analytical chemistry of freshwater humic substances

20
Analytica Chimica Acta 527 (2004) 105–124 Review Analytical chemistry of freshwater humic substances Suzanne McDonald, Andrea G. Bishop, Paul D. Prenzler, Kevin Robards School of Science and Technology, Faculty of Science and Agriculture, Charles Sturt University, Locked Bag 588, Wagga Wagga 2678, NSW, Australia Received 28 May 2004; received in revised form 6 October 2004; accepted 6 October 2004 Abstract Dissolved organic carbon (DOC) in aquatic environments represents one of the largest active organic carbon reservoirs in the biosphere. Current ideologies concerning the sources of DOC, how it is formed and utilized, and what determines the quality of DOC are examined. Humic substances can comprise a significant fraction of the DOC and developments in methods of analysis including the isolation and characterization of this fraction are reviewed. © 2004 Elsevier B.V. All rights reserved. Keywords: Dissolved organic carbon; Freshwater humic substance; Aquatic environment Contents 1. Introduction ........................................................................................................ 106 2. Sources, quality and bioavailability of DOC ........................................................................... 107 3. Formation of humic substances ...................................................................................... 109 3.1. Humic substances formed from lignin .......................................................................... 109 3.2. Humic substances formed by polymerization/condensation reactions .............................................. 110 4. Composition and structure of humic substances ........................................................................ 110 5. Methods of analysis ................................................................................................. 113 5.1. Isolation of humic substances .................................................................................. 113 5.2. Characterization of humic substances ........................................................................... 115 5.2.1. Size exclusion chromatography ........................................................................ 115 5.2.2. Nuclear magnetic resonance spectroscopy .............................................................. 115 5.2.3. Vibrational spectroscopy .............................................................................. 116 5.2.4. Differential scanning calorimetry ...................................................................... 117 5.2.5. Pyrolysis and related techniques ....................................................................... 117 5.2.6. Capillary electrophoresis .............................................................................. 118 5.2.7. Mass spectrometry techniques ......................................................................... 118 5.2.8. Fluorescence spectroscopy ............................................................................ 118 5.2.9. Field flow fractionation ............................................................................... 119 5.2.10. Microscopic techniques .............................................................................. 119 Corresponding author. Tel.: +61 2 6933 2539; fax: +61 2 6933 2737. E-mail address: [email protected] (K. Robards). 0003-2670/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2004.10.011

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  • Analytica Chimica Acta 527 (2004) 105124

    Review

    Analytical chemistry of freshwater humic substancesl D. Prenzler, Kevin Robards

    School of Science and Technology, Faculty of Science and Agriculture, Charles Sturt University, Locked Bag 588, Wagga Wagga 2678, NSW, AustraliaReceived 28 May 2004; received in revised form 6 October 2004; accepted 6 October 2004

    Abstract

    Dissolved organic carbon (DOC) in aquatic environments represents one of the largest active organic carbon reservoirs in the biosphere.Current ideologies concerning the sources of DOC, how it is formed and utilized, and what determines the quality of DOC are examined.Humic substances can comprise a significant fraction of the DOC and developments in methods of analysis including the isolation andcharacterization of this fraction are reviewed. 2004 Elsevier B.V. All rights reserved.

    Keywords: Dissolved organic carbon; Freshwater humic substance; Aquatic environment

    Contents

    1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1062. Sources, quality and bioavailability of DOC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1073. Formation of humic substances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109

    3.1. Humic substances formed from lignin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1093.2. Humic substances formed by polymerization/condensation reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110

    4. Composition and structure of humic substances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1105. Meth

    5.1.5.2.

    CorrespoE-mail a

    0003-2670/$doi:10.1016/jods of analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113Isolation of humic substances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113Characterization of humic substances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1155.2.1. Size exclusion chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1155.2.2. Nuclear magnetic resonance spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1155.2.3. Vibrational spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1165.2.4. Differential scanning calorimetry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1175.2.5. Pyrolysis and related techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1175.2.6. Capillary electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1185.2.7. Mass spectrometry techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1185.2.8. Fluorescence spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1185.2.9. Field flow fractionation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1195.2.10. Microscopic techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

    nding author. Tel.: +61 2 6933 2539; fax: +61 2 6933 2737.ddress: [email protected] (K. Robards).

    see front matter 2004 Elsevier B.V. All rights reserved..aca.2004.10.011Suzanne McDonald, Andrea G. Bishop, Pau

  • 106 S. McDonald et al. / Analytica Chimica Acta 527 (2004) 105124

    5.3. Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1205.3.1. Colour . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1205.3.2. UVvis absorption ratios . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

    . . . . . .

    6. Summa . . . . . .References . . . . . . .

    1. Introdu

    The amered to bethe biotic fdissolved frole in eneaquatic envganic carboof DOC inof CO2 car[6,7], affecganisms, ometal chela

    When reter, the termused interctween the twhen measas DOM [8samples wthe resultsbetween ththat DOMgeneral disrates the acthe carbon

    DOC ismatter thattion of DOCconductedmaking qua0.45m pocome undeof colloidaand rejectierational dorganic maoperationalticulate orgparticulatea compositing two pabiomoleculhydrates, presins [11]being a cat

    s orgaing yeefractnsistine rang14].umic sonentand h

    c subitionsare of

    ion, wDa inFulviolublerate mDa infractile in wmic suhemisic mahen reht is oas hubstanced. Ominedve bec subsa moleumicts, beial protal orgin wa

    n thee div

    pendety, an5.3.3. Chemiluminescence spectroscopy . . . . . . . . . . . . . . . . . . . . .ry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    ction

    ount and origin of organic matter can be consid-one of the most important factors in determiningunctioning of a riverine ecosystem [1,2], with theraction of organic matter (DOM) having a keyrgy flow [3]. Dissolved organic carbon (DOC) inironments represents one of the largest active or-n reservoirs in the biosphere [4], with the amountthe oceans alone being comparable to the amountbon in the atmosphere [5]. It is a vital resourceting food webs either directly, by uptake from or-r indirectly by mechanisms such as turbidity, pH,tion, and transport of contaminants.ferring to the dissolved fraction of organic mat-s DOM and DOC are applied, and are often

    hangeably [8] although there is a distinction be-wo terms. The dissolved fraction of organic matterured by chemical oxidation methods is referred to]. Many studies now rely on the combustion of

    ith the use of an automated carbon analyser, andare therefore reported as DOC [8]. Conversionse two measurements are conducted by assumingis 4550% organic carbon by mass [8]. As such,cussion about dissolved organic matter incorpo-ronym DOM while reference to specific results orcontent refers to DOC [9].operationally defined as the fraction of organicpasses through a 0.45m filter. While this defini-

    has been adopted and widely used, many studiesin the 1980s and early 1990s used other filter sizes,ntitative comparisons difficult [9]. The use of there size is one of convenience, and it has recentlyr criticism, as being inadequate for the removall species, and a compromise between flow rateon of clay minerals [10]. Nevertheless, the op-efinition of DOC has remained. Other forms oftter present in riverine environments may also bely defined by particle size, including coarse par-anic matter (CPOM, >1 mm in diameter) and fineorganic matter (FPOM,

  • S. McDonald et al. / Analytica Chimica Acta 527 (2004) 105124 107

    while allochthonous sources may include leachate from sur-rounding soils, grasses and inputs from riparian trees [21].This review examines analytical techniques for the isolation,characterizfraction ofing the souwhat determhumic fracalytical measpects of t

    2. Sources

    A numbpredict theand energyand limitatThe Riverdescribe thof communthis modelties along tresponse tovertebrate cof food soare stronglduces autolarge amou[22]. As thtrial organiproductionbecome incundergoesported dowreference tit has alsoecosystemsinconclusiv

    The nutity conceptnutrient spflow continbiological ppose, nutrieuse by organutrient avthe effects ogitudinal cpatterns anaxis depenriver chara

    In thesecontinuummade to inimportance

    by the development of the Flood Pulse Concept (FPC) [33].Floodplain-river interactions are important for exchangingparticulate and dissolved organic matter, with suspended

    entsoodplass arthat r

    ynamirge troble. Hatial as of othl (RPnd dirsent thmoden andropor

    m, becilatedbstanve co

    llochtn detethe qunsidetity ofine env

    carboic carConc1 tother hthe litedivid

    l qualfract

    actoryant too app

    ganicrties oy [44,istics iat havical prlthouging tho its rs [7].e bio

    ce of ie variwith o

    ost obolismation and quantification of the humic substancesDOC in freshwaters. Current ideologies concern-rces of DOC, how it is formed and utilized, andines the quality of DOC, with an emphasis on the

    tion are also examined. An appreciation of the an-thodology depends on an understanding of thesehe chemistry of humic substances.

    , quality and bioavailability of DOC

    er of models [2238] have been developed tointeractions between sources of organic matterflow through riverine ecosystems. The purposes

    ions of these models are summarized in Table 1.Continuum Concept (RCC) [22] was proposed toe predictable changes in structure and functionities longitudinally along a river continuum. In

    , the structure and function of biotic communi-he river continuum were predicted to develop inthe physical environment, with shifts in macroin-ommunities occurring in response to the location

    urces with stream size [22]. Headwater streamsy influenced by the riparian vegetation, which re-trophic production by shading, and contributesnts of allochthonous detritus, in the form of CPOMe stream size increases, the importance of terres-c inputs decreases, while autochthonous primaryand the transport of organic matter from upstreamreasingly important. The CPOM from upstream

    biological processing to form FPOM that is trans-nstream. The RCC was developed specifically ino natural, unperturbed stream ecosystems. Whilebeen hypothesized to be relevant to many altered

    [22], the results of numerous studies have beene [20,2326].

    rient spiralling concept and the serial discontinu-(SDC) are corollaries to the RCC [22,27]. The

    iralling concept [28] states that resources do notuously downstream, but are stored periodically inackages [27]. As the biological packages decom-nts are released and transported downstream fornisms, giving spatial and temporal components toailability in rivers [27]. The SDC [30] addressesf dams on rivers [31] as a discontinuity in the lon-

    ontinuum, which can shift the biotic and abioticd processes either up or down the stream-orderding on the dams location, purpose of operation,cteristics or the number of dams in a series.models, only the longitudinal changes in a riverare considered. An expansion of the SDC wasclude lateral and vertical connectivity [31]. Theof lateral connectivity was further emphasized

    sedimthe flbiomposesthe don ladictaof sprivermodetion arepreThiscarboare pstreaassim

    Surelatisus a

    Whetem,be coquanriverganicorganC/L.fromthe oto inoftentionalabilea refrresist

    Twof orpropeabilitacteries thphys[9] astanddue tment

    Thquenof thplexthat mmetaand nutrients being transported from the river toain, while organic detritus, algal and microbiale returned to the river channel [34]. The FPC pro-egular pulses of river discharge are a key factor incs of a river-floodplain system. The FPC is basedpical rivers where variations are said to be pre-

    owever, the concept understates the significancend temporal flow variability and predictability iner climatic regions [35]. The riverine productivity

    M) [38] hypothesized that autochthonous produc-ect organic inputs from the riparian zone togethere principal source of carbon driving food webs.

    l is based on large rivers in which autochthonousdirect allochthonous inputs from the riparian zonetionately more important than carbon from up-ause they are relatively labile and thus more easilyby heterotrophs.

    tial research has been conducted to determine thentribution and importance of autochtonous ver-honous inputs to various ecosystems [24,3941].rmining the value of a carbon source to an ecosys-ality and quantity of organic matter inputs must

    red. The question of what defines the quality andDOC is important in understanding its role in aironment. Quantity is a measure of how much or-n is present. It is typically measured as dissolvedbon, with measurements being expressed as mg

    entrations of DOC in natural fresh waters range60 mg C/L but are commonly 15 mg C/L [34]. Onand, quality [42,43] although frequently referredrature is much harder to define. Researchers have

    ed DOC into two components based on its nutri-ity [42] and importance to the food web [20] as aion that is available for uptake by organisms and

    or recalcitrant component that is generally morebiological attack [43].roaches have been used to determine the quality

    matter; direct measurement of selected chemicalf the DOC, or comparisons between its bioavail-

    45]. Defining quality in terms of the chemical char-s complicated by its vast heterogeneity. Most stud-e involved the determination of the chemical andoperties of DOC have been based on DOC in soilsh more attention is now directed towards under-e dynamics of DOC in riverine systems [4650]ole as a major energy source in aquatic environ-

    availability of DOC [51] is likely to be a conse-ts source(s), chemical composition, arrangementous functional groups, and the ability to com-ther chemicals. Traditionally, it has been acceptedf the turnover of DOC was accomplished via theof a small pool of labile, low molecular weight

  • 108 S. McDonald et al. / Analytica Chimica Acta 527 (2004) 105124

    (LMW), and structurally simple compounds within the DOC[18,34], despite the fact that a significant fraction of DOCis composed of high molecular weight (HMW) compounds[52]. There is increasing evidence, however, to suggest thatthe HMW compounds are also utilized [4,53]. In one study,the reactive pool of HMW DOC was in fact larger than thereactive pool of LMW DOC [4].

    The physical size of DOC is also an important factor in-fluencing microbial utilization, and attempts have been madeto classify DOC along a continuum of decreased biologi-cal reactivity [54]. The size continuum model [4] has beenused to describe the relationship between the size of organicmatter and its bioreactivity, relating the increasing reactiv-ity with decreasing size (from large, or HMW to small, orLMW) of organic matter and diagenetic state (from fresh toold). Riverine environments can be considered to be tran-sition zones typically dominated by diagenetically youngerorganic matter (

  • S. McDonald et al. / Analytica Chimica Acta 527 (2004) 105124 109

    time is necessary for organic matter to be considered refrac-tory. Residence times of centuries to millennia [64], monthsand years [65], weeks to months [66], or even days to months[67] have bfractory wdeciding otion on varthe heteroghas probaband one casame techncompoundsquantity ofbeen broadthe chemicderstandingin the envir

    3. Format

    Humic sof plant misms [64].to producemificationstabilizatio[72]. Tradistances inin soils, awas the adinto streamregarded awith fulvicing a largeboxylic anhumic acidmore aliphshown thatnature andaromaticitystances in sstances inhave someies, humicerated by acompounds[65].

    Despiteformation oHumic subnified tissuof simplering the degmay occurlignin in pl

    tion of plant biomass on the earths surface and are thereforethought to be likely sources of precursor material for humusformation [74].

    Humic

    e struion ofplant

    e planl alcoyl alcaturit

    predconifely equne unereals

    ropane[72].ant ster oficrob

    e themodelrtiallyxyl g

    ns proationd mo

    modelwhicholympeaksthe a

    e thishum

    ing eve inclance.accou

    propogh the

    46O10C52H

    er mobial a

    es maoxidi

    en confurtheotherhile tibe oneen considered. Uniformity in the definition of re-ould be useful when comparing studies, howevern appropriate time intervals or basing the defini-ious chemical properties may be difficult due toeneity of the substance. This lack of specificityly hindered efforts at generalizing across systems,nnot be sure that two investigators employing theique are actually working with the same group of

    [68]. Although considerable data exists on theDOC in rivers, only about one quarter of it has

    ly chemically characterized [56]. Understandingal structure of humic substances is important in un-

    the modes of interactions with other substancesonment [69].

    ion of humic substances

    ubstances are formed by the organic breakdownaterials [70,71] and the decay of dead organ-The process of degradation and mineralisationhumic substances is termed humification. Hu-

    has also been defined as being the prolongedn of organic substances against biodegradationtionally, it has been assumed that humic sub-river and lake waters were the same as those

    nd that the major source of humic substancesjacent soil, which was leached or eroded directlys during rainfall events [15]. Lignin has been

    s the primary source of soil humic substances,and humic acids originating from soil contain-

    proportion of aromatic (in particular benzenecar-d phenolic) compounds, whereas the fulvic ands from sediment and the aquagenic type wereatic [6,64]. Recent studies [15], however, havesoil humic substances are primarily aliphatic inthat lignin is probably not the major source ofin soil humic substances. In fact, humic sub-

    treams have different characteristics to humic sub-soils or the ocean, and marine humic substancesdefinite aromatic character [15]. In recent stud-substances, particularly fulvic acids, were gen-lgae and contributed to the multitude of diversethat comprised the dissolved humic substances

    intensive research, the reactions involved in thef humic substances remain largely unknown [73].stances are theorized to form directly from lig-es of plant material, or from the condensationlow molecular weight products generated dur-

    radation of macromolecules [74]. These processessimultaneously and randomly [75]. Cellulose andant structural material account for the largest por-

    3.1.

    Thpositsame

    in thnamysinapthe mligninfrommatepropaand ccyl punits

    Plnumband mscribfirstis pacarboactiomentsecon

    tionnolsand picantfromWhility increas

    or thsubst

    To[80]throu

    C52H

    Furthmicrostanctiallyoxygreactwith

    Wdescrsubstances formed from lignin

    cture of lignin is irregular (Fig. 1), with the com-lignin in different cells and cell complexes in thenot necessarily being uniform. Lignin is formedt by enzymatic dehydrogenation of various cin-hols, p-coumaryl alcohol, coniferyl alcohol andohol [76]. The variations of lignin will depend ony of the plant, but generally, gymnosperms containominantly guaiacyl propane monomers derivedryl alcohol. Angiosperm lignin contains approxi-

    al portions of guaiacyl propane units and syringylits derived from sinapyl alcohol [72]. Grassescontain approximately equal portions of guaia-, syringyl propane and p-hydroxyphenyl propane

    ructural material may be degraded by a limitedfungal species under aerobic conditions [77],

    es [74]. Two models have been proposed to de-formation of humic substances from lignin. Theis the lignin-degradation model in which lignindegraded to form smaller compounds such as

    roups, methoxyl groups and phenols. These re-ceed to form humic acids, and through frag-and oxidation to form fulvic acids [75]. Thedel is the lignin degradation and polymeriza-, where lignin is degraded to produce polyphe-then undergo enzymatic oxidation to quinones,

    erization to form humic substances [75]. Signif-in 13C NMR spectra have been shown to arise

    romatic rings present in lignin residues [78,79].model may account for the observed aromatic-ic substances, it does not account for the in-idence of aquatic humus being more aliphatic,

    usion of other elements such as nitrogen in the

    nt for the nitrogen found in humic acid, Waksmansed that modified lignins combine with proteinsSchiff reaction:

    (OCH3)COOH(OH)4CO + H2NRCOOH46O10(OCH3)COOH(OH)4C NRCOOH+ H2Odification to humic substances can occur due tond abiotic processing. Degradation of humic sub-y result in smaller biomolecules that can be par-zed to form a greater abundance of carboxylic andtaining functional groups that can then go on tor by microbial action or fractionation by sorptioninorganic compounds [74].he lignin conceptual model has been used toe possible pathway to the formation of humic

  • 110 S. McDonald et al. / Analytica Chimica Acta 527 (2004) 105124

    substancescontain lig

    3.2. Humicpolymeriza

    Degradasimple monisms. Thesform largedegradationpathways.jor componwhich are bin lignin decelerated bmetal oxidAnother poaction invosubstancescrobial decHowever, tsaturated coFig. 1. A schematic formula for a portion of aspen lignin

    , humification also occurs in plants that do notnin [81].

    substances formed bytion/condensation reactions

    tion of plant material may result in a variety ofomers that can be readily utilized by microorgan-

    e monomers can then undergo polymerization tor molecules that are more resistant to microbial

    [74]. The polymerization can occur via manyOne class of important monomers that are a ma-ent of humic substances [82] are polyphenols,oth synthesized by microorganisms and releasedgradation. The polymerization of phenols is ac-y the presence of transition metals in solution,es, and the presence of clays (Fig. 2, from [83]).ssible polymerization reaction is the Malliard re-lving the condensation of amino acids and relatedwith reducing sugars [74] that arise from the mi-omposition of cellulose and polypeptides [75].his reaction is less likely to occur under water-nditions [74] and it is not considered to be a major

    process in hhumic acidfree radicaseawater by

    While thjor functiocompletelyof humic sthough hument pathwaaquatic envmonomers

    position [7

    4. Compo

    Humic shumificatioactions betmetabolic pany structuflect structu(adapted from [74]).

    umification [81]. In the case of marine fulvic ands, a pathway has been proposed [84] involving thel cross-linking of unsaturated lipids released into

    algal growth.ese models account for the heterogeneity and ma-

    nal groups of humic substances [74], there is nosatisfactory model that describes the occurrence

    ubstances in diverse geologic environments. Al-ic substances are formed by a variety of differ-

    ys, they are thought to be relatively stable in theironment due to the idea that randomly orderedhave an inherent resistance to enzymatic decom-4].

    sition and structure of humic substances

    ubstances arise from the transformation (throughn) of biomolecules, or the build up from inter-ween small organic compounds released duringrocessing of natural macromolecules [10]. Thus,res formulated for humic substances should re-res that can occur in plants and microorganisms or

  • S. McDonald et al. / Analytica Chimica Acta 527 (2004) 105124 111

    their degradconstituentlose (non-csuch as tansugars, amsoluble comments, andare thoughhumic substans, suberaouter wallsarise from

    Fig. 3. Diagrafrom [88]).Fig. 2. Oxidative polymerization of phenol derivatives involving amin

    ation products [81]. The main classes of chemicals comprising plant litter are cellulose, hemicellu-ellulosic polysaccharides), phenolic compoundsnins and lignin, water-soluble compounds such asino acids and aliphatic acids, ether- and alcohol-

    pounds such as fats, oils, waxes, resins and pig-proteins. Other components of plant litter that

    t to be refractory and may therefore form part oftances include carbon black, sporopollenins, cu-ns, and algaenans that have been identified in theof a number of microalgae [85]. Compounds thatmicrobial constituents may include lipids, carbo-

    mmatic representation of the structure of humic acid (adapted

    hydrates, pies conductsoil and laktural featurinto the huand long-ctrast to the

    Fig. 4. Po sugar units (adapted from [83]).

    roteins, melanins and other polyketides [81]. Stud-ed by Wershaw et al. [86] on peat soil, agriculturale sediment, found that many of the chemical struc-es of the original plant material were incorporatedmic acid structure, including lignin, carbohydratehain aliphatic structural groups. This is in con-view that transformed soil organic matter bears

    roposed humic acid building block (adapted from [89]).

  • 112 S. McDonald et al. / Analytica Chimica Acta 527 (2004) 105124

    no morphological resemblance to the structures from whichit is derived [87].

    From these considerations, various structures have beenproposed ranging from basic conceptual structures (e.g.Fig. 3, from [88]), to more detailed structures based on py-rolytic degradation products, NMR, and computer modellingtechniques (e.g. Fig. 4, from [89]). However, the view thathumic substances are too complex to determine their chemi-cal structure, and that an average structure and functionalityshould in fact be used [16] is gaining popularity and thus theimportance of functional groups present in the humics mustnot be overlooked. Humic substances from different envi-ronments appear to have many characteristics in common al-though the relative abundance of functional groups may vary(Table 2). Humic substances contain a large number of ioniz-able sites [90] that give them the ability to complex metals andorganic compounds, and to act as pH buffers. The distributionof functional groups may also be important in determiningthe solubility and aggregation behaviour of humic substances[91].

    It is recognized that the composition of isolated/fractionated humic substances are a reflection of the sepa-ration procedure adopted [10], and that classical definitionsof humic sacid precipthey must aas aggregaally largerhydrogen bgroups andthe molecuamorphousaggregationdominate.

    Table 2Important fun

    Functional gr

    Acidic groupsCarboxylicPhenolic OEnolic hydQuinone

    Basic groupsAmineAmideImines

    Neutral groupAlcoholic OEtherKetoneAldehydeEster, lactoCyclic imid

    R is aliphatic r (S) maacid derivativ it is th

    Differences between conformations are apparent betweenwhat occurs naturally and what is isolated as a humic sub-stance, and this is reflected in the relative hydrophilic-ity/hydrophobicity of the substance before and after isolation.For example, humic substances in the natural environmenttend to exhibit hydrophilic properties due to the formation ofstrong hydrogen bonds with water molecules [93], however,after isolation, humic acid becomes more hydrophobic, andas such, is difficult to fully solubilise in water. Varying con-centrations of sodium hydroxide have therefore been used toeffectively disperse/dissolve the isolated samples, including0.002 M NaOH [94] and 0.001 M NaOH [95]. Humic acidhas also been solubilised in bi-distilled water with the use ofan ultrasonic bath [96].

    The conformation of humic substances in solution hasbeen the subject of much debate, and has been extensivelyreviewed [11,87]. In one model, humic substances are viewedas being macromolecular with random coil conformations insolution [75] that can change with pH, concentration andionic strength. As the pH increases, the acidic functionalgroups become less protonated, negative charges tend to repelone another, hydrogen-bonding decreases, and the moleculebegins to open, or uncoil [97]. Increases in the concentration,

    ring the pH or high ionic strength have been shown toote aggregation of fulvic acid in a solution of water, asured be fulvdroge

    ps exeof the

    ulvic aystemn altemolecubstances are operational only [11], with humicitating at pH < 2. For humic acids to precipitateggregate, giving a polydisperse system that existstes of various size [92]. Humic acids are gener-in size than fulvic acids. This is partly due to theonding between the phenolic hydroxyl functionalthe carboxylic acid functional groups present in

    le, and partly due to humic acids aggregating withsilica and clay material [92]. In the FA fraction,is not the case and monodisperse systems pre-

    ctional groups of DOC [10]oup Structure

    acid (Ar)RCO2HH ArOHrogen (Ar)RCH=CHOH

    Ar=O

    (Ar)RCH2NH2(Ar)RC=O(NHR)CH2=NH

    sH (Ar)RCH2OH

    (Ar)RCH2OCH2R(Ar)RC=O(R)(Ar)RC=O(H)

    ne (Ar)RC=O(OR)es (R)O=CNHC=O(R)

    backbone and Ar is aromatic ring. In addition to certain amino acids sulphues (Ar)RSO3H. It is notable that the backbone may be R or Ar but mostly

    loweprommeas

    of thof hygroutionsthat fous s

    AsupraWhere found

    90% of all dissolved organic carbonAquatic humic substances, phenolsAquatic humic substancesAquatic humic substances, quinones

    Amino acidsPeptides(Unstable, forming polymeric derivatives) humic substances

    Aquatic humic substances, sugarsAquatic humic substancesAquatic humic substances, volatiles, keto-acidsSugarsAquatic humic substances, hydroxy acids, tanninsAquatic humic substances

    y also occur as mercapto/thiol compounds (SH) and sulfoniceir combination.

    y fluorescence spectroscopy [98]. The behaviouric acid was thought to be due to a combinationn bonding, charge screening on dissociated acidrted by the background electrolyte, and interac-

    hydrophobic regions in fulvic acid, suggestingcid behaves like a flexible polyelectrolyte in aque-s [98].rnative model is that humic substances involveular associations of relatively small molecules

  • S. McDonald et al. / Analytica Chimica Acta 527 (2004) 105124 113

    held together by weak interactive forces. The evidence forthis model lies in a shift in molecular size from high to lowwhen organic acids were added to lower the pH of the hu-mic solutioacids disrupby weak dbonds, resu

    Anotheror pseudom[86]) and Wmic substaconventiontogether byexist as medegradationand chemica function oties of the iare components of plaand lipids)hydrophoblar function

    5. Method

    Differento differendefined smsugars, standata (e.g. [9tional and othe structur

    A varietacterize hu[100]. Duemodern anadevelopedutilize seveprehensivehumic andples are lowquired [101chemist hapropertiesyet, there imethods thof the struc

    5.1. Isolat

    Severalaration ofbeen separaing from lo

    lar weights (>1 kDa), derived from ultrafiltration techniques[4,5,52].

    Separation of DOM into its humic and non-humic compo-has been achieved by liquid extraction procedures [14],ption techniques, and the use of granular activated car-101]. The more frequently applied procedure, however,e separation of humic and non-humic fractions of DOCves the isolation and extraction of humic substances byption onto non-ionic or ion-exchange resins. Tradition-the isolation of humic substances has involved the useAmberlite XAD-8 resin (Fig. 5) [100]. At acidic con-s, all organic acids are fully protonated, non-ionic, and

    dsorb onto the surface of the resin. The organic acids aredesorbed from the resin with an alkaline solution (typ-

    with 0.1 M sodium hydroxide). These organic acidsferred to as humic substances or hydrophobic acids.er fractionations of the organic acids according to theirus hydrophilic and hydrophobic properties (Fig. 6, from) have also been achieved [104].e advantage of using the XAD-8 resin is that it was

    ted as the standard method for isolating and fraction-humic substances by the International Humic Sub-

    es Society [100,105], and has been the most frequentlymethod for the isolation and purification of humic sub-es. There are a number of disadvantages inherent withethod

    re, anddingificatiohe spo

    ceas

    s beec subified [

    Flow coil ande typesn [11]. It was proposed that the addition of organicted the supramolecular associations held together

    ispersive forces by the introduction of hydrogenlting in smaller subunits [11].viewpoint is that humic substances form micelles,icellar structures in solution. Wershaw (cited inershaw et al. (cited in [86]) proposed that hu-

    nces are mixtures that cannot be represented byal structural diagrams of functional groups heldcovalent bonds, but rather that humic substancesmbrane-like or micelle-like aggregates of plantproducts. They further proposed that physical

    al properties of these humic aggregates are moref the structure of the aggregates than of the proper-ndividual components. These ordered aggregatessed of the partially degraded molecular compo-nts (e.g. lignin fragments, carbohydrates, tannins

    , with the interior of the aggregates being moreic, and the exterior surfaces containing more po-alities such as carboxylic groups.

    s of analysis

    t methods of isolating humic substances may leadt characteristics of the substance [68]. For well-all compounds of DOC including amino acids anddard chemical analyses can provide concentration9]). However, for many larger compounds, func-perational definitions become more prevalent andes of the constituent compounds remain unknown.y of methods have been used to isolate and char-mic substances, and have been reviewed by Janosto the vast heterogeneity of the humic substances,lytical methods are continually being refined and

    in order to analyze them, and researchers tend toral techniques in tandem to generate a more com-understanding of the structure. Moreover, sincefulvic acid concentrations in natural water sam-, powerful techniques with high sensitivity are re-]. Nearly every method available to the analytical

    s been used in an attempt to unravel the complexand behaviour of humic substances [102], and ass no single analytical method or combination ofat can provide data for absolute characterizationture of humic materials.

    ion of humic substances

    procedures have been developed to achieve sep-DOM into fractions for further study. DOM hasted into nominal molecular weight cut-offs rang-w molecular weights (

  • 114 S. McDonald et al. / Analytica Chimica Acta 527 (2004) 105124

    hydrophobcomparisonthe bulk huslightly difchromatoglammoniumaquatic sam

    Alternatincluding tbut with vaexcessive bthe XAD-1of the slowcient elutiotion [107].to exhibitFig. 6. Flow chart showing the separation of humic substances into ac

    ichydrophilic sorptiondesorption properties into the XAD-8 resin, but with similar recoveries of

    mic material. However, it isolated systematicallyferent humic solutes as analysed by pyrolysisgasraphymass spectrometry, with offline tetramethy-

    hydroxide (TMAH) derivatisation of differentples [106].

    ive XAD type resins have been evaluated for use,he XAD-7, XAD-1, XAD-2 and XAD-4 [107],rying success. The XAD-7 resin may suffer fromleed problems, and the practical usefulness of, XAD-2 and XAD-4 resins is limited because

    diffusion-controlled adsorption and an ineffi-n process caused by charge-transfer complexa-In addition to this, XAD resins have been shownsize-exclusion effects, excluding some larger

    molecular[105].

    Alternatstances hamaterials [laminoethylate and conAn advantabe obtainedthat naturaltechnique dweight spemain XADidentical oanalyticalposition ofid, base and neutral fractions [103].

    weight components of aquatic humic substances

    ively, the weakly acidic character of humic sub-s been exploited by employing anion exchange108]. The weak anion exchange material, diethy-lcellulose (DEAE cellulose) has been used to iso-centrate humic substances from water [108,109].ge of using this method, is that organic acids canat neutral pH values, with high recoveries, and

    complex acids can be isolated whereas the XADoes not concentrate the colourless low molecular

    cific organic acids in water [108]. In general, thefractions and DEAE isolates consist of almost

    rganic compounds [100] as analysed by varioustechniques [110113], with the structural com-

    the humic solutes obtained by DEAE being a

  • S. McDonald et al. / Analytica Chimica Acta 527 (2004) 105124 115

    combination of the hydrophobic and hydrophilic acidic so-lutes obtained by the XAD technique [112].

    5.2. Chara

    Elemencontent [emg1 C1]of the bulkhumic subhas also bNMR techbination wtion as potances.

    5.2.1. SizeSize ex

    establishedstudy of hudetection [1mum absorspectrum [have beenSEC procestudy was cmum wave

    between 23sitivity for254 nm wasubstances

    Only reSEC to imacids. Othespray ionizpled plasmanalysers [[121].

    Many sthe molecuin order toand mobilishow diffeple, averagchanged wtection [12(HPSEC) imajor probmic fractiothe columnular weighdards are ua number ostandards tmic substacated the u

    sulfonates which are now routinely used to calibrate SECcolumns [123].

    Average molecular weights are determined rather thanfic moubstanes ofand wubstan

    n

    n

    i

    (h

    n

    i

    (n

    i

    e hi isving re rat

    cularispers

    graphg fron witionatisis shin thm boaccum

    ht randata as cont

    eight

    . Nucuclearwidel

    althouwithistribue NMionales, hor [126s thatNMRrstandeen umic s

    arily aas on

    humicituentical ecterization of humic substances

    tal composition (%, w/w), acidic functional groupq. mg1 C1], UVvis absorptivities [absorbance, and average molecular weights [114] are some

    measures that have been used to characterizestances. The composition of humic substanceseen partially described from fluorescence andniques. Frequently techniques are used in com-ith each other to provide as much informa-

    ssible about the characteristics of humic subs-

    exclusion chromatographyclusion chromatography (SEC) is a well-separation technique that has been applied to themic substances, traditionally with the use of UV15]. Humic substances however, possess no maxi-

    ption at any wavelength in the ultraviolet or visible116]. As a consequence, numerous wavelengthsused for detection [100]. In order to standardizedures for enhanced reproducibility of results, aonducted by Zhou et al. [117] to determine opti-

    lengths. The results showed reasonable detection0 nm and 280 nm. 230 nm provided better sen-solutions with a low carbon content, however,

    s recommended for the study of aquatic humic[117].cently have other techniques been coupled toprove the characterization of humic and fulvicr detectors that have been used include electro-ation-mass spectrometry [118], inductively cou-a-mass spectrometer [119], total organic carbon120], refractive index and fluorescence detectors

    ize exclusion separations are used to determinelar weight distribution of humic and fulvic acidsgain better understanding about their reactivity

    ty. The use of different detectors, however, mayrent molecular weight distributions. For exam-e molecular weights for fulvic and humic acidsith UV, refractive index and fluorescence de-1]. The high performance application of SECs the most extensively used technique [103]. Alem in using SEC to determine the size of hu-ns is the lack of adequate standards to calibrate

    (Table 3) [11,122]. Moreover, average molec-t values may vary depending upon which stan-sed to calibrate the column [103]. For instance,f investigators have shown that globular protein

    end to overestimate the molecular weights of hu-nces by a factor of 5 or more and have advo-se of random coil standards such as polystyrene

    specimic ssourc

    (Mn)mic s

    Mn =

    Mw =

    wheror ha

    ThmolepolydmatoelutinbutioFractanalylatedrandotivesweigTheetherular w

    5.2.2N

    beenandsiblethe din thfunctstancoccu

    inatethis,undehas bof huprimture,thatconstchemlecular weights due to the heterogeneity of hu-ces, and Table 4 highlights the variation betweenhumic substances as analysed by SEC. Numbereight-average (Mw) molecular weights for the hu-ces are determined using the following equations:

    i

    hi

    i/Mi)

    hiMi)

    hi

    the height of the HPSEC curve eluted at volume,etention time, i.io of the weight-average to number-averageweight gives an indication as to the analytesity. While SEC has been widely used, the chro-

    ic separation of the humic substances is poor,m the column as a broad, monomodal distri-h subtle shoulders and small sub-peaks [123].on of swamp humic substances and subsequentowed that carbohydrate macromolecules accumu-e highest molecular weight fractions suggestingnd cleavage on degradation [124]. Lignin deriva-

    ulated in fractions of intermediate molecularge suggesting some preferential bond cleavage.lso suggested that polyoxyethers or unsaturatedributed significantly to the oxygen in low molec-materials.

    lear magnetic resonance spectroscopymagnetic resonance (NMR) spectroscopy has

    y used for the study of humic substances [125],gh specific compound identification is not pos-this technique, information can be provided ontion of various functional groups present. PeaksR spectrum can usually be assigned to specificgroups in complex materials such as humic sub-wever, some overlap of resonance assignments can]. It is only when one type of compound predom-a well-defined signal can be observed. Despitetechniques have been valuable in advancing theing of humic substances. For example, 13C NMRsed to examine the aromaticity/aliphatic natureubstances, leading to the discovery that they areliphatic and not predominantly aromatic in na-ce thought [15]. NMR studies have also foundacids are composed of partially degraded plantcomponents [86], that fulvic acid is a separate

    ntity to humic acid [127], and that perhaps pheno-

  • 116 S. McDonald et al. / Analytica Chimica Acta 527 (2004) 105124

    Table 3Polymer standards utilized for SEC analysis of humic acid [121]Polymer standard Similarity to humic acid Difference from humic acid

    Globular proteins Water-soluble Overestimation of MwPolysaccharides Water-soluble component of HA Charge densitySodium polystyrene sulfonates Coil configuration Cross-linking, branching, aromatic carbon contentPoly(acrylic acid)s Acidity Branching

    lic carbons are minor rather than major components of humicsubstances [128].

    While 1H, 15N, and 13C NMR have been used for studyinghumic substances, 13C and 15N NMR have been utilized moreextensively due to 1H NMR being limited mostly to solutions.Solid-state 1H NMR spectra suffer from very poor resolution[128].

    13C NMR has several advantages over proton NMR interms of its power to elucidate organic and biochemical struc-tures, including the ability to provide information about thebackbone of molecules rather than the periphery [129]. Also,the chemical shift for 13C in most organic compounds is about200 ppm compared with 1015 ppm for the proton, and as aconsequence, there are less overlaps of peaks. When studyinghumic substances, solid state 13C NMR has been preferen-tially used 13 13conformatisolved humlattice relaxsignals inoverestimaaromatic ca

    15N NMthe forms oinantly in sabout nitro

    well resolved as that given by a 13C NMR due to its largequadrupole moment [126]. Higher field strengths are there-fore required to enhance the signal. Major peaks for amideand amino nitrogen in organic matter have been observedin 15N NMR spectra of humic substances [128]. From thesestudies, it is thought that the nitrogen was derived from pro-teins and survived the humification process to become incor-porated into the humic substance. Such a concept contrastswith the views that nitrogenous structures are incorporatedinto humic substances by condensation reactions such as theMaillard reaction [128].

    5.2.3. Vibrational spectroscopyInfrared spectroscopy (IR) has been widely used for the

    investigation of humic substances [131,132], with Fourierform infrared spectroscopy (FTIR) having a number ofntagesachievl-to-n

    arge af humi0 mg)gationever, m

    hat, bto el

    Table 4Weight averag arious s

    Sample

    Gota River H ormamGota River FA ormamNordic Refere n (200Nordic Refere n (200Nordic Refere dium aNordic Refere dium aLake Savojarv dium aLake Savojarv dium aLake Mekkoj dium aLake Mekkoj dium aSuwannee Riv Cl solutSuwannee Riv Cl solutSuwannee RivSuwannee RivAldrich humiSoil humic acPeat (well-huCompost (lignSoil HAover solution C NMR. In solution C NMR, theonal rigidity of aliphatic carbons in poorly dis-ic hydrophobic domains prevents the fast spin-ation of these carbons, thereby reducing spectral

    the aliphatic range [130]. As a consequence, antion of aliphatic carbons and underestimation ofrbons is given.R spectroscopy has also been used to characterizef nitrogen contained in humic substances predom-oils. While the use of 15N NMR gives informationgen functionality, the spectra obtained are not as

    transadvaity issignathe ltra o(11vestiHowlets tficult

    e and number average molecular weights of humic and fulvic acids from v

    Mn Mw Conditions for analysis

    A 1140 3062 254 nm detection, in dimethylf1034 2874 254 nm detection, in dimethylf

    nce HA 4410 11190 SEC with diode array detectionce FA 2180 3360 SEC with diode array detectionce HA 6204 23370 254 nm detection, in 10 mM sonce FA 4750 7428 254 nm detection, in 10 mM soi HA 6083 25171 254 nm detection, in 10 mM soi FA 5278 8022 254 nm detection, in 10 mM so

    arvi HA 9560 24419 254 nm detection, in 10 mM soarvi FA 6415 10819 254 nm detection, in 10 mM soer HA 2210 3670 254 nm detection, in 0.1 M Naer FA 1690 2180 254 nm detection, in 0.1 M Na

    er HA 2210 3330 254 nm detection, in 10 mM ammoniuer FA 1770 2640 254 nm detection, in 10 mM ammoniu

    c acid 2250 6060 254 nm detection, in 0.1 M NaCl solutid 3650 7760 254 nm detection, in 0.1 M NaCl solutmified) HA 7300 14900 200 nm detection, in 99% 5 mM phospin rich) HA 5700 12900 200 nm detection, in 99% 5 mM phosp

    3800 7900 200 nm detection, in 99% 5 mM phospover traditional IR techniques. Greater sensitiv-ed with increased energy throughput and a higher

    oise ratio [129]. The ease of obtaining IR spectra,mount of published information on the IR spec-c substances and the small sample size required,makes FTIR an attractive technique for the in-of functional groups in humic substances [132].ost studies are conducted using KBr pressed pel-

    ecause of its hygroscopic properties make it dif-iminate the interference of water bands in the

    ources

    Reference

    ideacetic acid (99 + 1, v/v) solution [121]ideacetic acid (99 + 1, v/v) solution [121]500 nm), in isopropanol/water (1:9) solution [118]500 nm), in isopropanol/water (1:9) solution [118]cetate [103]cetate [103]cetate [103]cetate [103]cetate [103]cetate [103]ion [29]ion [29]

    m bicarbonate in water/methanol (80:20) [115]m bicarbonate in water/methanol (80:20) [115]ion [29]ion [29]hate-buffered water/1% acetonitrile solution [122]hate-buffered water/1% acetonitrile solution [122]hate-buffered water/1% acetonitrile solution [122]

  • S. McDonald et al. / Analytica Chimica Acta 527 (2004) 105124 117

    humic substance spectra [131]. Also, under certain condi-tions, KBr has been reported to catalyze the decarboxylationof acidic functional groups, thereby altering the humic sub-stance [132by casting

    An alteflectance inConsidered[131], it ovlets [133].compost huity, a decrea decreaseages [134].DRIFTS ansubstancesfunctionalease of usepresent insearch hasquantitativetifying humdeveloped [quantify huin their nat

    Surfaceapplied to tto provideics [138]. Ssamples thaparticles (usurfaces ofassociatedtroscopy. Tsample sizespectra due[138140].of humic suabout the ican be provonto a silvboxylic groions can chcausing anor ammonimentary dasome ambithat the strupletely dete

    5.2.4. DiffDifferen

    been appliemetals andto the studyformation a

    from a glassy (condensed) to a rubbery (expanded) state, andaccordingly, the degree of increased mobility. Typically theglass transition temperature decreases with increased water

    nt ofnce oting the, andsubstae orga

    er anughtubstans ofSC prosubstaotherstudi

    c acidar patrent inamplecouldanceseraturwateraccha

    oil FAboundssignomati

    DSCquatificatioes [1r Jr.vely yic ma

    d glaorresp

    . Pyroermafrequ8,103dationeticallinformted weter (M

    is thvel), aecessa

    reflecare o

    be inreleas]. These problems may potentially be overcomefilms of the sample [132].rnative to transmission methods is diffuse re-frared Fourier transform spectroscopy (DRIFTS).to be one of the most sensitive infrared techniques

    ercomes the problems encountered with KBr pel-DRIFTS has been used to study the changes inmification, showing an increase in the aromatic-

    ase in the carboxylic acid functional groups, andin the aliphatic components of the compost as itFTIR has also been used to confirm these trends.d FTIR have also been used to compare humic

    from different environments, study the changes ingroups after irradiation (> 370 nm), explore theof IR, or simply examine the functional groups

    a sample [135137]. More recently however, re-focused on making FTIR and DRIFTS techniques

    as well as qualitative. A methodology for quan-ic substances using an internal standard has been132], while DRIFTS has also been used to rapidlymic substances or organic matter concentrations

    ural matrix [133].enhanced Raman spectroscopy (SERS) has beenhe study of organic matter and humic substancesdirect information on their structure and dynam-ERS involves obtaining vibrational spectra fromt are adsorbed onto the surface of colloidal metalsually silver, gold or copper), or on roughenedthese metals. There are a number of advantages

    with the use of SERS over normal Raman spec-he intensity of the signal is increased, a smalleris required, and there are no interferences in theto fluorescence emitted by the humic substancesBy examining SERS spectra of the adsorptionbstances onto the surface of metals, information

    nteraction between metals and humic substancesided. For example, the adsorption of humic acid

    er surface appears to occur through ionized car-ups [139]. The presence of co-adsorbed chlorideange the way in which humic acid may bond byadditional coordination through a terminal amineum group [139]. SERS spectra provide compli-ta to that obtained from IR. Still, there remainsguity in the assignment of peaks, due to the factcture of humic substances has still not been com-rmined [139].

    erential scanning calorimetrytial scanning calorimetry (DSC) has typicallyd to the study of pharmaceuticals, clays, minerals,polymers; more recently, it has also been appliedof humic substances [141,142]. DSC provides in-bout the transition temperature (Tg) of a substance

    contepreseaffecstancmicWhilpolymis thomic sregio

    Dmicwith[141]fulvisimildiffethe sternssubsttemptionpolysfor sfacewas a

    of arusingthat ahumisourc

    Weberelatiorganlinkeand c

    5.2.5Th

    been[10,5degratheorturaldetectromniqueng lenot ntiallywayscouldtheira biopolymer indicating a higher mobility in thef water [143]. Mobility is an important feature,e diffusion of smaller compounds into the sub-may have implications for the way in which hu-

    nces interact with the surrounding environment.nic matter is not strictly polymeric in form, thealogy is often applied to humic substances, andto be appropriate due to both polymers and hu-nces being macromolecular in nature and havingexpanded and condensed regions [144].vides limited information on the structure of hu-

    nces by itself, but may be useful when coupledtechniques. For example, Provenzano and Senesied a wide range of soil and aquatic humic ands (IHSS) and found that the DSC curves showedterns occurring at similar temperatures but withtensities depending on the origin and nature of. When used in combination with FTIR, the pat-be attributed to chemical changes in the humic

    . A strong endothermic peak appearing in the low-e region for all samples indicated a loss of hydra-and/or structural water, and of labile peripheralride chains [141]. The endotherm at about 623 Kwas ascribed to partial decarboxylation of sur-groups [141]. A broad exotherm around 773 K

    ed to thermal reactions involving rearrangementsc nuclei of the humic molecules [141]. Studies

    have also provided further evidence to suggestc humic and fulvic acids have a lower degree ofn compared to those arising from soil and peat

    41]. Through studies using DSC, Leboeuf and[142] viewed diagenesis as a process in whichoung, expanded, lightly cross-linked rubberytter is converted to more condensed, highly cross-ssy structure, having reduced molecular mobilityonding increased glass transition temperatures.

    lysis and related techniquesl degradation techniques such as pyrolysis haveently applied to the study of humic substances,106,111,145,146]. Pyrolysis involves the thermal

    of material into smaller components that can bey related back to the original sample, and struc-ation obtained. Pyrolysis products are typically

    ith gas chromatography (GC) and/or mass spec-S) combinations. An advantage with this tech-

    at only a small sample size is required (mg tond that extensive pre-treatment of the sample isry, however, the degradation products only par-t the source material, and their degradation path-ften unknown. Also, the structural interpretationscorrect if the products are altered before or aftere from the structure [147]. Further to this, there is

  • 118 S. McDonald et al. / Analytica Chimica Acta 527 (2004) 105124

    only partial combustion of the sample, with the volatile com-pounds obtained accounting for approximately 5070% ofthe original organic matter [148]. Limitations in the analysisof polar funlike decarbthe structu[111].

    The pyrpendent onof complex[103], how600 C havof humic s[147].

    Despiteful informaFor examphumic acidvealed thatplant cuticlPyrolysis oaliphatic prindicative o[145].

    5.2.6. CapThe mig

    trophoresistwo humictric field, thto-size ratihas provenbiomoleculturn, has inmic substa

    Separatipherogramacteristic dessential btion degreepeaks in thtents of agfrequentlywith borate1,2- and 1,3artifact forA recent cdemonstratnow used tstances. Thbetween auorganic mabetween thtinguishingences betwbeen used t

    [97] and differences have been detected in the migration be-haviour and UV absorption between humic and fulvic acids[95]. These differences were attributed to the degree of hu-

    ationtion iner ofn of thrsor sctivelubstan

    ], hoween so

    . Masass spas beemic s

    ave beof hum

    in thESI),desor

    ted lasus fa

    btainiiques

    ps in hn woioniz

    egatives [12dy comespiteass spave pmatt

    roscopt anyified

    S, inlic aciancesMAL

    omoleere n

    . Fluouorescof hunsiveare beubstadicate, and/on advaroscopctional groups due to unwanted thermal reactionsoxylation have led to the loss of information aboutral composition of the original macromolecule

    olysis behaviour of humic substances is highly de-the temperature selected. Pyrolysis temperaturesbiomaterials usually vary in the range 500800 Cever, temperature ranges between 500 C and

    e been suggested as being appropriate for the studyubstances, depending on the nature of the sample

    its limitations, pyrolysis has provided some use-tion about the composition of humic substances.le, detailed studies on the most resistant part of(hydrolyzed or persulfate oxidized residues) re-aliphatic biopolymers similar to those found in

    es could form part of the humic moiety [58,147].f DOC from various sources also yielded furans,oducts and nitrogen containing products that weref polysaccharides, lipids and proteins respectively

    illary electrophoresisration behaviour of molecules in capillary elec-(CE) depends on their charge-to-size ratio [95]. Ifsubstances exhibit the same behaviour in an elec-en they are likely to have a comparable charge-

    o. In the past decade, capillary electrophoresisits superiority for separating a wide variety of

    es such as peptides and proteins [97] which, inspired researchers to apply this technique to hu-

    nces [149151].on was typically poor and the typical electro-comprised a broad humic hump with few char-

    etails. High sample concentrations were generallyut concentrating samples increases the aggrega-

    of the possible individual humic materials ande electropherograms may represent different ex-gregation. Moreover, bare silica capillaries wereemployed and the best separations were achieved

    buffers [152] which are known to interact with-diols present in the humic mixture [151]. Hence,

    mation was a potential problem in such systems.omparative study of several CE techniques hased the potential of the technique [152] which iso provide chemical fingerprints of humic sub-ese fingerprints have been used to distinguishtochthonous and allochthonous sources of naturaltter (NOM) [149] by distinguishing differencese peak heights, however, that was the only dis-character of the electropherogram. Height differ-een the profiles of electropherograms have alsoo distinguish between young and old fulvic acids

    mificvarianumbtratioprecurespemic s[150betw

    5.2.7M

    that hof hular hysismadetion (laserassis

    Thfor otechngrouizatiomodethe nspecialrea

    Dby mMS hganicspectdetecidentMS/Mboxysubstusingmacr

    that w

    5.2.8Fl

    studypreheunitsmic sare inrings

    Aspect(for the young versus old comparison, [97]) and tothe aromatic content, the presence of a different

    UV active groups, or the variation in the concen-e individual fractions, caused by differences fromubstances or conditions during humification [95]y. Differences in structural characteristics of hu-ces have been attributed to their different origins

    ever, CE is not always able to pick up differencesme humic substances from different origins [95].

    s spectrometry techniquesectrometry (MS) is an important analytical tooln applied to the identification and characterizationubstances. Soft ionization techniques in particu-en found to be particularly suited to mass anal-ic substances [153], with much progress being

    e last decade with the use of electrospray ioniza-atmospheric pressure chemical ionization (APCI),ption-mass spectrometry (LD-MS) and matrix-er desorption/ionisation (MALDI) [125].r, ESI-MS has been the major analytical methodng molecular information by mass spectrometric[125]. The anticipated high content of carboxylicumic substances suggest that negative mode ion-uld be a more sensitive technique than positiveation, however, it has recently been observed thate ion mode tends to produce multiply charged5] that may be problematic when interpreting anplicated spectrum [154].

    the complications associated with analyses doneectrometry, both positive and negative mode ESI-rovided information on changes occurring in or-er after UV irradiation, when conventional UVy and size exclusion chromatography could notchanges [155]. Various compounds have been

    in IHSS soil and peat fulvic acids using ESI-cluding benzene, phenol, furan and thiophene car-ds [153]. Remmler et al. [156] fractionated humicusing ultrafiltration and analysed the fractionsDI. From this, they concluded that humic acid

    cules were composed of smaller building blocksot associated by covalent linkages [125].

    rescence spectroscopyence spectroscopy has also been applied to themic substances [98,157,158], and has been com-ly reviewed by Senesi [98]. Although fluorescinglieved to constitute only a minor component in hu-nces [157], the most efficient fluorophores in HSd to be variously substituted, condensed aromaticr highly unsaturated aliphatic chains [98,157].

    ntage of using fluorescence spectroscopy over UVy in the study of humic substances is the better

  • S. McDonald et al. / Analytica Chimica Acta 527 (2004) 105124 119

    resolution of the synchronous fluorescence spectra that leadsto better distinction between different samples [136,113]. Us-ing this technique, differences in fulvic acids from differentorigins havacid consismum intenof the samcharacteriznied by a nalso vary wof humificaarea under

    Fluorescstudy of huStudies bycapacity ofhigher molcarboxylicother applito monitorpollutants ssurably affIn one studsubstancesand solubil

    5.2.9. FielField flo

    niques thattechniquesthermal fietion and flolar, has beeand charact[161163].

    FFF hasical separasult of thechannel, aninjected intcarrier flowgles, whichwall. The cbrane thatpounds of ieluted accoysed with vsubstancestector at 25

    FlFF ofmolecularthe advantanamic size[168]. A posuch as lowmay occur

    has provided further characterisation of humic substances in-cluding their interactions with metals [162], and their hydro-dynamic behaviour and response to changes in ionic strength

    H whgation

    gth, anteractelow

    r fulviomolemono

    40 g m

    0. Miriouscterizs, trany dispopy (

    spechologlemener of

    or spoorks as and

    e nanous inoM isudy oganic. Usin

    ) was fa low

    sifiedpH, aner of 7ecausen soluscopyers a

    fect oe AFMolutionultieses o

    ated dbe theibutetheset varithe r

    gationft X-ing hufrome been detected [98]. Emission spectra of fulvict of a broad band of overall intensity with a maxi-sity wavelength that varies according to the originple, while excitation spectra of fulvic acids areed by one or more major peaks, often accompa-umber of secondary peaks and shoulders [98] thatith origin. A novel way of determining the degreetion has also been developed, by measuring thethe fluorescence spectra at 465 nm [159].ence spectroscopy has also been applied to themic substances and their interactions with metals.Wang et al. [158], found that most of the bindingLaurentian fulvic acid was associated with the

    ecular weight fraction (>30,000 Da, having loweracid groups), as fractionated by ultrafiltration. Incations, fluorescence spectroscopy has been usedpollution levels. Sorption of hydrophobic organicuch as DDT on humic substances [58] can mea-

    ect the solubility and transport of the pollutants.y, binding affinities of pyrene to different humichave been assessed by fluorescence quenching

    ity enhancement techniques [160].

    d ow fractionationw fractionation (FFF) comprises a family of tech-have been used to study macromolecules. Theseinclude sedimentation field flow fractionation,

    ld flow fractionation, steric field flow fractiona-w field-flow fractionation (FlFF). FlFF in particu-n used to study the molecular weight distributioneristics of humic substances from various sources

    been likened to a chromatographic-type analyt-tion [164], but with separation occurring as a re-differential flow of a solvent in an unobstructedd the nature of the sample. Briefly, a sample iso a thin ribbon-like channel containing a laminar. A cross flow, or field is applied at right an-compresses the components against the channel

    hannel wall is fitted with a semi-permeable mem-allows the cross flow to pass, but not the com-nterest [165]. The compounds of interest are thenrding to their diffusion characteristics, and anal-arious detectors [166] although NOM and humicare more commonly analysed with a UVvis de-4 nm [162,167,164].fers an alternative to SEC for characterizing theweight distribution of humic substances, and hasge of separating components by their hydrody-while not being affected by non-exclusion effectsssibility exists however, that at certain conditions

    ionic strengths, humic-membrane interactions[169]. FlFF in conjunction with other techniques

    and paggrestrenlar inues bRivemacr

    ablyto 16

    5.2.1Va

    charaniqueenergcrosc

    X-raymorpand enumblikenetwshapeto thvario

    SFthe stof or[174]filterwith(claswithamet

    Bing imicropolymthe efWhila res

    difficvolumestimalsocontrspiteheighlatingaggre

    Sostudyticlesen in solution. Studies have shown that increasingof humic substances occurs with increasing ionicd that the lowering of pH promotes intermolecu-

    ions with severe aggregation occurring at pH val-4 [168170]. FlFF has also shown that Suwannec acid standard consists mainly of relatively smallcules (1.52.5 nm in diameter) and is reason-disperse with a molecular weight range from 530ol1 [169].

    croscopic techniquesmicroscopic techniques have been employed toe humic substances. Among the available tech-smission electron microscopy (TEM) coupled toersive spectroscopy (EDS), scanning force mi-

    SFM), atomic force microscopy (AFM) and softtromicroscopy have been used to study the size,y, porosity, degree of aggregation, crystallinity,tal composition of single particles [171175]. A

    morphologies have been reported including torus-nge-like ring structures, chain-like assemblies,nd large humic molecule aggregates of differentsizes [176]. TEM-EDS allows resolution downmeter range [177], and has been used to studyrganicorganic matter interactions [175].a technique that has only recently been applied tof humic substances [176], allowing direct imagingmatter down to a resolution of several nanometersg this technique, filtered peat humic acid (1000 nmound to possess two categories of particles, thoseer bound diameter between 1.5 nm and 3.5 nmas humic acid molecules) that changed in heightd larger more inert particles with a spherical di-0160 nm [174].of its high resolution and the possibility of imag-tion or under ambient conditions, atomic force(AFM) is now widely employed for the study of

    nd has also been used for the systematic study off pH and ionic strength on humic substances [172].

    allows for imaging molecules and particles withof a fraction of a nanometer, there are inherent

    with determining accurate molecular heights andf particles. Lateral dimensions are largely over-ue to the geometry of the tip [178]. There maypossibility that the particles being examined mayto partial flattening by the AFM probe [172]. De-limitations, AFM has been used to obtain relativeations as a function of pH and ionic strength, re-elative hydrophobicity of the humic substance to

    properties [172].ray spectromicroscopy offers the advantage ofmic substances in situ, allowing imaging of par-submicrometer to several micrometers [171].

  • 120 S. McDonald et al. / Analytica Chimica Acta 527 (2004) 105124

    The possibility of observing humic substances in their nat-urally hydrated state has further advanced the study ofhumicionorganic interactions in solution [171], and their re-sponse undhumic acidticles, withoxidized st

    Resultsdent on thevarious artiformation (the particletion and aqsuch as shrticles mayfor preparaexample, smorphologcoated ontowith time.agglomerata transitionProcedureshandling anat least qua

    5.3. Quant

    Methodsubstancestion procedcharacterisacteristics

    5.3.1. ColoThe dif

    quantificatilimnologismeasure thphenolic comeasureme

    index for anatural wat

    The colsured by vicoloured soForel-Ule mHazen methstandard so[71]. AlthotrophotomeSpectrophothe measurin the visibused wavel

    Gilvin, or soluble humic colour is a major contributor oflight absorption in inland waters and is often the major sin-gle absorbing component [180]. Due to the consistent expo-

    al typentraticient

    lvin ind colo

    n and spectrun the 3wing e

    = a(e a()= abs

    iscrepto de

    tic humly mies andounds71]. C

    rd uniuation

    . UVnumbto pr

    contenatio of

    conterbingromatot indius revns [14e assulgates to c[182].m) ram) arive huweighs witheen shn notht [11n to he studase inhange6 ration to oes by fer oxic and anoxic conditions [179]. In one study,in the reduced form appeared as small dense par-much of the organic material being soluble. In theate, large loose aggregates were observed [179].obtained by microscopic methods are often depen-sample preparation steps involved, and may createfacts in the process [175]. Physico-chemical trans-adsorption, coagulation or redox modification) ofs may occur, caused by changes in pH, oxygena-uatic compound concentration. Physical artifactsinkage and the aggregation/disaggregation of par-also be produced as a result of the steps requiredtion of sections for TEM observations [175]. Fortudies by Mertig et al. [174] found that the filmy of a homogeneous layer of humic acid spin-an atomically flat mica substrate changed slowly

    As a result, humic acid could be viewed as beinges, when in fact, the agglomerates formed afterbetween a wetting and non-wetting phase [176].have been developed to help minimize sampled preparation, so that artifacts may be avoided orntified [175,177].

    ication

    s have been developed for quantifying humicin water, without the need for laborious isola-ures. These methods have primarily exploited thetics of colour, absorbance and fluorescence char-associated with humic substances.

    urficulty involved in the direct measurement andon of aquatic humic substances, has lead tots and oceanographers instead using assays thate properties of humic substances, such as colour,ntent, or tannin and lignin content [71]. Colournts are the most common, and provide a simple

    ssessing the concentration of humic substances iners [71].our of natural waters has traditionally been mea-sual comparisons between the water and variouslutions. Europeans used the methyl orange and theethods, whereas most North Americans used theod, which incorporates the use of platinum-cobaltlutions (mg/L), and records the results as Pt unitsugh visual colour techniques are still used, spec-tric methods are now more commonly utilized.tometric determination of colour in water involvesement of absorbance at one or more wavelengthsle or near UV regions, with the most frequentlyength being 440 nm [71].

    nenticonc

    coeffiof gisolvegilvition swithifollo

    a()whera(0)

    Dusedaquaquatestanccomption [towaof eq

    5.3.2A

    suredandthe rDOCabsothe adid nvarioorigi

    Thprommentratio665 n365 ngressularratiohas bshowweigshowIn ondecreatic cE4/Eshowstance absorption spectrum of humic substances, theiron can be measured in terms of the absorptionat a single wavelength [180]. The concentration

    terms of the absorption coefficient due to dis-ur at 440 nm is given the symbol g440 (m1). Bothoil humic substances give rise to a similar absorp-m, rising exponentially as wavelength diminishes50700 nm region, and can be represented by thequation [180]:0)e0.014(0)

    = absorption coefficient at any wavelength ();orption coefficient at reference wavelength (0)ancies may arise between the different methodsscribe the colour of natural waters and quantify

    ic substances. The Hazen method does not ade-mic the spectral properties of aquatic humic sub-

    often overestimates the concentration of humic, sometimes exceeding the total DOC concentra-uthbert and Giorgio [71] have provided a means

    fying the different methods by providing a seriess to convert standard Pt units to g440.

    vis absorption ratioser of UVvis absorption ratios have been mea-

    ovide information about the state of humificationt of humic material in the DOC. For example,the UV absorbance at 254 nm (UV254) with thent, provides an estimate of the abundance of UV

    species, and may also be used for comparison oficity of various humic materials [181]. This ratiocate any significant differences, however, betweenerse osmosis isolates taken from a range of aquatic9].umption that humic substances are polymers hasd the use of simple physical-chemical measure-

    haracterize humic substances, such as the E4/E6Frequently, the E4/E6 (absorbance at 465 nm and

    tio and E2/E3 (ratio of absorbances at 250 nm ande used to indicate an inverse relationship with pro-mification and increased condensation, or molec-t [183]. The correlation of the E2/E3 and E4/E6the condensed aromatic carbon content of a sampleown to be poor [11,75], and has been repeatedlyto hold the predicted relationship with molecular]. In contrast to this, numerous studies have beenold the predicted relationship [52,103,184186].y, an increase in the E4/E6 ratio occurred with anominal size fractions [183], indicating a system-in chemical composition. Also, an increase in the, and therefore a decrease in condensation, was

    ccur for the transformation of aquatic humic sub-ungi over a period of 28 days [186]. Moreover, the

  • S. McDonald et al. / Analytica Chimica Acta 527 (2004) 105124 121

    fractionation of slightly brackish HMW-DOC to LMW-DOCafter UV irradiation was indicated by an increased E2/E3 ra-tio [52].

    5.3.3. ChA recen

    ural waterflow injecthe oxidaticals suchClO, anding sensiti

    6. Summ

    Dissolvaquatic orto predictecosystem

    Whilequality ofhighly colthe poor quter, or wheriver contithat can bfor the humDOC [188food websquality ofautochthotions to thsearch. Itthe majorsystems.

    Severalmation ofbe conducassociatedknown abtion of aqis not clewith chansource ofsubstance

    To undeter, humicroundingthe studiegreatly adbon and hto characthas not bsubstance

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