Alternative Spectrofluorimetric Determination of Short-Chain Volatile

Anal. Chem. 2009, 81, 3063–3070

Alternative Spectrofluorimetric Determination of Short-Chain Volatile Fatty Acids in Aqueous Samples Fabien Robert-Peillard,† Edwin Palacio-Barco,† Yves Dudal,‡ Bruno Coulomb,† and Jean-Luc Boudenne*,† Laboratoire Chimie Provence-UMR 6264, Universite´ d’Aix-Marseille I, II, III-CNRS, 3 Place Victor Hugo, Case 29, 13331 Marseille Cedex 3, France, and INRA-SupAgro, Laboratoire Biogeóchimie du Sol et de la Rhizosphe`re-UMR1222, 2 Place Viala, Baˆtiment 12, 34080 Montpellier Cedex 2, France This paper presents a simple, rapid, and accurate method suitable for on-site measurement of short-chain volatile fatty acids (SCFA) in various environmental samples. This fluorimetric method involves a derivatization step of SCFA with N-(1-naphthyl)ethylenediamine (EDAN) and allows determination of acetic, butyric, propionic, valeric, lactic, succinic, and p-hydroxybenzoic acids in approximatively 10 min. To evaluate specificity and accuracy of the method, both laboratory-made waters and real samples ranging from wastewater plant and river to soils and composts have been tested. Good accuracy and correlation (r2 ) 0.9887) with HPIC determination have been obtained. The potential interference effect has been taken into account with compounds like humic substances, alcohols, amines, aldehydes, and metallic ions. This method seems thus well designed for the determination of total SCFA in waters, in the range 0.84-500 mg/L. Because this method seems well suited for following of anaerobic treatment, it has been calibrated versus acetic acid-equivalent. In anaerobic treatment processes (in sewage sludge treatment plants, in food industries, in industrial production of ethanol...), the short-chain volatile fatty acid (SCFA) concentration is usually the process control variable and is often determined by off-line procedures (sampling followed by laboratory analyses), inducing thus a delay for analysis results. Moreover, these procedures often involve chromatographic or potentiometric separation leading to nondirect, time-consuming, and material-demanding analyses that are not prone to fieldwork applications. Within the most reported methods are included ion-exclusion, liquid, and gas chromatography with sample pretreatment as distillation or headspace chromatography or with direct injection.1-4 In fermentation processes and anaerobic treatment of wastewaters or solid wastes, * Corresponding author. Phone +33-04-91106140. Fax +33-04-91106377. E-mail: [email protected]. † Laboratoire Chimie Provence-UMR 6264, Universite´ d’Aix-Marseille I, II, III-CNRS. ‡ INRA-SupAgro, Laboratoire Biogeóchimie du Sol et de la Rhizosphe`reUMR1222. (1) Larreta, J.; Vallejo, A.; Bilbao, U.; Usobiaga, A.; Arana, G.; Zuloaga, O. J. Sep. Sci. 2007, 30 (14), 2293–2304. (2) Lee, J. G.; Lee, C. G.; Kwag, J. J.; Rhee, M. S.; Buglass, A. J.; Lee, G. H. Chem. Anal. (Warsaw, Pol.) 2007, 52 (3), 411–422. 10.1021/ac802768u CCC: $40.75  2009 American Chemical Society Published on Web 03/24/2009

end-product (ethanol, methane...) or intermediate products (SCFA) inhibition occurs, which results in reduced process efficiency and stability; therefore control measures should be taken in continuous operation fermenters. End-product inhibition control cannot be achieved properly if on-line or on-site measurements of the product concentration are not performed.5 In this context, some researchers have proposed an adapted system for on-site analysis based on the titration method: the “5-point titration method” has been developped by Moosbrugger and co-workers,6,7 and an improvement of this method has then been proposed by Lahav et al.,8 involving eight pH observations. These titration methods suffer from many interferences, such as the presence of metallic ions which can form complexes with carboxylic groups,9 and are found to be either too elaborate or too approximate for general practical application. Recently, on-line systems allowing continuous determination of SCFA during anaerobic processes, by capillary gas chromatography10 or by headspace chromatography,11 have been proposed: these methods allow a qualitative and quantitative determination of the whole of SCFA present in reactors; Smyth et al.12 have proposed a rapid, direct, and low-cost method for the determination of volatile fatty esters and monoterpene alcohols in wines, by coupling near infrared spectroscopy and signal treatment. This last method allows a semiquantification of the whole esters or alcohols in samples. Our aim comes close to this last method: to design a simple, rapid, and low-cost method for the determination of total SCFA in aqueous samples, without chromatographic separation. Our aim is not to replace chromato(3) Siedlecka, E. M.; Kumirska, J.; Ossowski, T.; Glamowski, P.; Gołebiowski, M.; Gajdus, J.; Kaczynı`ski, Z.; Stepnowski, P. Pol. J. Environ. Stud. 2008, 17 (3), 351–356. (4) Martin Ruel, S.; Comeau, Y.; He´duit, A.; Deronzier, G.; Ginestet, P.; Audic, J.-M. Water Res. 2002, 36, 2337–2341. (5) Diamantis, V.; Melidis, P.; Aivasidis, A. Anal. Chim. Acta 2006, 573-574, 189–194. (6) Moosbrugger, R. E.; Wentzel, M. C.; Loewenthal, R. E.; Ekama, G. A.; Marais, G. V. R. Water SA 1993, 19 (1), 29–40. (7) Moosbrugger, R. E.; Wentzel, M. C.; Ekama, G. A.; Marais, G. V. R. Water Sci. Technol. 1993, 28 (2), 237–245. (8) Lahav, O.; Morgan, B. E.; Loewenthal, R. E. Environ. Sci. Technol. 2002, 36, 2736–2741. (9) Lahav, O.; Shlafman, E.; Cochva, M. Water SA 2005, 31 (4), 497–502. (10) Fischer, K. Anal. Chim. Acta 2002, 465, 157–173. (11) Boe, K.; Batstone, D. J.; Angelidaki, I. Biotechnol. Bioeng. 2007, 96 (4), 712–721. (12) Smyth, H. E.; Cozzolino, D.; Cynkar, W. U.; Dambergs, R. G.; Sefton, M.; Gishen, M. Anal. Bioanal. Chem. 2008, 390, 1911–1916.

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Scheme 1. Derivatization and Detection Pattern for Acetic Acida

a

1st Step: activation of carbon bearing -OH group with EDC. 2nd Step: fluorescent labelling with EDAN.

graphic methods, which will always be the reference methods if differentiation between SCFA is needed, but to propose a new approach, especially designed for determination of total SCFA with better accuracy at low concentrations than titrimetric methods, for field laboratory determinations. To achieve this goal, the intrinsic UV absorbance properties of the carboxyl group (around 205 nm) are clearly not suitable for a direct, sensitive, and specific detection. Therefore, most of detection methods of environmentally or physiologically important carboxylic acids are based on fluorescent tagging procedures, followed in all cases by chromatographic separation.13 In this paper, a direct spectrofluorimetric measurement of labeled carboxylic acids is proposed without any chromatographic separation needed. Numerous fluorescent labeling reagents can be found in the literature, depending on the reactive functional group, and are always coupled to chromatographic detection. The main classes of compounds are bromoalkyl,14 diazomethane,15 hydrazine,16 and amine17,18 reagents. Among these specific labels, amines are the most advantageous in terms of stability, reactivity, and solubility in aqueous medium. Conversion to amides bearing fluorophores can be achieved through condensation between the carboxyl moiety and the amine functional group. Both aliphatic (e.g., monodansyl cadaverine)19 and aromatic (e.g., Nile blue18 or 4-aminofluorescein)20 amines were previously used for labeling purposes, and the very first experimental observations (solubility, handling conditions, reactivity, commercial availability...) with (13) Toyo’oka, T. Anal. Chim. Acta 2002, 465, 111–130. (14) Takeshi, H.; Kamada, S.; Machida, M. Chem. Pharm. Bull. 1996, 44, 793– 799. (15) Schneede, J.; Mortensen, J. H.; Kvalheim, G.; Ueland, P. M. J. Chromatogr., A 1994, 669, 185–193. (16) Iwata, T.; Hirose, T.; Nakamura, M.; Yamaguchi, M. Analyst 1994, 119, 1747–1756. (17) Inoue, H.; Ikeno, M.; Ishii, Y.; Tsuruta, Y. J. Chromatogr., A 1998, 816, 137–143. (18) Rahavendran, S. V.; Karnes, H. T. Anal. Chem. 1997, 69, 3022–3027. (19) Lee, Y.-M.; Nakamura, H.; Nakajima, T. Anal. Sci. 1989, 5, 681–685. (20) Brando, T.; Pardin, C.; Prandi, J.; Puzo, G. J. Chromatogr., A 2002, 973, 203–210.

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these reagents prompted us to use N-(1-naphthyl)ethylenediamine (EDAN) for our labeling tests. EDAN is an aliphatic-type amine which has already been reported as an efficient labeling reagent by Kobayashi and Chiba,21 who performed derivatization and subsequent measurement of uronic acids and acidic polysaccharides. A prior activation step is generally needed to achieve efficient carboxylic acids derivatization, with various activating reagents such as carbodiimides,22 pyridinium salts,23 or disulfide reagents,24 depending on organic or aqueous reaction medium. N′-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC) seemed to be the reagent of choice for aqueous samples and was thus chosen for our study. The work presented here is a first though fundamental step to reach the ultimate goal of the designing of a new tool for onsite analysis. Indeed, the goal of this research is to conceive microplate assays for the detection of SCFA in various environmental samples. In this paper, the development and optimization of the derivatization and direct fluorimetric detection conditions of total small aliphatic carboxylic acids is described, according to experimental plan depicted in Scheme 1 (with an example of its application for acetic acid). Responses of various carboxylic acids as well as possible interferences caused by naturally occurring organic or mineral compounds found in real samples are also discussed. Validation and limitations of the method are finally assessed through selection and tests of various environmental samples (river, water-extracted soil and compost, water-diluted oxidized animal carcass residues, and sewage sludge samples). EXPERIMENTAL SECTION Materials, Reagent, and Instrumentation. EDC, EDAN, and methyl tert-butyl ether (MTBE) and propionic, butyric, valeric, (21) Kobayashi, M.; Chiba, Y. Anal. Biochem. 1994, 219, 189–194. (22) Sasamoto, K.; Ushijima, T.; Saito, M.; Ohkura, Y. Anal. Sci. 1996, 12, 189– 193. (23) Xiao-Lan, D.; Hua-Shan, Z.; Xiao-Feng, G.; Ying-Hua, D.; Hong, W. J. Chromatogr., A 2007, 1169, 77–85. (24) Narita, S.; Kitagawa, T. Anal. Sci. 1989, 5, 31–34.

isovaleric, formic, succinic, lactic (solution in water, 85%), 4-hydroxybenzoic, DL-malic, DL-tartaric, pyruvic, fumaric, and pcoumaric acids, humic acid sodium salt (technical grade), ethylamine (solution in water, 70 wt %), benzaldehyde, and butyraldehyde were purchased from Sigma-Aldrich. HOBT, dichloromethane, and phosphomolybdic acid hydrate were products of Fluka. Acetic, citric, oxalic, and ellagic acids and glycine, L-alanine, D-glucose, iron(III) chloride, and copper(II) sulfate were obtained from Acros. Sodium hydroxide 46/48% solution (analytical reagent grade) was purchased from Fisher Scientific. All other chemicals were of analytical grade. TLC aluminum sheets (silica gel 60 F254) 20 cm × 20 cm were products of Merck. Polyethersulfone (PES) filters (diameter, 47 mm; pore size, 0.8 µm) were obtained from Whatman Nucleopore. Fluorescence data acquisition was performed using a PerkinElmer LS-55 luminescence spectometer equipped with the FL Winlab software (4.00.02 version). A scan speed of 400 nm/min was used with a slit width of 5 nm for both excitation and emission. Readings took place in a 1 cm quartz cuvette of 4 mL volume. The fluorescence intensity from the emission spectra was expressed in absorbance units (a.u.). LC-MS analyses were performed with Elite Labchrom high-pressure binary pumps (VWR Hitachi), a Supelco C18 250 mm × 4.6 mm column, and an Esquire 6000 ion trap mass spectrometer equipped with an atmospheric pressure chemical ionization (APCI) source operated in positive polarity. High-pressure ionic chromatography (HPIC) determination of carboxylic acids and major anions have been conducted with an ICS-3000 (Dionex, Sunnyvale, CA), equipped with an SP gradient pump, a CD-25 conductivity detector, and an Ultimate3000 photodiode array UV-vis detector. An IonPac AS11-HC 4 mm × 250 mm column was used in the gradient mode (0.5-40 mM NaOH) at a flow rate of 1.5 mL/min. More details for HPIC determinations are indicated in the Supporting Information (see sections SI-1 and SI-2). Thin layer chromatography (TLC) analyses were performed on silica gel TLC plates, with ethyl acetate as the eluent and revelation under a UV lamp (254 nm) and the phosphomolybdic acid/ethanol reagent. Aqueous Sample Preparation Procedures. River samples (R1-R3) were collected along the Arc river watershed, located in highly urbanized and industrialized area, close to Aix-enProvence (Southeast of France), at its source (R1), and at upstream and downstream of a industrial water treatment plant (R2 and R3, respectively). These samples were directly filtered on 0.8 µm polyethersulfone (PES) filters. Soils samples (S1-S12) were collected from a control forest area (Maures Mountains, Southeast of France), submitted to various fire episodes, and having various total organic carbon (TOC) content. This area was used by different research teams working on the assessment of the impact of repeated forest fires on biodiversity and soils (More details at http://irise.mediasfrance.org/projet/index). Composts (C1-C6) were gratefully offered by a local manufacturer (Biotechna, Marseille, France), who produces these reusable wastes by composting of sewage sludges with green wastes. These samples were differentiated by their stages of maturation, ranging from 1 to 12 months; they were sieved at 15 mm before use. Soils and composts (whose physicochemical parameters are given in Table SI-3 in the Supporting Information) were extracted with ultrahigh quality water (10 g of samples in 50 mL water),

mechanically mixed with an automatic stirring table for 2 h, centrifuged at 6000 r/min for 15 min, and filtered through 0.8 µm PES filters. Sewage sludge samples (B1-B7) were collected in a local sewage sludge treatment plant (Marseille, Southeast of France) at different steps of the anaerobic methanogenic digestion (crude arrival at the plant, before and after fermentation tanks, final centrifuged treated sludges), centrifuged at 6000 r/min for 15 min and filtered through 0.8 µm PES filters. Water-diluted samples of oxidized animal carcass residues (O1-O5) were obtained from colleagues working on the wet air oxidation (WAO) of animal wastes:25 the final products of this oxidation process were mainly composed of acetic acid (refractory compound when WAO is carried out without catalysts).26 EDAN Solution Preparation Procedure. EDAN commercial reagent (98% grade) contains fluorescent impurities and needs thus to be quickly purified by acidic extraction before introduction in the reaction mixture. For preparation of about 400 µL of an EDAN-purified solution, 3 mg of EDAN are dissolved in 550 µL of UHQ water and acidified with 50 µL of HCl (1 M). This aqueous solution is then mixed with 600 µL of CH2Cl2, the biphasic system is vigorously stirred for 15 s and then allowed to decant until complete biphasic separation. The upper layer is the aqueous phase, further mentioned as the “EDAN solution”. Derivatization Procedure. To 1 mL of sample (carboxylic acid standard solutions, UHQ water for blank measurement or environmental aqueous samples prepared according to the abovedescribed procedure) in a 5 mL test tube was added KH2PO4 (6.8 mg) and N-hydroxybenzotriazole, HOBT, (3 mg). This latter product was added as a secondary activating reagent; the pH was adjusted to 3.5-4 (acidification with 0.2, 0.5, or 1 M HCl and basification with 0.2, 0.5, or 1 M NaOH, depending on the pH of the samples), EDC (3 mg) was added into the test tube, and the reaction mixture was stirred for 6 min. Addition of 100 µL of the EDAN solution (0.5 mg EDAN/100 µL, see preparation procedure above) was followed by a pH increase to 8-8.5 with 85 µL of 1 M NaOH. After an additional 4 min of stirring, the pH was decreased to 4.5-5 (with about 65 µL of 1 M HCl and further adjustment with 0.2 M HCl if necessary), followed by addition of 900 µL of tert-butyl-methyl ether and vigorous stirring of this biphasic mixture for 15 s. Subsequent decantation enabled collection of 600 µL of the organic supernatant layer that underwent another vigorous mixing/decantation step in a second test tube with 600 µL of a 50 mM phosphate buffer (pH 5). The fluorescence intensity of the final organic supernatant layer was measured after suitable dilution in a total volume of 3 mL of MTBE in the fluorescence cuvette (25- to 625-fold dilutions depending on the fluorescence intensity of the derivative compound), at λex ) 335 nm and λem ) 396 nm, whatever the carboxylic compound analyzed. All experiments with standard solutions were done in triplicate. RESULTS AND DISCUSSION Choice of Main Procedure Features and Parameters. The first experiments were conducted with acetic acid to test the feasibility of our fluorescence-based derivatization method. The (25) Barbati, S.; Fontanier, V.; Ambrosio, M. Ind. Eng. Chem. Res. 2008, 47, 2849–2854. (26) Debellefontaine, H.; Foussard, J.-N. Waste Manage. 2000, 20, 15–25.


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amidation reaction by EDAN was first performed in an organic medium (MeCN) using the classical EDC activation, with TLC analysis (eluent, ethyl acetate; revelation under UV lamp and phosphomolybdic acid detection) to check the condensation reaction effectiveness. Revelation of a new spot after reaction was indeed attributed to the formation of the acetic acid amidation product, which was characterized by a relative migration factor (Rf) of 0.22. A simple LC-MS analysis gave evidence of the expected compound ((M + H)+ ) 229) in positive APCI mode, details reported in section SI-4 in the Supporting Information). Optimization of the reaction and detection conditions in the aqueous phase was then first achieved thanks to observation of the TLC spot intensity variation (via phosphomolybdic acid revelation). Enhancing the activation step efficiency by the use of an additional activating reagent such as HOBT, which is a wellknown reagent for peptide synthesis and high-yielding amide formation,27 has been explored. Indeed, addition of HOBT to the reaction cocktail led to a clear improvement of the reaction yield estimated by TLC. Formation of the expected amide in aqueous phase was proven likewise by LC-MS. Room temperature conditions (20 °C) were selected for the initial temperature choice bearing in mind potential on-site further application, with TLC analyses also displaying similar results at 20 and 50 °C. Detection of the fluorescent amide products being obviously hindered by the simultaneous presence of the excess fluorescent amine reagent, a separation method of these two fluorescent compounds had to be designed through chemical or physicochemical means. Chemical methods include quenching by aromatic aldehydes (e.g., o-phthaldialdehyde)22 or other aromatic organic compounds (e.g., benzenesulfonyl chlorides). These compounds efficiently quenched the EDAN fluorescence, but experiments conducted after the aqueous derivatization procedure also proved quenching of the amidation product (TLC spot disappearance) and additional formation of other fluorescent byproduct formed in the reaction mixture. We thus focused on physicochemical extraction by an organic solvent, monitoring biphasic separation of EDAN (Rf 0) and condensation product (Rf 0.22) by TLC spotting. Taking advantage of the high affinity of aliphatic amines for acidic aqueous phase, we were able to isolate the amide compound in the organic layer by mixing an organic solvent with the resulting postacidified aqueous phase after the derivatization step with EDAN. Among all organic solvents tested (ether, dichloromethane, and hexane), MTBE exhibited the best properties for selective extraction of derivatized compounds and was thus selected. Its very low affinity for fluorescent EDAN enabled total elimination of this excess fluorescent reagent after only one extraction and one subsequent washing up of the organic layer with 50 mM phosphate buffer pH 5 (see Experimental Section for details). Figure 1 shows examples of the kind of emission fluorescent spectra obtained after application of these derivatization and detection conditions with an acetic acid solution (10 mg/L) and UHQ water (for blank measurement). The blank residual fluorescence is due to the unavoidable presence of fluorescent impurities in commercial EDAN reagents (maximum 98% grade). (27) Kawanishi, H.; Toyo’oka, T.; Ito, K.; Maeda, M.; Hamada, T.; Fukushima, T.; Kato, M.; Inagaki, S. J. Chromatogr., A 2006, 1132, 148–156.

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Figure 1. Examples of emission spectra obtained after derivatization procedure (λex ) 335 nm).

To summarize these initial experiments based mainly on TLC observations, five main parameters had to be further optimized: reaction medium pH values, reaction time of activating and derivatizing steps, and the pH conditions of the final aqueous reaction mixture for optimal organic extraction with MTBE. Optimization of the Selected Reaction Conditions. Results for optimization of previously mentioned key parameters are described in Figure 2. Optimization tests were conducted by keeping unmodified the other experimental conditions of the derivatization procedure detailed in the Experimental Section. As expected, while acidic conditions were needed for the activation step (optimal pH between 3.5 and 4, Figure 2A), nucleophilic replacement by EDAN preferentially occurred at basic pH, pH values between 8 and 9 being sufficient (Figure 2B). Reaction duration was suitable for the design of fast analyses, with activation and derivatization satisfactorily completed within 6 and 4 min, respectively (parts C and D of Figure 2). Afterward, organic extraction with MTBE was most efficiently performed by decreasing the pH to 4.5-5, as higher and lower pH led to higher blank and lower sample intensity (amidation products being slightly soluble in aqueous phase at low pH value, as observed by TLC), respectively (Figure 2E). Acetic Acid Calibration Curve. As it is well-known that acetic acid is the most frequently encountered carboxylic acid within soluble organic matter samples, it was selected as the reference for our study and its calibration curve will be used to transcript fluorescence emission intensities measured with real samples into carboxylic acid concentrations (expressed as acetic acid equivalents). This calibration curve, determined by application of the optimized procedure parameters, showed excellent linearity (r2 ) 0.9996) over a concentration range from 0 to 250 mg/L (Figure 3A). A more accurate curve is also shown in Figure 3B for concentration from 0 to 10 mg/L that enabled determination of a concentration detection limit (S/N ) 3, n ) 10) of 0.25 mg/L and a quantification limit (S/N ) 10) of 0.84 mg/L. Relative standard deviation (RSD) and confidence interval (R ) 0.05) were assessed on the 10 mg/L standard solution (RSD ) 11.1%; confidence interval, 787 < Ifluo < 911, n ) 10).28,29 Other calibration points were done in triplicates. An additional calibration curve ranging from 0 to 1000 mg/L is also given in section SI-5 (28) Iwasawa, T.; Wash, P.; Gibson, C.; Rebek, J., Jr. Tetrahedron 2007, 63, 6506–6511. (29) Tedder, J. M.; Nechvatal, A.; Murray, A. W. Amino-Acids and Proteins. In Basic Organic Chemistry; John Wiley & Sons: London, 1972; pp 305342.

Figure 2. Optimization of key reaction conditions. (A) activation step pH; (B) fluorescent labeling step pH; (C) activation step duration; (D) fluorescent labeling step duration; and (E) extraction step pH.

in the Supporting Information, exhibiting the limit of our methodology for concentrations over 500 mg/L. If samples contain more than 500 mg/L SCFA, expressed in acetic acid-equivalents, a dilution of the sample will thus be necessary. Application of the Procedure to Various Carboxylic Acids. The same derivatization procedure was applied to a large number of carboxylic acids detectable in aqueous environmental samples. A wide variety of carboxylic acids was tested, from mono-, di-, or tricarboxylic aliphatic acids, including compounds containing unsaturated carbons and hydroxy groups, aromatic, and cinnamic acids. Indeed, the procedure developed intended to be used for SCFA determination; it was thus necessary to determine potential interferences resulting from the presence of other carboxylic

compounds, commonly found in environmental samples. All these compounds were tested from a 10 mg/L prepared standard solution. Bearing in mind that their intrinsic concentrations in aqueous samples are reported to be several order of magnitude lower than SCFAs,6 it is assumed that if this 10 mg/L concentration does not interfere with the fluorescence response of SCFAs, no further interferences will occur. Results of these experiments are listed in Table 1. Acetic, propionic, butyric, and valeric acids (entries 1-4) resulted in the most intense responses with close values between around 800 and 1000. Formic acid (entry 5) displayed almost no fluorescent response, as well as most of the other aliphatic compounds (entries 6-13), with the exception of lactic (entry 7) and succinic acid (entry 9), with a fluorescence Analytical Chemistry, Vol. 81, No. 8, April 15, 2009

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Figure 3. Acetic acid calibration curves: (A) range, 0-250 mg/L; (B) range, 0-10 mg/L. Table 1. Carboxylic Acid Fluorescence Responses after Optimized Derivatization carboxylic entry acid 10 mg/L 1 2 3 4 5 6 7 8 a

acetic propionic butyric valeric formic pyruvic lactic oxalic

fluorescence intensity (a.u.)a entry 850 950 1030 820 110 70 210 70

9 10 11 12 13 14 15 16

carboxylic acid 10 mg/L

fluorescence intensity (a.u.)a

succinic malic tartaric citric fumaric coumaric p-OH benzoic ellagic

300 85 75 90 80 70 140 75

Table 2. Fluorescence Response of Potential Interfering Organic Compounds entry

standard solution

fluorescence intensity (a.u.)a

1 2 3 4 5 6 7 8

acetic acid 10 mg/L humic acid 200 mg/L humic acid 500 mg/L glycine 100 mg/L alanine 100 mg/L glucose 100 mg/L benzaldehyde 100 mg/L butyraldehyde 100 mg/L

850 75 95 75 85 160 125 170

a

Uncorrected fluorescence intensity; blank value ) 65.

Uncorrected fluorescence intensity; blank value ) 65.

intensity of 210 and 300, respectively.Two reactions are involved during the derivatization procedure The first one, carried out at pH ) 3.8, aims to increase the reactivity of the carbonyl carbon as an electrophile. Operating at pH ) 3.8 enables protonation of the carbonyl oxygen and mainly for acids whose pKa values are around 4.8. This explains the reactivity of mainly acetic, propionic, butyric, and valeric acids, compared to the low reactivity of other acids having lower pKa values (pyruvic, 2.9; citric, 3.1; tartaric, 3.0...). Once the carbonyl oxygen is protonated, EDC is added to the mixture to activate the carbonyl carbon. HOBT is then added to activate the carbonyl activated. The second step consists in the actual amidation: the hydroxyl group is replaced by the EDAN molecule. It is known that nucleophile agents (EDAN in our case) react more readily with unhindered carbonyl groups, and this explain the poor reactivities of malic, citric, ellagic, and coumaric acids, which are branched or aromatic carboxylic acids. As stated by Iwasawa et al.,28 carboxylic acids are especially difficult to place in a sterically demanding environment; their oxygens are exposed and convoluted molecular architectures must be created to bring intramolecular elements near them in space. Moreover, the chemoselectivity of amidation reactions are highly determined by the electronic properties of the substituents in the R-position to the carboxylic group:29 presence of electron donor groups in the R-position of most of di- and tricarboxylic acids tested may impair the reaction of amidation. The whole parameters explain the best efficiency of the reaction toward linear, unsubstituted, short-chain aliphatic acids (from C2 to C5) and having pKa values around 4.8. 3068


These experimental comparisons thus clearly proved that our analytical procedure seemed particularly suitable for the detection of SCFAs, which are both predominant and the most reactive carboxylic acids. More evidence is given in section SI-6 in the Supporting Information, where calibration curves and slopes of the corresponding linear regression are depicted for the abovecited reactive carboxylic acids. Evaluation of the Possible Interferences Due to Organic or Mineral Compounds. Real environmental samples are complex mixtures with a great variety of organic and mineral compounds. They can potentially (i) either react with the fluorescent amine to give an interfering fluorescence response (for example naturally occurring organic compounds with reactive functionnal groups, such as humic acid, amino acids, carbohydrates, or aldehydes; fluorescent intensities after derivatization with standard solutions of some of these compounds and comparison with acetic acid are described in Table 2) or (ii) interfere with the derivatization and detection of carboxylic acids (alcohols, amines, and metallic cations); fluorescent intensities after derivatization of standard solutions containing 10 mg/L acetic acid and the potentially interfering organic or mineral compounds are listed in Table 3. Table 2 gives clear evidence that fluorescent readings are very little affected by hypothetical interferences caused by noncarboxylic acid organic compounds (entries 2-8). These latter ones were tested with highly concentrated standard solutions that exhibited low fluorescent response compared to an acetic acid standard solution prepared in a relatively low concentration. Fluorescence intensities resulting from derivatization with humic acid standards were very low even with concentration up to 500 mg/L (entries 2 and 3), probably caused by poorly reactive sterically hindered aromatic acid groups. The same results were observed with amino acids (entries 4 and 5), presumably

Table 3. Assessment of Possible Fluorescent Interferences Due to Organic or Mineral Compounds

entry

interfering compound added to a solution of acetic acid 10 mg/L

fluorescent intensity (a.u.)a

∆If (%)b

1 2 3 4 5

ethanol [100 mg/L]c ethylamine [100 mg/L]c Fe3+ [500 mg/L]c Cu2+ [500 mg/L]c

850 830 810 910 855

0 2.3 4.7 7.0 0.6

a Uncorrected fluorescence intensity. b Compared to a pure solution of acetic acid 10 mg/L (entry 1). c Final concentration in the solution with acetic acid.

accounted for by low pKa values of 2.3 of their carboxylic protons. Aldehyde-containing compounds and glucose gave as expected a small fluorescent response but still unsignificant compared to acetic acid (entries 6-8). Experimental measurement of the concentration of SCFAs could be also modified by disturbances either during derivatization or during the detection step. Table 3 displays results obtained with acetic acid standard solutions enriched with an excess of potentially interfering materials (over 10-fold excess). Nucleophilic ethanol (entry 2) and ethylamine (entry 3) could generate competitive nucleophilic displacement with EDAN. Iron and copper salts (entries 4 and 5, respectively), known to have strong affinities for carboxylic groups, have to be tested for both possible inhibition of the activation step caused by carboxylic metal complexation and the detection step through fluorescent quenching. None of these attempts gave a significant fluorescence decrease, and it was therefore concluded that the presence of these materials in real samples should not result in major analytical interferences. It must also be pointed out that natural fluorescence of underivatized aqueous samples (due to humic acids and aromatic compounds) had no effect on the measured fluorescence after derivatization and extraction with MTBE. Indeed, naturally fluorescent compounds have low solubility in MTBE under the extraction conditions used in the procedure. Application to Real Sample Analysis. Validation of our methodology was assessed by application of the procedure to aqueous samples originating from various sources: river, soils, composts, oxidized animal carcass residues, and sewage sludges. Each sample was previously quantitatively analyzed by HPIC, and their acid carboxylic contents were then expressed as acetic acidequivalents, by multiplying their carboxylic acid contents (in milligrams per liter) by the following ratio: molecular weight of acetic acid/molecular weight of the considered carboxylic acid (more details in section SI-7 in the Supporting Information). Results of our subsequent fluorimetric measurements (expressed as acetic acid-equivalents, based on the calibration curves for acetic acid displayed in Figure 3) were plotted versus HPIC conductometric measurements (Figure 4). The regression line shows a good regression coefficient, a very low intercept, and an overall slope satisfactorily close to unity, proving accuracy of our alternative methodology. It was also demonstrated by the calculation of the w quotient according to the French Agency of Normalization,

Figure 4. Real sample analysis.

AFNOR,30 assessing the accuracy of an alternative method compared to a reference method. To meet the requirement of this standard, each method has to be applied on various samples and differences between both measurements for each sample are worked out. w is the quotient of the differences average and the differences standard deviation, and accuracy of the alternative method can be certified if w e 3 (R ) 0.01). Our measurements led to a w quotient of 2.72, further supporting our previous conclusions about the accuracy of our methodology. These results on real samples confirm the promising results obtained after determination of the acetic acid linear calibration curve and of the seemingly nonexistence of potential interferences due to various intrinsic sample components. Measurements were particularly accurate for samples that had the highest SCFA content (>15 mg/L), exemplified by sewage sludges. This fast and direct determination of SCFAs in such samples can therefore be of a great interest, as SCFA concentration monitoring is routinely used as a physicochemical indicator of organic matter digestion in sewage sludge treatment plants. As expected, SCFA concentrations clearly decrease during anaerobic digestion, our fluorimetric methodology displaying a dramatic drop from 83.5 to 7.1 mg/L for corresponding samples before and after digestion, in agreement with HPIC monitoring (77.5 and 7.4 mg/L, respectively). Development of automated microplate analyses is currently underway in the laboratory to fully exploit the potential of this effective methodology for field laboratory measurements. The objective is to propose an integrated microplate, ready-to-use (that is to say with wells containing each of the reagents presented in this paper and ready to be completed with the sample to be analyzed) and able to be handled by current microplate readers. This kind of analytical tool could provide very interesting alternatives to expensive and time-consuming chromatographic methods and therefore lead to new prospects in the field of on-site analyses. (30) AFNOR (Agence Française de Normalisation). Qualite´ de l’Eau-Protocole d’E´valuation d’une Me´thode Alternative d’Analyse Physico-Chimique Quantitative par Rapport a` une Me´thode de Re´fe´rence [Water Quality-Protocol of Assesment of a Quantitative Physico-Chemical Alternative Method versus a Reference Method]; Norm no. XPT 90-210, 1999.


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ACKNOWLEDGMENT The French Research Agency (ANR) provided support through the Programme “Ecosphe`re Continentale: risques environnementaux_2006”, and the French Environment and Energy Management Agency (ADEME) has financially supported this research as well. Fabien Robert-Peillard was supported by a fellowship of ADEME.

soils and samples, SCFA content in samples analyzed, calculations to express results in acetic equivalents, application of the method to main SCFAs found during anaerobic fermentations, and HPLC-MS analysis. This material is available free of charge via the Internet at http://pubs.acs.org.

SUPPORTING INFORMATION AVAILABLE Material used, description of HPIC system and analytical features of HPIC calibration, physicochemical characterization of

Received for review December 31, 2008. Accepted March 4, 2009.

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AC802768U

Alternative Spectrofluorimetric Determination of Short-Chain Volatile

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