FTIR-spectroscopic characterization of humic acids and humin fractions obtained by advanced NaOH, Na4P2O7, and Na2CO3 extraction procedures

$Page 1: FTIR-spectroscopic characterization of humic acids and humin fractions obtained by advanced NaOH, Na4P2O7, and Na2CO3 extraction procedures$
FTIR-spectroscopic characterization of humic acids and humin fractionsobtained by advanced NaOH, Na4P2O7, and Na2CO3 extraction proceduresMichael Tatzber1*, Michael Stemmer2, Heide Spiegel2, Christian Katzlberger2, Georg Haberhauer3, Axel Mentler1, andMartin H. Gerzabek1

1 Institute of Soil Research, Department of Forest and Soil Sciences, University of Natural Resources and Applied LifeSciences, Peter Jordan Straße 82, 1190 Vienna, Austria

2 Austrian Agency for Health and Food Safety, Vienna, Spargelfeldstraße 191, 1226 Vienna, Austria3 ARC Seibersdorf Research GmbH, Department of Environmental Research, 2444 Seibersdorf, Austria

Accepted December 1, 2006

SummaryAim of our study was the development of the methodologicalbasis for the characterization of humic fractions of a long-term field experiment. Humic acids (HAs) were extractedfrom three layers of a nontilled soil using three differentextractants (1 M NaOH, 0.1 M Na4P2O7, 1 M Na2CO3), andthe humin fraction was enriched. NaOH as extractant forFTIR analysis of humic substances yields higher resolved IRspectra, especially in the important regions of stretchingvibrations including aromatic and aliphatic groups and in thefingerprint area including amides, aliphats, and aromats thanthe other extractants. The NaOH extraction has lower extrac-tion yields as compared to Na4P2O7 and Na2CO3 and repre-sents a different part of the soil organic matter (SOM). This isreflected by lower C : N ratios and higher E4 : E6 and fulvicacid–to–humic acid ratios as compared to the other extrac-tants. The FTIR band areas of HA fraction obtained by NaOHshowed an increase of the aromatic and carbonyl groups anda decrease of amide groups with increasing soil depth. Ali-phatic groups showed contradicting results: The bands of the

stretching vibrations increased, and the band of the bendingvibrations decreased. We assume that band interactions inthe bending vibrations were responsible for that phenomenonunder the assumption of an increase of aliphatic groups withincreasing soil depth. The IR bands of the enriched huminfraction showed a decreasing trend in case of both aliphaticbands deriving from stretching vibrations and an increase ofaromatic characteristics with depth. Our study led to the con-clusion that HA fractions obtained by 1 M NaOH represent asmall and dynamic fraction indicated by the measured yieldsin combination with values of Nt, C : N, E4 : E6 ratios, andratios of fulvic acids (FA) to HA. The humin fraction has ahigh contribution to the total organic C and represents a morestabilized fraction of SOM which still shows changes in itsaromatic and aliphatic characteristics with soil depth.

Key words: HA extractions / FTIR spectroscopy / humin fraction /E4 : E6 / FA : HA ratio

2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 1436-8730/07/0408-522

522 DOI: 10.1002/jpln.200622082 J. Plant Nutr. Soil Sci. 2007, 170, 522–529

1 Introduction

The characterization of soil organic matter (SOM) can bedone both in the soild phase and by analyzing soil extracts.The latter method is especially used to characterize humicfractions which are known to have manifold functions in phy-sical, chemical, and biological soil properties. The majorhumic fractions of SOM are fulvic acids (FAs, soluble both inthe extraxt and after acidification), humic acids (HAs, solublein the extract and precipitated after acidification; when redis-solved in base and electrolyte is added, gray HAs are precipi-tated and brown HAs stay in solution) and humin, whichrepresents the insoluble fraction (Schnitzer and Khan, 1978).The concentration of NaOH as extracting agent affects theyield of the extracted HAs as well as its ash content (Schnit-zer and Khan, 1978). There are some reactions induced byNaOH in organic matter (OM) like the Kolbe reaction (carriedout in the laboratory at a temperature of 125°C): R–O�Na+ +CO2 → HO–R–CO�

2 Na+

In this kind of reaction, carboxyl groups are induced intosodium salts of phenols to produce sodium carboxylate salts.This reaction can very probably occur between phenol or qui-

none groups in humic structures when they are exposed toatmospheric CO2 in a strongly alkaline environment (Headand Zhou, 2000). Other reactions mentioned in the literatureare mainly processes like auto-oxidation, breakdown ofhumic macromolecules, and condensation of amino-carbo-nyls (Rosa et al., 2005) as well as reactions like hydrolysis ofester groups (resulting in alcohol derivates and acidic com-pounds) or the base-catalyzed aldol condensation reaction(resulting partly in a-b unsaturated ketones) (Vollhardt andSchore, 1995). Furthermore, there are many examples forthe use of Na4P2O7 (Gerzabek et al., 1992, 1997; Novotny etal., 1999) as milder agents for extracting HAs. However, theextraction with Na4P2O7 might also lead to changes in the HAstructure due to incorporation of orthophosphate (PO3�

4 ) inthe lower–molecular weight fractions of HAs and pyropho-sphate (P2O4�

7 ) in the fractions of higher molecular weight(Francioso et al., 1998).

The relatively simple and cheap Fourier-transform infrared(FTIR) spectroscopy is increasingly used for the characteri-zation of SOM, e.g., organic surface layers of forest soils(Haberhauer et al., 1998, 2000; Wang et al., 2004) or in long-term field experiments including evaluation of humic extracts(Ellerbrock and Kaiser, 2005; Kaiser and Ellerbrock, 2005).The advantage of FTIR spectroscopy is the direct informationabout functional groups in the SOM fraction analyzed.

* Correspondence: Dr. M. Tatzber,e-mail: [email protected]

Since humins are relatively seldom analyzed (Preston andNewman, 1995; Song et al., 2005), there are no standardmethods for their characterization, and various methods sta-ted in literature like Rumpel et al. (1998) or Eusterhues et al.(2003) can be used as a possible basis for methodologicaldevelopments.

The objective of our study was to develop the methodologicalbasis for the characterization of SOM, especially HAs and thehumin fraction, of a long-term field experiment in Lower Aus-tria. We focussed on an optimized method for extraction ofhumic substances and their characterization using FTIRspectroscopy. In combination with basic parameters like Corgand Nt content, E4 : E6 values, ratios of FAs to HAs, yields ofthe humic substances, and ash content of the enriched huminfraction, this approach should be a basis for essential infor-mation about the impact of soil tillage on the dynamics ofhumic substances. A second objective was to characterizethe insoluble fraction (humins) by FTIR spectroscopy.

2 Materials and methods

2.1 Soil sampling

Soil samples were taken from a long-term tillage field experi-ment, which was initiated in 1988 close to Fuchsenbigl in theLower Austrian “Marchfeld” (Spiegel et al., 2002). In 2004,we collected samples from the 0–10, 10–20, and 20–30 cmlayers from the minimum-tilled soils. Subsequently, the soilswere sieved to 2 mm, air-dried, and stored at room tempera-ture. The soil is classified as a fine-sandy loamy Haplic Cher-nozem (clay 22%, silt 41%, sand 37%, pH(CaCl2) 7.65,organic C 1.19%, total N 0.16%). The average annual tem-perature of the field site is 9.4°C, the mean annual precipita-tion is 529 mm.

2.2 Extraction of HAs with 1 M NaOH

This method was developed based on Schnitzer and Khan(1978), where many different methods are discussed. Weused 1 M NaOH instead of 0.1 M or 0.5 M NaOH and did notwork under N2 atmosphere. Ten grams of each sample weremixed with 190 mL of 1 M NaOH in a 250 mL centrifuging bot-tle, shaken over night on a horizontal shaker, and centrifugedat 25,900 g (this centrifugal force was always taken) for30 min. The supernatant was decanted and stored. This pro-cedure was repeated twice. Five milliliters of each solutionwere used for E4 : E6 measurements. Subsequently, 16 mL ofconc. HCl were added to the solution of the first extraction toprecipitate the HAs, centrifuged for 20 min, and the superna-tant discarded (5 mL were used for the measurements of theFA : HA ratio). The centrifuged HAs were dissolved in thesolution of the second extraction, and the procedure wasrepeated. The same approach was performed with the solu-tion of the third extraction. The precipitated HAs were trans-ferred with 5 mL of 1 M NaOH into a 30 mL centrifuging tube,refilled with H2O to approx. 29 mL and 0.4 mL of 37% HCl toretrieve the HAs via centrifuging for 20 min. The supernatantwas discarded. This procedure was repeated twice. In a nextstep, the HAs were dissolved in 1 mL of 1 M NaOH, refilled

with H2O to approx. 30 mL and precipitated with five drops of37% HCl. This solution was also centrifuged for 20 min andthe supernatant discarded. Finally, the HAs were washedwith 30 mL of H2O and three drops of 37% HCl. The HAswere transferred with a little amount of H2O into 50 mL plasticbottles and freeze-dried. The dried samples were stored in anexiccator. The extractions were carried out three times foreach sample.

2.3 Extraction of HAs with 0.1 M Na4P2O7

We used 0.1 M Na4P2O7 referring to García and Hernandez(1996) and Xing et al. (2005). Our method differed from bothdistinctly. Ten grams of each sample were mixed with 190 mLof 0.1 M Na4P2O7 in a 250 mL centrifuging bottle and shakenover night on a horizontal shaker. The remaining procedureof the extraction was performed in a similar way as describedfor 1 M NaOH. Instead of 16 mL, 6.4 mL of conc. HCl wereused to precipitate HAs.

The collected HAs were dissolved in 10 mL of 0.1 M Na4P2O7via shaking over night and transferred into 30 mL centrifugetubes with 3 × 5 mL of 0.1 M Na4P2O7; the precipitation was car-ried out with 0.9 mL of 37% HCl followed by centrifugation for20 min. We washed the HAs by dissolving them in 5 mL of 0.1 MNa4P2O7, refilling them to about 30 mL with water, and precipi-tating them with 0.5 mL of conc. HCl. After centrifugation for20 min, the supernatant was decanted and discarded. This pro-cedure was performed three times. In a further step, the HAswere dissolved in 1 mL of 0.1 M Na4P2O7, refilled to approx.30 mL, precipitated with 0.2 mL of 37% HCl, and centrifuged for20 min. The supernatant was decanted and discarded. Finally,the HAs were elutriated in water with three drops of conc. HCland centrifuged in the common manner. The HAs were trans-ferred with as less water as possible in 50 mL bottles andfreeze-dried. They were stored in an exiccator.

2.4 Extraction of HAs with 1 M Na2CO3

We are not aware of any published method of extractions ofHAs dealing with 1 M Na2CO3. However, a similar extractionreagent, NaHCO3, was mentioned by Ziechmann (1980). Theextraction was performed similar to the extraction with 0.1 MNa4P2O7. The precipitation was accomplished by 32–35 mLof conc. HCl and needed much patience because of a highformation of lather, which also caused slight losses of HAs.Therefore, the solutions were shared in two bottles each. Thewashing of the HAs was performed in a first step via combin-ing them into one bottle, which took about 30 mL of 1 MNa2CO3, refilling them up to 190 mL, and precipitating themwith 5 mL of conc. HCl. The second step was to transfer themin 30 mL tubes with about 5 mL of 1 M Na2CO3, refill the solu-tion to about 30 mL, and precipitate the HAs with 1 mL ofconc. HCl. The third step was the dissolution of the HAs with5 mL of 1 M Na2CO3 and their subsequent precipitation with1 mL of conc. HCl. Finally, the HAs were elutriated in a solu-tion of H2O with three drops of conc. HCl, centrifuged for20 min, and the supernatant decanted and discarded. Theextracted and washed HAs were freeze-dried and stored inan exiccator.

2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.plant-soil.com

J. Plant Nutr. Soil Sci. 2007, 170, 522–529 FTIR-spectroscopic characterization of humin fractions 523

2.4 Enrichment of the humin fraction

Seventy milliliters of H2O were added to 10 g of each bulk soilsample. Subsequently, 6 mL of conc. HCl were added. Wewaited approx. 4 h until the formation of CO2 was completelyfinished. The following addition of 1 mL of conc. HCl showedno further formation of CO2. This mixture was refilled withH2O to 200 mL, centrifuged for 30 min, and the supernatantdecanted. This procedure was repeated with 3 mL of conc.HCl to remove CaCl2.

To separate the OM from clay minerals and silicates, the car-bonate-free samples were shaken at room temperature in200 mL of 10% HF solution (10% HF was chosen in accor-dance to Rumpel et al. (1998) and Eusterhues et al. (2003))for 5 h on a horizontal shaker and subsequently centrifugedfor 30 min; the supernatant was decanted. This procedurewas repeated twice whereas the times of shaking were 15 hin case of the second and 24 h in case of the third time.

The carbonate-free and HF-treated samples were shaken in190 mL of a solution of both 0.1 M Na4P2O7 and 1 M NaOHon a horizontal shaker over night at room temperature. Thissolution was centrifuged for 30 min and the supernatant de-canted. This procedure was repeated three times. The lasttreatment was performed with the addition of 150 mL of asolution of both 0.1 M Na4P2O7 and 1 M NaOH and 40 mL ofwater. The remaining treatment was analogue to the proce-dures described before. After these treatments, mixed crys-tals of Na4P2O7 and NaOH were formed. To remove thesecrystals, 190 mL of water at a temperature of 50°C wereadded and shaken on the horizontal shaker over night. Thesolution was centrifuged for 30 min and the supernatant de-canted. This treatment was repeated once.

We observed gray-colored solutions and assumed that grayHAs (Gerzabek, 1991) were left. Therefore, we decided totreat the soil samples with separate solutions of NaOH andNa4P2O7. The samples were shaken on a horizontal shakerin 0.05 M Na4P2O7 at room temperature for 1 h, centrifugedfor 30 min, and the supernatant decanted. This treatment wasrepeated with 0.05 M NaOH and three times with 0.005 MNa4P2O7, whereas in case of the treatment with 0.005 MNa4P2O7, the first shaking time was 1 h, the second one 75 min,and the third time 210 min. We used 190 mL of H2O to wash theenriched humin fraction by shaking over night, centrifuging for30 min and decanted supernatants; this treatment was repeatedtwice. The obtained enriched humin fractions were transferredwith a little amount of H2O into 50 mL plastic bottles and freeze-dried. The samples were stored in an exiccator.

2.5 Measurement of the ash content of theenriched humin fraction

An amount of 200–300 mg of enriched humin fraction wastransferred into ashing crucibles and heated for 1 h at 600°C.This temperature was kept for 2 h. Fourier-transform infraredspectra of the ashes were measured to exclude any systema-tic errors in the band areas deriving from left over inorganicmaterial.

2.6 Measurement of organic C and total N

Organic-C and total N content of HAs and enriched huminswere measured after dry combustion at 1050°C using aCarlo-Erba Total Analyzer. In the case of the bulk soil, sam-ples were treated with 0.1 M HCl to get rid of carbonate C.

2.7 Measurements of E4 : E6 and the FA : HA ratios

The basic concept was taken from Gerzabek et al. (1993)and modified. One milliliter of the extract was mixed with5 mL of NaOH/glycine buffer (pH 10). The photometric meas-urements were performed with the extracts at 400 nm (E1)and 600 nm (E2) as well as with the precipitated extracts(without HAs) at 400 nm (E3). The photometric measure-ments were performed with an Agilent 8453 UV-visible spec-trometer.

2.8 Fourier-transform infrared spectroscopy

Our method was based on Haberhauer and Gerzabek(1999). Fourier-transform infrared analyses of soils werebased on KBr pellets, which were produced by finally mixingan exactly weighed amount (about 1 mg of enriched huminfraction, 2 mg of ashes of the humin fraction, and 0.5 mgextracted HAs) with an exactly weighed amount (about200 mg) of KBr (FTIR Grade) in a swinging mill. To increasethe accuracy of the sample content in the pellet, mixing ofsample with KBr was performed in two steps using a solidsolution of HA or enriched humin fraction in KBr (1/10, m/m)as an intermediate step. The final mixture was pressed toobtain a transparent pellet. The FTIR measurements wereperformed with a Perkin Elmer Paragon 500 Spectrometer inthe mid IR area from 400 to 4000 cm–1. The recording wasperformed with a resolution of 4 cm–1, weak apodization, and16 scans per sample. Each spectrum was corrected againstpure KBr and the ambient air as a background spectrum. Theband evaluations were performed with start and end basispoints and are consistent with the method of tangential baselines. The observed dimension of the units of the peak area isA cm–1 or A cm–1 mg–1 when normalizing the values on mgdried HA. The comparably small standard deviation showedthat this approach is acceptable.

2.9 Statistical evaluations

Statistics were performed using univariate ANOVA withScheffè test as post-hoc test. A probability level of a = 0.05was used to accept significance of differences.

3 Results

The yields of HA fractions obtained by 1M NaOH were signifi-cantly higher in the top layer compared to the lower ones(Tab. 1). Extracting with NaOH or Na2CO3 led to significantlylower yields than extracting with sodium pyrophosphate.Higher N contents resulted in lower C : N ratios of NaOH-extracted HAs, indicating a younger age of these fractions.The ratios of photometric absorptions of buffered extractionsolutions at 400 and 600 nm (E4 : E6) and FA : HA ratios


524 Tatzber, Stemmer, Spiegel, Katzlberger, Haberhauer, Mentler, Gerzabek J. Plant Nutr. Soil Sci. 2007, 170, 522–529

show distinct differences between the fractions obtained byNaOH extraction and the ones obtained by the other extrac-tion methods. Ratios of E4 : E6 of NaOH extracts are approxi-mately twice that of the other extracts, FA : HA ratios are upto 10-fold higher. This seems to be a consistent result asE4 : E6 ratios decrease with an increasing degree of polymeri-zation (Gerzabek, 1991). The comparatively low degree ofpolymerization suggests the younger age of the HAs ob-tained by NaOH extraction. Humic acid carbon obtained by

NaOH extraction strongly decreased in relation to the totalorganic C with increasing soil depth. On the other hand, the Ccontent of the enriched humin fraction slightly increases withdepth. This indicates a rather fast depletion of the NaOH-extracted HAs with depth. The humin fractions exhibit a rela-tive lower Corg content than the HAs. A reason for this unex-pected result could be an increased oxygen content due to in-creased degree of oxidation. However, humin fractions rep-resent about 50% of total soil Corg.


cm-1

1000200030004000

% T

40

60

80

100

120

140

160

0.1 M Na4P

2O

7

1 M Na2CO3

1 M NaOH

I

II

III

IV

V VI

VII

VIIIIX

X

XI

XII

XIII

XIVXV

XVI

XVII

XVIII

XIX

XX XXIFigure 1: FTIR spectra ofhumic substances extractedfrom the 0–10 cm layer of aminimum-tilled soil with differ-ent extraction procedures.Peak assignment according toTab. 3. For comparison pur-poses, %T values for Na2CO3and Na4P2O7 were increasedby adding 20% and 40%,respectively.

Table 1: Total amounts of organic carbon (Corg) and nitrogen (Nt) and C : N ratios in bulk soil and soil extracts and yields of soil extracts in threedepth layers of the minimum-tillage soil from the long-term field experiment (data are given as arithmetic means ± standard deviation; n = 3 forNaOH extract, n = 2 for humin extract, and n = 1 for Na4P2O7 and Na2CO3 extract). Data from NaOH extraction followed by different letters aresignificantly different (p ≤ 0.5).

Fraction* Soildepth[cm]

Bulk soilCorg[g kg–1]

Extracted FractionYield[g kg–1]

Photometrical resultsE4 : E6 FA : HA

Corg[g kg–1 [% of bulkfraction] soil Corg]

Nt[g kg–1

fraction]

C : N

HA(1 M NaOH)

0–10 19.6 ± 0.6 1.7 ± 0.1a 10.7 ± 0.3a 0.95 ± 0.07a 508 ± 25a 4.4 ± 0.2a 49 ± 8a 10.6 ± 1.3a10–20 15.1 ± 0.6 0.8 ± 0.0b 9.6 ± 0.7a 1.11 ± 0.16a 500 ± 19a 2.6 ± 0.1b 45 ± 1a 11.0 ± 0.4a20–30 15.5 ± 0.7 0.8 ± 0.1b 9.9 ± 0.1a 1.16 ± 0.12a 490 ± 17a 2.4 ± 0.3b 43 ± 3a 11.6 ± 1.3a

HA (0.1 MNa4P2O7)

0–10 19.6 ± 0.6 5.0 4.1 0.13 516 13.3 37 14.110–20 15.1 ± 0.6 4.1 4.0 0.13 532 14.5 37 14.220–30 15.5 ± 0.7 4.1 4.0 0.12 519 13.8 36 14.6

HA (1 MNa2CO3)

0–10 19.6 ± 0.6 1.8 5.0 0.27 457 4.3 31 14.610–20 15.1 ± 0.6 1.3 4.8 0.29 509 4.4 33 15.220–30 15.5 ± 0.7 1.4 4.7 0.31 467 4.3 30 15.6

Humins 0–10 19.6 ± 0.6 20.5 ± 0.4** – – 434 ± 31 45 ± 2 29 ± 2 15.1 ± 210–20 15.1 ± 0.6 19.4 ± 1.0** – – 401 ± 37 51 ± 6 27 ± 2 14.8 ± 220–30 15.5 ± 0.7 19.1 ± 0.2** -- – 417 ± 32 51 ± 5 35 ± 7 12.1 ± 3

* Humic acid fractions obtained by extraction with:** Values exclude ash contents (%): (58.3 ± 0.6) for 0–10 cm, (59.4 ± 0.7) for 10–20 cm, and (59.5 ± 0.1) for 20–30 cm.


Figure 1 shows FTIR spectra of HA fractions (from the0–10 cm layer) obtained by different extraction methods.Humic acids obtained by 1 M NaOH showed stronger alipha-tic and aromatic bands at 2850–3000cm–1 and 3030 cm–1

and a higher resolved fingerprint area (from 1600 to900 cm–1) than those extracted with Na4P2O7 or Na2CO3.Additionally, the absorption bands <1700 cm–1 show a differ-ent pattern for NaOH extracts compared to Na4P2O7 orNa2CO3 extracts. The band number VI appears as an at leastcommensurate band in case of extraction with NaOH,strongly reduced in case of Na4P2O7-extracted HAs, and as a

shoulder in case of Na2CO3-extracted HAs. Table 2 includesa list of the bands given in Fig. 1 (I–XXI) and their assign-ments to functional groups according to literature. It has to benoted that OH-deformation bands can appear from 1410 to1260 cm–1, two ester bands appear between 1330 and1050 cm–1, and fingerprint bands of aromats appear from1225 to 950 cm–1. It is possible, that all bands in the areafrom 1410 to 950 cm–1 could be influenced by these vibra-tions and, therefore, the assignment of these bands has to behandled with care.

Table 2: Band assignments of HA FTIR spectra.

Number Location [cm–1] AssignmentI 3700–2200 OH, small contributions might come from –NH3

+, –NH2+, –CO–NH2, –CO–NH–, S–H, and P–H b

II 3050 Aromatic groups a

III, IV 2920, 2850 Aliphatic groups a, the second part (= IV) also includes methoxy groups b

V 1700 Carbonylvibrationsofcarboxylgroups,aldehydesandketonesa,contributionsmightcomefromcarbonylgroupsofestersb

VI 1650 C=Oofamidegroups(amideIband)andC=Oofquinoneketonesa,contributions:nitrates(R–O–NO2),nitrites(R–O–NO)b

VII 1550 (shoulder) COO– , –C–NO2, and C=C b

VIII 1500 N–H, C=N (amide II band), and C=C a

IX 1450 Aliphatic C–H a

X 1420 C=N of primary amides (amide III band) a, contributions are possible from groups like COO– b

XI 1360 –CO–CH3 and possibly from nitrate b

XII 1315 Sulfonegroups(SO2); thiscouldbeoneof the twobands thatshouldappear fromesters inanareafrom1330–1050cm–1b

XIII 1270 (shoulder) Nitrates (R–O–NO2), =C–O–C groups, and possibly P=O vibrations b, C–O of phenolic groups c

XIV 1210 C–O and OH of COOH, C–O of aryl ethers and phenols a, possibly P–O–aryl and tertiary alcohols b

XV 1120 C–OH of aliphatic OH–groups a, sulfones b

XVI 1075 C–O of alcohols and C–O of aliphatic ethers b

XVII 1020 C–OofpolysaccharidesorsimilarsubstancesandSi–Oofsilicatesa,possiblecontributions:S=O,P–O–alkyl,or=C–O–Cb

XVIII 900 (shoulder) R2C=CH2 groups b

XIX 873–728 Aromatic C–H a, less substituted rings appear at less wave numbers b

XX 794 Benzene rings with 2 or 3 adjacent H and/or an isolated H, R2C=CRH groups, and nitrates b

XXI 766 sp3-CH2 and benzene rings with 4 and/or 5 adjacent H b

a Senesi et al. (2003)b Hesse (2005)c Francioso et al. (1998)

Band number

II III

IV V VI

VII

VII

I

IX X XI

XII

XII

I

XIV XV

XV

I

XV

II

XV

III

XIX XX

XX

I

A c

m-1

(m

g-1

HA

d.w

.)

0

2

4

6

8

10

0 - 10 cm

10 - 20 cm

20 - 30 cm

Figure 2: Band areas (absorbancecm–1

(mg HA d.w.)–1) within FTIR spectra ofHAs extracted with 1M NaOH from aminimum-tilled soil at different soil depths(peak assignment according to Tab. 3). n= 3, arithmetic means plus standarddeviation, values followed by differentsigns are significantly different (p ≤ 0.5).


Figure 2 illustrates evaluated bands of FTIR spectra of HAsextracted with NaOH based on HA dry weight. The assign-ments of the band numbers (BN) I–XXI are given in Tab. 2.The band areas (A cm–1 mg–1) of BN II, III, V, XI, and XVIIIincrease significantly from the uppermost to the deeperlayers. BN IV, close to BN III, shows only a significant differ-ence between the toplayer and the 20–30 cm layer. BN VI,VII, VIII, XIII, XIV, XVI, XVII, XIX, and XXI show no significantdifferences between the layers. BN IX, X, XII, XV, and XXdecrease significantly from the top- to the deeper layers.

Figure 3 presents the FTIR spectrum of the enriched huminfraction of the uppermost layer of a minimum-tilled soil. Theobserved spectra indicate that the most significant bandsappropriate for investigations are the aliphatic bands at 2920and 2850 cm–1 and possibly the aromatic band. The mea-surement of only two samples each inhibited making anANOVA for determining their significances. Therefore, onlytrends can be listed here. The bands at 1580 cm–1 and1360 cm–1 showed no trends. The other bands showed thefollowing trends: A decrease in case of the aliphates I bandwith increasing soil depth (all units in A cm–1 mg–1): 0.81 ±0.02 for 0–10 cm, 0.625 ± 0.005 for 10–20 cm, and 0.59 ±0.02 for 20–30 cm. Also a decrease of the aliphates II band:0.1835 ± 0.0002 for 0–10 cm, 0.145 ± 0.002 for 10–20 cm,and 0.141 ± 0.008 for 20–30 cm. In contrast, the aromaticband showed an increase with increasing soil depth: 0.130 ±0.002 for 0–10 cm, 0.14 ± 0.004 for 10–20 cm, and 0.145 ±0.011 for 20–30 cm.

4 Discussion

NaOH as extractant of humic substances for FTIR analysisyields better resolved IR spectra, especially in the importantregions of stretching vibrations including aromatic and alipha-

tic groups and in the fingerprint area including amides, ali-phats, and aromats. An additional question is, however, if thedifferences in the FTIR spectra of extracted HAs observed forthe three different extraction methods could be due to analteration of the extracted humic substances by the extractantitself, as some authors claim (Gerzabek, 1991; Head andZhou, 2000; Rosa et al., 2005). These processes shouldmainly affect the oxidized area at about 1700 cm–1, wherecarbonyl and carboxyl groups appear, but not the entire spec-trum. Furthermore, all extracted HAs should be influenced ina similar way. Our conclusion is, that NaOH extraction doesnot influence the HA characteristics concerning those spec-tral regions we used for further interpretations.

The NaOH extraction has lower extraction yields as compar-ed to Na4P2O7 and Na2CO3 and represents a different part ofthe SOM. These results suggest that 1 M NaOH at leastpartly leads to precipitation of gray HAs, which might explainthe lower yield in comparison to the other extractants. Thiswas also evident from the HA color, which was brown in caseof NaOH vs. gray-black in case of the other extractants. Thisis also reflected by smaller C : N ratios and higher E4 : E6 andFA : HA ratios as compared to the other extractants. A possi-ble reason for low amount of HA fractions obtained withNaOH extractions could be that the gray HAs are not solublein solutions with a high concentration of diluted electrolyte(Gerzabek, 1991). The brown HA fractions are soluble underthese conditions.

The evaluation of the FTIR band areas of HAs extracted withNaOH showed an increase of the aromatic and carbonylgroups and a decrease of amide groups with increasing soildepth. Aliphatic groups showed contradicting results: Thebands of the stretching vibrations increased, and the band ofthe bending vibrations decreased. We assume that band


cm-1

1000200030004000

% T

0

20

40

60

80

100

Aliphats I

Aliphatic C-H,C=Nof primary amides (amide III band),

COO-, phenolic OH and C-O

O-H stretching, N-H stretching

Aromatic C-H

Si-O ofsilicate impurities

Aliphats IIAromats

Background of inorganic material

Aromatic C=C, COO-, C=C (aromatic),

C=N (amide II band), C=O of COOH,

aldehydes and ketones Figure 3: FTIR spectrum of theenriched humin fraction from the0–10 cm layer of a minimum-tilledsoil. The band interpretations fol-low Xing et al. (2005) combinedwith Senesi et al. (2003). Forcomparison purposes, %T valuesfor the inorganic background wereincreased by adding 5%, respec-tively.


interactions in the bending vibrations were responsible forthat phenomenon based on the observed increase of alipha-tic groups with increasing soil depth. Spaccini et al. (2001) aswell as Capriel et al. (1995) used the bands around2920 cm–1 for their investigations. The reason for selectingthis band is that stretching vibrations are the more robustones. Our results suggest that the amount of aliphatic groupswithin brown HAs increased during decomposition. Theexplanation for that could be the formation of micelles of HAsas some authors suggest (Terashima et al., 2004; Sierra etal., 2005). The increase of aromatic and carboxylic propertiesof the HAs with depth is in line with studies from Haberhaueret al. (1998). However, it has to be mentioned that condensa-tions of amino carbonyls and aldols as well as hydrolysis ofester groups can lead to artifacts when groups with a distinctbehavior react quantitatively under build up of a new group.The involved bands would be BN V to VIII, BN X to XVIII, andBN XX. These processes should lead to a kind of alterationor enhancement of the significances of changes. On the otherhand, those reactions should influence the HAs of all soillayers in the same way. The problem seems to be the inse-curity of contributions from originally different functionalgroups. Using 1 M NaOH, aldol condensations should leadfrom carbonyl groups to groups like R2C=CR2 or R2C=CRH(Vollhardt and Schore, 1995). We observed a band withinthese groups, BN XX, showing a decrease. This suggeststhat these processes should not influence the results signifi-cantly. The increase of the bands deriving from aromaticstretching vibrations both for HAs and the enriched huminfraction with depth seems plausible as well.

The IR bands of the enriched humin fraction show anincrease of aromatic characteristics with depth. The aliphaticbands decrease with increasing soil depth. This phenomenonmight be caused by the fact that aliphatic moieties could becomparatively easily available for microorganisms—similar toamide groups. For larger molecules (like in the humin frac-tion), it might be more difficult to form micelles where aliphaticgroups could be protected.

The overall assessment of the three layers of the nontilledsoil leads to the conclusion that HAs extracted with 1 MNaOH represent a small and dynamic fraction indicated bythe measured yields together with values of Nt, C : N, E4 : E6,and FA : HA ratios. The humin fraction has a high contributionto the total organic carbon and represents—in opposite to theNaOH-extracted Has—a probably older and more stabilizedfraction of OM which still shows changes in its aromatic andaliphatic characteristics with soil depth.

Acknowledgments

This work was financed by the Austrian Science Foundation(FWF), project number P16667-B06. We are also grateful toDr. Andrea Watzinger and Dr. Sabine Klepsch for their help inso many situations as well as to Prof. Ingrid Kögel-Knabnerand Prof. Werner Mikenda for valuable discussion. We aremuch obliged to Dipl. Ing. Ewald Brauner, Ruth Pöll, andElisabeth Cuncl for performing the elemental analyses.

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FTIR-spectroscopic characterization of humic acids and humin fractions obtained by advanced NaOH, Na4P2O7, and Na2CO3 extraction procedures