DIVISION S-3-SOIL MICROBIOLOGY& BIOCHEMISTRY

Low-Temperature Ashing of Humic and Fulvic AcidC. R. De Kimpe* and M. Schnitzer

ABSTRACTLow temperature (<100 °C) ashing (LTA) has been proposed for

removing organic matter from mineral soil constituents under rela-tively mild conditions. However, little is known about the chemicalreactions governing organic-matter removal by this method. Thisstudy was conducted to evaluate the nature of residual materials atvarious stages of the LTA process. The LTA procedure was appliedto a humic acid extracted from a lignite and a fulvic acid extractedfrom a Spodosol Bh horizon. Residues were recovered at variousstages of weight loss and characterized by chemical, infrared (IR),and 13C nuclear magnetic resonance (NMR) analyses. Chemicalanalyses showed, on a moisture- and ash-free basis, relatively smalldecreases in C for both the humic and fulvic acid with increasingexposure to LTA. The humic acid, however, lost more than half ofits N after IS min of LTA, whereas the N content of the fulvic acidmore than doubled after 3 min of LTA and tripled after 30 min ofexposure. These increases in N appeared to be due to the oxidationof NH3 and other N forms to NO3, which was adsorbed on mineralcomponents of the fulvic acid. The IR and 13C NMR spectra of theresidues at different stages of LTA were similar, which suggestedthat LTA decomposed the organic matter homogeneously by a sur-face reaction. Possible uses of LTA include the removal of organicmatter from soils without damaging mineral components, the prep-aration of soil samples of uniform mineral composition but withdifferent organic-matter contents, and the partial degradation of soilorganic matter in structural studies on these materials.

LOW-TEMPERATURE ASHING has been developed asa procedure for removing organic materials from

inorganic specimens with minimal or no damage tothe inorganic constituents (Marcoen, 1975). Damageto inorganics is minimized because surface tempera-tures below 100 °C can be maintained by using lowpower levels (Thomas and Hollahan, 1974). In thistechnique, a stream of excited O2, produced by a high-frequency electromagnetic field, is the oxidizing agent.The excited oxygen breaks C-C, C-H, and C-N bonds,and the oxidation products (CO, CO2, NO, NO2, andH2O) are removed with a vacuum pump. The methodhas been applied to studies on oil shales (Smith andFuta, 1974), coals (Soong and Gluskoter, 1977), bio-logical materials (Gleit and Holland, 1962), and forthe preparation of samples for scanning electron mi-croscopy (Sullivan and Koppi, 1987). The LTA pro-cedure has also found applications in soil science forthe pretreatment of samples prior to mineralogicalanalyses (Marcoen, 1975) and for the determinationof the organic-matter content of soils (Delecour et al,Land Resource Research Centre, K.W. Neatby Bldg., Central Ex-perimental Farm, Agriculture Canada, Ottawa, ON K1A OC6, Can-ada. Contr. no. 89-34. Received 20 Apr. 1989. "Corresponding au-thor.

Published in Soil Sci. Soc. Am. J. 54:399-403 (1990).

1975). In the latter applications, the soil organic-mat-ter contents, as determined by LTA, equalled thoseobtained by wet oxidation with K2Cr2O7 solution, al-though the organic-matter contents of some soils wereunderestimated because of weight gains attributed tothe formation of solid residues. The latter werethought to be NO3 adsorbed by the residual mineralfraction (Marcoen and Delecour, 1976). Recently, thesolid residue was unambiguously identified as NO3(Miles and Neville, 1989, unpublished data), whichcould easily be removed by washing the sample withwater.

The literature contains little information on themechanism of soil organic-matter removal by LTA,although information on controlled thermal degra-dation is available (Schnitzer and Hofiman, 1964; Ko-dama and Schnitzer, 1970). The objective of this re-search was to obtain a better understanding of thechemical mechanism(s) governing LTA of soil organicmatter. In contrast with thermal degradation andchemical oxidation, LTA is a relatively mild proce-dure. We were especially interested in finding out howLTA decomposes soil organic matter and whether,aside from its use for total degradation, it could alsobe used for the partial degradation of soil organics instructural studies on these materials. In order to ob-tain the desired information, we applied LTA to ahumic acid (HA) and a fulvic acid (FA) and charac-terized the residual materials at various stages of theprocess by chemical and spectroscopic methods.

MATERIALS AND METHODSThe HA that we employed was extracted from a leonardite

lignite obtained from an oxidized coal seam of the GascoyneMine, Bowman County, North Dakota. This HA is a high-C HA that is difficult to decompose by thermal and wet-chemical methods. Methods of extraction, separation, andpurification were identical to those described by Calderoniand Schnitzer (1984). Briefly, 100 g of air-dry leonardite wasshaken for 16 h at room temperature with 1 L of 1.0 M HC1to remove alkali earth metals. Following separation of thesupernatant by centrifugation at 850g for 1 h, the residuewas washed once with distilled H2O and then neutralizedwith 1 M NaOH to pH 7. This required 0.15 L of 1.0 ATNaOH. To the resulting suspension, 1 L of 0.1 M NaOHwas added and the system shaken intermittently under N2at room temperature for 24 h. The alkaline residue was sep-arated from the supernatant by centrifugation, acidified with6 M HC1 to pH 1, and allowed to stand at room temperaturefor 24 h. The supernatant (FA) was separated from the co-agulate (HA) by centrifugation. Suspended clays were re-moved from the HA by dissolving the HA in a minimumvolume of 0.1 M KOH under N2 and then adding KC1 tomake the system 0.3 M in K. After allowing to stand for 4h, suspended solids were removed by centrifugation. Theclear HA solution was acidified to pH 1, and the HA allowed

399

400 SOIL SCI. SOC. AM. J., VOL. 30, MARCH-APRIL 1990

Table 1. Chemical properties of untreated and low-temperature ashing (LTA) treated humic acid (HA) and fulvic acid (FA) samples.Time of

exposure toSample no. LTA Weight loss

Moisture-free basis

Ash

Moisture and ash-free basis

C N C/N

-gkg'1

HA12345

Mean ± SDLSD (0.05)

0153346

100

017294379

580.0584.0579.0571.0506.0

564.0 ± 32.857.5

17.07.07.06.06.0

8.6 ± 4.78.3

20.024.028.035.096.0

40.6 ±31.555.2

613.0614.0608.0598.0570.0

601.0 ± 18.232.0

18.07.08.06.06.0

9.0 ± 5.18.9

348876

10095

FA12345

Mean ± SDLSD (0.05)

035

1030

017324363

402.0381.0340.0302.0188.0

322.6 ± 84.9148.4

4.09.08.08.07.0

7.2 ± 1.93.4

215.0259.0317.0381.0588.0

352.0 ± 146.0254.6

546.0545.0525.0512.0473.0

520.2 ± 30.052.7

6.013.013.013.018.0

12.6 ± 4.37.5

9142403926

to coagulate. The latter was separated by centrifugation andshaken three successive times for 24 h at room temperaturewith 250 mL of HC1-HF (5 mL cone. HC1 + 5 mL 52% HF+ 990 mL distilled H2O) solution until the ash content re-mained constant. Following this, the HA was freeze-driedand then dried in a vacuum desiccator over P2O5 at roomtemperature.

The FA was extracted from the Bh horizon of an Anna-dale soil, a Spodosol on Prince Edward Island, Canada. Thissoil was selected because the organic matter in this horizonis mainly FA, and because we had studied this particularFA over a period of many years and had accumulated in-formation that would help us in the interpretation of theresults of this investigation. The FA was extracted accordingto the procedures described by Schnitzer and Skinner (1968).Briefly, 100 g of soil was extracted by shaking with 1 L of0.5 M NaOH under N2 for 24 h. The alkaline extract wasacidified with 2 M HC1 to pH 2 and allowed to stand atroom temperature for 24 h. Coagulated HA was separatedfrom soluble FA by centrifugation. The FA was passed onlyonce over a column of Amberlite IR-120 exchange resin(Mallinckrodt Chemical Works, St. Louis, MO) in the Hform. The effluent was then freeze-dried. No attempt wasmade to lower the ash content of the FA to less than 21.5%because we were interested in observing the effect of a rel-atively high ash content on the LTA process.

The LTA procedure was done in a four-chamber LTA 504low-temperature asher (LFE Corp., Waltham, MA). Samplesof extracted HA or FA were placed in quartz boats and thefour chambers were evacuated to 0.133 kPa. Subsequently,O2 was admitted into the asher at a rate of 300 mL/min. Togenerate excited O2, the radiofrequency generator of the in-strument was adjusted to 13.56 MHz, and operated with apower input of 300 W and a reflected power of 8 W. Sampleswere ashed to the desired weight losses (Table 1).

Residues were analyzed by IR spectroscopy on a Beckman4250 IR spectrophotometer (Beckman Instruments, Fuller-ton, CA). The samples were prepared as KBr pellets at adilution of 1 mg sample in 400 mg KBr. Solid-state 13CNMR spectra were recorded at a frequency of 45.28 MHzon a Bruker CXP NMR spectrometer (Bruker GmbH, Karls-ruhe, W. Germany) equipped with a Doty Scientific Probe(Doty Scientific, Columbia, SC). Single-shot cross-polariza-tion contacts of 2 ms were used with matching radiofre-quency field amplitudes of 75 KHz. Up to 120 000 500-worddecays were co-added with a delay time of 1 s. These werezero filled to 4000 data points before Fourier transforma-tion. Magic angle spinning rates were 4 KHz.

To facilitate the analysis of the 13C NMR data, the spectrawere divided into the following regions: 0 to 40 ppm (C instraight-chain, branched, or cyclic alkanes and alkanoicacids); 40 to 60 ppm (C in branched aliphatics, amino acids,and OCH3 groups); 60 to 105 ppm (C in carbohydrates andin branched aliphatics containing C bonded to OH, etheroxygens, or occurring in five- or six-membered rings bondedto O); 105 to 150 ppm (aromatic C); 150 to 170 ppm (phe-nolic C); and 170 to 190 ppm (C in CO2H groups). Areaswere measured with an integrator. Aromaticities were cal-culated by expressing aromatic C (105-170 ppm) as percentof aliphatic C (0-105 ppm) + aromatic C. Uncertainty inarea measurement was approximately ± 10% and dependedboth on the signal to noise (S/N) ratio and on the extent ofoverlap of spectral regions.

Residues were also analyzed chemically for C by dry com-bustion (Morris and Schnitzer, 1967) and N by the micro-Kjeldahl method (Hesse, 1971). Coefficients of variabilityfor replicate analyses were 0.1% for C and 0.1% for N. Mois-ture in each sample was determined by heating samples at105 °C for 24 h, and ash by ignition at 750 °C for 4 h. Coef-ficients of variability for replicate moisture and ash analyseswere 0.5%. Three batches of the leonardite and the SpodosolBh horizon were extracted. There were no significant chem-ical differences between the extracts. All chemicals andspectroscopic determinations were done in triplicate. Co-efficients of variability for replicate analyses are listed sep-arately for each determination.

In addition to mean and standard deviation (SD), the leastsignificant difference (LSD, 0.05) was computed for each setof data in Table 1. The LSD was calculated according toSteel and Torrie (1960) from the following equation:

( 5 - 1 [1]

where £3 is the standard deviation of the mean differenceand 5 is the number of measurements.

LSD (0.05) = 2.776 Sa [2]where 2.776 is the relevant factor derived from a t table(Steel and Torrie, 1960).

RESULTS AND DISCUSSIONWeight Loss and Chemical Analysis of the Residues

A blue color persisted for about 150 min with HAand 50 min with FA samples in the LTA chambers,

DE KIMPE & SCHNITZER: LOW-TEMPERATURE ASHING OF ORGANIC ACIDS 401

98%

4000 3500 3000 2500 2000 1500WAVENUMBER (cm

1000 500

Fig. 1. Infared spectra of humic acid residues at different weightlosses.

then the color progressively returned to the violet ofthe pure O2 discharge, indicating that the organic-mat-ter oxidation had been completed. Complete oxida-tion as determined by chemical analyses was attainedwith 600 min for the HA samples and 300 min for theFA samples. In separate runs, subsamples were re-moved at 17, 29, 43, and 79% weight loss for the HAand at 17, 32, 43, and 63% weight loss for the FA inorder to determine the characteristics of the residues.

As shown in Table 1, the ash content of the HA andFA residues increased with time of exposure to LTA.On a moisture-free basis, the C and N content of theHA tended to decrease with time of exposure. In thecase of the FA residues, C decreased but N first in-creased and then decreased slightly. The most inter-esting data in Table 1 are those expressed on a mois-ture- and ash-free basis. These data show that thedecrease in C content of the HA residues was not sta-tistically significant up to 79% weight loss or 100 minof exposure. By contrast, the N content of the HAresidues declined significantly after only 15 min ofexposure. As far as the FA residues were concerned,statistically significant C losses were observed after 30min of exposure at 63% weight loss, while substantialincreases in N were already observed after only 3 minof exposure. These increases became statistically sig-nificant after 30 min exposure to LTA and 63% weightloss.

These data show that: (i) C components of the HAand FA are appreciably more stable to LTA exposurethan the majority of the N components, and (ii) thatC components and one-half to two-thirds of the Ncomponents in these materials appear to occur as sep-arate entities because the behavior to LTA of most ofthe N differs from that of C.

It may be pertinent to compare the LTA results inTable 1 with thermogravimetric data on very similarmaterials reported some time ago (Schnitzer and Hoff-man, 1964). When HA or FA is heated on a ther-mobalance from room temperature to 540 °C, the Ccontent of the residues starts at 50 to 60% and in-creases to over 90%, while the O2 content starts at 30to 40% and drops to 0%. The main reaction governingthe thermal decomposition of humic substances was

UJO

i

4000 3500 3000 2500 2000 1500 1000 500WAVENUMBER (cm-1)

Fig. 2. Infared spectra of fulvic acid residues at different weightlosses.

found to be dehydration. Somewhat surprisingly, thedata in Table 1 indicate that the mechanism of LTAof humic substances differs from that of thermal deg-radation. No large C losses are observed and, as willbe shown in the following sections, IR and 13C NMRspectra of the residues exposed to LTA for differentlengths of time do not show any significant structuralchanges in the HA and FA. Finally C/N ratios of theHA (Table 1) tended to increase with time of exposurebecause more N than C was lost. The reverse was ob-served for the FA residues. Reasons for this will beoffered later.

Infrared SpectroscopyThe original HA sample was characterized by a

broad OH band at 3430 cmr1, small aliphatic peaks at2920 and 2850 cm-1, strong C=O of COOH andCOO" carboxyl bands at 1720 and 1610 cm-1, respec-tively, and two broad bands at 1400 and 1200 cm-1

(Fig. 1). This spectrum changed little as a result ofLTA up to 79% weight loss, in agreement with chem-ical analyses (Table 1). The mineral residue at 98%weight loss still contained 2% ash in spite of prolongedtreatment with dilute HC1-HF solution. The IR spec-trum of this material (Fig. 1) showed distinctive fea-tures such as a strong OH band, a H2O deformationband at 1640 cnr1, complex SiO vibrations between1200 and 1000 cm-1, and a Si-O-Al deformation bandaround 600 cm"1. The IR spectrum of the original FA(Fig. 2) showed a broad OH band at 3420 cm-1, smallaliphatic peaks at 2920 and 2850 cm-1, a carboxylictrough near 2550 cm-1, strong C=O of COOH andCOO- bands at 1720 and 1620 cm-1, respectively, aphenolic OH band at 1250 cm-1, two bands charac-teristic of mineral components, a SiO vibration bandat 1020 cnr1, and a complex Si-O-Al deformationband at 475 cm"1 (Fig. 2). During LTA, the spectrumremained more or less unchanged, except for the de-velopment of a shoulder at 3620 cm-1, correspondingto the OH vibration of a micaceous mineral (Kodama,1985), associated with a SiO vibration band at 1040cm-1 and lattice deformation bands around 475 cm"1.These bands correspond to the mineral componentand developed as LTA progressed. Furthermore, start-

402 SOIL SCI. SOC. AM. J., VOL. 30, MARCH-APRIL 1990

Table 2. Distribution of C in untreated humic acid and low-temper-ature ashed bumic acid residues.

Table 3. Distribution of C in untreated fulvic acid and low-temper-ature ashed fulvic acid residues.

SamplefChemical shift range

ppm

0- 4040- 6060-105

105-150150-170170-190190-230Aliphatic C

(0-105 ppm)Aromatic C

(105-170 ppm)Aromatic! ty

0%

26.76.04.3

44.89.57.80.9

37.0

54.359.5

17%

28.19.83.9

40.57.87.82.0

41.8

48.353.6

29%

25.08.36.5

41.77.49.31.9

39.8

49.156.0

43%————

26.810.33.1

40.27.2

11.31.0

40.2

47.454.1

79%

23.67.63.5

45.19.78.32.1

34.7

54.861.2

Mean± SD

26.08.44.3

42.58.38.91.6

38.7

50.856.9

±±±±±++

±

±±

1.81.71.32.31.21.50.6

2.8

3.53.3

t Sample removed at the indicated weight loss.

ing from Sample 3 at 32% weight loss, a small peakappeared at 1384 cm"1, which became sharper andmore intense in Sample 5 at 63% weight loss. Thispeak corresponded to NO3 (Miles and Neville, 1989,unpublished data) retained by the mineral fraction.No NO3 retention was observed in the HA residues,even after 90 h of LTA treatment and 95% weight loss.

17%

29%

43%

79%

SamplefChemical shift range

ppm

0- 4040- 6060-105

105-150150-165165-190Aliphatic C

(0-105 ppm)Aromatic C

(105-165 ppm)Aromaticityt Sample removed at the

0%

17.911.621.428.68.0

12.5

50.9

36.641.8

17%

13.611.821.829.18.2

15.5

47.2

37.344.1

32%

15.010.323.429.97.5

14.0

48.7

37.443.4

43%———

15.512.723.926.88.5

12.7

52.1

35.340.4

63%

17.712.922.624.29.7712.9

53.2

33.938.9

Mean± SD

15.9 ± 1.811.9 ± 1.022.6 ± 1.027.7 + 2.38.4 + 0.8

13.5 + 1.2

50.4 + 2.5

36.1 ± 1.541.7 ± 2.1

indicated weight loss.

The presence of NO3 in the FA-sample residues isclearly related to the high ash contents of these resi-dues; some of these ash components are capable offixing NO3.

Carbon-13 Nuclear Magnetic ResonanceSpectroscopy

The HA spectrum showed two major peaks at 30(aliphatic C) and 129 (aromatic C) ppm, and a smallerone at 168 ppm (CO2H-C) (Fig. 3). The overall con-figuration of the spectra changed little as the result ofthe LTA treatment. Only slight variations were ob-

17%

32%

43%

63%

250 200 150 100 50

PPMFig. 3. Carbon-13 nuclear magnetic resonance spectra of humic acid

residues at different weight losses.

250 200 150

PPM

100 50

Fig. 4. Carbon-13 nuclear magnetic resonance spectra of fulvic acidresidues at different weight losses.

DE KIMPE & SCHNITZER: LOW-TEMPERATURE ASHING OF ORGANIC ACIDS 403

served in the relative intensities of the two majorpeaks. When expressed as percentages of total C in thedifferent chemical shift ranges, there were only minorvariations among the untreated and LTA-treated sam-ples (Table 2). Only aliphatic C, aromatic C, and thearomaticity of the most oxidized sample, at 79%weight loss, fell outside the range of the mean value± 1 SD.

The NMR spectra of the FA residues (Fig. 4) werenot as well denned because of the interference by min-eral components. Four peaks were recognized, how-ever, at 34 and 74 ppm in the aliphatic C region andat 127 and 173 ppm in the aromatic C region. Again,only minor differences in the distribution of C withincreasing exposure to LTA were detected (Table 3).Only the most oxidized sample, at 63% weight loss,differed from the other FA residues in the relative per-centages of aliphatic and aromatic C, and also in thearomaticity. However, this could be related, at leastpartly, to interferences with 13C NMR signals, whichbecame more prominent at high weight loss, by min-eral components.

CONCLUSIONSThe chemical, IR, and C13 NMR data show that HA

and FA residues subjected to prolonged treatmentswith LTA have essentially the same chemical struc-ture as the initial materials. While most N compo-nents of these materials appear to be more susceptibleto oxidation by LTA than do the C components, thereare no indications of specific degradation reactionslike those occurring during thermal degradation. Dur-ing the latter process, one can observe the decompo-sition of COOH groups, OH groups, and, finally, ofthe "nuclei" of humic substances. With LTA, thesetypes of degradations do not seem to occur.

When minerals capable of fixing NO3 are present inthe organic matter, the N present initially in differentorganic forms or as NH3 can be oxidized by LTA toNO3, which is then fixed by these minerals. This re-action has occurred in the FA residues.

It appears that the oxidation of HA and FA by LTAis a homogeneous surface reaction by which moleculesof humic substances are stripped off, layer by layer,without altering or damaging the residual layers. Thisprocess can be compared with peeling off onion skins.Because LTA is a relatively mild procedure, it seems

to be suitable for the removal of organic matter fromsoils without damaging mineral components, and forthe preparation of soil samples of uniform mineralcomposition but with different organic-matter con-tents. Another possible application is the preparationof partially degraded soil organic matter that couldfacilitate structural studies on these materials.

ACKNOWLEDGMENTSThe authors wish to thank H. Lie for the FA separation,

and M. McGrath and D. Tutte for the IR and chemicalanalyses, respectively.