leachates, or wastewater) are also stored at sub-zero temperatures. , , ,
Sample preparation procedures are determined by target analytes, their concentrations, the sample matrix, and the analytical instrumentation used for measurement. Procedures can range from a simple filtration to a sequence of steps which may include filtration, centrifugation, extraction, concentration, removal of interfering compounds, and derivatization. Simple uncomplicated sample preparation procedures are preferred since they are less prone to errors, less susceptible to contamination, and less time consuming. Sample preparation can be quite labor-intensive although on-line procedures have also been developed.42,107,108
Samples with high solids contents such as, wastewater or landfill leachates are often centrifuged before the supernatant is processed further.29,90 - 92 Soil solutions are obtained most commonly by centrifugation of moist soil, usually followed by filtration and sometimes further clean-up steps.110-113,189 Methods involving direct injections of aqueous samples into AEC, IEC, or GC require the filtration of turbid samples, usually through filters with a standard pore size of 0.45 /Am.70 For some samples such as ultraclean water or drinking water this step may be omitted.18,42,43,86 Polycarbonate filters and cellulose acetate filters may be used for filtration of samples with low organic acids concentrations since these only leach moderate amounts of organic acids which can be removed by flushing with deionized water (100 ml for polycarbonate, 500 ml for cellulose acetate) prior to sample filtration.103 Glass fiber filters are not recommended since these release high concentrations of organic acids and colloids.103
2. Extraction/Concentration/Clean up
Extractions are employed either to concentrate organic acids, to remove interfering compounds, or to obtain solutions of organic acids from sampling devices such as air filters.
Air sampling involves trapping of organic acids from the gaseous phase, the aqueous phase, and from airborne particulate matter. Different devices allow for the collection of organic acids either separated out by phase or as a total of all phases combined. If alkaline filters or denuders are used as the main trapping device, sample preparation consists in extraction with either deionized water or eluent followed by preservation.55,60 Filters which are used to collect particulate organic acids prior to sampling the gaseous and aqueous phases may also be extracted by first wetting them with methanol and then extracting them with either deionized water or eluent.60 Several applications use ultrasound for enhancing the extraction of organic acids from particulates trapped on filters.61,63 However, it is reported that extended use of ultrasound may lead to analytical errors and simply soaking filters in water is preferred for organic acids extraction.58,62
A concentration step may be required for sample matrices with relatively low organic acid concentrations such as rain, drinking water, or seawater. Different approaches to concentrate samples have been taken depending on sample matrix, the specific organic acids to be analyzed, and analytical instrumentation.
When using AEC most aqueous matrices can be measured directly.38'42_44'68'70_72'86 In cases where concentration is required, a concentrator column, usually an anion exchange resin, may be used on-line. This approach works well for samples with low inorganic anion concentrations6,18,84 but problems arise (e.g., breakthrough) when even moderate concentrations of inorganic anions are present (Section II.C.2.a). An alternative for increasing method sensitivity in AEC is large volume
injections of up to 1 ml which rely on the so-called "relaunch" effect (Section II.C.2.a), , , meaning that in high capacity columns, analytes are collected as a relatively small sample band at the start of the column during injection, resulting in chromatograms without significant peak broadening.129-131
When using GC organic acids are typically transferred into an organic solvent followed by derivatization, which makes organic acids suitable for GC measurement. Exceptions are direct aqueous injections of water samples (e.g., wastewater) onto specialty GC columns (Section II.C.1). Transfer into organic solvent may be achieved by liquid-liquid extraction, evaporation at high pH and subsequent solvent addition, solid-phase extraction on anion exchange resins and subsequent elution with solvents, or aqueous derivatization followed by extraction (Section II.B.3.a).
Liquid-liquid extraction of short-chain organic acids, ketoacids, or dicarboxylic acids result in low and often unreproducible extraction yields due to the hydrophilic character of the analytes.132 However, some authors report reproducible results for short-chain acids at mg/l concentrations after liquid-liquid extraction at pH 2, although extraction yields remain low.48,119 Note also that organic solvents, namely diethylether, may be contaminated with organic acids.106 An unusual variation in liquid-liquid extraction is the use of tri-w-octylphosphine oxide (TOPO) in methyl-ieri-butylether (MtBE) to enhance extraction yields, e.g., of acrylic acid in marine waters50 and of organic acids in aqueous solutions obtained from air collection chambers.4 TOPO's very low solubility in water and its high polarity make it suitable for extraction of polar compounds. The extraction yield for acrylic acid was 40% and its detection limit after derivatization with pentafluorobenzyl bromide was estimated to be 3 nM.50
Organic acids including saturated and unsaturated mono- and dicarboxylic and also keto- and hydroxy acids may be concentrated by first adjusting the sample pH to approximately 9, thus
ensuring dissociation of the acids. Next, samples are concentrated by using a rotary evaporator and then blown off to dryness with nitrogen. The dry extract is redissolved in solvent and derivatized. When determining monocarboxylic acids in rain, good recoveries of 73 to 107% were reported.74
Solid-phase extraction on anion exchange resins is very rarely used.132 Recoveries for the organic acids may show great variability which may be caused by incomplete removal from the resin, especially at low concentrations, or it may be attributed to breakthrough of the carboxylate ions during the extraction process if the matrix contains significant amounts of inorganic anions.
A unique approach has been taken to concentrate nanomolar concentrations of organic acids in seawater by employing static or dynamic diffusion using membranes.16 Although time consuming, this procedure ensures removal of the majority of salts which might interfere with subsequent analysis if using, for example, AEC. However, in this application the concentrated acids have been measured by GC/FID (flame ionization detector) although AEC could have been employed equally as well.
Complex matrices such as soil solutions or landfill leachates usually require clean up where interfering compounds are removed prior to organic acid analysis. Procedures used include removal of humin-like substance by passing through special cartridges27,29 or precipitation after
acidification, 9 removal of carbonate by purging samples after their acidification, ' and distillation when determining VFA.54'118'121 Although pretreatment procedures are quite time consuming, only a few attempts have been made to automate these. Removal of Hg2+, which was initially added for preservation purposes, has been automated by putting a cation exchanger in-line between the autosampler and injection loop prior to AEC injection.42 Another very interesting online procedure for soil solutions combines clean up and extraction. Samples are first passed through a cation exchanger to trap metal ions and then through an anion exchanger after acidification to a pH value of 1 to remove interfering anions. This is followed by extraction of the organic acids into a TOPO-impregnated liquid membrane and their subsequent trapping in NaOH solution followed by AEC.108 Pretreatment and chromatographic analysis take 35 min per sample. This method has been applied successfully in a study investigating low molecular organic acids at /¿molar concentrations in soil solutions of beech forest.107
Derivatizations are used to make the analytes suitable for a chosen instrumentation and to increase the sensitivity of the overall method. Ideally, derivatizations should be specific to the compounds of interest. They should not result in any by-product formation and achieve reproducible, preferably high yields. The excess reagent should not interfere with the determination of the derivative and the procedure should be quick and easy to execute. Although many derivatizations are available for organic acids,133-136 only relatively few are used in the analysis of organic acids in environmental matrices.
a. Gas Chromatography (GC)
Many short-chain organic acids are thermostable and sufficiently volatile, thus fulfilling key requirements for GC measurement. However, their high polarity leads to severe peak tailing when employing standard capillary columns. Only when using specialty columns is it possible to analyze arange of these acids directly (Section II.C.1). Organic acids are therefore often derivatized to their less polar corresponding esters prior to their measurement on standard GC columns. Most derivatizations take place in nonaqueous solutions and organic acids have thus to be transferred into suitable solvents by either a concentration step or an extraction procedure (Section II.B.2) prior to their derivatization.
Esterification with alcohols using the Lewis catalyst BF3 is a well-established procedure.133 Organic acids are derivatized with BF3/butanol to their corresponding butylesters and then extracted into hexane. For dicarboxylic acids derivatizations, excess butanol was removed to avoid
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