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III. Humic Substances: Analytical Characteristics

III. Humic Substances: Analytical Characteristics

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12



M. SCHNITZER

Table I

Analytical Characteristics of a Haploboroll

HA and a Spodosol FA

HA

Element (g kgϪ1)

564

55

41

11

329

Functional groups (cmol kgϪ1)

Total acidity

660

COOH

450

Phenolic OH

210

Alcoholic OH

280

Quinonoid CuO

250

Ketonic CuO

190

OCH3

30

E4 /E6

4.3



C

H

N

S

O



FA



509

33

7

3

448

1240

910

330

360

60

250

10

7.1



all of the O in the FA is similarly distributed. The E4 /E6 ratio of the FA is almost

twice as high as that of the HA, which means that the FA has a lower particle or

molecular weight than the HA (Chen et al., 1976).



B. INFRARED (IR) AND FOURIER TRANSFORM

IR SPECTROSCOPY

IR and FTIR spectra of humic substances show bands at 3400 cmϪ1 (H-bonded

OH), 2900 cmϪ1 (aliphatic C–H stretch), 1725 cmϪ1 (CuO of CO2H, CuO stretch

of ketonic CuO), 1630 cmϪ1 (COOϪ, CuO of carbonyl and quinone), 1450 cmϪ1

(aliphatic C–H), 1400 cmϪ1 (COOϪ), 1200 cmϪ1 (C–O stretch or OH deformation

of CO2H), and 1050 cmϪ1 (Si–O of silicates). The bands are usually broad because

of the extensive overlapping of individual adsorbances. IR and FTIR spectra reflect

the preponderance of oxygen-containing functional groups, i.e., CO2H, OH, and

CuO in humic materials. While IR and FTIR spectra provide worthwhile information on the functional groups and their participation in metal–humic interactions,

they tell us little about the chemical structure of humic materials.

Celi et al. (1997b) applied FTIR to the analysis of CO2H groups in a number of

HAs. Concentrations of CO2H groups in HAs were determined directly from FTIR

spectra by totaling adsorbances at 1720–1710 cmϪ1 (CO2H) and 1620–1600

cmϪ1 (COOϪ). Good correlations were found between total carboxyl groups determined by FTIR, a wet chemical method, and by 13C NMR. Thus, depending on



A LIFETIME PERSPECTIVE



13



the equipment and facilities available, soil chemists have a choice of methods that

can be used for measuring CO2H groups in HAs.



C.



13C



NMR SPECTROSCOPY



Until about the mid-1980s, when the use of liquid- and solid-state 13C NMR became more widespread, most soil chemists thought that the chemical structure of

humic substances was predominantly aromatic. 13C NMR demonstrated that

aliphatic structures in humic substances were often as important, and sometimes

even more important than aromatic structures (Schnitzer and Preston, 1986; Wilson, 1987; Norwood, 1988; Schnitzer, 1991). This was a very significant development in our understanding of the chemistry of humic substances. Aromaticities

of HAs extracted from soils of widely differing pedological origins range from 30

to 60% (Schnitzer, 1991). A substantial portion of aliphatic carbons in HAs consists of paraffinic carbons. Of considerable interest are the prominent resonances

in both liquid- and solid-state 13C NMR spectra of humic substances near 130 and

132 ppm, which can be assigned to C in aromatic rings that are not substituted by

strong electron donors such as O and N but by C. Alkaylaromatics are typical structures that produce such resonances (Breitmaier and Voelter, 1978).

Of special interest in the context of this discussion is a comparison of solid-state

13C NMR spectra of a HA (extracted from the Ah horizon of a Haploboroll) and a

FA (extracted from the Bh horizon of a Spodosol). The HA spectrum in Fig. 4 shows

several distinct peaks in the aliphatic (0–105 ppm), aromatic (106–150 ppm), phe-



Figure 4 Solid-state 13C NMR spectra of HA (extracted from the Ah horizon of a Haploboroll)

and FA (extracted from the Bh horizon of a Spodosol).



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M. SCHNITZER



nolic (155–160 ppm), and carboxyl (170–180 ppm) regions. The signals at 17, 21,

25, 27, and 31 ppm are likely due to alkyl C. The resonance at 17 ppm is characteristic of terminal CH3 groups and that at 31 ppm of (CH2)n in straight paraffinic

chains. The resonance at 40 ppm could also include contributions from both alkyl

and amino acid C. The broad signal at 53 ppm and the sharper one at 59 ppm may

be due to C in OCH3. Amino acid C may also contribute in this region (Breitmaier

and Voelter, 1978). Carbohydrates in HA would be expected to produce signals in

the 60 to 65, 70 to 80, and 90 to 104 ppm regions, although other types of aliphatic

C bonded to O could also do so. The aromatic region contains a relatively sharp maximum near 130 ppm due to alkyl aromatics. The peak at 155 ppm indicates the presence of O- and N-substituted aromatic C (phenolic OH and/or NH2 bonded to an

aromatic C). The broad signal near 180 ppm is due to C in CO2H groups, although

amides and esters could also contribute to this resonance.

The 13C NMR spectrum of the FA (Fig. 4) consists of a number of aliphatic resonances in the 20- to 50-ppm region, followed by signals from C in OCH3 groups,

amino acids, and carbohydrates between 50 and 85 ppm. Broad signals between

130 and 133 ppm indicate the presence of C in alkyl aromatics. The strong signal

between 170 and 180 ppm shows the presence of C in CO2H groups. In general,

fewer sharp signals are observed in the 13C NMR spectrum of the FA than in that

of the HA, possibly because of more H bonding in the FA.

13C NMR data for HA and FA are summarized in Table II in terms of the distribution of C in the different spectral regions. An examination of data in Table II

shows a similar C distribution in the two humic fractions. HA is slightly more aro-



Table II

Distribution of C (%) in a Haploboroll HA and a Spodosol FA as Determined

by 13C NMR

% of C

Chemical shift range (ppm)



HA



FA



0–40

41–60

61–105

106–150

151–170

171–190

Aliphatic C (0–105 ppm)

Aromatic C (106–150 ppm)

Phenolic C (151–170 ppm)

Aromaticitya



24.0

12.5

13.5

35.0

4.5

10.5

50.0

35.0

4.5

44.1



15.6

12.8

19.3

30.3

3.7

18.3

47.7

30.3

3.7

41.6



((Aromatic C ϩ phenolic C) /(Aromatic C ϩ phenolic C ϩ aliphatic C)) x 100.



a



A LIFETIME PERSPECTIVE



15



matic than FA, but FA is richer in CO2H groups, which appears to be the main difference between the two humic substances. Other differences are that HA is richer in paraffinic C but poorer in carbohydrate C than FA. However, on the whole,

the main structural features, as well as aromaticity and aliphaticity, are similar so

that HA and FA have similar chemical structures. These findings disagree with

those of Sprengel (1826) and other earlier workers who thought that the chemical

structures of HA and FA were quite different from each other.

Little is known about the chemical structure of humin, which is that portion of

SOM that stays behind after extraction of the soil with dilute alkali. In a more recent investigation, Preston et al. (1989) deashed the surface layer of Bainsville soil

with aqueous HCl/HF (1.16 M HCl and 2.88 M HF) at room temperature for a prolonged period of time. With progressive deashing, the humin became more soluble in 0.5 M NaOH. After extensive deashing the solid-state 13C NMR spectrum

was similar to that of HA extracted from the same soil. This suggests that humin

is essentially HA bound strongly to soil minerals.

Valuable information on the chemical structure of humic substances can be obtained by combining 13C NMR with chemical methods. In this manner, effects on

the chemical structure of humic substances of different extractants, methylation,

hydrolysis, oxidation, and reduction can be evaluated. Spectra shown in Fig. 5 illustrate this point.

Figure 5a shows the solution-state 13C NMR spectrum of HA extracted from the

Ah horizon of a Haploboroll from northern Alberta (Preston and Schnitzer, 1984).

The presence of aliphatic C (i.e., C in straight chain, branched and cyclic alkanes,

alkanoic acids, and other aliphatic compounds) is indicated by signals in the 10to 40-ppm region of the spectrum. Carbon in proteinaceous materials (amino acids,

peptides, and proteins) exhibits resonances between 40 and 60 ppm, whereas C in

OCH3 groups shows signals near 56 ppm. Signals between 61 and 105 ppm arise

from C in carbohydrates. Resonances between 106 and 150 ppm are due to aromatic C whereas those between 150 and 160 ppm arise from phenolic C. The strong

signal between 170 and 180 ppm comes mainly from C in CO2H groups, with possibly some contributions of C in esters and amides.

Figure 5b shows the solution-state 13C NMR spectrum of the same HA after hydrolysis for 24 hr with hot 6 M HCl. Most of the resonances in the 40- to 105-ppm

region arising from C in proteinaceous materials and carbohydrates are no longer

observed because of the hydrolytic removal of these materials by the hot acid.

Also, the intensity of the signal between 170 and 180 ppm, due largely to C in

CO2H groups, has been reduced because of partial decarboxylation of these groups

by the strong acid. However, intensities of the two principal HA components, i.e.,

aliphatic C (10 –60 ppm) and aromatic C (106 –150 ppm), remain undiminished.

Thus, by removing carbohydrates and proteinaceous components, acid hydrolysis

“purifies” the HA and allows us to study the remaining aliphatic and aromatic components in greater detail.



16



M. SCHNITZER



Figure 5 Solution-state 13C NMR spectra of a HA extracted from the Ah horizon of a Haploboroll

before (a) and after (b) hydrolysis with hot 6 M HCl. From Schnitzer (1991), with permission of the

publisher.



D. ELECTRON SPIN RESONANCE SPECTROSCOPY

Electron spin Resonance (ESR) spectroscopy measures free radicals (unpaired

electrons) in humic substances. It has been known since the early 1960s (Rex,

1960; Steelink and Tollin, 1962) that humic substances contain free radicals that

may participate in a wide variety of organic–organic and organic–inorganic interactions. The theory and a number of applications of ESR spectroscopy have been

described by Atherton (1973). The ESR spectrum of a typical HA is shown in Fig.

6. The spectrum consists of a single line devoid of hyperfine splitting. From the

spectrum (by comparison with a standard), the number of free radicals per unit

weight as well as the g value (the spectroscopic splitting constant) and also the line

width can be calculated. From the magnitudes of the g values for humic materials,

with most ranging from 2.0038 to 2.0042 (Senesi and Schnitzer, 1977), it appears

that prominent free radicals in humic materials are semiquinones or substituted



A LIFETIME PERSPECTIVE



Figure 6



17



ESR spectrum of a typical HA.



semiquinones. There are two types of free radicals in these materials: (a) permanent free radicals with long lifetimes and (b) transient free radicals with relatively short lives (several hours). Transient free radicals in humic materials can be generated in relatively high concentrations by chemical reduction, irradiation, or

increase in pH (Senesi and Schnitzer, 1977). Permanent free radicals, in contrast,

appear to be stabilized by the complex chemical structures of humic materials,

which can act both as electron donors and as electron acceptors. These oxidation–

reduction reactions are reversible and can be assumed to proceed in the following

manner:



18



M. SCHNITZER



The semiquinone (shown in the center) can be produced either by the reduction

of a quinone or by the oxidation of a phenol. Under alkaline conditions, a semiquinone anion and the semiquinone dianion are formed. Except for indicating the

presence of phenols, semiquinones, and quinones in humic materials, ESR spectroscopy has so far contributed little to our understanding of the structural chemistry of these substances. The main reason for this is that it has been difficult to

split the signal. However, significant new information has been generated by ESR

on the symmetry and coordination of metals in metal–humic complexes (Cheshire

et al., 1977; Lakatos et al., 1977; Senesi et al., 1977; McBride, 1978; Boyd et al.,

1981; Cheshire and Senesi, 1998). HAs and FAs interact with paramagnetic metal ions to form a variety of metal–organic complexes. These metal ions include

Cu2+, (VO)2+, Mn2+, Mo(V) and Mo(III), Cr3+, and Fe3+. ESR parameters of complexes formed between these metal ions and HA and FA have been described in

considerable detail by Cheshire and Senesi (1998). Especially interesting is the

ESR spectrum of a Fe3+ –FA complex (Fig. 7) (Senesi et al., 1977; Cheshire and

Senesi, 1998). The signal at gϳ 2.0 (resonance C) generally arises from Fe3+ ions

in close proximity to each other. This signal is removed easily by reduction with

hydrazine. From Mössbauer studies, it is suggested that this type of Fe3+ is held

in octahedral coordination on external HA and FA surfaces.

The signal at g~ 4.3 (resonance A) is assigned to Fe3+ in an organic complex,

in which high-spin Fe3+ ions occupy sites of approximately orthorhombic sym-



Figure 7



ESR spectrum of a Fe3+ –FA complex.



A LIFETIME PERSPECTIVE



19



metry. Thus, ESR spectroscopy can provide important information on the coordination and symmetry of paramagnetic metal ions complexed by humic substances.



E. ELECTRON MICROSCOPY

Particle sizes and shapes of HAs and FAs seen under the electron microscope

are affected by pH, electrolyte concentration, and HA and FA concentrations (Chen

and Schnitzer, 1989).

Freeze dried at pH 2, FA produces a scanning electron microscopy (SEM) micrograph (Fig. 8a) that exhibits elongated fibers and bundles of fibers (Chen and

Schnitzer, 1976b; Chen et al., 1976). The fibers are either linear or curved, ranging up to 6–7 ␮m in length and 120–400 nm in thickness. At pH 4 (Fig. 8b), the

fibers tend to become thinner and the bundles of fibers more prominent. The

lengths of the fibers remain unchanged, but their thicknesses are reduced to 120–

200 nm. At pH 6 (Fig. 8c), the fibers diminish in numbers and thicknesses and a

greater proportion of the FA occurs in bundles of closely knit fibers. A pH 7 (Fig.

8d), a fine network of tightly meshed fibers with parallel orientation is observed.

At pH 8 (Fig. 8e), the fine network turns into a sheet-like structure of varying thicknesses. At pH 9 (Fig. 8f), the sheets tend to thicken. At pH 10 (Fig. 8e), the SEM

micrograph shows homogeneous grain-like particles.

Because of solubility constraints, the effect of pH on the structure of HA over

the pH range 6 to 10 is similar to that observed on FA (Chen and Schnitzer, 1976b).

Effects of increasing concentrations of neutral salts in aqueous solutions of HA

and FA on their SEM micrographs following freeze-drying were studied by Ghosh

and Schnitzer (1982). Fiber thicknesses increase but particle orientation gradually decreases with increases in salt concentrations. These effects are similar to those

observed when the pH is decreased from neutrality to the moderately acidic region. Thus, both pH and elevated salt concentrations affect the dissociation and

conformation of HAs and FAs in a similar manner.

Effects of varying the concentrations of HA and FA in solution prior to freezedrying on the sizes and shapes of the micromolecules under transmission electron

microscopy were examined by Stevenson and Schnitzer (1982). Single drops of

dilute aqueous HA and FA solutions, adjusted to different pH values, were spread

uniformly on strips of freshly cleaned mica. The solution-bearing strips were tilted and then frozen rapidly in Freon. This allowed the surface film to run toward

the lower edges of the mica and form a concentration gradient. The smallest individual particles that appear in the dilute areas are spheroidal with diameters ranging from 9 to 27 nm. Spheroids tend to coalesce to form round-shaped aggregates

or linear chain-like structures. At higher concentrations, the spheroids and chainlike structures form flat, elongated, multibranched fibers (Fig. 9), similar to those

observed earlier by Chen and Schnitzer (1976b). The width of the fibers (or filaments) ranges from 20 to 100 nm. At higher concentrations, parallel arrays of fibers



Figure 8 Scanning electron micrograph of a freeze-dried FA at various pH values. From Chen and Schnitzer (1976b), with permission of the publisher.



A LIFETIME PERSPECTIVE



21



Figure 9 Transmission electron micrograph of a 0.01% HA solution. From Schnitzer (1991), with

permission of the publisher.



tend to coalesce to form sheet-like structures. Spheroids observed at low concentrations are larger and range in diameters from 12 to 50 nm. With increasing concentrations of HA and FA, spheroids coalesce into aggregates of spheroids and then

into chain-like structures, followed by fibers and flattened sheets.

An interesting conclusion drawn by Chen and Schnitzer (1989) is that humic

substances in aqueous solutions act like flexible, linear polymers rather than spheroids. When sample concentrations are high and the pH of the solution is low or

when appreciable amounts of neutral electrolytes are present, humic particles may

assume a spheroidal shape. At low concentrations and neutral to basic pH, the particles stretch, forming slightly coiled fibers. The latter consist of a large number of

oriented molecules. At low solute concentration, low ionic strength, and high pH,

the coils stretch further, the filamentous structure disintegrates, and complete dispersion takes place. If, under similar conditions, the concentration of humic substances is high, sheet-like structures may be formed due to coagulation.



IV. CHEMICAL STRUCTURE

OF HUMIC SUBSTANCES

A. OXIDATIVE DEGRADATION

One of the most useful methods for obtaining information on the chemical structure of complex organic substances is oxidative degradation. Over the course of

20 years, my co-workers and I have investigated the oxidative degradation of HAs,



22



M. SCHNITZER



FAs, humins, and whole soils using a variety of methods. These included the oxidation of methylated and unmethylated humic substances with an alkaline KMnO4

solution (Schnitzer and Khan, 1978; Griffith and Schnitzer, 1989). The somewhat

milder oxidation with alkaline CuO, as well as the sequential oxidation with CuONaOH ϩ KMnO4 and with CuO-NaOH ϩ KMnO4 ϩ H2O2 solutions, has also

been employed (Schnitzer and Khan, 1978; Schnitzer, 1978). Humic substances

have also been degraded under acidic conditions with peracetic acid and nitric acid

(Schnitzer, 1978). Other oxidants used include alkaline nitrobenzene, sodium

hypochlorite, and H2O2 solutions (Schnitzer and Khan, 1978). Degradations with

Na2S and phenol have also been carried out (Hayes and O’Callaghan, 1989).

Major compounds produced by the oxidation of methylated and unmethylated

humic substances from widely differing pedological and geographical origins under alkaline as well as under acidic conditions are aliphatic carboxylic, phenolic,

and benzenecarboxylic acids (Schnitzer and Khan, 1978; Schnitzer, 1978; Griffith

and Schnitzer, 1989).

Among aliphatic oxidation products are mono-, di-, tri-, and tetracarboxylic

acids. Major aromatic oxidation products are benzenecarboxylic acids such as the

tri, tetra, penta, and hexa forms (Fig. 10), whereas phenolic acids include compounds containing between one and three OH groups and between one and five

CO2H groups per aromatic ring (Fig. 11).

From the oxidation products identified and from 13C NMR spectra of humic

substances, it appears that aromatic rings are cross-linked by paraffinic chains (Fig.

12). On oxidation, the aliphatic carbons closest to the rings become the C of CO2H

groups and remain bonded to the rings whereas the other carbons in the aliphatic

chains are oxidized to either aliphatic acids or CO2. The formation of CO2 from



Figure 10



Major benzenecarboxylic oxidation products.



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