methods for analysis of aquatic humic substances

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Methods for Analysis of Aquatic Humic SubstancesM. Klavins a , L. Eglite a & J. Serzane aa Department of Environmental Sciences, University of Latvia, Raina Blvd. 19, LV 1586, Riga,LatviaPublished online: 03 Jun 2010.

To cite this article: M. Klavins , L. Eglite & J. Serzane (1999) Methods for Analysis of Aquatic Humic Substances, CriticalReviews in Analytical Chemistry, 29:3, 187-193, DOI: 10.1080/10408349891199383

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Critical Reviews in Analytical Chemistry, 29(3):187–193 (1999)

I. INTRODUCTION

Among the various naturally occurring or-ganic substances, humic substances (HS) are themost widespread. Humic substances form most ofthe organic component of soil, peat, lignite, natu-ral waters, and their sediments. Up to 70% of thesoil organic carbon and up to 90% of dissolvedorganic carbon may occur in the form of humicsubstances. Humic substances influence ground-water properties and the process of formation offossil fuels; however, in concert they play a majorrole in the global carbon geochemical cycle. Theglobal pool of humic matter is an important com-ponent in the formation of atmospheric carbondioxide. The estimated amount of carbon in theatmosphere is 638 to 702 × 1015 g C, which is inthe same range as carbon accumulated as soilhumus (700 to 3000 × 1015 g C), peat humus(250× 1015 g C), and aquatic humic substances(850 to 1700 × 1015 g C) (Klavins, 1998). Re-cently, it has been estimated that the global trans-port of organic carbon to oceans is about 0.4 to0.9 × 1015 g C yr.–1 (Hope et al., 1994).

The roles of humic substances is determinedby their formation during the humification (de-cay) process of living matter. Humic substancesform an intermediate phase in the transformationprocess of living matter (organic carbon reser-voir) that continue in the organic carbon cycle orbe deposited (as fossil materials). Humic sub-

stances are known to complex heavy metals andpersistent organic xenobiotics. The interaction ofhumic substances with xenobiotics may modifythe uptake and toxicity of these compounds byliving organisms and affect the fate of pollutantsin the environment. Considering these aspects theroles of humic substances in the environmentsmay be regarded as important. Humus propertiesdepend on its sources. Depending on the charac-ter of humification process, the greatest reser-voirs of humic matter are distinguished as soil,peat, and aquatic humic substances (AHS).

The present level of knowledge indicates thathumic substances form one of the most importantgroups of biomolecules. However, their proper-ties are confusing from the viewpoint ofmonodisciplinary sciences — chemistry, biology,and Earth sciences. Humic substances can be re-garded as one of the chemical anomalies. Thisattitude is evident even from the definition ofhumic substances (Stevenson, 1982). The mostcommon definition is (Humic Substances, 1985):“Humic substances are a general category of natu-rally occurring, biogenic, heterogeneous organicsubstances that can generally be characterized asbeing yellow to black in color, of high molecularweight, and refractory. Humic substances can bedivided in three fractions:

• Humin is the fraction of humic substances thatis not soluble in water at any pH

Methods for Analysis of Aquatic HumicSubstances

M. Klavins, L. Eglite, and J. SerzaneDepartment of Environmental Sciences, University of Latvia, Raina Blvd. 19, LV 1586, Riga, Latvia

ABSTRACT: Methods designed for analysis of aquatic humic substances are summarized and compared. A needto evaluate just the concentration of true humic substances, rather than summary parameters, characterizingdissolved organic substances such as COD, DOC, TOC, and others is stressed. Possible future trends in the studiesof dissolved humic substances are touched on.

KEY WORDS: humic substances, analysis, natural waters.

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• Humic acid (HA) is the fraction of humic sub-stances that is not soluble in water under acidicconditions (below pH 2), but becomes solubleat greater pH

• Fulvic acid (FA) is the fraction of humic sub-stances that is soluble under all pH conditions

The main forms of organic carbon (and hu-mic substances) in aquatic environments are dis-solved and particulate (colloidal) organic matter.

The division between dissolved organic matter(DOM) and particulate organic matter (POM) isgenerally made on the basis of whether it passesthrough a 0.45-µm filter (Figure 4). The genera-tion of these forms proceeds during the genesisand transformation of organic matter and is linkedwith processes such as decomposition of livingmatter, association of organic molecules with in-organic particulate matter and aggregation ofsmaller particles.

N atural organic matter (decomposition products , metabolites of l iving organic matter)

Nonhumic substances Humic substances(know n cl asses of organi c compounds) (pigmented refrac tory polym ers )

F ul vic acids H umic ac ids Humin (soluble at any pH ) (soluble if pH >2) (insol ubl e)

FIGURE 2. Size range of particulate and dissolved organic matter and carbon compounds in natural waters. AA,amino acids; CH, carbohydrates; DOM, dissolved organic matter; POM, particulate organic matter; Fa, fatty acids;Ha, hydrophilic acids; HC, hydrocarbons. (Adapted from Thurman, 1985a.)

FIGURE 1. Classification of humic substances.

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Aquatic humic substances originate mostlyfrom three processes:

1. Allochthonous aquatic humic substancesderived from terrestrial organic matter

2. Autochthonous aquatic humic substancesderived from in situ biological production

3. Humic substances derived from agricultural,domestic, and industrial activities (Degens,1982)

Contemporary structural models considershumic substances as polifunctional macromol-ecules (Figure 3).

The use of a wide variety of analytical meth-ods, including degradative and nondegradativeapproaches, to characterize Suwanee River fulvicacids, led to a model proposed by Leenheer et al.(1989, 1995b). This model shows the most pos-sible average structural formulas (Figure 3) andalso considers the possible precursors in the hu-mification process. The aromatic and aliphaticstructures in the proposed formulas indicate hy-pothetical residues of parent compounds, such aslignins, tannins, carbohydrates, and lipids, whichare incorporated into the structure of humic sub-stances. The multitude of possible links of thesestructural units and their biodegradation degreecan explain the large diversity of structural for-mulas for the possible humus structure. The pro-posed structures at best can explain the actuallyfound physical and chemical properties of humicsubstances isolated from the given environments,but the coexistence of HS as complexes withmineral particles is neglected.

Despite the great role of aquatic humic sub-stances in the environment, many aspects of theirfunctions and properties are still unclear, includ-ing the analytical determination of AHS.

II. PRINCIPLES FOR ANALYSIS OFHUMIC SUBSTANCES

A commonly required water characteristic isthe concentration of aquatic humic substances.Despite comparatively high concentrations innatural waters and yet higher concentrations ofhumic substances in waste waters, their analyticaldetermination is, however, problematic. This issurprising, regarding the importance of humicsubstances in geochemical cycles of elements andmetals, in other xenobiotic speciation, and as in-dicators of biological processes in waterbodies.In the early period of humus investigations, AHShas been characterized by spectrophotometricaldetermination of water color, chemical oxygendemand (COD), and biological oxygen demand(BOD). Improved instrumental techniques andorganic carbon analysers allows the evaluation ofthe concentrations of humic substances as organiccarbon content (TOC, total organic carbon, DOC,dissolved organic carbon, POC, particulate or-ganic carbon) in waters. Contemporary recom-mended methods are based on such approaches.

O

O

O

HOOC

O

HO

O O

C H3C OOH

C OOH

O

A

OO C OOH

O

O

OH

O

HOOC OHO

O

C OOH

COOH

OH

B

O

OO COOH

C OOH

O OH C = O

OH

O

HOOC OH

OC OOH

C

FIGURE 3. Three structural models (A-C) of fulvicacid molecules from the Suwannee River (Leenheer etal., 1995).

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However, all of these analytical methods provideonly indirect measures of humus concentration inwater.

The problems of analytical determination ofconcentrations of aquatic humic substances in

natural waters are closely related to their proper-ties and the lack of strict boundaries betweenhumic and nonhumic substances. The fundamen-tal properties that may be used for analytical de-termination of AHS are (Table 1):

TABLE 1

Major Functional Groups and Building Blocks of Aquatic Humic and Associated Substances

Loosely bound substances

Metal ions: (Ca+2, Fe+3, Na+ etc.), adsorbed inorganic particulate materials, low molecularweight organic substances

Functional groups

–COOH Carboxyl groups

–OH Hydroxylgroups

>C=O Carbonylgroups

–O– Ether groups

–OCH3 Methoxylgroups

–CH3 Methylgroups

–C(N Cyanogroups

–SH, –SO3H Sulphur containing groups

–NH2, >N– Amino groups

Major structures of aquatic humic substances

–CH2–CH2–, –CH = CH–, –CH2 –NH2 Aliphatic structuresO H

CO O HPhenoles, phenolecarboxylic acids

COOH

COOHBenzenecarboxylic acids

Aromatic and polyaromatic structures

O O Quinones

O

OO

O Oxygen containing heterocycles

NNNN Nitrogen containing heterocycles

S Sulphur containing heterocycles

Pentoses and hexoses Carbohydrates

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• Organic carbon as a building element of humicmatter (they are organic substances)

• Their acidic character (humic substances arepolycationites)

• Ability of humic substances to form insolublesalts with some cations

• The presence of chromophores and fluorophoresin their structure

• Presence of hydrophobic structural units in theirstructure

The most common method for the analysis ofhumic substances in surface waters is the determi-nation of organic carbon (COD, BOD, TOC, DOC,POC). This is a very powerful and widely usedmethod to study organic matter in natural waters(Datsko, 1959; Degens, 1982; Coel et al., 1984Agren, 1991), but it neglects the differences be-tween low molecular organic substances and hu-mic substances.

III. CONTEMPORARY ANALYTICALMETHODS OF HUMIC SUBSTANCES

The sorption of humic substances in theUV-Vis spectral region allows this characteristicand water color to be used for their analyticaldetermination. Water color has been determinedfor many years and hence may be used for trendanalysis of humus concentrations in surface wa-ters. Associated analytical problems are sensitivityand interference, particularly from iron and polyphe-nolic substances (lignin). A more powerful methodis the fluorimetric method, which allows the deter-mination of lignin sulfonates and humic substancessimultaneously in waters. The fluorimetric methodhas been used to measure fulvic acids in seawater,after preconcentration on DEAE cellulose and sub-sequent desorption and final fluorimetric determi-nation (Ivanova et al., 1986). Also, simultaneousabsorbance measurements at two wavelengths canbe used to determine humic acid and iron concen-trations in natural waters (Carpenter and Smith,1984). This method requires sample volumes ofless than 15 ml and is sensitive enough for directapplication to most natural waters (limit of detec-tion is 0.01 mg/l for humic acid).

To isolate humic matter from a pool of or-ganic and inorganic substances and to separate

humic acids from fulvic acids, chromatographicmethods are most successful. Their application isbased on the use of polymeric anionites (contain-ing tertiary amino groups or diethylamino-ethylgroups) or on the use of hydrophobic sor-bents (styrene-divinylbenzene or acrylic acidcopolymers). Functional groups in these sorbentsmay interact with carboxylgroups or hydrophobicstructural units in humic substances, allowing theirselective isolation from aquatic environments andtheir separation from accompanying substances.An example of this method is based on the isola-tion of humic substances from waters by theirchromatographic sorption on DEAE cellulose,elution precipitation of humic acids at pH 2, andspectrophotometric determination of both humicand fulvic acids (Krasiukov and Lapin, 1988).Nonionic macroreticular sorbents (Amberlite XADresins) have been proposed for analysis of humicsubstances (Sorouradin et al., 1993). Analyticaldetermination of humic substances has beenachieved by sorption and desorption with subse-quent spectrophotometric analysis. To achievequantitative desorption, elution of polymers withsodium dodecyl sulfate solution in strongly alka-line media was used.

Methods based on use of DEAE or XADsorbents for analytical determination of AHS arenow standard methods (Standard Methods, 1992).Aquatic humic substances are concentrated bycolumn chromatography on DEAE cellulose orXAD resin, then desorbed and measured as dis-solved organic carbon concentrations (Figure 4).The estimated detection limit is 1.1 mg/l for a 50-ml sample. A recommended analytical methodbased on the use of sorbents is included in theappendix.

Thin layer chromatography on silica gel plateshas also been used successfully to analyze humicsubstances in surface waters after their pre-con-centration on Amberlite XAD 4 column (Katalan-Macan et al., 1992).

Later developed method is based on the oxi-dation of humic acid with hydrogen peroxide inthe presence of formaldehyde in alkaline solution(Kitano et al., 1994). This method is sensitive andselective (detection limit 50 ppb and relative stan-dard deviation for five measurements 1.8%) forthe determination of HA in natural waters withlittle interference from substances common in

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natural waters. The method can also be realizedcontinuously. Humic acids in natural waters havebeen analysed also by chemiluminiscence pro-duced by the reaction between humic acid andNaBrO in alkaline solution (Hu et al., 1994).

Enzyme-linked immunoassay for the deter-mination of humic acids in natural waters hasbeen proposed (Ulrich et al., 1993) using humicacid coupled to snail Helix pomatia hemocyaninand a humic acid horseradish peroxidase conju-gate as a tracer. This detection method hasachieved high sensitivity (detection limit was es-timated to be 0.5 µg/l) and allowed to elucidatestructural differences among humic substances ofdifferent origin.

Because suspended particles in surface wa-ters are covered by humic substances, it is impor-tant determine their amount. This can be achievedby selective desorption of humic substances fromparticulate matter (Hiraide et al., 1988). It hasbeen found that the major part of metals on or insuspended particles exist as humic complexes(Hiraide, 1992).

IV. CONCLUSIONS

Unless substantial efforts to develop analyti-cal methods for determination of humic substancesin surface waters, the problem still is open andversatile, efficient, and unified methods areneeded. As the most promising approaches can beregarded use of chromatographic techniques, as

well as different approaches based on pre-concentraiton of humic substances.

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Carpenter, P.D., Smith, J.D. (1984) Simultaneous spectro-photometric determination of humic acid and iron inwater. Anal. Chim. Acta, 159, 299–308.

Coel, J.J., McDowell, W.H., Likens, G.E. (1984) Sourcesand molecular weight of “dissolved” organic carbon inan oligotrophic lake. Oikos, 42, 1–9.

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FIGURE 4. Sequence of steps for analysis of aquatic humic substances.

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methods for analysis of aquatic humic substances

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