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Determination of low molecular weight organic acids in soil solution by HPLC
Talanta 48 (1999) 173 – 179
Determination of low molecular weight organic acids in soil solution by HPLC Patrick A.W. van Hees a,*, Johan Dahle´n b, Ulla S. Lundstro¨m a, Hans Bore´n a, Bert Allard b a b
Deptartment of Chemistry and Process Technology, Mid Sweden Uni6ersity, 851 70 Sunds6all, Sweden Department of Water and En6ironmental Studies, Uni6ersity of Linko¨ping, 581 83 Linko¨ping, Sweden Received 14 November 1997; received in revised form 30 April 1998; accepted 18 June 1998
Abstract An HPLC method employing an ion exclusion column was developed for the determination of low molecular weight organic acids in soil solution. The method includes extensive sample pretreatment using ultrafiltration and cation exchange. The method showed linear calibration graphs (r\ 0.99) and the limits of detection in the range 0.1–26 mM. The recovery of eleven added acids ranged from 89 to 102%. Soil solutions of five horizons of a podzolised soil were analysed. The results showed that these compounds made up 1 – 3% of the dissolved organic carbon and 0–14% of the acidity. Identification of the major acids was also carried out by capillary zone electrophoresis. © 1999 Published by Elsevier Science B.V. All rights reserved. Keywords: HPLC; Organic acids; Soil solution; Capillary zone electrophoresis
1. Introduction Low molecular weight (LMW) organic acids are produced during degradation of organic material in soils, e.g. example litter and dead roots by fungi and bacteria [1]. Furthermore, exudates from different fungi and the roots of a number of plants may contain acids of this kind [2,3]. These acids may also participate in polymerisation reactions [4]. A number of different aliphatic acids have been found in forest soils, e.g. oxalic, citric, formic, acetic, malic, lactic, and fumaric acids * Corresponding author. Tel.: +46 60 148493; fax: + 46 60 148802; e-mail: [email protected]
[1,5,6]. The total content of these acids is estimated to account for 2–10% of the total dissolved organic carbon (DOC) in spodosols (e.g. podzols) [1,6]. Many low molecular weight acids have high stability constants with different metal ions [7]. It has been found that up to 37% of the Al in the soil solution of an O-horizon of a podzolised soil was bound to these compounds [5]. Due to their complexing ability they are believed to be involved in the translocation of Al and Fe in the podzolisation process [8], and can also enhance the weathering of primary minerals [9]. In addition their role as possible Al detoxifiers in forest soils has been discussed [7].
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Different HPLC techniques have been used for the separation of aliphatic acids in soil and litter extracts/solutions. For example, ion exchange chromatography (IEC) has been employed in some cases [6,10,11]. This technique has low limits of detection, but, for example, unsuitable capacity factors may cause problems [10]. Ion exclusion HPLC columns in combination with UV detection have also been used [1,7,12]. Although this type of column offers good selectivity, the determination can be interfered by unretained substances which cause a large void peak with tailing. Capillary electrophoresis and gas chromatography are two other techniques that successfully have been applied to similar samples [13,14]. The aim of this study has been to develop an HPLC method for the qualitative and quantitative determination of low molecular weight organic acids in the soil solution. To ensure the identification of the acids capillary electrophoresis was employed.
2. Experimental
2.1. Site A sediment soil was sampled at Hyytia¨la¨ Forestry Field Station in Finland, (61°48’N, 24°19’E) for the determination of LMW acids. The site was forested with Norway spruce (Picea abies) and Scots pine (Pinus syl6estris) about 100 years old. The field layer consisted of Vaccinium myrtillus and moss. The soil was podzolised and the soil horizons had a thickness of about: O:10 cm, E:15 cm and B: 40 cm.
2.2. Instruments A Beckman J2-21 centrifuge (Beckman, Spinco Division, Palo Alto, CA) was used for preparing the soil solution. DOC in the soil solutions was determined using a Shimadzu 5000 (Shimadzu, Osaka, Japan). The HPLC system (Shimadzu LC10, Shimadzu, Osaka, Japan) featured a diode array detector. Detection was performed at 210 nm. A column oven (Pharmacia HPLC column oven 2155, Pharmacia, Uppsala, Sweden) was also
mounted. Data acquisition was performed using the Class VP 5.0 (Shimadzu) computer program. A Supelcogel C610-H (Supelco, Richmond, PA, USA) ion exclusion column (300× 7.8 mm) was employed for separation of the small organic acids. Ultrafiltration was carried out using a stirred cell Amicon 8050 with Diaflo YM-1 filters (cut-off size 1000 Da) (Amicon, Beverly, MA). Capillary zone electrophoresis (CZE) analysis was performed on a Quanta 4000 (Waters, Milford, MA). Titration was performed employing an ABU900 Autoburette system controlled by a TIM900 Titration Manager (Radiometer, Copenhagen, Denmark).
2.3. Chemicals All organic acids (present in their acid form or as Na/K salts) were of analytical reagent grade with the exception of lithium lactate (GR). All acids were obtained from Aldrich (Steinheim, Germany) and Merck (Darmstadt, Germany). Phosphoric acid (85%) (AR) was purchased from Aldrich. The Bio-Rex cation exchange resin was bought from Bio-Rad (Hercules, CA). 1,2,4-Benzenetricarboxylic acid was obtained from Janssen (Beerse, Belgium), tetradecyltrimethylammonium bromide (TTAB) from Sigma (Labkemi, Stockholm, Sweden) and octanesulfonate from Fluka (Buchs, Switzerland). The water used was ion exchanged and run through a Milli-Q system (Millipore, Bedford, MA).
2.4. Soil solutions The centrifugation drainage technique and sampling procedure described by Giesler and Lundstro¨m [15] were used to obtain the soil solutions. When sampling the mor (O-horizon), the upper organic soil was removed and divided into two layers. The green parts were removed. The B1 and B2 samples were taken at 0–5 and 5–10 cm depth in the illuvial horizon, respectively. The soil samples were centrifuged for 30 min at a speed of 14000 rpm. After centrifugation the collected centrifugates were filtered through a 0.45 mm filter (Millex-HV, Millipore) and the pH was then determined. The soil solution was finally transferred
P.A.W. 6an Hees et al. / Talanta 48 (1999) 173–179
to 10 ml polypropylene tubes and stored in a freezer until further analysis.
2.5. Sample preparation and HPLC analysis A stirred ultrafiltration cell (model 8050, Amicon) equipped with a Diaflo YM-1 filter was used for removing interfering compounds. The device was pressurised by N2 gas at a pressure of 3–3.5 bar, and the filtrate was collected in a tube surrounded by ice. The sample (10 ml) was filtered twice and the filters were changed between the two runs. Before use the ultra filters were immersed for at least 1 h in firstly 0.1 M NaOH, secondly 5% (w/w) NaCl, and finally rinsed twice in Milli-Q water. This cleaning procedure was needed to ensure the removal of, especially, lactic acid which otherwise could contaminate the sample. A blank was run and no interfering peaks were found. The sample was then acidified by means of 0.5 M H2SO4 to pH3, and run through a cation exchanger (0.4 ml Bio-Rex 70, 200 – 400 mesh) in the H + form. The first 1 ml of eluate after the pH change was discarded and then a sample of approximately 4 ml was collected. The sample was immediately run on the HPLC column. The mobile phase consisted of 0.2% (v/v) H3PO4 and the flow rate was 0.5 ml min − 1. The injection volume was 50 ml. First one run was carried out with a column oven temperature of 30°C. A second run was then performed at 60°C when citric, shikimic and lactic acids were determined. This procedure was required due to the overlapping of lactic and shikimic acids at 30°C and the appearance of a ghost peak interfering the determination of citric acid. The acids were calibrated using standards which had been treated in the same way.
2.6. CZE analysis CZE analysis was carried out according to [16] and was performed on a Quanta 4000. An 82-cm (74-cm to detector) fused-silica capillary (J&W Scientific, Folsom, CA) with an i. d. of 75 mm and a carrier electrolyte consisting of 5 mM 1,2,4-benzenetricarboxylic acid with 0.5 mM TTAB (pH 8)
175
were used. TTAB was added to reverse the electroosmotic flow. Injection was performed by electrokinetic injection at − 5 kV for 45 s or by hydrostatic injection at 100 mm for 30 s, and the applied separation voltage was − 15 kV. The analytes were detected by indirect UV detection at 254 nm. Octanesulfonate, which acts as a terminating electrolyte, was added to the samples to final concentrations of 70 mM to achieve an isotachophoretic steady state during the sample introduction.
2.7. Titrations of soil solution The sample was first acidified to pHB 2.5 and run through a cation exchanger. It was then bubbled with N2 for 15 min and titrated by means of 0.01 M NaOH. The evaluation was made using the procedure outlined by Molvæsmyr and Lund [17] based on the Gran extrapolation giving the concentrations of strong (SA) and weak acids (WA).
3. Results and discussion
3.1. Calibration and performance A satisfactory separation of a wide range of acids could be achieved (Table 1). The performance of the HPLC method was evaluated with regard to limits of detection, calibration and recoveries of standard additions. Citric, shikimic and lactic acids were determined at 60°C, all other acids at 30°C. The results are presented in Table 1. Succinic acid was also tested and it was found that this acid had very similar retention times to shikimic acid, both at 30 and 60°C. However, succinic acid could not be found in the samples when running CZE. All calibration graphs were linear and had a correlation coefficient r\ 0.99. Two or three replicates were run and each standard was injected twice. In the case of fumaric, shikimic and oxalic acids lower concentrations of the standards were used due to its high UV-absorbance. Recovery studies were performed by standard addition of all acids to three samples of the E-horizon.
50, 250, 500 50, 250, 500 50, 250, 500 5, 25, 50 50, 250, 500 50, 250, 500 50, 250, 500 50, 250, 500 5, 10, 25 50, 250, 500 5, 10, 25 50, 250, 500
Acetic Citric Formic Fumaric Lactic Malic Malonic Oxalic
3 2 3 2 2 3 3 3 2 3 2 3
Replicates (n)
0.994 1.000 0.998 0.992 0.995 0.998 0.996 0.998 0.993 0.998 0.994 0.993
Corrolation coefficient (r)
25.7 0.2 4.0
23.9 3.1 16.0 0.1 7.1 6.1 8.1 0.4
LOD (mM)
Retention time (min) 21.42 12.04b 19.98 24.25 17.95b 14.67 15.36 10.09 26.00 16.94b 13.25
Recovery (n =3) (standard addition 100 mM)a (%) 93 9 8 99 9 5 95 9 7 102 9 3 97 9 6 105 97 100 97 89 915 98 98 101 93 100 9 7
a
Confidence limits (95%) of standard addition to sample. In the case of fumaric and shikimic acids 10mM; b determined at 60°C, all other acids at 30°C.
Propionic Shikimic Tartaric
Concentration (mM)
Acid
Table 1 Calibration of organic acids
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P.A.W. 6an Hees et al. / Talanta 48 (1999) 173–179
177
Table 2 HPLC determinations and titration data of the soil solutions from Hyytia¨la¨
pH Acetic acid (mM) Citric acid (mM) Fumaric acid (mM) Lactic acid (mM) Oxalic acid (mM) Shikimic acid (mM) DOC (mM) %LMW acids of DOC WA(tot)a (meq l−1) %LMW acids (meq l−1) of WA(tot)
O1
O2
E
B1
B2
3.76 174+28 80+6 2.4+5 27+41 7.2+2 21.4+2 32.33 3.3 3.44 14.0
3. 53 67+42 27+7 1.3+6 n.d. 4. 5+3 5.1+2 22.08 1.6 2.22 7.5
3. 83 — 19+6 1.3+5 n.d. 4.1+3 1.6+2 4.94 3.1 0.96 7.7
4. 85 — — 1.1+6 n.d. 8.5+3 1.7+2 3.13 1.2 0.42 5.0
5.99 — — — n.d. tr. 1.3+2 1.02 0.9 0.37 0.3
Data is given for pH, concentrations+confidence limits(95%), DOC, and total WA (WA(tot)) content. n.d., Not detectable, area of peak smaller than intercept of calibration equation or below LOD; tr., traces, peak identified but not integrated. a In the case of O1 SA is included.
Good recovery values were obtained for all acids. The poorer value for oxalic acid can partly be explained by the larger variance and the fact that this acid elutes close to the void peak. A correct determination of this acid might be difficult when analysing samples with very high DOC levels (\ 1000 ppm). The limits of detection (LOD), calculated as the concentration corresponding to three times the background noise, were in the range 0.1–26 mM. The values were in accordance with the UV absorbance of the different acids, resulting in better limits of detection for the acids with higher absorbance. The repeatability for natural samples was checked by performing duplicate analyses for two samples (see below), and was estimated as the pooled S.D. for the replicates. The deviation was in the range 0.03 – 1.40 mM for the commonly detected acids (citric, shikimic, oxalic and fumaric acids). The variance for acids found in natural samples was not significantly greater than for standards as evaluated by F-tests [18].
3.2. Application and identification Soil solutions from five soil layers were analysed for LMW organic acids, SA and WA content using the methods described above (Table
2). A comparison of a chromatogram before and after the sample clean-up procedure is shown in Fig. 1. The acids were identified by comparing retention times and where possible, due to the void peak, UV spectra. In the case of oxalic, citric and fumaric acids these were also confirmed by standard additions. In order to obtain an independent identification of the organic acids, some of the samples were also analysed by CZE. Identification was made by retention time and by spiking the samples with a small amount of organic acid stock solution. By this procedure citric, fumaric, lactic, oxalic, acetic and shikimic acids were tentatively identified in the CZE electropherograms. Although the analysis was performed on a qualitative basis it could be established that the concentrations determined by the HPLC application were of the same order of magnitude as compared to the CZE analysis. An electropherogram of the O2 horizon solution is showed in Fig. 2. Two replicates of the O1 and E horizons including pretreatment and HPLC analysis were made, but single analyses were carried out for the other samples. Each sample was injected twice on the HPLC column. Several different acids were identified, of which all have been reported earlier [1,5,19]. Shikimic acid and traces of oxalic acid
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P.A.W. 6an Hees et al. / Talanta 48 (1999) 173–179
Fig. 1. HPLC chromatogram (run at 30°C) of an O2 soil solution before (a) and after pretreatment (b). The insert picture in (b) shows an enlargement of the chromatogram.
were found in all horizons. Citric and fumaric acids were also observed in most samples. As would have been expected the concentrations decrease in the deeper soil layers. A small increase of oxalic and shikimic acids was however observed in the B1 horizon. Concerning the acidity, the fraction of the LMW organic acids made up 0.3 – 14.0% of the WA(tot). SA was only found in O1, and was
included in the WA(tot) value because experiments showed that for citric and oxalic acids the carboxyl group with the lowest pKa was not included in the WA(tot) measurement. The percentage of LMW acids decreases through the soil profile. Regarding the DOC, LMW acids made up 0.9–3.3%. Also the fraction of the DOC consisting of LMW acids generally decreased in the deeper horizons.
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179
Fig. 2. CZE electropherogram of an O2 soil solution after pretreatment. Electrokinetic injection. Peaks: 1, oxalic; 2, citric; 3, fumaric; 4, acetic; 5, shikimic acids.
Acknowledgements This study was financially supported by the Swedish Natural Science Research Council (NFR). The authors would like to thank A.-M. van Hees, M. Nystro¨m and V. Stigsson for assistance. Dr H. Ilvesniemi is acknowledged for providing facilities for the field work and L. Backman for soil sampling.
References [1] T.R. Fox, N.B. Comerford, Soil Sci. Soc. Am. J 54 (1990) 1139. [2] L. Stro¨m, T. Olsson, G. Tyler, Plant Soil 167 (1994) 239. [3] T. Wickman, Weathering assessment and nutrient availability in coniferous forests, PhD dissertation, Royal Institute of Technology, Stockholm, 1996. [4] A.A. Pohlman, J.G. McColl, Soil Sci. Soc. Am. J. 53 (1989) 686. [5] P.A.W. van Hees, A.-M.T. Andersson, U.S. Lundsto¨m, Chemosphere 33 (1996) 1951. [6] A.J. Krzyszowska, M.J. Blaylock, G.F. Vance, M.B. David, Soil Sci. Soc. Am. J. 60 (1996) 1565.
.
[7] N.V. Hue, G.R. Craddock, F. Adams, Soil Sci. Soc. Am. J. 50 (1986) 28. [8] U.S. Lundstro¨m, N. van Breemen, A.G. Jongmans, Eur. J. Soil Sci. 46 (1995) 489. [9] U. Lundstro¨m, L.-O. O8 hman, J. Soil Sci. 41 (1990) 359. [10] R. Baziramakenga, R.R. Simard, G.D. Leroux, Soil Biol. Biochem. 27 (1995) 349. ˚ . Jo¨nsson, G. Tyler, Soil. Biol. [11] Y. Shen, L. Stro¨m, J.-A Biochem. 28 (1996) 1163. [12] A.A. Pohlman, J.G. McColl, Soil Sci. Soc. Am. J. 52 (1988) 265. [13] O. Deveˆvre, J. Garbaye, B. Botton, Mycol. Res. 100 (1996) 1367. [14] A.M. Szmigielska, K.C.J. Van Rees, G. Cieslinski, P.M. Huang, Commun. Soil Sci. Plant Anal. 28 (1997) 99. [15] R. Giesler, U.S. Lundstro¨m, Soil Sci. Am. J. 57 (1993) 1283. [16] J. Dahle´n, S. Bertilsson, C. Pettersson, Environ. Int. 22 (1996) 501. [17] K. Molvæsmyr, W. Lund, Water Res. 17 (1983) 303. [18] J.C. Miller, J.N. Miller, Statistics for Analytical Chemistry, 3rd ed., Ellis Horwood, Chichester, 1993, p. 60. [19] J.G. McColl, A.A. Pohlman, J.M. Jersak, S.C. Tam, R.R. Northup, Organics and metal solubility in California forest soils, in: S.P. Gessel et al. (Eds.), Sustained Productivity of Forest Soils, Proc. 7th North American Forest Soils Conf., Vancouver, 24 – 28 July 1990. Faculty of Forestry Publication, University of British Columbia, 1990, pp. 178 – 195.
Determination of low molecular weight organic acids in soil solution by HPLC Patrick A.W. van Hees a,*, Johan Dahle´n b, Ulla S. Lundstro¨m a, Hans Bore´n a, Bert Allard b a b
Deptartment of Chemistry and Process Technology, Mid Sweden Uni6ersity, 851 70 Sunds6all, Sweden Department of Water and En6ironmental Studies, Uni6ersity of Linko¨ping, 581 83 Linko¨ping, Sweden Received 14 November 1997; received in revised form 30 April 1998; accepted 18 June 1998
Abstract An HPLC method employing an ion exclusion column was developed for the determination of low molecular weight organic acids in soil solution. The method includes extensive sample pretreatment using ultrafiltration and cation exchange. The method showed linear calibration graphs (r\ 0.99) and the limits of detection in the range 0.1–26 mM. The recovery of eleven added acids ranged from 89 to 102%. Soil solutions of five horizons of a podzolised soil were analysed. The results showed that these compounds made up 1 – 3% of the dissolved organic carbon and 0–14% of the acidity. Identification of the major acids was also carried out by capillary zone electrophoresis. © 1999 Published by Elsevier Science B.V. All rights reserved. Keywords: HPLC; Organic acids; Soil solution; Capillary zone electrophoresis
1. Introduction Low molecular weight (LMW) organic acids are produced during degradation of organic material in soils, e.g. example litter and dead roots by fungi and bacteria [1]. Furthermore, exudates from different fungi and the roots of a number of plants may contain acids of this kind [2,3]. These acids may also participate in polymerisation reactions [4]. A number of different aliphatic acids have been found in forest soils, e.g. oxalic, citric, formic, acetic, malic, lactic, and fumaric acids * Corresponding author. Tel.: +46 60 148493; fax: + 46 60 148802; e-mail: [email protected]
[1,5,6]. The total content of these acids is estimated to account for 2–10% of the total dissolved organic carbon (DOC) in spodosols (e.g. podzols) [1,6]. Many low molecular weight acids have high stability constants with different metal ions [7]. It has been found that up to 37% of the Al in the soil solution of an O-horizon of a podzolised soil was bound to these compounds [5]. Due to their complexing ability they are believed to be involved in the translocation of Al and Fe in the podzolisation process [8], and can also enhance the weathering of primary minerals [9]. In addition their role as possible Al detoxifiers in forest soils has been discussed [7].
0039-9140/99/$ - see front matter © 1999 Published by Elsevier Science B.V. All rights reserved. PII S0039-9140(98)00236-7
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Different HPLC techniques have been used for the separation of aliphatic acids in soil and litter extracts/solutions. For example, ion exchange chromatography (IEC) has been employed in some cases [6,10,11]. This technique has low limits of detection, but, for example, unsuitable capacity factors may cause problems [10]. Ion exclusion HPLC columns in combination with UV detection have also been used [1,7,12]. Although this type of column offers good selectivity, the determination can be interfered by unretained substances which cause a large void peak with tailing. Capillary electrophoresis and gas chromatography are two other techniques that successfully have been applied to similar samples [13,14]. The aim of this study has been to develop an HPLC method for the qualitative and quantitative determination of low molecular weight organic acids in the soil solution. To ensure the identification of the acids capillary electrophoresis was employed.
2. Experimental
2.1. Site A sediment soil was sampled at Hyytia¨la¨ Forestry Field Station in Finland, (61°48’N, 24°19’E) for the determination of LMW acids. The site was forested with Norway spruce (Picea abies) and Scots pine (Pinus syl6estris) about 100 years old. The field layer consisted of Vaccinium myrtillus and moss. The soil was podzolised and the soil horizons had a thickness of about: O:10 cm, E:15 cm and B: 40 cm.
2.2. Instruments A Beckman J2-21 centrifuge (Beckman, Spinco Division, Palo Alto, CA) was used for preparing the soil solution. DOC in the soil solutions was determined using a Shimadzu 5000 (Shimadzu, Osaka, Japan). The HPLC system (Shimadzu LC10, Shimadzu, Osaka, Japan) featured a diode array detector. Detection was performed at 210 nm. A column oven (Pharmacia HPLC column oven 2155, Pharmacia, Uppsala, Sweden) was also
mounted. Data acquisition was performed using the Class VP 5.0 (Shimadzu) computer program. A Supelcogel C610-H (Supelco, Richmond, PA, USA) ion exclusion column (300× 7.8 mm) was employed for separation of the small organic acids. Ultrafiltration was carried out using a stirred cell Amicon 8050 with Diaflo YM-1 filters (cut-off size 1000 Da) (Amicon, Beverly, MA). Capillary zone electrophoresis (CZE) analysis was performed on a Quanta 4000 (Waters, Milford, MA). Titration was performed employing an ABU900 Autoburette system controlled by a TIM900 Titration Manager (Radiometer, Copenhagen, Denmark).
2.3. Chemicals All organic acids (present in their acid form or as Na/K salts) were of analytical reagent grade with the exception of lithium lactate (GR). All acids were obtained from Aldrich (Steinheim, Germany) and Merck (Darmstadt, Germany). Phosphoric acid (85%) (AR) was purchased from Aldrich. The Bio-Rex cation exchange resin was bought from Bio-Rad (Hercules, CA). 1,2,4-Benzenetricarboxylic acid was obtained from Janssen (Beerse, Belgium), tetradecyltrimethylammonium bromide (TTAB) from Sigma (Labkemi, Stockholm, Sweden) and octanesulfonate from Fluka (Buchs, Switzerland). The water used was ion exchanged and run through a Milli-Q system (Millipore, Bedford, MA).
2.4. Soil solutions The centrifugation drainage technique and sampling procedure described by Giesler and Lundstro¨m [15] were used to obtain the soil solutions. When sampling the mor (O-horizon), the upper organic soil was removed and divided into two layers. The green parts were removed. The B1 and B2 samples were taken at 0–5 and 5–10 cm depth in the illuvial horizon, respectively. The soil samples were centrifuged for 30 min at a speed of 14000 rpm. After centrifugation the collected centrifugates were filtered through a 0.45 mm filter (Millex-HV, Millipore) and the pH was then determined. The soil solution was finally transferred
P.A.W. 6an Hees et al. / Talanta 48 (1999) 173–179
to 10 ml polypropylene tubes and stored in a freezer until further analysis.
2.5. Sample preparation and HPLC analysis A stirred ultrafiltration cell (model 8050, Amicon) equipped with a Diaflo YM-1 filter was used for removing interfering compounds. The device was pressurised by N2 gas at a pressure of 3–3.5 bar, and the filtrate was collected in a tube surrounded by ice. The sample (10 ml) was filtered twice and the filters were changed between the two runs. Before use the ultra filters were immersed for at least 1 h in firstly 0.1 M NaOH, secondly 5% (w/w) NaCl, and finally rinsed twice in Milli-Q water. This cleaning procedure was needed to ensure the removal of, especially, lactic acid which otherwise could contaminate the sample. A blank was run and no interfering peaks were found. The sample was then acidified by means of 0.5 M H2SO4 to pH3, and run through a cation exchanger (0.4 ml Bio-Rex 70, 200 – 400 mesh) in the H + form. The first 1 ml of eluate after the pH change was discarded and then a sample of approximately 4 ml was collected. The sample was immediately run on the HPLC column. The mobile phase consisted of 0.2% (v/v) H3PO4 and the flow rate was 0.5 ml min − 1. The injection volume was 50 ml. First one run was carried out with a column oven temperature of 30°C. A second run was then performed at 60°C when citric, shikimic and lactic acids were determined. This procedure was required due to the overlapping of lactic and shikimic acids at 30°C and the appearance of a ghost peak interfering the determination of citric acid. The acids were calibrated using standards which had been treated in the same way.
2.6. CZE analysis CZE analysis was carried out according to [16] and was performed on a Quanta 4000. An 82-cm (74-cm to detector) fused-silica capillary (J&W Scientific, Folsom, CA) with an i. d. of 75 mm and a carrier electrolyte consisting of 5 mM 1,2,4-benzenetricarboxylic acid with 0.5 mM TTAB (pH 8)
175
were used. TTAB was added to reverse the electroosmotic flow. Injection was performed by electrokinetic injection at − 5 kV for 45 s or by hydrostatic injection at 100 mm for 30 s, and the applied separation voltage was − 15 kV. The analytes were detected by indirect UV detection at 254 nm. Octanesulfonate, which acts as a terminating electrolyte, was added to the samples to final concentrations of 70 mM to achieve an isotachophoretic steady state during the sample introduction.
2.7. Titrations of soil solution The sample was first acidified to pHB 2.5 and run through a cation exchanger. It was then bubbled with N2 for 15 min and titrated by means of 0.01 M NaOH. The evaluation was made using the procedure outlined by Molvæsmyr and Lund [17] based on the Gran extrapolation giving the concentrations of strong (SA) and weak acids (WA).
3. Results and discussion
3.1. Calibration and performance A satisfactory separation of a wide range of acids could be achieved (Table 1). The performance of the HPLC method was evaluated with regard to limits of detection, calibration and recoveries of standard additions. Citric, shikimic and lactic acids were determined at 60°C, all other acids at 30°C. The results are presented in Table 1. Succinic acid was also tested and it was found that this acid had very similar retention times to shikimic acid, both at 30 and 60°C. However, succinic acid could not be found in the samples when running CZE. All calibration graphs were linear and had a correlation coefficient r\ 0.99. Two or three replicates were run and each standard was injected twice. In the case of fumaric, shikimic and oxalic acids lower concentrations of the standards were used due to its high UV-absorbance. Recovery studies were performed by standard addition of all acids to three samples of the E-horizon.
50, 250, 500 50, 250, 500 50, 250, 500 5, 25, 50 50, 250, 500 50, 250, 500 50, 250, 500 50, 250, 500 5, 10, 25 50, 250, 500 5, 10, 25 50, 250, 500
Acetic Citric Formic Fumaric Lactic Malic Malonic Oxalic
3 2 3 2 2 3 3 3 2 3 2 3
Replicates (n)
0.994 1.000 0.998 0.992 0.995 0.998 0.996 0.998 0.993 0.998 0.994 0.993
Corrolation coefficient (r)
25.7 0.2 4.0
23.9 3.1 16.0 0.1 7.1 6.1 8.1 0.4
LOD (mM)
Retention time (min) 21.42 12.04b 19.98 24.25 17.95b 14.67 15.36 10.09 26.00 16.94b 13.25
Recovery (n =3) (standard addition 100 mM)a (%) 93 9 8 99 9 5 95 9 7 102 9 3 97 9 6 105 97 100 97 89 915 98 98 101 93 100 9 7
a
Confidence limits (95%) of standard addition to sample. In the case of fumaric and shikimic acids 10mM; b determined at 60°C, all other acids at 30°C.
Propionic Shikimic Tartaric
Concentration (mM)
Acid
Table 1 Calibration of organic acids
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Table 2 HPLC determinations and titration data of the soil solutions from Hyytia¨la¨
pH Acetic acid (mM) Citric acid (mM) Fumaric acid (mM) Lactic acid (mM) Oxalic acid (mM) Shikimic acid (mM) DOC (mM) %LMW acids of DOC WA(tot)a (meq l−1) %LMW acids (meq l−1) of WA(tot)
O1
O2
E
B1
B2
3.76 174+28 80+6 2.4+5 27+41 7.2+2 21.4+2 32.33 3.3 3.44 14.0
3. 53 67+42 27+7 1.3+6 n.d. 4. 5+3 5.1+2 22.08 1.6 2.22 7.5
3. 83 — 19+6 1.3+5 n.d. 4.1+3 1.6+2 4.94 3.1 0.96 7.7
4. 85 — — 1.1+6 n.d. 8.5+3 1.7+2 3.13 1.2 0.42 5.0
5.99 — — — n.d. tr. 1.3+2 1.02 0.9 0.37 0.3
Data is given for pH, concentrations+confidence limits(95%), DOC, and total WA (WA(tot)) content. n.d., Not detectable, area of peak smaller than intercept of calibration equation or below LOD; tr., traces, peak identified but not integrated. a In the case of O1 SA is included.
Good recovery values were obtained for all acids. The poorer value for oxalic acid can partly be explained by the larger variance and the fact that this acid elutes close to the void peak. A correct determination of this acid might be difficult when analysing samples with very high DOC levels (\ 1000 ppm). The limits of detection (LOD), calculated as the concentration corresponding to three times the background noise, were in the range 0.1–26 mM. The values were in accordance with the UV absorbance of the different acids, resulting in better limits of detection for the acids with higher absorbance. The repeatability for natural samples was checked by performing duplicate analyses for two samples (see below), and was estimated as the pooled S.D. for the replicates. The deviation was in the range 0.03 – 1.40 mM for the commonly detected acids (citric, shikimic, oxalic and fumaric acids). The variance for acids found in natural samples was not significantly greater than for standards as evaluated by F-tests [18].
3.2. Application and identification Soil solutions from five soil layers were analysed for LMW organic acids, SA and WA content using the methods described above (Table
2). A comparison of a chromatogram before and after the sample clean-up procedure is shown in Fig. 1. The acids were identified by comparing retention times and where possible, due to the void peak, UV spectra. In the case of oxalic, citric and fumaric acids these were also confirmed by standard additions. In order to obtain an independent identification of the organic acids, some of the samples were also analysed by CZE. Identification was made by retention time and by spiking the samples with a small amount of organic acid stock solution. By this procedure citric, fumaric, lactic, oxalic, acetic and shikimic acids were tentatively identified in the CZE electropherograms. Although the analysis was performed on a qualitative basis it could be established that the concentrations determined by the HPLC application were of the same order of magnitude as compared to the CZE analysis. An electropherogram of the O2 horizon solution is showed in Fig. 2. Two replicates of the O1 and E horizons including pretreatment and HPLC analysis were made, but single analyses were carried out for the other samples. Each sample was injected twice on the HPLC column. Several different acids were identified, of which all have been reported earlier [1,5,19]. Shikimic acid and traces of oxalic acid
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Fig. 1. HPLC chromatogram (run at 30°C) of an O2 soil solution before (a) and after pretreatment (b). The insert picture in (b) shows an enlargement of the chromatogram.
were found in all horizons. Citric and fumaric acids were also observed in most samples. As would have been expected the concentrations decrease in the deeper soil layers. A small increase of oxalic and shikimic acids was however observed in the B1 horizon. Concerning the acidity, the fraction of the LMW organic acids made up 0.3 – 14.0% of the WA(tot). SA was only found in O1, and was
included in the WA(tot) value because experiments showed that for citric and oxalic acids the carboxyl group with the lowest pKa was not included in the WA(tot) measurement. The percentage of LMW acids decreases through the soil profile. Regarding the DOC, LMW acids made up 0.9–3.3%. Also the fraction of the DOC consisting of LMW acids generally decreased in the deeper horizons.
P.A.W. 6an Hees et al. / Talanta 48 (1999) 173–179
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Fig. 2. CZE electropherogram of an O2 soil solution after pretreatment. Electrokinetic injection. Peaks: 1, oxalic; 2, citric; 3, fumaric; 4, acetic; 5, shikimic acids.
Acknowledgements This study was financially supported by the Swedish Natural Science Research Council (NFR). The authors would like to thank A.-M. van Hees, M. Nystro¨m and V. Stigsson for assistance. Dr H. Ilvesniemi is acknowledged for providing facilities for the field work and L. Backman for soil sampling.
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