An electron microscopic examination of fulvic acid

Geoderma, 13 (1975) 279--287 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

AN ELECTRON MICROSCOPIC EXAMINATION OF FULVIC ACID 1

M. SCHNITZER and H. KODAMA

Soil Research Institute, Agriculture Canada, Ottawa, Ont. (Canada) (Received June 4, 1974; accepted for publication December 9, 1974)

ABSTRACT Schnitzer, M. and Kodama, H., 1975. An electron microscopic examination of fulvic acid. Geoderma, 13: 279--287. An electron microscopic examination of fulvic acid shows that the crystallinity, shapes, dimensions and extent of aggregation vary with pH. At pH 2.5, three types of particles can be observed: small spheroids (15--20 A in diameter), aggregates of spheroids (200--300 A in diameter) and an amorphous material of low contrast, perforated by voids, 500--1,100 A in diameter. The spheroidal aggregates tend to form elongated, irregularly shaped structures, 20,000--30,000 A long. At pH 3.5, which is the natural pH of a dilute fulvic acid solution, electron micrographs show a sponge-like structure of variable thickness (100--300 A ), punctured by voids, 2 0 0 - 1 , 0 0 0 A in diameter. At pH 4.5 and higher, electron micrographs show fiat sheet-like lamellae of very low contrast, perforated by voids, 2 0 0 - 2 , 0 0 0 A in diameter. Electron diffraction patterns show crystallinity in fulvic acid aggregates formed at pH 2.5 only but not in those formed at pH 3.5. The electron microscopic data are in harm o n y with X-ray, chemical and spectroscopic data on the same fulvic acid, and point to a relatively " o p e n " structure, perforated by voids of varying dimensions which can trap or fix organic and inorganic compounds that fit into the voids, provided that the charges are complementary.

INTRODUCTION

Electron microscopy permits direct observations of the shapes and dimensions of relatively large molecules. The method has been applied by several workers ( Flaig and Beutelspacher, 1951; Visser, 1963; Wiesemuller, 1965; Khan, 1971; Orlov and Glebova, 1972} to humic acids. To the best of our knowledge no systematic investigation of this nature has ever been done on fulvic acid (FA). In view of the importance of FA in the complexing of metals, hydrous oxides, clays and also of organic compounds, including toxic pollutants (Schnitzer and Khan, 1972}, we decided to initiate such a study, hoping that it would yield meaningful information on the shapes, dimensions and structural arrangements of FA particles or possibly "molecules". 1Contribution No. 498.

280 MATERIALS AND METHODS

FA The FA originated from the Bh horizon of the Armadale soil, an imperfectly drained Podzol in Prince Edward Island, Canada. Methods of extraction and purification were the same as those described previously (Schnitzer and Skinner, 1968}. The purified FA contained 1.00% ash and, on a moisture- and ash-free basis (Schnitzer, 1974): 50.92% C, 3.34% H, 0.74% N, 0.26% S and 44.74% O. Functional group analysis showed 9.1 me CO2H, 3.3 me phenolic OH, 3.6 me alcoholic OH, 0.6 me quinoid C=O, 2.5 me ketonic C=O, and 0.1 me OCH3 per g. The number-average molecular weight (Mn) measured by vapor pressure osmometry was 951. The molecular formula calculated from these data was C2sH16(CO:H)8(OH)7(CO)3.

Electron microscopic investigations Specimens were prepared by spotting 0.01% (w/w) aqueous FA solutions, either not adjusted or adjusted to the desired pH value with dilute HC1 or NaOH before spotting, on copper or gold grids coated with thin carbon films, and allowing the spots to dry at room temperature. The prepared specimens were stored in a vacuum desiccator over P~O5 at room temperature until examined under the electron microscope. The latter was a Philips EM 300 instrument, which was operated at 80 kV. Muscovite was used as standard for calibrating d-spacings in electron diffraction measurements. RESULTS

FA at pH 2.5 Micrographs a to c in Fig.1 were taken at increasing levels of magnification on FA adjusted to pH 2.5 and spotted on copper grids. At the lowest magnification (la), three types of particles are visible: small spheroids, aggregates of spheroids, and an amorphous material of low contrast, in which the larger spheroidal aggregates appear to be embedded and which acts like glue in favouring the formation of elongated, irregularly shaped structures (20,000-30,000 A in length) perforated by voids. At higher magnification (lb), the shapes of the three major components become more distinct. The highest magnification ( l c ) shows that in the larger aggregates (200--300 A in diameter), smaller particles appear to be held together by films (of water or hydrogenbonding) of slightly lower contrast. The spheroidal shape of the smallest particles (15--20 A in diameter) becomes apparent again, as does the presence of irregularly shaped voids of different dimensions (500--1,100 A in diameter) in the amorphous material. Soil scientists have known for a long time that humic substances are coagulated at low pH and dispersed at higher pH levels,

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so that the observed aggregation of the spheroidal FA particles at pH 2.5 is not unexpected. We realize that drying may change the shapes and dimensions of the FA particles and that these may be different in solution. Since FA is known to interact with copper (Schnitzer and Hansen, 1970),

Fig.1. Electron micrographs of FA at pH 2.5: a, bright-field, lowest magnification; b, bright. field, intermediate magnification; c, bright-field, highest magnification; d, bright-field, different aggregates but similar magnification as a; e, electron diffraction pattern of aggregates in lower left corner of d; f, dark-field, using a strong diffraction spot in second inner ring in e.

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we spotted FA adjusted to pH 2.5, on gold grids and t o o k micrographs at different magnifications. The resulting micrographs were very similar to those taken on copper grids, so that the type of metal from which the grid was made did not appear to have any effect on the electron microscopy of FA. Aggregates in the lower left corner of l d were examined for crystallinity by electron diffraction analysis. Fig.le is the resulting electron diffraction pattern, which shows the presence of crystalline materials plus other component(s) that produce diffuse patterns. In order to obtain further information on the crystalline materials responsible for the diffraction spots, we t o o k the dark field image (l f) of one strong diffraction spot in the second inner ring (see Table I, No. 2, d = 2.12 h ) , rich in diffraction spots (le). A comparison of Figs.ld and I f shows that most small spheroidal particles are crystalline. TABLEI Electron diffraction data for FA aggregates prepared at pH 2.5 No. of rings

Feature of ring

d (A)

I

1 2 3 4 5 6 7 8 9

diffuse spotty spotty diffuse spotty diffuse spotty spotty spotty

2.5 2.12 1.82 1.5 1.28 1.2 1.10 0.84 0.76

weak strong medium strong very weak strong weak medium strong medium weak weak

The electron diffraction data are listed in Table I. Most of the spacings resemble those of disordered carbon (Frondel and Marvin, 1967), except for the basal spacing. Since this conformation was only clearly observed at pH 2.5, it appears to us that low pH favours the formation of crystalline structures from at least parts or certain components of FA "molecules" or aggregates. FA at p H 3.5 Electron micrographs of FA at pH 3.5 are shown in Figs.2a--2d. A 0.01% solution of FA in distilled water has a pH of 3.5, so that no acid or base was added. For these reasons, micrographs taken at this pH are probably the most informative ones. The micrographs show a sponge-like structure of variable thickness, perforated by voids of widely differing sizes {200--1,000 A in diamFig. 2. Electron micrographs of FA at pH 3.5: a, bright-field, lowest magnification; b, brightfield, intermediate magnification; c, bright-field, still higher magnification; d, bright-field, highest magnification; e, electron diffraction pattern of FA aggregates; f and g, shadowgraphs of aggregates.

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eter). Within the general structure, small, single spheroidal particles, 20--30 A in diameter (Figs.2c and 2d), can be discerned. At a shadowing angle of 12 °, lengths of shadows for FA particles at pH 3.5 ranged from 500 to 1,500 A (Figs.2f and 2g). From the shadowing angle and length of shadows we calculated particle heights of 100--300 A. Fig.2e is the electron diffraction pattern of FA particles at pH 3.5. The pattern consists of two diffused rings, with maxima at around 2.1 and 1.2 A. These spacings again suggest a structure that resembles that of disordered carbon (Kodama and Schnitzer, 1967). F A at higher p H

At pH 4.5 and higher, FA produces electron micrographs that show flat, sheet-like lamellae of very low contrast, perforated by voids (200--2,000 A in diameter) (Figs.3a--3c). It is possible that the particle shape is distorted on drying and that particles may be shaped differently in solution. Electron micrographs taken at higher pH are difficult to interpret. They give the impression that at neutral pH and higher, FA is highly dispersed and on drying forms thin sheets. Whether or not the latter perforate during drying, remains to be investigated. DISCUSSION

The micrographs presented herein show at pH 2.5, the presence of elongated, irregularly shaped aggregates, 20,000--30,000 A in length, perforated by voids, 200--300 A in diameter. The smallest particles are spheroids, 15--20 A in diameter, which tend to form aggregates, ranging from 200 to 300 )k in diameter. Electron diffraction analyses indicated the occurrence of crystalline materials in the aggregates prepared at pH 2.5. Probably the most interesting data are those for FA at pH 3.5. This is the natural pH of this material and no extraneous reagents were added. Electron micrographs run on this material show a sponge-like structure, 100--300 A thick, perforated by voids of differing dimensions that range from 200 to 1,100 A. The smallest particles that we could find under these experimental conditions ranged from 20 to 30 A. Judging from the measured molecular weight (Mn = 951) and from X-ray data (Kodama and Schnitzer, 1967), it is likely that the smallest particles are FA "molecules" rather than aggregates. At pH 3.5, no crystalline materials were detected by electron diffraction analysis. At higher pH, FA becomes highly dispersed and produces micrographs with very low contrast. Under the most favourable conditions one can see flat, sheet-like, very thin lamellae perforated by voids, 200--2,000 £ in diameter.

Fig.3. Electron micrographs of FA at higher pH: a, bright-field, pH 7.5; b and c, brightfield, pH 8.5.

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Thus, depending on the pH, the crystallinity, shapes and dimensions of FA particles vary. If we assume that electron micrographs at pH 3.5 are most representative of conditions that prevail in acid soils such as in Spodosols, then the most outstanding features exhibited by FA are: (1) a sponge-like, polymeric structure, perforated by voids or holes of relatively large dimensions; (2) the occurrence in the polymeric structure apparently in free form, of very small spheroids (20--30 A in diameter) and of aggregates of spheroids; and (3) the structure is sensitive to relatively small changes in pH, aggregating when the pH is lowered and dispersing when it is raised by even only one pH unit. Any proposed structure for FA must take these observations into account. The electron microscopic results reported herein are in harmony with data obtained by Kodama and Schnitzer (1967) who concluded from X-ray analyses, that the carbon skeleton of FA consisted of a broken network of poorly condensed aromatic rings with appreciable numbers of disordered aliphatic chains or alicyclic structures around the edges of the aromatic layers. Thus, FA has a relatively " o p e n " structure which allows for the presence of a large number of voids. More recently, Schnitzer {1971, see Schnitzer and Khan, 1972) has proposed on the basis of extensive chemical investigations that FA consists of phenolic and benzenecarboxylic acids, which are its "building blocks", and that these are joined by relatively weak bonds such as hydrogen-bonding and Van der Waal's forces to form a polymeric structure of considerable stability. This t y p e of structural arrangement would be expected to be sensitive to changes in pH, salt concentration and valence of cations, in accordance with observations made by soil scientists over many years. One of the most interesting characteristics of the proposed structure is that it is punctured by voids or holes of different dimensions which can trap or fix organic molecules and inorganic c o m p o u n d s provided that these have the proper molecular sizes to fit into the holes and also that the electrostatic charges in the holes and on the trapped compounds are complementary. It is n o t e w o r t h y that three independent approaches, that is, X-ray analysis, chemical and spectroscopic methods and electron microscopy all point to the same type of general structure for FA, which is essentially a relatively loose association of molecules. What is needed n o w is information on the shapes and dimensions of FA particles and aggregates as they occur in solution.

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287 Kodama, H. and Schnitzer, M., 1967. X-ray studies of fulvic acid, a soil humic compound. Fuel, 4 6 : 8 7 - - 9 4 Orlov, D.S. and Glebova, G.I., 1972. Electronmicroscopic investigations of humic acids. Agrochemistry, 7 : 131--136 (in Russian) Schnitzer, M., 1974. Alkaline cupric oxide oxidation of a methylated fulvic acid. Soil Biol. Biochem., 6 : 1 - - 6 Schnitzer, M. and Hansen, E.H., 1970. Organo-metallic interactions in soils: 8. An evaluation of methods for the determination of stability constants of metal--fulvic acid complexes. Soil Sci., 1 0 9 : 3 3 3 - - 3 4 0 Schnitzer, M. and Khan, S.U., 1972. Humic Substances in the Environment. Marcel Dekker, New York, N.Y., 327 pp. Schnitzer, M. and Skinner, S., 1968. Alkali versus acid extraction of soil organic matter. Soil Sci., 1 0 5 : 3 9 2 - - 3 9 6 Visser, S., 1963. Electronmicroscopic and electron-diffraction patterns of humic acid. Soil Sci., 9 6 : 3 5 3 - - 3 5 6 Wiesemuller, W., 1965. Untersuchungen iiber die Fraktionierung der organischen Bodensubstanz. Albrecht-Thaer-Archiv, 9 : 4 1 9 - - 4 3 6