref. 9). (iv) XAD 7 is superior to XAD 8 in terms of compound retention and physical stability.

(v) Filtration (0.025 p) prior to XAD resin treatment is essential for complete recovery of fulvic acids. (vi) Desorption at pH 6.5 (pH - stat conditions) releases 98% of adsorbed acids; the remainder can be released at pH IO. Scheme

1

LEACHING # 9 pH 1.7

ACIDIFICATION,

#+ ADSORPTION,

XAD-7

K7 DESORPTION,

pH 6.5

9 CATION

#

EXCHANGE +

and

9 FREEZE

concentrate

+

DRY

filter

centrifuge 0.02S~

TABLE 1 Elemental composition (%) and ash content (%) for fulvic acids isolated from the IHSS peat sample.

FsE?OnAsh Pyrophos0.2 phate/ XAD 7 Alkali/ 2.3 XAD8 -

C

H

N

P

Al

Ca

Fe

si

Na

49.3

3.7

2.3

0.06

0.01

0.01

0.05

0.06 0.02 0.05

50.9

3.3

2.3

0.00

0.02

0.31

0.03

0.17 0.44 0.07

The ash content and elemental composition of fulvic acids isolated (from the International Humic Substances Society reference peat sample) by the

K

6

pyrophosphate - XAD 7 method (IHSSP) and the alkali - XAD 8 method (IHSSA) (ref. IO) are compared in Table 1. EFFECT OF ALKALI TREATMENT Fulvic acids isolated from IHSS peat by the pyrophosphate - XAD method were reacted with 0.1 M NaOH for 4 or 72 h. at room temperature, with rigorous exclusion of oxygen. The products were isolated after ion exchange in a closed system under N2, followed by freeze drying. These treated and untreated acids were compared with those isolated by the alkali - XAD 8 method. The molecular weights, equivalent weights and pK, values for the untreated and treated fulvic acids were determined. The presence of hydrolysis products arising from possible ester saponification under alkaline conditions was assessed by g.1.c. analysis of hydrophobic compounds generated. Hvdrolvsis oroduc& Drifting and hysteresis have been noted in the pH titration curves for fulvic acids (ref. 11). The drifting is related to the presence of ash and colloids in the fulvic acids used by many workers, and is easily eliminated by use of low ash colloid-free samples. However, hysteresis is observed when the upper pH limit in the titration is above 8.7; it can be rationalised in terms of ester hydrolysis of fulvic acids. The most definitive way of establishing that hydrolysis has occurred is to isolate hydrolysis fragments from the bulk sample and characterise them. A fulvic acid solution was adjusted to pH 6.5 and non-ionic components removed on a Cl8 Sep-Pak cartridge. The sample was hydrolysed (pH 13) for 72 h., the pH lowered to 6.5, and non-ionic hydrolysis products removed on the C,s Sep-Pak; these were subsequently eluted with acetone and the eluate analysed by capillary g.1.c. (Fig. I). The presence of 3 non-ionic hydrolysis products is indicated (#I); one has an elution volume similar to vanillin, another similar to butanol or pentanol. The products detected represent in total only 0.3% of the sample mass; however it is probable that hydrolysis also leads to low molecular weight products which are ionic at pH 6.5, and to high molecular weight ionic or non-ionic components.

PHENOL J VANILUN 1,5 -DIHYDROXY+,

NAPTHALENE

LL

0 hr

bLA ,

,

72 hr

s 1 mg ml-’

5 min

Fig. 1 Glc traces of fulvic acid hydrolysis products in acetone, using a Shimadzu GC 9A instrument with SPB-5 glass capillary column (30 m x 0.75 mm ID). Temperature program = l OO-250% at 100 mint, then 250%. Standards (S) 1 mg ml-t. ent weroht -remen& Table 2 summarises the equivalent weights (mass per mot of -COOH groups) determined for samples isolated from soil and peat, before and after alkaline (72 h.) hydrolysis. The equivalent weight was calculated from the carboxyl end point in the pH titration curve (pH g. 74, as determined by Gran’s analysis of data (ref. 12). All titrations were done under N2 in 0.10 M KCI.

8

TABLE 2 Equivalent weights g (mol COOH)-1 for soil and peat fulvic acids before and after alkaline hydrolysis. Sample

Alkali(72 h.) -

Bh (podzol) IHSS (peat)

163+5 139zt4

149f4 131 f4

137f4

Alkali treatment (72 h.) of pyrophosphate extracted fulvic acids caused a significant decrease (6-9%) in equivalent weight. This is consistent with a net generation of titratable carboxyl groups by hydrolysis of esters. A less prolonged reaction with alkali (c. 16 h.), during the alkali - XAD 8 extraction of fulvic acid from the IHSS peat, did not produce a significant change in equivalent weight. oK, measurement8 The fulvic acid titration curve can be expressed empirically in terms of a group of monoprotic acids, the groups having mean pKt values, pKt . ... ..pK. (ref. 13). This interpretation requires a non-linear least squares analysis of titration data. In this work we found a model involving 4 monoprotic acids gave the best least squares fit. The predicted effect of an increased density of carboxyl groups (as resulting from ester hydrolysis under alkaline conditions) is a decrease in pKt , pK2 .. . and an increase in . .. pK,f , pK, (ref. 14). Results in Table 3 show the mean pt$ values and the percentage of carboxyl groups in each grouping of monoprotic acids for fulvic acid extracted from IHSS peat. It is clear that the majority of carboxyl groups in non-hydrolysed samples have low pKt values. For 4 samples we have studied pKt was in the range 2.3 - 2.7 and this dissociation involved 50-70% of carboxyl groups (IHSS peat, pKt 2.63 (70%)). These groups are substantially ionised in dilute aqueous solution and contribute to the solubility of fulvic acid.

9

TABLE 3 Protonation constants (+-0.04) for fulvic acids extracted from IHSS peat, modelled as 4 monoprotic acid groupmgs. PKt PKI PK2 PK3 PKA

Pvroohosohate ext act o Alkalir(ni hn)

Alkali extraction

2.63 4.67 5.57 6.50

3.02 4.82 5.91 7.19

(70%) (17%) (7%) (6%)

3.20 5.03 5.61 7.03

(76%) (10%) (9%) (5%)

(60%) (23%) (10%) (7%)

The effect of alkali treatment was to increase p& (+ 0.5) but also to increase pKt (+ 0.6). The latter change was not predicted. One reaction which could contribute to this change is the formation of a peptide (pKt 9. 3.2) from condensation of an amino acid (pKt c. 2.6) and an (amino acid) ester under mild alkaline conditions (ref. 15 ). R.COOR’ + R”CH(NH,).COOH 4 R.CONH.CH(R”).COOH The nitrogen content of the IHSS sample is sufficient for one carboxyl group in five to be part of an amino acid. A less prolonged exposure to alkali in extraction of fulvic acid from the IHSS peat caused an intermediate change in pKl. Metal comolex formation The relative binding strengths of the alkali- and pyrophosphate-extracted fulvic acids for copper ions were compared by measuring the free metal concentration as a function of pH, using a copper ion selective electrode. In Fig. 2 the percentage free metal is plotted against pH. For both samples complexing commenced at pH 3, and 90% of the copper was complexed at pH 7 for the concentrations of ligand (1.8 x 10-4 M carboxyl) and metal (4 x 1O-5M) chosen. However, from pH 4 to 7 the curve for the alkali extracted sample is displaced to lower pH by 0.4 pH. This indicates stronger binding by the alkali extracted sample. The curves were determined for equal concentrations of carboxyl group and for a carboxyl/copper ratio of 4.5; if they were determined for equal masses of fulvic acid a slightly greater separation would be observed. The result is consistent with the observation that alkali-extracted fulvic acids form more stable copper complexes than do water extracted acids (ref. 16). The shift to lower pH is consistent with an increase in the density of donor groups (increased incidence of potentially polydentate binding sites), as may arise from ester hydrolysis. However, it is not consistent with conversion of amino acids to peptides

(weaker complexing) (ref. 17), although our modelling calculations indicate that citrateand malonate-type groupings make the major contribution to copper(U) complexing. For comparison, the copper binding curves determined for citric, malonic and salicylic acids at the same carboxyl group concentration (carboxyl/copper 4.5) are shown in Fig. 2.

MALONIC

41

ACID

’ 3

8 4

5

6

7

PH Fig. 2 Copper binding curves for fulvic, citric, malonic and salicylic acias (1.8 x 1O-4M COOH); [CUE+]= 4.0 x 10-s M, p = 0.1 M (KNOs), 250 C. IHSSA, IHSSP: alkali and pyrophosphate extracts.

1

Fulvic acid is known to contain fluorescent centres. This fluorescence can be quenched in the presence of paramagnetic ions such as Cu*+ (ref. 18). The relationship between these fluorescent centres and metal binding sites has been studied by monitoring the fluorescence quenching as a function of pH for copper - fulvic acid solutions. The fluorescence was measured relative to ligand at the same pH. Quenching began as complexing commenced, c. pH 3, indicating that some fluorescent centres are in close proximity to, or are part of, the strongest binding sites. In the presence of metal the fluorescence decreased with increasing pH (and complexing), but did not decay to zero as complexing approached 100%. The ratio of complexed copper to quenched fluorescence was the same for both the alkali extracted and pyrophosphate extracted fulvic acids up to pH 5. Above pH 5 quenching levelled off at approximately 50%. CONCLUSIONS Alkali treatment of fulvic acids initially extracted under mild conditions (acid pyrophosphate, pH 2-4) lead to significant changes in physical properties. The titratable acidity per gram increased by 6-9% (a decrease in mass per carboxyl group). There was a significant decrease in the acidity (ApK, + 0.6) for the most acidic carboxyl groups (5070% of total). Some low molecular weight nonionic hydrolysis products were identified after alkali treatment, suggesting some ester saponification. The properties of fulvic acids extracted from the International Humic Substances Society reference peat sample by pyrophosphate - XAD and alkali-XAD methods differed in accordance with the described effects of alkali treatment. The alkali extracted fulvic acids formed more stable complexes with copper( REFERENCES M. Schnitzer and S.U. Khan, Humic Substances in the Environment, Marcel Dekker, New York, 1972. G.R. Aiken, E.M. Thurman, R.L. Malcolm and H.F. Walton, Comparison of XAD macroporous resins for the concentration of fulvic acids from aqueous solution, Anal. Chem., a (1979) 1799-1803. M.L. Mihailovic and 2. Cekovic, Oxidation and Reduction of Phenols, in: S. Patai (Ed.), The Chemistry of the Hydroxyl Group, Part I, Wiley Interscience, London, 1971. R.S. Swift and A.M. Posner, Autoxidation of humic acid under alkaline conditions, J. Soil Sci., B (1972) 381-393. H.K.J. Powell and MC. Taylor, Interactions of iron and iron(lll) with gallic acid and its homologues: a potentiometric and spectrophotometric study, Aust. J. Chem.,s (1982) 739-56. J.L. Frahn, The photochemical decomposition of the citrate-ferric ion complex: a study of the reaction products by paper ionophoresis, Aust. J. Chem., u(l958) 399405.

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7 T.D. Waite and F.M.M. Morel, Photoreductive dissolution of colloidal iron oxide: effect of citrate, J. Colloid Interface Sci., m (1) (1984) 121-137. 8 E.R. Thurman, Organic Geochemistry of Natural Waters, Martinus Nijhoff/Dr W. Junk Publishers, Dordrecht, Netherlands, 1985. 9 J.E. Gregor and H.K.J. Powell, Acid pyrophosphate extraction of soil fulvic acids, (J. Soil Science), in press. 10 E.M. Thurman and R.L. Malcolm, Preparative isolation of aquatic humic substances, Environ. Sci. Technol., ti (1981) 463-466. 11 H. Davis and C.J.B. Mott, Titrations of fulvic acid and fractions. 1. Interactions influencing the dissociation/reprotonation equilibria, J. Soil Sci., 2 (1981) 379-391. 12 G. Gran, Determination of the equivalence point in potentiometric titrations. Part II. Analyst (London), Zz (1952) 661-671. 13 N. Paxeus and M. Wedborg, Acid-base properties of aquatic fulvic acid, Anal. Chim. Acta, m (1985) 87-98. 14 S.D. Young, B.W. Bathe, D. Welch and H.A. Anderson, Analysis of the potentiometric titration of natural and synthetic polycarboxylates, J. Soil Sci., 2 (1981) 579-592. 15 D.P.N. Satchel1 and R.S. Satchell, Substitution in the groups COOH and COOR, in: S. Patai (Ed.), The chemistry of carboxylic acids and esters, Interscience Publishers, London, (1969), 375-452. 16 J.R. Sanders and C. Bloomfield, The influence of pH, ionic strength and reactant concentrations on copper complexing by humified organic matter, J. Soil Sci.,a (1980) 53-63. 17 D.D. Perrin, ‘Stability Constants of metal-ion complexes, Part 8, Organic Ligands, Pergamon, Oxford, 1979. 18 K. Ghosh and M. Schnitzer, Fluorescence excitation spectra and viscosity behaviour ;02f;lvrc acid and Its copper and Iron complexes, So11Sci. Sot. Amer. J., s (1981) - .