environmental analysis of aliphatic carboxylic acids by ion-exclusion chromatography

Analytica Chimica Acta 465 (2002) 157–173

Review

Environmental analysis of aliphatic carboxylic acidsby ion-exclusion chromatography

Klaus Fischer∗University of Trier, FB VI, Geography/Geosciences, Analytical and Ecological Chemistry,

Universitätsring 15, D-54286 Trier, Germany

Accepted 6 March 2002

Abstract

During the last decades, ion-exclusion chromatography has been developed to one of the leading analytical techniques forthe separation and determination of short-chain aliphatic carboxylic acids in various matrices. Meanwhile, environmentalapplications including soil, air, ground water, drinking water, geological materials, sewage, waste and biomass are numerous.After a brief overview over actual research topics linked with the environmental occurrence of organic acids, the workingprinciples of ion-exclusion chromatography, etc. are outlined. Furthermore, methods for sample clean-up and solute enrichmentare detailed. Then a synopsis of environmental ion-exclusion chromatography applications of the last 10 years follows includinga tabulated compilation of experimental conditions used. Finally, benefits and shortcomings of the analytical methods areassessed under the viewpoint of their practical implication and actual trends are highlighted.© 2002 Elsevier Science B.V. All rights reserved.

Keywords: Aliphatic carboxylic acids; Volatile fatty acids; Ion-exclusion chromatography; Environmental analysis; Soil; Water; Air; Waste;Biomass residues

1. Introduction

The occurrence of aliphatic carboxylic acids inthe environment stems from a multitude of biolog-ical sources, geological process, (bio)geochemicalreactions and from anthropogenic emissions. Manyorganisms, including bacteria, fungi and algae as wellas higher plants and animals, excrete organic acidsinto their habitats. Additionally, the degradation andhumification of dead biomass releases significantamounts of organic acids. Important anthropogenicemission sources are residues and byproducts fromagriculture and food processing, sewages, organic

∗ Fax: +49-651-201-3617.E-mail address: [email protected] (K. Fischer).

wastes and various products of their technical treat-ment and disposal (e.g. sewage sludge and landfillleachates). Photochemical and biochemical transfor-mations of released hydrocarbons, biocides and ofother organic pollutants play a part in the generationof aliphatic acids in the environment.

As a consequence, organic acids are ubiquitous inthe ecosphere. Therefore, the environmental analy-sis of this class of compounds comprises almost allrelevant compartments and matrices including theenvironmental technical sphere.

Scientific questions addressed to the environmentaloccurrence and distribution of aliphatic carboxylicacids are numerous. Central topics are, amongstothers, their contribution to the organic carbonpool of various compartments, their integration into

0003-2670/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved.PII: S0003-2670(02)00204-0

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biogeochemical carbon cycles, their ecological func-tions and their possible interactions with metal ionsand minerals.

Further general aspects are distribution and trans-fer processes between different phases in environmen-tal compartments, biological sources, (bio)degradationand seasonal variations in the occurrence of organicacids and of their metal compounds[1–5].

In environmental analytics, as an applied scientificdiscipline, the development, adaptation and applica-tion of a selected analytical procedure is always aresponse to a specific scientific demand. The mainresearch topics in the field of environmental analyticsof aliphatic carboxylic acids, which are specifyingdifferent analytical tasks, objectives and strategies,are summarized inTable 1.

Principally various analytical techniques, e.g.ion-exchange chromatography[24,25], ion pairchromatography[26,27], gas chromatography (GC)[15,28,29]and capillary zone electrophoresis[30–32]are suited for the environmental analysis of aliphaticcarboxylic acids. The use of ion-exclusion chromatog-raphy for this analytical task goes back to the 1970s[33]. Arguments for the application of ion-exclusionchromatography are: (i) excellent suitability foraqueous matrices; (ii) no loss of very hydrophilicor volatile compounds; (iii) no thermal stress forthermolabile compounds; (iv) uncomplicated sam-ple pre-treatment, no pre-column derivatization re-quired; (v) almost no interferences with inorganicions, except from very weakly dissociated acids; (vi)combination with other chromatographic techniques,especially with ion-exchange chromatography, toachieve two-dimensional separations is possible[34].

This review pursues the purpose to provide anoverview over ion-exclusion chromatography ap-plications in the field of environmental analysis ofaliphatic carboxylic acids within the last decade, fo-cusing on the experimental conditions applied andon the analytical efficiency achieved. After a shortintroduction into the principles of ion-exclusion chro-matography, sample clean-up and solute enrichmentmethods are concerned. Then a synopsis of relevantion-exclusion chromatography applications follows.Finally advantages and disadvantages of the methodsare summarized, comparisons with alternative analyt-ical techniques are made and actual developments arediscussed.

2. Principles of ion-exclusion chromatography

Since several reviews have been published on ion-exclusion chromatography[35–42], this section is re-stricted to briefly summarize the ground-laying prin-ciples of ion-exclusion chromatography as far as theyare essential for the understanding of chromatographicprocesses involved in the separation of organic acids.

The characteristic feature of ion-exclusion chro-matography is that the sign of the electric charge ofthe dissociated functional groups on the stationaryphase (an ion-exchange resin) is the same as that ofthe (potential) ionic compounds to be separated. Itfollows that negatively charged ions, e.g. organic acidanions, are separated on cation-exchange resins func-tionalized with anionic groups, e.g. sulfonate and car-boxylate groups. A deciding factor is the establisheddissociation equilibrium between the neutral, undisso-ciated form and the corresponding anionic form of theacidic solute. Inter alia this equilibrium depends onthe acidity and activity of the analyte, on the protonactivity and electrolyte content of the mobile phaseand on its dielectric constant. The more the analytesare dissociated the more they are excluded from thestationary phase by electrostatic repulsion (“Donnanexclusion”). The undissociated analyte species canpenetrate through the Donnan membrane and interactwith the stationary phase, causing retardation relatedto the flow of eluent molecules.

It is supposed that retention is achieved by the fol-lowing mechanisms depending on analyte structureand properties of the stationary phase:

• hydrophobic (“reversed phase”) interactions (ad-sorption);

• polar interactions such as hydrogen bonding(“normal phase”);

• �–� electron interactions.

Besides, electrostatic exclusion size exclusion con-tributes to analyte separation.

Since several mechanisms take place in the separa-tion of the solutes (“mixed-mode” separation process),their relative contributions to the overall retentionprocess depend on the structural and functional prop-erties of the chromatographic resin. The strength ofDonnan exclusion effects increases with increasingion-exchange capacity and cross-linking. Microp-orous beads are characterized by low porosity, lack of

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internal pore structure and low surface areas, henceretention of solute molecules by adsorptive interac-tions and Van der Waals forces is less pronounced.Using an unbuffered mobile phase, it was foundthat retention of monocarboxylic acids by hydropho-bic adsorption increases following the sequence:sulfonated silica gel< polymethacrylate resin<poly(styrene–divinylbenzene) stationary phase[40].

Hydrogen bonding occurs between the functionalresin groups and hydroxy groups or undissociated acidgroups of the solutes. Weak carboxylate groups, e.g. asconstituents of methacrylate resins, are suited for hy-drogen bonding especially. Consequently, cross-linkedpoly(styrene–methacrylate–divinylbenzene) co-poly-mers functionalized with sulfonate and carboxylategroups show unique features in the separation of hy-droxycarboxylic and Krebs cycle acids[43,44]. Thearomatic units of polymeric resins are able to exertstrong �–� electron interactions with aromatic andunsaturated solutes.

Several criteria, such as acidity, polarity, solvationproperties and compatibility with detection mode(e.g. UV transparency, low conductance, formation ofweakly dissociated ion pairs with suppressor reagentand volatility) regulate the choice of the mobile phase.Mostly eluents prepared from strong mineral or or-ganic acids (HCl, H2SO4 and aliphatic sulfonic acids)were employed. The application of moderate to weakorganic acids (perfluorinated aliphatic carboxylicacids, aromatic acids) is also common. It could also bedemonstrated, that principally non-acidic hydrophiliceluents, such as polyols, sugars and (poly)alcoholsare suited for the separation of monocarboxylic acids[45–47].

Measurement of direct UV absorbance of wave-lengths between 200 and 220 nm and of analyte con-ductance after passing a suppressor column are byfar the most employed detection modes in environ-mental analysis of aliphatic acids with ion-exclusionchromatography. Preferably HCl, heptafluorobutanoicacid (HpFBA), tridecafluoroheptanoic acid (TDFA)and aliphatic sulfonic acids (methylsulfonic acid(MSA), hexylsulfonic acid (HSA) and octylsulfonicacid (OSA)[48]) are used for suppressed conductivitydetection. Usually, the suppressor reagent is tetra-butylammonium hydroxide (TBAOH). At constantflow and concentration of the acidic eluents and ofthe suppressor reagent, the background conductance

decreases following the sequence (reverse increase ofthe detection sensitivity): HCl> MSA > HSA >

OSA ≈ TDFA.For non-suppressed conductivity detection, eluents

made from aqueous dilutions of weak aromatic acids(benzoic acid, phthalic acid) are preferred.

Direct UV detection is usually accomplished withH2SO4 as eluent. In a few cases, MSA is used[49],whereas aromatic acids are preferred for indirect UVdetection[50].

3. Sample clean-up and analyte enrichment

Since environmental samples from different originsare diverse in their physical and chemical properties,various matrix specific sample pre-treatment and ana-lyte enrichment methods have been developed.

Frequently sample pre-treatment methods forion-exclusion chromatography analysis of aliphaticacids are concerned with the following issues:

• removal of particles and microorganisms (samplesterilization);

• removal of high molecular weight molecules andof strongly adsorbing compounds, e.g. humic andfulvic acids, polyphenols, etc.;

• improvement of the detection of moderate organicacids, i.e. oxalic acid, by reduction of the concen-tration of inorganic ions;

• carbonate removal.

In a few investigations, metal ions and/or aminoacids are eliminated from the sample prior to analysisadditionally[8,51–53].

3.1. Soil solutions and related matrices

Prevailing sampling techniques and devices to ob-tain soil solutions are centrifugation and collectionthrough suction units, i.e. suction cups and freelydraining lysimeters. Effects of the sampling methodon the sample composition, which will not be dis-cussed here further, are often critically mentioned inthe literature[54–56].

In some cases sample pre-treatment was restrictedto membrane filtration (method SP1), applying filterdisks made from cellulose acetate or nylon. Pore diam-eters between 0.45 and 0.2�m were selected[57–61].

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Several times the extraction of organic acids withorganic solvents (ethylacetate or diethylether) is de-scribed (method SP2), sometimes preceded by acoarse fractionation of the dissolved organic carbon(DOC) content into fractions differing in acid/baseproperties and in polarity[62]. The organic extractof the acidified (pH 2.0–3.0) soil water is evaporatedto dryness under vacuum and the solid residue isre-dissolved either in the ion-exclusion chromatog-raphy eluent or in distilled water. Simultaneouslythis treatment facilitates the enrichment of analytes[63–66].

Various authors removed interfering organic com-pounds by ultrafiltration through 1 or 10 kDa filters,e.g. Diaflo YM-1 or -10 (Amicon) under pressure withnitrogen gas[67,68], partially followed by an elimi-nation of metal ions using cation-exchange columnsor cartridges (method SP3)[8,51,53,68].

A common sample pre-treatment method, suitedfor the enrichment of carboxylic acids under certainconditions too, is the application of anion-exchangeresins or cartridges (method SP4), such as Bio-RadAG1-X8 [69–72], Dowex-2[68], Amberlite IRA-400(poly(styrene–divinylbenzene) matrix with quaternaryammonium group)[73], Waters IC anion concentratorcolumn (methacrylate resin)[74], DEAE-cellulose[67], Bond Elut SAX cartridges (trimethylpropylam-monium group with Cl− as counter ion)[75] or silicagel cartridges chemically modified with quaternaryammonium functions[76]. The majority of the ex-change materials are supplied in the hydroxide formand transformed in the formate or bicarbonate formprior to use. Usually a quantitative binding of thecarboxylic acids to the anion-exchangers is intended,however, one application aimed at a reduction of theconcentration of inorganic anions without impact onthe concentration level of carboxylate ions[70]. Inthis case, the selection of appropriate reaction con-ditions was controlled by a comparison of analytedetection signals from spiked and non-spiked soil andstandard solutions.

Despite the principally existing potential for ana-lyte enrichment, anion-exchange resins were appliedfor DOC fractionation and sample purification mainly.Elution of the analytes is accomplished by passing so-lutions of HCl (0.5 or 1.0 M), H2S04 (0.5 M) or formicacid (2.0 or 6.0 M) through the resin bed, occasionallyfollowed by evaporation to dryness and re-dissolution

of the residue in the ion-exclusion chromatographyeluent.

The efficiency of carboxylate enrichment byanion-exchange resins depends strongly on samplecomposition. Usually, anions of strong mineral acidsare bound preferentially. Hence, the total exchangecapacity which has to be provided for the treatmentof a given sample volume must be carefully adaptedto the total amount of exchangeable charges or, if acertain selectivity is required, to the pH-dependent ra-tio of charges borne by organic and inorganic anions.Haddad and Jackson recommend analyte enrichmentby anion-exchangers only for solutions character-ized by low levels of inorganic anions, e.g. polar icesamples[74].

3.2. Waste related samples and biomass hydrolysates

Typically, the sample matrix contains high salt con-centrations, moderate to high concentrations of variousgroups of low molecular weight organic compounds(aliphatic and aromatic carboxylic acids, amino acids,sugars, amines, alcohols, aldehydes, etc.) and signif-icant amounts of polymers (polysaccharides) and ofother high molecular weight components (proteins,polyphenols, humic acids and humin-like compounds,etc.). Depending on sample origin significant amountsof xenobiotics (phthalates, alkylbenzolsulfonates,non-ionic tensides, etc.) are present as well.

Sample pre-treatment is directed towards reductionof salt concentrations, prevention of column foulingand carbonate elimination. Analyte enrichment is nota task.

An usual treatment scheme (method SP5) com-prises dilution, centrifugation or coarse filtrationthrough filter papers or glass fiber filters, filtrationthrough membrane filters (<0.45�m) and removalof strongly adsorbing compounds by SPE cartridges,e.g. Dionex On-Guard-P (polyvinylpyrrolidone resin)[77]. Carbonate elimination is accomplished by acid-ification of the sample, preferentially by addition ofthe acidic ion-exclusion chromatography eluent andsubsequent ultrasonic agitation[78].

3.3. Air (gas phase, particles and precipitation)

For sampling of volatile fatty acids in the atmo-spheric gas phase several specific techniques were


developed whose common principle is to induce andto enhance analyte transfer from the gas phase intoa liquid (aqueous) phase (method SP6). The liquidphase can have the physical form of a voluminoussolution (connection of several impingers in series[79]), of a film (annular denuder technique[80]) ormist (aqueous scrubber technique applying a mistchamber[81]). To scavenge the organic acids ef-fectively a base (KOH or Na2CO3) is added to theliquid. The various sampling techniques are referredand compared in detail by Keene et al.[82].

Alternatively organic acids can be trapped from airby a C 18 cartridge coated with a KOH/methanol film.Analyte elution is achieved with water[83].

In combination with the impinger sampling pro-cedure, a special analyte enrichment system wasdeveloped, connected on-line to a chromatographicsystem[79]. Organic acids were enriched by a selec-tive permeation through a liquid membrane (porousPTFE membrane, pore size 0.2�m, impregnated witha solution of tri-n-octylphosphine oxide in di-n-hexylether). Details of the liquid membrane extractiontechnique are given in[84,85]. The same enrich-ment method was applied for the determination ofcarboxylic acids in soil solutions by ion-exchangechromatography[86,87].

In order to measure aliphatic acids in precipi-tation, an on-line pre-concentration technique wasemployed analogously to that described as “methodSP4”. An anion concentration column filled with low-capacity anion-exchange resin obtained from Sykamor from Dionex (HPIC-AG1, exchange capacity30 meq. g−1, particle size 25�m) was mounted be-tween the sample loop and the analytical column[88].The analytes were recovered by flushing the concen-tration column with the ion-exclusion chromatogra-phy eluent (10 mM H2SO4) and directly transferredto the exclusion column.

4. Synopsis of environmental ion-exclusionchromatography applications

Environmental ion-exclusion chromatography ap-plications of the last 10 years (some pioneering olderpublications are considered as well) are covered andscreened with regard to the detailed analytical instru-mentation and experimental conditions. The analytical

data are indexed (categories: sample type, samplepreparation procedures, column type, eluent compo-sition and flow rate, column temperature, detectionmode, detected solutes), presented inTable 2 andarranged according to the sampled environmentalcompartments and environmental technical materials.The detected analytes were marked by a numericalcode and listed inTable 3.

The gained overview of experimental conditionsreflects to a certain extent several shortcomings oneencounters searching analytical data. For instance,occasionally the analytical equipment and the chro-matographic conditions are incompletely or impre-cisely described. Often only the deduced analyticalresults are given without presenting chromatograms,calibration functions or statistical data evaluation. Onthe other hand, this might be an indication for diverseanalytical quality standards and traditions of differentscientific subject areas.

As will be gathered fromTable 2, in total 36aliphatic carboxylic acids were detected by ion-exclusion chromatography in various environmentalmatrices. The diversity of detected compounds wasrelatively low in the air samples, but some of the re-lated studies were intentionally restricted to measureformic and acetic acids. The group of investigatedcompounds comprises monocarboxylic acids up to sixcarbon atoms and multifunctional acids up to sevencarbon atoms.

In a few cases the missing detection of analytes withhigher carbon numbers might belong to the inherentlimitations of the analytical technique (strong retar-dation of these compounds, relatively low detectionsensitivities, depending on the detection mode). Thecomparison with results of other analytical methods,e.g. GC, applied for the same matrices (sometimesfor an identical sample) provides some evidence,that aliphatic acids with longer carbon chains do notcontribute to the sample composition to a significantextend normally[6,65,89–91].

The concentrations of aliphatic carboxylic acidsdetermined by ion-exclusion chromatography in thedifferent environmental matrices span an enormousrange. Concentrations of gaseous compounds rangefrom 0.1 ppb (remote “pristine” atmospheres) to40 ppb (urban air). For organic acids in airborneaerosols, concentrations between 20 and 400 ng m−3

were reported. Depending on the specific properties

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Table 3List of detected aliphatic carboxylic acids

Code Acid Code Acid

1 Formic 5-1 n-Valeric2-1 Acetic 5-2 Isovaleric2-2 Oxalic 5-3 Glutaric2-3 Glycolic 5-4 2-Oxoglutaric2-4 Glyoxylic 5-5 Methylsuccinic3-1 Propionic 6-1 n-Hexanoic3-2 Malonic 6-2 Isohexanoic3-3 Lactic 6-3 Adipic3-4 Glyceric 6-4 Citric3-5 Pyruvic 6-5 Isocitric4-1 n-Butyric 6-6 cis-Aconitic4-2 Isobutyric 6-7 trans-Aconitic4-3 Succinic 6-8 Gluconic4-4 Malic 6-9 Glucaric4-5 Tartaric 6-10 Isosaccharinic4-6 Threonic 7-1 Quinic4-7 Maleic 7-2 Shikimic4-8 Fumaric4-9 Methylmalonic

of soil sample and on the sampling or leaching tech-nique used, individual acids in soil centrifugates andsoil leachates were found in concentrations between0.1 and 100�M. Concentrations up to 75 mM weredetermined for volatile fatty acids in the seepage ofdumped sewage sludge and compost piles. Lactic acidreaches concentrations of about 0.5 M in grass silagejuices.

As Table 2reveals, most of the environmental anal-ysis was done using UV detection at wavelengths ofor around 210 nm. Suppressed conductivity detectionwas also frequently used, but not for soil analysis,whereas RI detection was seldom applied. ApplyingUV detection, unsaturated compounds and compoundswith additional chromophoric groups have essentiallylower detection limits than monofunctional carboxylicacids. According toTable 4, the detection limits offumaric, shikimic, aconitic and oxalic acids are about20–1000-fold lower than the simple fatty acids. In con-trast to this, conductivity detection offers a much moreuniform sensitivity. Here, the detector response in-creases with increasing number of carboxylic groups.An often obeyed secondary rule is the increase ofthe detection sensitivity with increasing analyte acid-ity. For instance, the detection limits of tricarboxylicacids, i.e. citric and isocitric acid, are about 4–6-foldlower than monocarboxylic acids[77]. Some authors

have noticed that suppressed conductivity detection of-fers a higher sensitivity for monofunctional carboxylicacids than UV absorbance does at around 210 nm[92].Sometimes difficulties were stated arising from thelarge difference in UV absorption of carboxylic acidsin such a way that the very intense detection signalsof unsaturated compounds obscured the peaks of acidswith lower absorption coefficients[72]. However, UVdetection was found advantageous investigating pro-pionic acid in carbonate containing samples, becausecarbonate does not absorb UV radiation from the rel-evant spectral range, excluding interferences with thepropionate peak[92]. As a consequence, some investi-gations were conducted connecting a suppressed con-ductivity detector in tandem with an UV detector[92]or performing parallel chromatographic runs applyingboth detectors in different ion-exclusion chromatogra-phy systems[93–95].

Analyte identification is usually achieved inion-exclusion chromatography by comparison withretention times of standard compounds. This methodrequires an experience-based hypothesis regardingthe probable sample composition. Scaled by the ef-fective plate numbers obtainable for volatile fattyacids under optimized conditions, the separation effi-ciency of ion-exclusion chromatography columns isby far less than capillary columns in GC and evenless than advanced anion-exchange columns. Reflect-ing the limited separation efficiency of ion-exclusionchromatography columns, one has to expect insuf-ficient resolution and co-elution of various groupsof analytes with similar chromatographic behavior,precluding an unambiguous substance identificationvia retention time match. In this respect, it is surpris-ing that the majority of the included environmentalapplications of ion-exclusion chromatography doesnot face this problem. The composition of co-elutingsubstance groups depends on column selectivity, chro-matographic conditions and on the concentration ofinterfering, non-target matrix components (inorganicanions, unresolved DOC background, etc.). Severaltimes an insufficient resolution of the following sub-stance combinations was reported to create analyticalproblems:

• combinations of compounds having low capac-ity factors (k′ < 0.5) under selected chromato-graphic conditions, e.g. oxalic, dihydroxymalonic,

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hydroxymalonic, maleic, 2-ketoglutaric acids (sep-aration from each other and from the void volume);

• fumaric/acetic acids;• succinic/glycolic acids.

Several strategies are followed to increase thecertainty of analyte identification, to improve chro-matographic resolution or the enlarge the analyticalinformation yield.

4.1. Control of ion-exclusion chromatographyresults by independent analytical methods

Presumably due to the considerable additional ex-penditure required this strategy is not often pursuedand marks sometimes a change between the preferredanalytical techniques, e.g. switch from ion-exclusionchromatography to GC applications[72].

Two applications utilize enzymatic conversionfor substance identification[67,96]. Amongst othersglyceric, fumaric and malic acids were enzymaticallytransformed and the reaction solutions were ana-lyzed by ion-exclusion chromatography again[67].Other techniques consulted include capillary zoneelectrophoresis[51], GC [65,72], high-performanceanion-exchange chromatography (HPAEC)[81] anda colorimetric assay for citric acid[65].

Fig. 1. Organic acids in a process water from a biowaste fermentation plant. Analytical conditions: Merck Polyspher OA-HY column andguard column; separation temperature, 45◦C; eluent, 5 mM H2SO4; flow, 0.5 ml min−1; UV detection, 210 nm. Peaks (acids): (1) void; (2)oxalic; (3) pyruvic; (4) glyoxylic; (5) glyceric; (6) unknown; (7) lactic; (8) formic; (9) acetic; (10) propionic; (11 and 12) unknown; (13)n-butyric; (14) isovaleric; (15 and 16) unknown. From[93], with permission.

4.2. Sample clean-up to remove interferingcompounds

Improved analyte resolution was achieved aftertreatment of samples with anion-exchange resins(method SP4, seeSection 3.1and[70,75]).

As Figs. 1 and 2reveal, improvement of analyteseparation due to the elimination of interfering matrixcomponents can follow from a sample fractionationby means of gel permeation chromatography.Fig. 1displays a chromatogram of a process water from anaerobic tank of a biowaste fermentation plant prior toGPC fractionation[93]. Sample pre-treatment was ac-cording to method SP5. A high content of UV absorb-ing matrix compounds leads to a strongly tailing voidpeak. Oxalic acid is almost completely covered bythis peak.Fig. 2 shows a chromatogram recorded un-der identical ion-exclusion chromatography conditionsfrom the main GPC fraction containing short-chainaliphatic acids[97]. GPC separation was achieved witha Sephadex gel G-10-120 and a KCl eluent (0.01 M).The void peak is now drastically reduced and its tailingis eliminated. Oxalic acid is detectable without inter-ferences from matrix components and all analytes arebaseline resolved. Since GPC fractionation is accom-panied by sample dilution the concentrations of sometrace compounds are now below the detection limits.


Fig. 2. Organic acids in a GPC fraction of a process water from a biowaste fermentation plant. Analytical conditions: GPC Sephadex gelG-10-120; eluent, 0.01 M KCl. Ion-exclusion chromatography as forFig. 1. Peaks (acids): (1) void; (2) oxalic; (3) pyruvic; (4) lactic; (5)formic; (6) acetic; (7) propionic. From[97], with permission.

4.3. Multiplicate analysis at differentchromatographic conditions

Eluent pH and column temperature are decidingfactors regulating column selectivity, efficiency andanalyte retention[44,92,98,99]. For the separation ofacidic soil leachates and centrifugates on a HamiltonPRP-X 300 column, H2SO4 was applied as eluent inconcentrations of 0.5 mM (pH 3.0) and 10 mM (pH1.7) [57].

Two concentrations of HpFBA (pH 3.1, 0.9 mM;pH 2.5, 3.2 mM) were adjusted for the separation oforganic acids in silage effluents on a Dionex IonPacHPICE-AS5 column at 10◦C [77]. Overall better peakshapes and detection sensitivities were reached at pH3.1, however, for the separation of tartaric, formic andmalonic acids a lower eluent pH was necessary.

Duplicate analysis of soil centrifugates were per-formed on a Shimadzu SCR-102H column at tempera-tures combinations of 30/60◦C[51] and 30/70◦C[53],respectively. Temperatures of 60 and 70◦C resulted ina better separation of citric, lactic and shikimic acids.

A simultaneous variation of eluent pH and columntemperature was proved to be advantageous for thedetermination of sugar acids (glyceric, threonic, glu-conic and glucaric acids) and of other carboxylic acidsin hydrolysates from carbohydrate containing residues[22].

Various landfill leachates and similar samples wereinvestigated applying two ion-exclusion chromatog-raphy systems, differing in the chromatographic se-lectivity and in the detection mode mainly[93,94].Chromatograms were recorded under variation of thecolumn temperature and of the eluent pH (Figs. 3 and4). Due to the variant analyte retention and elutionsequences offered by both ion-exclusion chromatog-raphy systems and separation conditions, complemen-tary chromatographic informations were obtained,increasing both the number of separated compoundsand the certainty of their identification.

A few environmental applications struggle with UVdetection limits of volatile fatty acids, di- and tricar-boxylic acids ascertained too high for their specificpurposes (investigation of soil waters and precipi-tation). With enrichment on an anion concentrationcolumn, detection limits of 0.1–0.4�M or lowerwere reached for glyoxylic, glycolic, formic, aceticand propionic acids[88]. Applying the membraneenrichment technique, detection limits of 0.5�M forpyruvic, malic, lactic and butyric acids and of 0.1�Mfor formic acid were attained[79].

Calibration of organic acids in the concentrationrange between 0.01 and 1 mM (UV detection) or2.0 and 200 mg l−1 (suppressed conductivity detec-tion) was found to yield functions of high linearity[100,101].


Fig. 3. Organic acids in a compost seepage. Analytical conditions as forFig. 1. Peaks (acids): (1) void; (2) pyruvic; (3) glyoxylic; (4)glyceric; (5) lactic and succinic; (6) glycolic; (7) acetic and glutaric; (8) adipic; (9) propionic; (10 and 11) unknown; (12) isobutyric; (13)n-butyric; (14) isovaleric; (15 and 16) unknown; (17)n-valeric; (18 and 19) unknown. From[94], with permission.

The quantitation of standard compounds at concen-tration levels of 10.0 or 50.0 mg l−1 led to relativestandard deviations (R.S.D.) of 2% or less (n = 3 or6) typically [78,101].

RSD values of the determination of citric, mal-onic, formic and succinic acids in soil centrifugates at

Fig. 4. Organic acids in a compost seepage. Analytical conditions as forFig. 1, except: eluent, 50 mM H2SO4; column temperature, 10◦C.Peaks (acids): (1) void; (2) pyruvic; (3) glyceric; (4) glycolic; (5) succinic and lactic; (6) acetic; (7) propionic and glutaric; (8) isobutyric;(9) n-butyric; (10–12) unknown. From[94], with permission.

concentrations below 0.1 mM were determined torange between 3.1 and 7.5% (n = 5) [70].

Recoveries of various organic acids in soil solu-tions after spiking (concentration increases of 0.1 or0.01 mM in the case of fumaric and shikimic acids)were evaluated to span from 89 to 102%[51]. The


relatively low recovery of oxalic acid (89%) was at-tributed to its incomplete separation from the voidpeak. Recoveries of formic, acetic, propionic, butyricand lactic acids at the 20 mg l−1 level in environmentalmicrocosm samples were between 80% (butyric acid)and 102% (formic acid)[101].

5. Assessment of the performance ofenvironmental ion-exclusion chromatographyapplications and actual analytical trends

As it was to be expected most of the practitioners as-sessed ion-exclusion chromatography as a reliable andefficient method well suited for their analytical tasks.The main advantages offered by this method have beenquoted in the introduction section already. Some otherpractical benefits were stated by several authors suchas low susceptibility to column fouling and to matrixinterferences even in contact with highly loaded sam-ples, i.e. sewage, high analytical precision and highreproducibility of quantitative data, calculated on thebasis of linear calibration functions.

It is obvious that the evaluation of environmentalanalytical methods are closely related to the researchobjectives pursued, the range and diversity of com-pounds to be detected simultaneously, the required de-tection limits and the physical and chemical sampleproperties. As already mentioned, repeatedly stateddrawbacks are insufficient peak resolution, very lowcapacity factors of moderate organic acids and incom-plete separation of these acids from the void peak,inadequate detection limits and low peak capacities.To overcome these difficulties, mostly substantial im-provements of the analytical procedure were achieved,e.g. careful optimization and combination of chro-matographic conditions, specific sample clean-up andanalyte enrichment, control by independent analyticaltechniques, etc. In a few cases, it was decided to re-place ion-exclusion chromatography by GC. Despiteoverall satisfying results achieved with the GC meth-ods selected, significant losses of non-volatile acids(oxalic and citric) and of volatile acids (acetic, pro-pionic and butyric acids) during sample preparation(extraction with organic solvents or enrichment onanion-exchange membranes) were ascertained[65,72].

Since a few years, HPAEC is on the way to competesuccessfully with ion-exclusion chromatography for

organic acid analysis. An increasing number of appli-cations for soil[86,87,113–116], air [17,18,117,118],surface and drinking waters[119] and for other en-vironmental matrices[19,24,120–123]is published.Technological development and commercializationof new anion-exchange materials is considerablyfaster than ion-exclusion chromatography resins andtheir selectivity for organic acid analysis was en-hanced in the last years. Modern anion-exchangepackings offer high column efficiency and the sep-aration of mono- and dicarboxylic acids togetherwith inorganic ions is possible. Due to the combi-nation of highly efficient stationary phases, smallercolumn diameter and very efficient suppressor re-generation techniques, usually the ion-exchangemode provides better peak shape and lower back-ground conductance than ion-exclusion chromatog-raphy, yielding higher detection sensitivities[122].A special advantage of the anion-exchange materi-als is their capability of gradient elution, combinedwith complete solvent compatibility. This enhanceschromatographic versatility and increases peak ca-pacity.

In view of this situation, the future importance ofion-exclusion chromatography in the field of organicacid analysis seems to depend on the ability to re-alize its inherent potentials and to evolve a new andup-to-date column generation. Briefly some possiblestarting points are outlined.

(a) As shown by Morris and Fritz, macroporous resinsprovide an interesting alternative to “classical”gel-type columns[45]. Separation speed is in-creased, column dimensions are reduced and gra-dient elutions with organic solvents is possible.Adding a chemical moderator to the eluent, theresin properties are altered to match the separa-tion conditions required. A complementary strat-egy might be to create the structural prerequisitesto accentuate and to fortify the “mixed-mode” sep-aration mechanism of ion-exclusion chromatogra-phy. Conceivable is a dynamic regulation of thecation-exchange capacity of the column packingsor the introduction of additional polar groups en-hancing “normal phase” interactions.

(b) Nowadays hyphenation with mass spectrometry isa “must”. Actually several approaches towards thisgoal are followed[123,124].


(c) Several attempts to increase the sensitivity ofconductimetric detection are promising. Usingnon-acidic eluents containing alcohols, sugarsor polyols, low background conductivity andsub-micromolar detection limits were reached forhomologous fatty acids[45–47].

These potentials, together with the characteristicfeatures of ion-exclusion chromatography, may lead toa situation where the recently stated “ongoing replace-ment of high-performance ion-exclusion chromatog-raphy by HPAEC”[25] ends up with a new allocationof tasks between these related chromatographic tech-niques.

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environmental analysis of aliphatic carboxylic acids by ion-exclusion chromatography

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