Basic research conducted on humic acid (HA) in tropical soils is limited. A study was conducted to characterize HA

extracted from tropical peat by elemental and functional group analyses, infrared (IR) spectra and by direct

examination on HA using scanning electron microscopy (SEM). From the elemental analysis it was found that C

ranged from 40.26 - 53.26%, H 4.84 - 6.36%,038.39 - 51.62%, N 1.51- 2.64%, and P 0.02 - 0.06%. Studies of

functional groups indicated that the carboxyl group present in HA of tropical peat was about twice as high as that of

phenolic hydroxyl groups. From IR spectra, the HA had an aromatic character. Results of IR analysis also exhibited

consistently similar spectra, demonstrating that regardless of place of origin, the extracted humic compound had a

similar structure and was composed of the same functional groups. Electron microscopic examination of dried HA

showed a sub-angular blocky shape which varied in size. All extracted HA exhibited similar form

Balasundram, Siva Kumar

Manas, Abdul Rahman

Mohd Hanif, Ahmad Husni

Sani Babu, Shanti Devi

Universiti Putra Malaysia Institutional Repository

Pertanika J. Trop. Agric. Sci. 19(2/3): 189-196 (1996)ISSN: 0126-6128© Penerbit Universiti Pertanian MalaysiaPhysico-cheDlical Attributes of HUDlic Acid Extracted froDlTropical PectM.H.A HUSNI, SHANTI DEVI, ABD.RAHMAN MANAS l and K.B. SIVADepartment of Soil ScienceFaculty of AgricultureUniversiti Pertanian Malaysia43400 Serdang, Selangor, Malaysia1Department of ChemistryFaculty of Science and Environmental StudiesUniversiti Pertanian Malaysia43400 Serdang, Selangor, MalaysiaKeywords: humic acid, tropical peat, infrared spectraABSTRAKKaJian asas mengenai asid humik pada gambut tropika adalaiz terhad. Satu kaJian mengenai ciri-ciri asid humikyang diekstrak dari gambut tropika telah diJalankan dengan menganalisis elemen dan kumpulan berfungsi, spektruminframerah dan uJian terus pada asid humik dengan menggunakan mikroskop elektron imbasan. Berdasarkan kepadaanalisis elemen, Jumlah Cyang didapati adalah 40.26 - 53.26%, H 4.84 - 6.36%, 038.39 - 51.62%, N 1.51 -2.64%, dan P 0.02 - 0.06%. KaJian dalam kumpulan berfungsi menunJukkan kumpulan karboksil yang wuJuddalam asid humik di gambut tropika adalah dua kali ganda lebih banyak daripada kumpulan hidroksil fenolik.Daripada spektrum inframerah, asid humik didapati mempunyai ciri-ciri aromatik. Analisis inframerah JugamenunJukkan spektrum yang sama dan selaras tanpa mengira perbezaan tempat asalnya, kompaun humik yangdiekstrak mempunyai struktur dan komposisi kumpulan berfungsi yang sama. Pemeriksaan menerusi mikroskopelektron pada asid humik kering menunJukkan bentuk yang berblok dan separa berliku dengan saiz yang berbeza.Semua asid humik yang diekstrak menunJukkan bentuk yang sama.ABSTRACTBasic research conducted on humic acid (HA) in tropical soils is limited. A study was conducted to characterize HAextracted from tropical peat by elemental and functional group analyses, infrared (IR) spectra and by directexamination on HA using scanning electron microscopy (SEM). From the elemental analysis it was found that Crangedfrom 40.26 - 53.26%, H 4.84 - 6.36%,038.39 - 51.62%, N 1.51- 2.64%, and P 0.02 - 0.06%. Studies offunctional groups indicated that the carboxyl group present in HA of tropical peat was about twice as high as that ofphenolic hydroxyl groups. From IR spectra, the HA had an aromatic character. Results ofIR analysis also exhibitedconsistently similar spectra, demonstrating that regardless ofplace of origin, the extracted humic compound had asimilar structure and was composed of the same functional groups. Electron microscopic examination of dried HAshowed a sub-angular blocky shape which varied in size. All extracted HA exhibited similar form.INTRODUCTIONHumic acid (HA), defined as the fractionthat is soluble in dilute base and coagulatedupon acidification of the alkaline extract, isthe main component of humic fraction inboth temperate and tropical peat (Nor-hayati 1989; Garcia et ai. 1991). HA iscomprised of highly reactive polymericcompounds of varying molecular weight,containing C, H, 0, and N, and itsreactivity is influenced by the nature andamount of oxygen-containing functionalgroups (Stevenson and Goh 1972).According to Cheshire et ai. (1967) ,M.HA HUSNI, SHANTI OEVI, ABO.RAHMAN MANAS AND K.B. SIVApeat humic acids have 52% C, 5.1 % H,2.2% Nand 40.3% 0 content. A study byVisser (1987) on humic acids extractedfrom decomposing plant and peat coresamples showed content of 34-55% C, 5-11 % Hand 40-55% 0, different from thevalues of mineral soils 55-60% C, 3-5% H,2-6% Nand 30-40% 0 (Kononova 1961).HA extracted from Sphagnum and Carexpeat showed 49.7 and 55.4% C, 6.0 and5.0% H, 42.0 and 35.5% 0, 2.3 and 3.5% Ncontent, respectively (Garcia et at. 1991).IR spectra of HA extracted fromtemperate peat showed aromatic characterand great similarity among different sam-ples ofHA (Visser 1987; Garcia et at. 1991).Studies by Norhayati (1989) also indicatedthat the HA extracted from tropical peatexhibited very similar IR spectra.SEM provides a non-destructive meth-od for direct recording of the shape of tinyhumic particles (Bohr and Hughes 1971;Orlov and Glebova 1972). Ghosh andSchnitzer (1982) found under SEM, humicacids to consist of small discrete sphenoid,flattened aggregates, linear chain assem-blies of these aggregates and flattenedfilaments.There is a dearth of information on thestudy of elemental composition and reac-tivity of HA, and its structure under SEM.This study was therefore carried out tocharacterize the HA extracted from tropi-cal peat by elemental and functional groupanalyses, its IR spectra and its structure bySEM.MATERIALS AND METHODSFourteen samples from major peat areas ofMalaysia were used in this experiment,twelve from the west coast of PeninsularMalaysia and two from Sarawak (peatsamples 5 and 6). Percentage of organicmatter was determined as loss on ignitionfrom sample maintained at 500°C for 4 h(Storer 1984).Humic acid was extracted with 0.2 MN aOH using a soil to extractant ratio of1: 10. The mixture was shaken continuouslyon a mechanical shaker for 24 h and thesoluble organic matter was separated fromthe peat soil by centrifugation at 10,000rpm for 15 min. The residue was washedwith distilled water in a ratio of 1:5 andwashing water separated from the residueby centrifugation. The combined super-natants were acidified with concentratedH 2S04 to pH 1.0 and allowed to stand for24 h. The coagulated humic acid (HA)precipitate was purified by re-precipitationand re-dissolution using H 2S04 andN aOH, respectively. The alkaline extractwas then shaken for 48 h with excess HCl-HF mixture (0.5%, v/v). The acid mixturewas removed by centrifugation and theresidue thoroughly washed with distilledwater until the filtrate became free ofchloride. The humic acid was then dia-lyzed against distilled water until there wasno colour change in the outer solution(distilled water) and finally frozen anddried at ambient temperature (Schnitzer1982; Norhayati and Verloo 1984).Carbon, Hand N were determined bydry combustion using a CHN analyser(VARIO-EL), 0 was calculated by differ-ence and P by dry ashing at 500°C anddetermined colorimetrically at 882 nm(Murphy and Riley 1962). Carboxylgroups were measured by Ca(OAch meth-od (Schnitzer and Gupta 1965) and thephenolic hydroxyl groups by the colori-metric method using Folin Cicocalteu'sreagent (Tsutsuki and Kuwatsuka 1978).These determinations were represented asthe average result of two duplicate samples.IR spectra were recorded on a Beck-man Fourier transfer IR spectrophotometerusing the KBr pellet technique. Prior to theanalyses, the HA with the KBr was storedfor at least 48 h in a desiccator to preventabsorption of atmospheric moisture.190 PERTANlKA J. TROP. AGRIC. SCI. VOL. 19 NO. 2/3, 1996PHYSICO-CHEMICAL ATTRIBUTES OF HUMIC ACID EXTRACTED FROM TROPICAL PECTFor SEM examination, the dried HAobtained directly after extraction wasattached to AI-stubs with the aid ofcolloidal silver paint. To make the speci-mens surface conducting, they were coatedwith a layer of gold-palladium. Theprepared specimen were examined on aSEM aOEL 35C) operated at 25kV.RESULTS AND DISCUSSIONThe organic matter contents per 100 g peatof the 14 peat samples studied ranged from44 - 97% (Table 1). Percentage of HAobtained ranged from 8.30 - 39.52/100 gpeat.The data on the elemental compositionof HA in terms of percentage weight arepresented in Table 2. Carbon contentranged from 40.26 - 53.26%, H 4.84 -6.3%, 0 38.39 - 51.62%, N 1.51 - 2.64%and P 0.02 - 0.06% with means of 49.34%C, 5.64% H, 43.05% 0, 1.99% Nand0.03% P. Comparing these with theelementary composition of HA reportedby Visser (1987) and Garcia et at. (1991)indicated that our results fell within thereported range for temperate peat.The analysis of the major oxygen-containing functional groups is shown inTable 3. The carboxyl group of HAextracted from tropical peat ranges from2.50 - 3.10 meq/g of HA while phenolichydroxyl groups range from 0.99 - 1. 75meq/g of HA. Mean values of functionalgroups showed 2.84 meq COOH and 1.36meq phenolic hydroxyl/g of HA. Thecarboxyl groups present in HA are morethan twice as numerous as in the phenolichydroxyl groups, implying that the pH-dependent charge in peat is controlled bycarboxyl groups.IR spectra and the absorption bands ofHA extracted from peat are shown in Fig. 1and Table 4, respectively. The broadabsorption band at 3550-3200 cm-1 regioncan be attributed to H-bonded OH groups,while peaks between 2920-2860 cm-1 aredue to aliphatic C-H stretch. According toTABLE 1Classification and yield of HA from peatPeatSample 1C1assification Depth (cm)2 O.M.3 HAo. (%) (%)1 A 0-15 44 21.232 A 0-15 82 26.883 A 0-15 86 18.304 B 15-30 88 25.785 A 0-15 90 23.786 B 15-30 94 8.407 A 0-15 97 35.678 A 0-15 97 20.689 B 0-15 76 23.4510 A 0-20 94 36.5211 A 20-40 97 12.3812 B 0-15 89 19.8213 B 20-40 97 8.3014 A 0-15 96 26.921 A Typic Tropohemists, B Typic Tropofibrists2 Percentage of organic matter3 % Humic acid (HA) per 100 g peatPERTANIKA J. TROP. AGRIC. SCI. VOL. 19 NO. 2/3, 1996 191192M.H.A HUSNI, SHANTI DEVI, ABD.RAHMAN MANAS AND K.B. SIVATABLE 2Elemental analysis of HA extracted from tropical peatPeat Elements (%)SampleNo. C H 0 N PI 51.93 5.14 40.52 2.37 0.062 52.01 5.62 39.88 2.45 0.043 45.75 6.36 45.26 2.59 0.054 51.25 5.23 41.33 2.16 0.045 53.39 5.59 38.34 2.64 0.056 44.39 6.23 47.72 1.63 0.037 51.04 5.22 41.98 1.74 0.028 47.80 5.77 44.68 1. 73 0.029 49.76 4.84 43.88 1.51 0.0210 50.52 5.14 42.14 2.18 0.0311 49.07 5.78 43.48 1.65 0.0212 50.00 5.67 42.16 1. 71 0.0213 53.56 5.91 38.88 1.64 0.0214 40.26 6.32 51.60 1.84 0.02Range 40.26- 4.84- 38.39- 1.51- 0.02-53.56 6.36 51.62 2.64 0.06Mean 49.34 5.64 43.05 1.99 0.03TABLE 3Analysis of oxygen-containing functional groups of HA extractedfrom tropical peatPeat Functional groups (meqjg of HA)SampleNo. COOH PhenolOH1 2.90 1.362 2.75 1.073 2.95 1.124 2.75 1.395 2.80 0.996 3.10 1.567 2.50 1.228 2.80 1.449 2.90 1.4010 3.00 1.7511 3.00 1.4312 2.85 1.5413 2.95 1.5914 2.50 1.22Range 2.50-3.10 0.99-1. 75Mean 2.84 1.36PERTANIKA J. TROP. AGRIC. SCI. VOL. 19 NO. 2/3, 1996PHYSICO-CHEMICAL ATTRIBUTES OF HUMIC ACID EXTRACTED FROM TROPICAL PECTTABLE 4Absorption bands of infrared spectra of HA from tropical peatBands 3550- 2920- 1725- 1690- 1550- 1470- 1280-(cm- 1)* 3200 2860 1705 1600 1500 1430 1200PeatSampleNo.Relative intensity1# +s m m a w w2 m m a w w3 m w a w w4 m m a w w5 m m a w w6 m m a w w7 m w w w w8 m m w w w9 m m w w w10 m w w w w11 m m w w w12 m m w w w13 m m w w w13 m m w w w14 m m w w w* 3550-3200 (hydrogen-bonded OH); 2920-2860 (aliphatic C-H stretch); 1725-1705 (C = 0ofCOOH and ketones); 1690-1600 (C=O stretch of quinones, C=O and/or C=O ofHconjugated ketones); 1550-1500 (aromatic ring stretch, P- and 0- distributed); 1470-1430(aromatic ring stretch, O-distributed); 1280-1200 (C-O stretch, and deformation ofCOOH,CoO stretch of aryl ethers)+ (s) strong; (m) medium; (w) weak; (a) absent# Sample number for HA extracted from respective peat soilBellamy (1969), bands in the region of3000-2500 cm-1 were produced by mostcarboxylic acids and arose from vibration ofintermolecular OR...O band. Therefore,Schnitzer and Griffith (1975) indicatedthat the O-H stretch « 2800 em-I) was atest for the occurrence ofH-bonded GOOHgroups. However, in this study only amediurn peak occurred at this regionsimilar to the results of Norhayati (1989).Norhayati (1989) showed that higheramounts of carboxyl groups were present inHA molecules. Similar results were ob-tained in this study, where most of the RAspectrum exhibited a medium peak in the1725-1705 cm-1 region (band for carbonylgroup) with strong peaks in the 1690-1600cm-1region (band for carbonyl group).The spectra also showed weak bands at1550-1500 cm- 1 and 1470-1430 em-I.Similar results were obtained by Garcia etai. (1991), where the above bands meantfor HA extracted with alkali disappearedfor HA extracted with pyrophosphatereagents in both Sphagnum and Garexpeat. This indicated that HA extractedwith alkali extractant exhibited strongeraromatic characteristics than that usingpyrophosphate reagent.A weak band obtained for all samplesin the 1289-1200 cm-1 region is attributedto OH, from GOOH deformation. OtherPERTANIKA J. TROP. AGRIC. SCI. VOL. 19 NO. 2/3, 1996 193M.H.A HUSNI, SHANTI DEVI, ABD.RAHMAN MANAS AND K.B. SIVAHA ofSoil123454000 3000 2000 1600 1200 800 400cm-1Fig. 1. Infrared spectra of HA extracted fromtropical peatbands, such as 1440, 1125 and 1034-1080cm- l were also observed. However, all thesebands were of weak intensity, and wereabsent in some samples (Fig. 1). Bandsoccurring at 1440 cm- l indicated the pre-sence of the carboxyl anion (Schnitzer 'andGriffin 1975); 1125 and 1034-1080 cm- lindicated aromatic ring P- and 0- disub-stituted, c-o-o stretch of aryl ethers andC-O stretch, aromatic ether, hydratedpolyols and carbohydrates, respectively(Garcia et al. 1991). Thus, this studydemonstrates that HA of tropical peat hasa greater amount of carboxyl groups.The IR spectra of the HA extractedfrom peat ofdiverse origin displayed similarbands and indicated the presence of similarstructures, diff~ring mainly in the numberof functional groups. The results corre-sponded with those of Norhayati (1989).The shape and particle arrangement ofHA under SEM are shown in Plate 1. TheHA materials are sub-angular blocky, varyin size, and are irregular in shape (Plate 1a).Plate 1b shows the plate-like structure ofHAsurface upon higher magnification (1 ~m).Plate 1. Scanning electron micrographs of shape andparticle size humic acid)' a) top: sub-angular blocky whichvary in size and shape) b) bottom: plate like structure ofHA surface.194 PERTANlKA J. TROP. AGRIC. SCI. VOL. 19 NO. 2/3, 1996PHYSICO-CHEMICAL ATIRIBUTES OF HUMIC ACID EXTRACTED FROM TROPICAL PEeTCONCLUSIONThe elemental analysis of HA showed Cranged from 40.26 - 53.26%, H 4.84 - 6.36%,o 38.39 - 51.62%, N 1.51 - 2.64% and P0.02 - 0.06%. Studies of the functionalgroups indicated that carboxyl groupspresent in HA of tropical peat are abouttwo times more numerous than phenolichydroxyl groups. From IR spectra, the HAobtained is of aromatic character. The IRanalyses also exhibited consistently similarspectra, demonstrating the possibility thatregardless of sample site, the extracted HAhas a similar structure and is composed ofthe same functional groups. SEM examina-tion of dried HA showed sub-angularblocky shape of varying size was similarfor all the HA extracted. These resultssupport those obtained by Norhayati(1989) for Malaysian peat, and demon-strate the similarity between tropical andtemperate peats regardless of plant originor climate.ACKNOWLEDGEMENTThe authors wish to thank the NationalCouncil for Scientific Research and Devel-opment, Malaysia, Program 1-07-05-047for financial support, and Mr. Mohd FuziMohd. Sharifof Soil Fertility Section for histechnical assistance.REFERENCESBELLAMY, LJ. 1969. Advances in Infrared GroupFrequencies. London: Methuen.BOHR, B.F. and R.E. HUGHES. 1971. Scanningelectron microscopy of clays and clay mine-rals. Clays and Clay Minerals 19: 49-53.CHESHIRE, M.V., P.A. CRANWELL, C.P.FALSHAW, A.]. FLOYD and R.D.HOWARTH. 1967. Humic acid. Tetrahedron23: 1669-1682.GARCIA, D., j. CEGARRA. M.P. BERNAL and A.NAVARRO. 1991. Comparative evaluation ofmethods employing alkali and sodium pyr-ophosphate to extract humic substances frompeat. Commun. Soil Sci. Plant Anal. 24(13/14):1481-1494.GHOSH, K. and M. SCHNITZER. 1975. Analyticalcharacteristics of humic acid and fulvic acidsextracted from tropical volcanic soils. Soil Sci.Soc. Am. Proc. 39: 1961-867.KO ONOVA, M.M. 1961. Soil Organic Matter. NewYork: Pergamon Press.MURPHY, J. and PJ. RILEY. 1962. A modifiedsingle solution method for the determinationof phosphate in natural waters. Anal. Chem.Acta 27: 31-36.NORHAYATI, M. 1989. Composition of organicmaterials of peat soils in Peninsular Malaysia.p. 81-87. In Recent Development in SoilGenesis and Classification. ]. Rubber InstituteResearch Malaysia 32(1): 40-49.NORHAYATI, M. and M. VERLOO, 1984. Cha-racterization of organic matter in four soils ofPeninsular Malaysia. II. Physical and chemi-cal analysis of humic substances. ]. RubberResearch Institute Malaysia 32(1): 40-49.ORLOV, D.S. and G.I. GLEBOVA. 1972. Electron-microscopic investigations of humic acids.Agrochemistry 7: 131-136 [Russian].SCHNITZER, M. 1982. Organic matter character-ization p. 581-594. In Methods of Soil Ana!Jsis.ed. A.A. Page et al. Part 2, 2nd edn.Agronomy Monograph 9. Madison, WI:ASA.SCHNITZER, M. and S.M. GRIFFITH. 1975.ovel methods for estimating hydrogen-bonded C02H groups in humic substances.Can. ]. Soil Sci. 55: 491-493.SCHNITZER, M. and D.C. GUPTA. 1965. Deter-mination of acidity in soil organic matter. SoilSci. Soc. Am. Proc. 29: 274-277.STEVENSON, F.j. and K.M. GOH. 1972. Infraredspectra of humic and fulvic acids and theirmethylated derivatives. Evidence for nonspe-cificity of analytical methods for oxygen-containing functional groups. Soil Sci. 113(5):334-345.STORER, D.A. 1984. A simple high samplevolume ashing procedure for determinationof soil organic matter. Commun. Soil Sci. PlantAnal. 15(7): 759-772.TSUTSUKI, K. and S. KUWATSUKA. 1978. Che-mical studies on soil humic acids. II. Com-position of oxygen-containing functionalgroups of humic acids. Soil Sci. Plant Nutr.24: 547-560.PERTANIKA J. TROP. AGRIC. SCI. VOL. 19 NO. 2/3, 1996 195M.H.A HUSNI, SHANTI OEVI, ABO.RAHMAN MANAS AND K.B. SlYAVISSER, S.A. 1987. Crystal formation by lowmolecular weight fulvic avid and humic acids.Sci. Total Environment 62: 129-138.(Received 1 August 1996)(Accepted 31 January 1997)196 PERTANIKA J. TROP. AGRIC. SCI. VOL. 19 NO. 2/3, 1996

Physico-chemical attributes of humic acid extracted from

tropical pect

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Physico-chemical attributes of humic acid extracted from tropical pect

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