mass spectrometry

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Journal of Chromatography A, 1171 (2007) 112–123

Determination of functionalised carboxylic acids in atmospheric particles and cloud water using capillary electrophoresis/mass spectrometry Dominik van Pinxteren, Hartmut Herrmann ∗ Leibniz-Institut f¨ur Troposph¨arenforschung (IfT), Permoserstrasse 15, 04318 Leipzig, Germany Received 8 June 2007; received in revised form 24 August 2007; accepted 10 September 2007 Available online 14 September 2007

Abstract A capillary electrophoresis/electrospray ionisation mass spectrometry (CE/ESI-MS) method was developed for the determination of 38 organic acids in atmospheric particles and cloud water. The target analytes include many functionalised carboxylic acids, such as carboxylic acids with additional oxo-, hydroxy- or nitro-groups. These compounds are of large interest as their determination might give new insights into the atmospheric multiphase chemistry. OASIS HLB sorbent material (Waters) was used to extract and enrich polar carboxylic acids from aqueous solutions with recoveries greater than 80% for most analytes. Relative standard deviations in the range of 4–20% for peak areas (n = 5), including the SPE step, and 0.2–0.5% (n = 8) for migration times were found. The limits of detection (S/N = 3) ranged from 0.005 to 0.6 ␮mol l−1 for an ion-trap mass spectrometer and from 0.0004 to 0.08 ␮mol l−1 for a time-of-flight mass spectrometer. These detection limits translate into atmospheric concentrations in the low pg m−3 range based on the experimental conditions in this study. Severe matrix effects were observed for real samples, arising from complex co-extracted organic material. However, using the method of standard addition, most of the analytes could successfully be quantified in samples of ambient particles and cloud water with concentrations in the low ng m−3 to high pg m−3 range. These results demonstrate the suitability of the proposed method for the determination of a wide range of polar carboxylic acids at low concentrations in complex samples of different atmospheric phases. © 2007 Elsevier B.V. All rights reserved. Keywords: CE/MS; SPE; Experimental design; Organic acids; Organic aerosol

1. Introduction Atmospheric particles contain a significant amount of organic compounds, besides inorganic substances. In contrast to the well characterised inorganic fraction, the composition of the organic carbon (OC) is still largely unknown and only a small fraction of it could be identified so far [1,2]. The determination of single organic species is therefore a major task in current atmospheric research [1,3,4]. Among the identified compounds of organic particle constituents, carboxylic acids usually represent a major fraction [5]. Most of the literature studies performed up to now were focused on particle phase straight-chain mono- and dicarboxylic acids, usually with 2–10 C-atoms (e.g. [6–11]). Only very few measurements exist for functionalised carboxylic acids, i.e. monoor dicarboxylic acids with additional functional groups, such as

∗

Corresponding author. Tel.: +49 341 2352446; fax: +49 341 2352325. E-mail address: [email protected] (H. Herrmann).

0021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2007.09.021

oxo-, hydroxy- or nitro-groups. However, a large fraction of particulate OC is known to consist of highly oxidised compounds [12]. Therefore, a determination of functionalised carboxylic acids can be expected to extend the knowledge of the chemical composition of particulate OC. Additionally, functionalised carboxylic acids are likely to be produced in oxidation reactions in the atmosphere. Potential sources could be radical reactions in the liquid phase, leading to hydroxylated or oxygenated acids [13]. A study of functionalised carboxylic acids could therefore give new insights into tropospheric multiphase chemistry. For detailed studies of the OC composition, gas chromatography/mass spectrometry (GC/MS) is commonly used as an analytical technique. For GC/MS, polar compounds such as carboxylic acids need to be derivatised to more volatile products (e.g. esters), which makes the sample preparation time consuming. It also increases the risk of losses of the volatile esters during sample enrichment, which is usually done by volume reduction under vacuum [14–17]. Therefore, modern liquid phase separation techniques such as high performance liquid chromatography (HPLC) or capillary electrophoresis (CE)

D. van Pinxteren, H. Herrmann / J. Chromatogr. A 1171 (2007) 112–123

coupled to mass spectrometry (MS) are attractive alternatives to GC/MS determinations for the determination of polar compounds [18–23]. For ionisable compounds, CE is a very well suited tool due to the high separation efficiency, low sample consumption, short analytical times and low operation costs [24]. In combination with indirect UV detection it is a well established technique for the determination of short-chain organic acids in ambient particles (e.g. [2,25–27]). The aim of the present work is to develop and characterise a method for the determination of functionalised carboxylic acids in atmospheric particles and cloud water, using capillary electrophoresis/mass spectrometry (CE/MS). 2. Experimental 2.1. Chemicals The chemicals used in this work were obtained from the following suppliers: ammonium hydroxide solution (25%), acetic acid (>99.5%), and 2-propanol (>99.8%) from Fluka (Munich, Germany), methanol (Chromasolv, >99.0%), aqueous sodium hydroxide (NaOH, 1 mol l−1 ), and hydrochloric acid (HCl, 1 mol l−1 ) from Riedel-de Ha¨en (Munich, Germany). All carboxylic acids were obtained from Sigma–Aldrich (Munich, Germany). Their purity was always better than 97%, except 5oxoazelaic acid (96%), 2-hydroxy-3-nitrobenzoic acid (<95%), and 2-octenoic acid (technical grade, 85%). 5 ␮mol l−1 stock solutions of each carboxylic acid were prepared in 10 ␮mol l−1 aqueous NaOH and stored at −20 ◦ C. Standard mixtures were prepared in deionised water (Milli-Q, Millipore, Schwalbach, Germany) and stored at 4 ◦ C. 2.2. Sampling and sample extraction Sampling of atmospheric particulate matter with an upper aerodynamic diameter of 10 ␮m (PM10 ) was performed on quartz fibre filters (MK 360, Munktell, Sweden), which were placed into a Digitel DHA-80 filter sampler (Riemer, Hausen, Germany). The sampler was operated at a flow rate of 0.5 m3 min−1 for 24 h per sample. Before use, the filters were heated for 24 h at 105 ◦ C to reduce the blank content of carbonaceous material. The samples used in this study were taken during summer/autumn 2005 at the research station Melpitz, approximately 50 km northeast of Leipzig, Germany, in a rural area. After sampling, the filters were stored at 4 ◦ C. For extraction of water soluble carboxylic acids, 30 pieces (78.5 mm2 each) were punched out of the quartz filter and placed into a disposable 5 ml syringe (Omnifix, Braun, Melsungen, Germany). Deionised water (5 ml) was added and the solution was shaken at 900 rounds per minute for 2 h. The extract was then filtered through a pre-cleaned syringe filter (Acrodisc 13, Pall, Dreieich, Germany) and an aliquot (1–5 ml) was used for solid phase extraction (SPE, see Section 2.5). Cloud water samples were taken during the FEBUKO field campaign [28] using the Caltech Active Strand Cloudwater Collector 2 [29]. The sampling time was 2 h and the sampled cloud water was stored at −20◦ C until analysis. Cloud water sam-

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ples were filtered through a syringe filter just before the SPE step. 2.3. CE/MS CE separations were carried out in an Agilent 3D CE instrument (Waldbronn, Germany) using fused silica capillaries of 60–80 cm length, 50 ␮m inner diameter, and 360 ␮m outer diameter (CS-Chromatographie Service, Langerwehe, Germany). The polyimide coating at both ends of the capillary was removed (1–2 cm) by exposing into a flame. A 20 ␮mol l−1 ammonium acetate solution with 10% methanol (v/v) and a pH of 9.1 (adjusted with NH4 OH) was used as background electrolyte (BGE). It was freshly prepared on a weekly basis and stored at room temperature. Hydrodynamic injection was performed applying 40–50 mbar for 10–20 s, depending on capillary length. The separation was carried out at 30 kV and 20 ◦ C. In between two measurements the capillary was rinsed for 3 min with freshly replenished BGE. New capillaries were conditioned before connection to the electrospray ionisation (ESI) source by flushing with HCl (1 mol l−1 ), NaOH (1 mol l−1 ), and BGE. At the start of each measurement day, the capillary was flushed with deionised water and BGE. Two types of mass spectrometers were used in this study: An Esquire 3000plus ion-trap mass spectrometer (ITMS), and a micrOTOF time-of-flight mass spectrometer (TOFMS, both Bruker Daltonics, Bremen, Germany). The coupling of CE with MS was done via the commercially available ESI interfaces. The sheath liquid was delivered by a syringe pump (Cole-Parmer, Illinois, USA) with a flow rate of 4 ␮l min−1 . Nitrogen was used as nebulising gas at pressures of 0.3 bar (4 psi) for ITMS and 0.1 bar for TOFMS. The dry gas (nitrogen) was delivered at 8 l min−1 and a temperature of 200 ◦ C (ITMS) or 150 ◦ C (TOFMS). ESI was operated in negative mode with a voltage of 4.5 kV applied at the MS inlet capillary and an endplate offset of −500 V. The scanning mass range was from m/z 50 to 500 for both mass spectrometers. Due to the soft ionisation conditions of ESI, all analytes were detected as [M − H]− quasi-molecular ions. For exact mass measurements, the TOFMS was calibrated by injecting a small volume of lithium acetate solution (20 ␮mol l−1 , 10 mbar, 5 s) just before the injection of the sample. The lithium cations induce peaks of lithium acetate cluster ions at an early migration time, which can be used for the internal mass calibration of the TOFMS. From the exact mass for a given peak the most probable elemental composition of the compound was calculated by the instrument software (Bruker Daltonics microTOF Data Analysis 3.2). 2.4. Optimisation of ESI The Design-Expert 6 software (Stat-Ease, Minneapolis, USA) was used for creating experimental designs and for data processing. A two-level fractional factorial design was used to determine the parameters which significantly influence the intensity of the ESI-MS signals. The design consisted of 16 analysis runs with differently combined settings of 5 factors. These factors are listed together with their lower (−1) and higher levels

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Table 1 Factors and their corresponding levels used in the experimental designs Factor

Unit

−␣

(a) Fractional factorial design Fraction isopropanol % in sheath liquid Flow rate sheath ␮l min−1 liquid Pressure nebulising bar gas ◦C Temperature dry gas Flow rate dry gas l min−1 (b) Central composite design Fraction isopropanol % in sheath liquid Pressure nebulising bar gas

−1

0

+1

50

80

4

8

0.2

0.5

100 4 30 0.1

40 0.2

2.6. Method validation +␣

200 8 65 0.4

90 0.6

100 0.7

(+1) in Table 1(a). In a second step the significant parameters were optimised applying a central composite design (CCD). It consisted of 13 analysis runs which were carried out in a randomised order. The factors of the CCD are given in Table 1(b) together with their different levels (−␣, −1, 0, +1, +␣). The sum of peak heights of eight model compounds (heptenoic acid, 7oxooctanoic acid, 2-hydroxyhexanoic acid, 4-hydroxybenzoic acid, 2-isopropylmalic acid, phthalic acid, azelaic acid) was chosen as response for both designs. A quadratic polynomial model was fitted to the experimental results and validated using the built-in statistical tests of the Design-Expert software. 2.5. SPE and enrichment For SPE, different sorbents were tested for this study: ENVI18 (C18, Supelco, Munich, Germany, 100 mg sorbent mass, 1 ml volume), Bondelut18 (C18, Varian, Darmstadt, Germany, 100 mg, 1 ml), OASIS HLB (polymer, Waters, Eschborn, Germany, 10 mg, 1 ml), Bondelut PPL (polymer, Varian, 50 mg, 1 ml), Nexus (polymer, Varian, 60 mg, 3 ml), FOCUS (polymer, Varian, 10 mg, 1 ml), and StrataX (polymer, Phenomenex, Aschaffenburg, Germany, 30 mg, 1 ml). Before sample application, the cartridges were conditioned with 1 ml methanol and equilibrated with 1 ml HCl (pH = 1 or 2). After sample application, the cartridges were rinsed with 1 ml HCl (pH = 1 or 2). The adsorbed analytes were eluted with 1 ml solvent into conically shaped glass vials (custom made). Analyte breakthrough and recoveries were determined by comparing the peak areas of the effluent and eluate with the peak areas from corresponding standard mixtures. For enrichment, the eluate was evaporated under a gentle stream of nitrogen to near dryness and transferred to a conically shaped CE-vial (250 ␮l, PP, Agilent, Waldbronn, Germany). The walls of the glass vial were rinsed three times with 50 ␮l methanol and the combined solutions were further evaporated to a final volume of 10–20 ␮l. To avoid wall adsorption losses, the walls of the CE-vial were carefully rinsed with the concentrated eluate before analysis.

The repeatability of peak areas (PA) and migration times (MT), as well as the SPE recoveries were determined with 5 ␮mol l−1 standard solutions (pH = 1 for SPE) using the ITMS as a detector. Calibration curves were recorded including the SPE and solvent evaporation steps. Standard solutions (5 ml, pH = 1) at five concentration levels were concentrated by the above described procedure and analysed using CE/TOFMS. The concentration levels were chosen to roughly meet the expected concentrations of the analytes in real samples. Adipic acid-d8 was used as internal standard (IS) to account for slight variations in the final volume of the extract. The limits of detection (LODs) were determined by analysing a dilution series of standard mixtures. The LOD was defined as the concentration at which the analyte peak in the corresponding extracted ion electropherogram (XIE) shows a signal to noise ratio of 3 (S/N = 3). The mass window of the XIE was set to ±0.5 Da (ITMS) and ±0.01 Da (TOFMS). For some m/z the narrow TOFMS mass window resulted in XIEs with no noise at all (flat baseline). In these cases the S/N could not be calculated and the LOD was estimated as the lowest concentration resulting in a clearly detectable peak. To allow a comparison with analytical techniques other than CE/MS, the liquid phase LODs were transformed into the corresponding atmospheric concentrations of the compounds. A final volume of 20 ␮l after evaporation of the SPE extract was assumed for this calculation and the recovery of the SPE step was considered. The LOD as atmospheric concentration furthermore depends on the sampling rate of the filter sampler (0.5 m3 min−1 ), the sampling time (24 h), the extracted filter area (15%), the solvent volume for filter extraction (5 ml), and the aliquot of the filter extract used for the SPE step (1 ml in the case of real samples). These sampling and sample preparation conditions result in a theoretical sampled air volume of 21.6 m3 being enriched in each concentrated SPE extract. 2.7. Matrix effects To study matrix effects for real samples, five filter samples from the Melpitz site with different concentrations of water soluble organic carbon (WSOC; 0.9, 3.4, 3.9, 4.5, and 5.5 ␮g m−3 ) were chosen. The WSOC concentrations served as indicators for the matrix concentration and were determined using a TOC-analyser (TOC-VCPH , Shimadzu, Duisburg, Germany). At different points of the enrichment process (after filter extraction with 5 ml H2 O, after SPE elution with 1 ml methanol, after volume reduction to approximately 150 ␮l, and after volume reduction to approximately 20 ␮l) 18 ␮l of the corresponding extract were spiked with 2 ␮l of a standard solution containing different carboxylic acids at a concentration of 50 ␮mol l−1 each. The spiked compounds were not present in the original sample extracts and covered a wide range of migration times. Thus, by comparing the PA of the spiked samples with the ones obtained from a matrix-free standard solution (18 ␮l methanol + 2 ␮l standard), the ionisation of carboxylic acids at different times during the separation could be studied.

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Fig. 1. Half normal probability plot with both significant effects marked.

3. Results and discussion 3.1. Optimisation 3.1.1. Background electrolyte In former studies of our group [19], a 20 mM ammonium acetate BGE with 10% (v/v) methanol and a pH of 9.1 resulted in good separation efficiency for acidic compounds. In the present work, slight variations of the ammonium acetate concentration (10–30 mM), the methanol fraction (0–15%), and the pH value (8 and 10) did not lead to significant improvements in the resolution of individual acids in a standard mixture. Therefore, this BGE was used for all further studies. 3.1.2. Electrospray ionisation The optimisation of electrospray parameters was conducted in two steps by applying experimental designs. The studies were carried out on both the ITMS and the TOFMS instruments. As the results did not differ between the instruments, only the data obtained with the TOFMS are reported here. In a first step, five factors were tested for their influence on signal intensity using a factorial design (see Section 2.4 and Table 1(a)). In Fig. 1, the results of this screening study are shown as half-normal probability plot. Random effects will follow a standard normal distribution and form a straight line in the probability plot. As can be seen, only the fraction of isopropanol in the sheath liquid and the pressure of the nebulising gas had a non-random, and therefore significant effect on the peak heights. The effect of the isopropanol fraction was pos-

itive, i.e. higher fractions led to higher signal intensity, while a higher nebulising pressure led to lower signal intensity. The remaining three factors did not strongly influence the peak intensity within the tested range. Their values were set arbitrarily (see Section 2.3). The two significant factors were optimised in a central composite design (Section 2.4 and Table 1(b)). In Fig. 2, the response surface of the sum of peak heights versus the two tested factors is shown. As can be seen, the peak intensities significantly increase towards higher isopropanol fractions and lower nebulising gas pressures. Interestingly, at low to intermediate isopropanol fractions the nebulising pressure does not have a large influence on the peak heights. At high isopropanol fractions, however, there is a sharp decrease in peak heights with increasing nebulising pressure. The optimal values were found to be 100% isopropanol as sheath liquid and 0.1 bar nebulising gas pressure. A pure organic sheath liquid can sometimes cause problems in electrospray ionisation due to its low electrical conductance. However, in our case the pure organic solvent did not affect the stability of the electrospray. The high pH of the BGE used resulted in a high electroosmotic flow in the CE capillary. This likely led to an efficient mixing of the sheath liquid with the aqueous BGE at the tip of the electrospray needle and thereby a sufficient electrical conductance. As mentioned above, the results of the optimisation studies for the ITMS were qualitatively the same. Due to a slightly different geometry of the ion source, the optimal nebulising pressure was 4 psi (ca. 0.3 bar) for this instrument. The optimised values were used for all further studies.

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Fig. 2. Response surface plot of the central composite design. Points represent experimental results on the design levels.

3.1.3. Solid phase extraction For matrix removal and analyte enrichment an SPE procedure was developed. In a first step of preliminary tests the adsorption efficiency of the OASIS HLB sorbent was studied using a standard mixture of model compounds (glutaric, adipic, pimelic, suberic, azelaic, terephthalic, octanoic, decanoic, benzoic, hydroxybenzoic, and methylbenzoic acid, 20 ␮mol l−1 each) at different pH values of 2, 4, and 10. It was found that all of the tested compounds were quantitatively retained at a pH of 2. At pH = 4 some of the compounds and at pH = 10 nearly all of the compounds showed significant breakthrough. Only the two most hydrophobic compounds in the standard mixture were still retained at the basic pH value. The pKa values of the model compounds lie between 3.5 and 5. From the results it can be seen that especially the more polar acids (more than one functional group) are effectively adsorbed only in their undissociated form. Therefore, the pH of all solutions was adjusted to a value of 2 or lower for all further SPE studies. In a next step, the desorption efficiency was studied for different methanol/water volume fractions (20/80, 40/60, 60/40, 80/20, and 100/0) in the desorption solution. Highest recoveries were obtained for methanol fractions of 80–100%. As a high organic fraction facilitates the subsequent volume reduction of the eluate, 100% methanol was used in all further studies to desorb the carboxylic acids from the SPE sorbent. In order to find the best suited sorbent material for the extraction of all analytes, a comparison of seven commercially available SPE cartridges was performed (see Section 2.5). For

all analytes except glutaric, dimethylmalonic, and 3-hydroxy3-methylglutaric acid (which were not yet included into the standard mixture at this stage of the study) the recoveries from the different sorbents were determined. In Fig. 3, the results are summarised in a box-whisker-plot. For some of the most polar compounds (e.g. hydroxy- and oxopentanoic acid) very poor recoveries were observed for the C18 materials and for one of the polymeric phases (FOCUS). The other four tested polymeric materials showed comparably high recoveries for most of the compounds, with OASIS HLB resulting in a slightly higher average recovery than the others. Therefore, this sorbent was chosen for all further studies.

Fig. 3. Comparison of analyte recoveries for different commercially available SPE sorbents.

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Fig. 4. Standard electropherogram with XIEs for all analytes (5 ␮mol l−1 , except nos. 14, 15, and 16: 1 ␮mol l−1 ). For peak labelling see Table 2. Due to the high noise level of m/z 141 (octenoic acid, peak no. 2, see Section 3.2.3), the baseline of this XIE is only shown between 5 and 6 min.

3.2. Method validation In qualitative studies a number of particle and cloud water samples were analysed with the described SPE CE/TOFMS method. A large number of peaks were detected which could tentatively be attributed to different functionalised mono- and dicarboxylic acids, based on the exact mass results of the TOFMS determinations and the migration time information. However, a reliable identification of many of the detected peaks was hindered due to the lack of authentic standards. The 38 compounds which were positively confirmed by comparing migration times in the real samples with those of authentic standard solutions are listed in Table 2. An electropherogram containing all analytes is shown in Fig. 4. As can be seen, some peaks partly overlap. By comparing PA of a standard mixture with single compound solutions no significant interferences were observed. The results of the method validation for these analytes are reported in the following sections.

3.2.2. Solid phase extraction and calibration The recoveries for the SPE step are given in Table 2 as averages of five extractions. Except for the most polar compounds (3-hydroxy-3-methylglutaric acid, glutaric acid, 4-oxopentanoic acid, methylsuccinic acid, and methylenesuccinic acid) the recoveries were better than 80%, which confirms the suitability of the chosen sorbent to efficiently adsorb polar organic acids from aqueous solutions. The PA RSDs for the SPE step were in the range of 4–20% and usually not much different from the values obtained by analysing the original aqueous standard (see Section 3.2.1). This shows that the SPE step did not deteriorate the precision of the method. The coefficients of determination (R2 ) for the linear regression lines of the calibration experiments are given in Table 2. The obtained values were between 0.9774 and 0.9999 and can be considered satisfactory and better. This again confirms the suitability of SPE CE/ESI-MS for the determination of small carboxylic acids from aqueous samples.

3.2.1. Repeatability The relative standard deviations (RSD) for both the MT and the PA are given in Table 2 for all analytes. The repeatability of the MT was very good with values below 0.5%. The repeatability of the PA was in the range of 4–21% which is acceptable for ESI. Iinuma and Herrmann [19] obtained RSDs of 0.3–0.4% for MT and RSDs of 3.6–8.8% for PA for the analysis of aromatic acids using the same instrumental equipment as in this study. For the determination of five different C4-carboxylic acids with CE/MS, RSD values of 0.2–0.6% for MT and 1.4–4.2% for PA were reported [30]. Better PA repeatability might be a result of the high analyte concentrations used by these authors (54–86 ␮mol l−1 ). RSDs of 4.5–15.2% for PA were reported for the determination of haloacetic acids using CE/MS in a non-aqueous electrolyte [31]. These data confirm that the repeatability of the separation and ionisation in this study compares very well to the results obtained for similar analytes using CE/MS.

3.2.3. Limits of detection The LODs for all analytes are given in Table 2. As can be seen, the LODs from TOFMS measurements are a factor of 3–125 lower than the corresponding ones obtained by ITMS. The much smaller mass window of the TOFMS (see Section 2.6) results in much lower noise in the XIE and therefore better S/N and lower LODs. The lowest LODs were obtained for hydroxy-nitrobenzoic acids which could be detected down to concentrations of 0.4 nmol l−1 . Octenoic acid (m/z 141) showed a rather high LOD. This was due to a very high noise level for m/z 141, resulting from the Na(CH3 COO)2 − -cluster ion. Trace amounts of sodium ions are usually present in the BGE, resulting from leaching of glassware. A literature comparison of the LODs is only possible for structurally similar compounds, as the analytes reported in this study have not been measured by CE/MS before. For aromatic acids from biomass burning LODs between 0.1 and 1.0 ␮mol l−1

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Table 2 Figures of merit m/z

Compound (peak no.)

a b

8-Hydroxyoctanoic acid (1) 2-Octenoic acid (2) 7-Oxooctanoic acid (3) 6-Oxoheptanoic acid (4) 2-Hydroxyhexanoic acid (5) 6-Heptenoic acid (6) trans-Cinnamic acid (7) 4-Oxohexanoic acid (8) 2-Hydroxypentanoic acid (9) 3-Hydroxybenzoic acid (10) 4-Hydroxybenzoic acid (11) 4-Oxopentanoic acid (12) Benzoic acid (13) 2-Hydroxy-4-nitrobenzoic acid (14) 2-Hydroxy-3-nitrobenzoic acid (15) 2-Hydroxy-5-nitrobenzoic acid (16) 2-Hydroxybenzoic acid (17) Sebabic acid (18) 3-Oxosebabic acid (19) Azelaic acid (20) 5-Oxoazelaic acid (21) Suberic acid (22) 3,3-Dimethylglutaric acid (23) Pimelic acid (24) 2,2-Dimethylglutaric acid (25) 4-Methylphthalic acid (26) 2-Isopropylmalic acid (27) Homophthalic acid (28) Adipic acid (29) 4-Oxopimelic acid (30) 3-Hydroxy-3-methylglutaric acid (31) 4-Hydroxy-3-nitrobenzoic acid (32) Phthalic acid (33) Terephthalic acid (34) Glutaric acid (35) Methylsuccinic acid (36) Dimethylmalonic acid (37) Methylenesuccinic acid (38)

S/N = 3. Calculated for a sampled air volume of 21.6 m3 .

Solid phase extraction (n = 5)

Calibration

Limits of detection

MT (min)

RSD MT (%)

RSD PA (%)

Recovery (%)

RSD PA (%)

Concentration range (␮mol l−1 )

R2

CE/ITMSa (␮mol l−1 )

CE/TOFMSa (␮mol l−1 )

CE/ITMSb (pg m−3 )

CE/TOFMSb (pg m−3 )

5.5 5.6 5.8 6.0 6.1 6.1 6.1 6.3 6.4 6.4 6.7 6.8 6.8 6.9 7.0 7.1 7.2 8.1 8.4 8.6 8.9 9.1 9.5 9.7 10.0 10.1 10.1 10.6 10.7 10.8 11.2 11.4 11.5 11.8 12.3 12.4 12.7 14.4

0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.2 0.3 0.3 0.3 0.2 0.3 0.3 0.3 0.3 0.3 0.4 0.4 0.3 0.5 0.4 0.4 0.4 0.4 0.3 0.5 0.5 0.5 0.4 0.5 0.5 0.5 0.5

15 21 8 15 14 18 15 10 8 16 13 4 10 18 14 15 12 16 7 11 11 8 9 6 6 11 11 11 11 9 5 7 9 8 15 8 8 8

88 104 98 90 88 83 95 98 79 102 106 42 88 89 87 101 92 90 103 95 88 84 94 102 95 91 91 93 100 82 6 97 88 99 39 57 94 63

13 16 13 12 12 10 14 18 8 15 15 15 11 18 15 14 9 17 8 4 15 14 12 6 6 17 18 17 10 8 11 13 9 5 20 10 12 12

0.004–0.1 0.01–0.25 0.004–0.1 0.002–0.05 0.001–0.025 0.02–0.5 0.004–0.1 0.01–0.25 0.001–0.025 0.001–0.025 0.002–0.05 0.004–0.1 0.001–0.025 0.001–0.025 0.001–0.025 0.001–0.025 0.001–0.025 0.002–0.05 0.02–0.5 0.04–1 0.1–2.5 0.01–0.25 0.004–0.1 0.004–0.1 0.001–0.025 0.01–0.25 0.02–0.5 0.004–0.1 0.1–2.5 0.04–1 0.004–0.1 0.001–0.025 0.04–1 0.004–0.1 0.01–0.25 0.01–0.25 0.002–0.05 0.001–0.025

0.9975 0.9991 0.9999 0.9946 0.9984 0.9906 0.9990 0.9959 0.9945 0.9993 0.9906 0.9774 0.9947 0.9998 0.9967 0.9977 0.9945 0.9939 0.9996 0.9999 0.9998 0.9977 0.9993 0.9992 0.9988 0.9992 0.9991 0.9992 0.9999 0.9995 0.9944 0.9861 0.9983 0.9963 0.9994 0.9992 0.9990 0.9968

0.4 >5 0.4 0.5 0.1 0.5 0.2 0.4 0.2 0.3 0.2 0.5 0.2 0.005 0.005 0.005 0.05 0.2 0.2 0.1 0.6 0.2 0.1 0.3 0.5 0.08 0.2 0.1 0.5 0.4 0.1 0.05 0.1 0.2 0.5 0.3 0.1 0.5

0.004 0.8 0.04 0.08 0.006 0.004 0.04 0.08 0.006 0.02 0.01 0.04 0.006 0.0004 0.0004 0.0004 0.004 0.02 0.03 0.03 0.08 0.02 0.003 0.008 0.02 0.02 0.04 0.02 0.02 0.08 0.008 0.002 0.03 0.04 0.05 0.03 0.01 0.01

67 >627 59 74 14 71 29 49 27 37 24 126 26 0.9 1 0.8 7 41 39 18 127 38 16 44 77 15 36 18 67 78 248 9 17 31 156 64 13 95

0.7 100 6 12 0.8 0.6 6 10 0.8 3 1 10 0.8 0.1 0.1 0.1 0.6 4 6 6 17 4 0.5 1 3 4 7 4 3 16 20 0.3 5 6 16 6 1 2

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159.103 141.092 157.087 143.071 131.071 127.077 147.045 129.056 117.056 137.024 137.024 115.040 121.030 182.010 182.010 182.010 137.024 201.113 215.093 187.098 201.077 173.082 159.066 159.066 159.066 179.035 175.061 179.035 145.051 173.046 161.046 182.010 165.019 165.019 131.035 131.035 131.035 129.019

Repeatability (n = 8)

D. van Pinxteren, H. Herrmann / J. Chromatogr. A 1171 (2007) 112–123

were reported [19]. Haloacetic acids were determined by CE/MS with LODs between 0.5 and 5.4 ␮mol l−1 [31]. For five different C4 dicarboxylic acids LODs between ca. 6 and 30 ␮mol l−1 were obtained [30] and for C3–C4 dicarboxylic acids LODs of 10–86 ␮mol l−1 were reported [32]. In the same work [32] glutaric acid was determined with an LOD of 30 ␮mol l−1 , which is considerably higher than the values of 0.5 ␮mol l−1 (ITMS) and 0.05 ␮mol l−1 (TOFMS) obtained in this study. This comparison shows that the LODs reached with CE/TOFMS under optimised conditions in this study are the best ones which have been reported for carboxylic acid analysis by CE/MS so far. The atmospheric LODs given in Table 2 are based on sampled air volume of 21.6 m3 as described in Section 2.6. As can be seen, the atmospheric LODs are below 0.25 ng m−3 for all analytes (except octenoic acid) for CE/ITMS measurements. Taking advantage of the high sensitivity of the TOFMS, even values in the sub-pg m−3 range can be reached for some analytes. It must be noted, however, that the calculation of these values is based on the results of aqueous standard measurements. Matrix effects may lead to higher LODs for real samples due to ion suppression (see Section 3.3). However, even assuming the LODs to be one order of magnitude higher due to ion suppression, the values presented in Table 2 are still very low. This can be shown by comparing the LODs obtained by SPE CE/MS with literature data obtained by different analytical techniques. As most of the compounds in this study have not been determined in field studies of organic particles or cloud water, the comparison is limited to some dicarboxylic acids and two oxo-acids. Additionally, the LODs for the studied compounds were reported only in a few publications. These data are summarised in Table 3. For a reasonable comparison, the LODs were translated to 21.6 m3 sampled air volume using the information about sampling and sample preparation given in the respective work. As can be seen, the LODs obtained by the frequently applied GC/MS methods are in most cases substantially higher than the ones obtained in this study. The very scarce data available for ion chromatography (IC) and high performance liquid chromatography (HPLC) are even higher. This is mainly due to the fact that these methods do not include a preconcentration step, unlike the GC/MS methods and the method described in this work.

119

The literature comparison reveals that the combination of SPE and CE/TOFMS forms probably the most sensitive method to date for the determination of a wide range of small carboxylic acids from atmospheric samples. 3.3. Matrix effects Matrix effects for real sample measurements were studied at different stages of the enrichment process using samples with different matrix concentrations (see Section 2.7). The results of these studies are shown in Fig. 5. As can be seen from Fig. 5a, the PA ratios (spiked samples vs. aqueous standards) were close to unity for all spiked compounds in the 1 ml methanol sample extract directly after the SPE step. This means, no matrix effects were observed at this stage of the sample enrichment, regardless of the matrix concentration in the sample (indicated by the WSOC concentration of the respective sample). However, at this stage hardly any analytes were directly detectable in the extract, either. After the blow-down to 150 ␮l the PA ratios were found to be clearly below unity for all spiked compounds besides cyclopropane-1,1-dicarboxylic acid and 2,5dihydroxyterephthalic acid (Fig. 5b). Furthermore, the PA ratios decreased with increasing WSOC concentration in the samples. This indicates that co-extracted organic matrix substances suppressed the ionisation of the spiked model compounds. The higher the matrix concentration was, the more drastic ion suppression was observed. The same effect can be seen from Fig. 5c and d within the enrichment process of the samples. The more dilute extracts (5 ml aqueous filter extract and 1 ml methanolic SPE extract) did not exhibit significant ion suppression, while with increasing enrichment the PA ratios strongly decreased. The exceptions, again, were cyclopropane-1,1-dicarboxylic acid and 2,5-dihydroxyterephthalic acid. The ion suppression was most likely induced by higher-molecular-weight compounds (HMWC) of complex structure, which are usually present in atmospheric particles and were highly abundant in the enriched samples. These HMWC were released from the CE capillary in broad “humps” over a wide range of migration times. At the late migration time at which cyclopropane-1,1-dicarboxylic acid and 2,5-dihydroxyterephthalic acid were detected, however, their abundance was very low. Therefore, no ion suppression

Table 3 Comparison of LODs with literature data, translated to 21.6 m3 sampled air volume and appropriately rounded

Glutaric acid Methyl succinic acid Adipic acid Pimelic acid Phthalic acid Suberic acid Azelaic acid Sebabic acid 4-Oxopimelic acid 4-Oxopentanoic acid a

CE/ITMS (this work) (pg m−3 )

CE/TOFMS (this work) (pg m−3 )

GC/MS [9] (pg m−3 )

GC/MS [52,53] (pg m−3 )

GC/MS [54,55] (pg m−3 )

156 64 67 44 17 38 18 41 78 126

16 6 3 1 5 4 6 4 16 10

1970 1620 2890 1850 12300 1160 1620 1740

(ca. 40)a (ca. 40)a (ca. 40)a (ca. 40)a (ca. 200)a (ca. 40)a (ca. 40)a (ca. 40)a (ca. 40)a

ca. 470–2310 ca. 470–2310 ca. 470–2310 ca. 470–2310 ca. 470–2310 ca. 470–2310 ca. 470–2310

2430

Sampled air volume not given, no translation to 21.6 m3 possible.

GC/MS [56] (pg m−3 )

ca. 7

ca. 7

IC [57] (pg m−3 )

HPLC [58] (␮g m−3 )

4580

26 29 38

4580

120

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Fig. 5. Ion suppression for spiked compounds against WSOC concentration (a and b) and enrichment factor of the sample (c and d). Number in parentheses is migration time of the compound in min.

was observed for these model compounds. The origin and the nature of such HMWC in atmospheric particles are unclear at present [33]. From the results of this study, however, it seems that they play a critical role in the quantitative determination of organic aerosol constituents by ESI-based techniques. Attempts to separate HMWC from small carboxylic acids by a modified SPE procedure or by an ion exchange clean-up were not successful. 3.4. Analysis of real samples One ambient particle sample and one cloud water sample were analysed with the developed method. Due to the severe matrix effects observed, the quantification of the analytes was done by the method of standard addition. In Fig. 6, the base peak electropherogram (BPE) for m/z 50–500 and the XIEs for all analytes detected in the ambient particle sample are shown. The quantitative results of the measurements are given in Table 4. The highest concentrations in the real samples were found for aliphatic dicarboxylic acids (C5–C9). Among all carboxylic acids this substance group has been most frequently determined in atmospheric particles, usually by GC/MS (e.g. [6–11]). Fewer measurements exist for atmospheric liquid phases such as snow, ice, and rain [34–37]. Cloud water concentrations of C6–C9 dicarboxylic acids were published only twice in the literature [28,38]. Additionally, glutaric acid was determined in two studies [39,40]. Relatively high concentrations in the real samples were also found for some aromatic dicarboxylic acids. Phthalic acid

showed substantial blank concentrations on a blank quartz filter. As no blank correction was performed for these example measurements, the true concentration of phthalic acid is lower than the value reported in Table 4. Similarly to the aliphatic dicarboxylic acids, phthalic acid has been determined frequently in different atmospheric phases (see references above). Fewer measurements exist for the particulate concentrations of terephthalic acid and 4-methylphthalic acid (e.g. [8,41–43]). Liquid phase determinations of 4-methylphthalic acid were reported only for rainwater [35,36]. Terephthalic acid has never been measured in atmospheric liquid phases. Among the oxo-dicarboxylic acids, 4-oxopimelic acid has been determined in particles (e.g. [6,7,43,44]) and rainwater [37]. Particulate concentrations of further oxo-dicarboxylic acids have been reported only once [21]. Liquid phase concentrations of further oxo-dicarboxylic acids are not available in the literature. At least for three of these interesting compounds concentrations can be obtained in future field studies using the proposed method. The lack of commercially available standards unfortunately hinders the reliable quantification of a larger number of oxo-dicarboxylic acids. Lack of standards poses a problem for other functionalised dicarboxylic acids, too. However, one hydroxylated C7-dicarboxylic acid and one unsaturated C5-dicarboxylic acid could be quantified in the samples. The unsaturated C5-dicarboxylic acid was determined as methylenesuccinic acid. As isomeric structures cannot always be resolved by the CE method, the corresponding peak could also be methylmaleic acid. This compound has been found in both particulate (e.g. [45,46]) and aqueous atmospheric phases (e.g.

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121

Fig. 6. Base peak electropherogram and extracted ion electropherograms for a PM10 filter sample. Corresponding concentrations are given in Table 4.

[34,36]). Hydroxylated dicarboxylic acids with more than five C-atoms have rarely been determined in the atmosphere [43,47]. Among the functionalised monocarboxylic acids oxomonocarboxylic acids have been determined frequently in particles (e.g. [9,15,41,43,46]) and ice, snow, and rainwater [34,36,37]. C3–C6 hydroxy-monocarboxylic acids were found only in particles [43]. Few concentrations of particulate 4hydroxybenzoic acid [48–50] and 3-hydroxybenzoic acid [48]

are available in the literature. 2-Hydroxybenzoic acid was found in our samples. The compound class of hydroxy-nitrobenzoic acids is reported for the first time in atmospheric particles and cloud water. This short literature overview demonstrates the scarce body of atmospheric concentration data for many of the described functionalised carboxylic acids. In future field measurements the proposed CE/MS method will therefore allow to study these compounds in more detail.

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Table 4 Results from real sample measurements Compound

PM10 (ng m−3 )

Cloud water (␮mol l−1 )

Cloud water (ng m−3 )

Hydroxy-monocarboxylic acids 2-Hydroxypentanoic acid 2-Hydroxyhexanoic acid 8-Hydroxyoctanoic acid

0.2 n.d. n.d.

n.d. n.d. n.d.

n.d. n.d. n.d.

Oxo-monocarboxylic acids 4-Oxopentanoic acid 4-Oxohexanoic acid 6-Oxoheptanoic acid 7-Oxooctanoic acid

n.d. n.d. 2.9 n.d.

n.d. n.d. n.d. n.d.

n.d. n.d. n.d. n.d.

Unsaturated monocarboxylic acids 6-Heptenoic acid n.d. 2-Octenoic acid n.d.

n.d. n.d.

n.d. n.d.

Aromatic monocarboxylic acids Benzoic acid trans-Cinnamic acid

0.1 0.03

4.3 1.8

Aromatic hydroxy-monocarboxylic acids 2-Hydroxybenzoic acid 0.1 3-Hydroxybenzoic acid n.d. 4-Hydroxybenzoic acid n.d.

n.d. n.d. n.d.

n.d. n.d. n.d.

Aromatic hydroxy-nitro-monocarboxylic acids 2-Hydroxy-3-nitrobenzoic acid 0.3 2-Hydroxy-4-nitrobenzoic acid n.d. 2-Hydroxy-5-nitrobenzoic acid 0.4 4-Hydroxy-3-nitrobenzoic acid 0.3

0.03 0.001 0.03 0.01

2.5 0.05 2.1 0.7

Dicarboxylic acids Glutaric acid Methylsuccinic acid Dimethylmalonic acid Adipic acid Pimelic acid 2,2-Dimethylglutaric acid 3,3-Dimethylglutaric acid Suberic acid Azelaic acid Sebabic acid

6.8 6.6 0.1 13.8 1.6 n.d. 0.1 2.1 2.1 n.d.

0.4 n.d. 0.01 0.8 0.1 n.d. 0.01 0.04 0.02 n.d.

19.8 n.d. 0.7 47.8 5.5 n.d. 0.5 2.9 1.4 n.d.

Hydroxy-dicarboxylic acids 3-Hydroxy-3-methylglutaric acidn.d. 2-Isopropylmalic acid 0.5

n.d. 0.01

n.d. 1.0

Oxo-dicarboxylic acids 4-Oxopimelic acid 5-Oxoazelaic acid 3-Oxosebabic acid

8.7 2.7 n.d.

0.1 0.1 n.d.

5.0 9.2 n.d.

Unsaturated dicarboxylic acids Methylenesuccinic acid

0.3

0.1

3.1

Aromatic dicarboxylic acids Homophthalic acid Phthalic acid Terephthalic acid 4-Methylphthalic acid

1.1 12.4 4.0 2.4

0.1 0.9 n.d. 0.2

4.7 61.0 n.d. 11.1

0.4 0.2

n.d.: not detected.

4. Conclusions A method has been developed to determine the concentrations of 38 carboxylic acids in aqueous extracts of atmospheric particles and cloud water using CE/MS and SPE for sample

enrichment. The method shows excellent LODs, high separation efficiency, and good repeatability. A successful application of the method to complex real samples demonstrated that CE/MS in combination with SPE forms an attractive alternative to more established derivatisation-based GC/MS techniques. The proposed method offers the possibility to determine a large number of functionalised carboxylic acids within short analysis times. This will allow conducting comprehensive field studies and might lead to interesting new insights into the atmospheric chemistry of this poorly studied compound class. Future improvements of the method could lie in a selective separation of matrix compounds from the analytes to overcome the observed problems with ion suppression and permit a less laborious quantification. Membrane extraction techniques might be a promising option for this purpose [51]. An online implementation of the SPE step could further simplify the handling of high sample numbers in future field studies.

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