Anal Bioanal Chem (2002) 372 : 630638 DOI 10.

1007/s00216-002-1246-6

S P E C I A L I S S U E PA P E R

V. Van den Bergh H. Coeckelberghs I. Vanhees R. De Boer F. Compernolle C. Vinckier

HPLCMS determination of the oxidation products of the reaction between - and -pinene and OH radicals
Received: 7 December 2001 / Revised: 27 December 2001 / Accepted: 13 January 2002 / Published online: 28 February 2002 Springer-Verlag 2002

Abstract Biogenic non-methane hydrocarbons such as isoprene, -pinene, and -pinene, are emitted by forests in very large quantities. To evaluate the role of - and pinene and their contribution to the global production of trace gases and especially aerosol precursors, a study of the oxidation mechanism of - and -pinene with hydroxyl radicals must be conducted. The degradation products of both monoterpenes with hydroxyl radicals were identified and quantified in a fastflow reactor. The products were collected on a liquid-nitrogen trap coated with a 2,4-DNPH solution to which two internal standards (benzaldehyde-2,4-DNPH and tolualdehyde-2,4-DNPH) had been added. The collection method was based on the in situ conversion of aldehyde and/or ketone compounds to their 2,4dinitrophenylhydrazone derivatives. The derivatives were analyzed by HPLCMS using APCI(). TIC chromatograms and mass spectral data for the various oxidation products are presented. For -pinene, pinonaldehyde is the most important degradation product, with smaller amounts of acetone, formaldehyde, campholenealdehyde, and acetaldehyde. For -pinene, nopinone and formaldehyde are the most abundant products, of almost equal importance, whereas acetone and acetaldehyde are minor compounds. Keywords Monoterpenes DNPH HPLCMS Product yields Fast-flow reactor

monoterpenes. Because these monoterpenes are unsaturated hydrocarbons, they are highly reactive towards ozone, OH, and the nitrate radical; as a result of these reactions, these hydrocarbons might affect the concentration of trace compounds in the atmosphere on a global scale [4]. These trace compounds can be divided into three categories volatile compounds such as carbon monoxide and carbon dioxide [5, 6, 7], semi-volatile compounds such as formaldehyde, acetone [8, 9], pinonaldehyde [6, 9, 10, 11, 12] and nopinone [6, 10, 11, 12]. Monoterpenes are also known to be involved in the production of atmospheric aerosols [6, 13, 14, 15]. Because the overall chemistry in the atmosphere is far too complex to be studied in situ, experiments must be performed on a laboratory scale. To establish the degradation paths and to determine the product yields of the /-pinene/OH reaction, measurements were conducted in a fast-flow reactor with a clean OH radical source. In this way the reaction with hydroxyl radicals could be separated from other primary reactions with ozone or nitrate radicals. In this paper results from the fast-flow reactor studies of monoterpene oxidation are described. The OH oxidation of - and -pinene has been studied in the presence of NOx. The semi-volatile oxidation products have been identified and quantified for the /-pinene/OH reaction.

Experimental Introduction
Biogenic non-methane hydrocarbons are emitted by forests in very large quantities which are an order of magnitude larger than anthropogenic volatile hydrocarbon emissions [1, 2, 3]. A large contribution can be attributed to the
V. Van den Bergh () H. Coeckelberghs I. Vanhees R. De Boer F. Compernolle C. Vinckier Department of Chemistry, KULeuven, Celestijnenlaan 200F, 3001 Leuven, Belgium e-mail: viviane.vandenbergh@chem.kuleuven.ac.be Fast-flow reactor The fast-flow reactor technique with its clean OH radical source was selected to simplify the reaction chemistry. Details of the experimental technique have been published elsewhere [16, 17]. The fast-flow reactor consists of a quartz tube with an internal diameter of 2.8 cm and a length of 70 cm. By means of an oil rotary pump with a nominal pump capacity of approximately 12 m3 h1, a flow velocity of 3.81 m s1 was obtained with helium as carrier gas. The OH radicals were generated by the titration reaction H+NO2OH+NO. The H atoms needed for this reaction were produced by means of a microwave discharge in an H2/He mixture. In all experiments the H2 concentration was 1.371013 molecules cm3

631 and the power of the discharge was 100 W (except for the blank, when the discharge was turned off). The experiments were conducted at total pressures of 50 and 100 Torr (1 Torr=133.322 Pa) helium containing 20% oxygen. Hydroxyl radicals were produced in an upstream zone in the fast-flow reactor by reacting excess hydrogen atoms with NO2 at a concentration of 3.621012 molecules cm3. In the downstream zone, addition of - or -pinene was achieved by allowing a fraction of the carrier gas helium to flow through a vessel containing the - or -pinene; this resulted in a concentration of - or -pinene in the reactor in the range from 1012 to 1013 molecules cm3. The amount of - and -pinene consumed was determined from the weight difference of the vessel before and after the experiments. Sampling method The semi-volatile products were collected over a period of 5 to 6 h on a liquid-nitrogen (LN2) trap, installed at the downstream end of the reactor; the batch samples were subsequently analyzed by HPLC. The LN2 trap was coated with a solution containing 2,4dinitrophenylhydrazine, sulfuric acid, dichloromethane, and acetonitrile (ACN). Because of this coating solution all the aldehyde and ketone compounds formed in the /-pinene/OH oxidation reaction were converted to their 2,4-dinitrophenylhydrazone derivatives. This is the most commonly applied method for the determination of carbonyl compounds in environmental samples [18, 19, 20, 21, 22, 23, 24]. Details of the sampling method are given elsewhere [25, 26]. In these collection experiments, the solution contains two internal standards (I.S.): benzaldehyde-2,4-DNPH and tolualdehyde-2,4-DNPH. These two internal standards were needed because treating the trap could result in loss of coating material, and subsequent loss of oxidation products. To correct this problem, benzaldehyde-2,4-DNPH was added to the solution before coating of the LN2 trap, whereas tolualdehyde-2,4-DNPH was added to the solution after rinsing the collected residues from the trap. Adding these two I.S. at the same concentration enables correction for possible losses of the first I.S. benzaldehyde-2,4-DNPH. It is assumed in this work that the oxidation products are lost to the same extent as the first I.S. When the collection experiment was complete the LN2 trap was brought to atmospheric pressure for further preparation of the collected samples. For the small carbonyl compounds, e.g. formaldehyde, the derivatization process at room temperature has been shown to be complete after 10 min [27]. Because these volatile carbonyl compounds are trapped in an organic medium in which they are very soluble, there will be no loss during warming up of the collected samples. It was checked that for the oxidation products of -pinene the derivatization reaction was complete within 2 h at room temperature. When the solution was not analyzed immediately it was kept in a refrigerator. For -pinene the derivatization of nopinone is much slower. HPLC measurements of the solution were conducted over a period of 24 h. This revealed that nopinone is completely derivatized only after 15 h. When collection experiments were performed on -pinene the solution was kept at room temperature overnight before analysis. Method of analysis HPLCMS The carbonyl-2,4-DNPH derivatives were analyzed by high-performance liquid chromatographymass spectrometry (HPLCMS) with atmospheric pressure chemical ionization (APCI). The negative ion mode was selected for the measurements [24]. The 2,4-DNPH solutions were separated on a cc Nucleosil 100 C18 column (250 mm length3 mm inner diameter, 5 m particles) using the HewlettPackard 1100 HPLC instrument. Separations were performed at 35 C using a mobile phase gradient from 5% acetonitrile (ACN)/95% water to 84% ACN/16% water in 50 min, followed by 10 min isocratic elution. The flow rate was 0.6 mL min1 and the sample injected was 10 L, by means of a Rheodyne injector. A diode array detector (DAD) and the total ion current (TIC) signal of a triple quadrupole mass spectrometer (MS) (Quattro II, Micromass, Manchester) were available for detection. The DAD was set at a wavelength of 360 nm. The mass spectrum corresponding to each peak in the TIC chromatogram was compared with spectra of reference materials which were obtained from commercial sources or by synthesis. Reagents The reagents supplied as gas mixtures were: NO2 (0.1%) in helium (Oxhydrique), H2 (0.1%) in helium (Praxair), O2 with a purity of 99.998% (LAir Liquide), and He with a purity of 99.995% (LAir Liquide). The purities of the liquid reagents - and -pinene (Aldrich) were 98% and 99%, respectively. The solvents used were acetonitrile HPLC grade (Biosolve) and dichloromethane p.a. (Merck). 2,4-DNPH was recrystallized from ethanol, rinsed with ethanol, dried in a dessicator, and analyzed by HPLC for possible carbonyl impurities. Formaldehyde-, acetaldehyde-, acetone-, benzaldehyde-, and tolualdehyde-2,4-DNPH, purity 99%, were obtained from Supelco. Nopinone-, myrtanal-, trans-3-hydroxynopinone-, perillaldehyde, campholenealdehyde-, and pinonaldehyde-di-2,4DNPH were synthesized. Calibration solutions For quantitative measurements calibration curves were constructed, using the DAD as detector, for formaldehyde-, acetaldehyde-, acetone-, campholenealdehyde-, nopinone-, and pinonaldehyde-di-2,4DNPH. Benzaldehyde-2,4-DNPH was used as internal standard.

Results
Identification of reaction products As described above, the collection method is based on the conversion of the aldehydes and/or ketones to 2,4-dinitrophenylhydrazone derivatives. The aldehydes and ketones formed by oxidation of - and -pinene were collected on the LN2 trap coated with a solution of 2,4-DNPH. After collection the trap was rinsed with ACN and the solution was further analyzed by HPLCMS. First a blank experiment was run in the absence of or -pinene and OH radicals. The resulting HPLC chromatogram is shown in Fig. 1. When -pinene was reacted with OH radicals at 50 Torr pinonaldehyde-di-2,4-DNPH (tR=50.0 min, Mr=528) was identified as the main product. Other identified products shown in Fig. 2 are campholenealdehyde-2,4-DNPH (tR= 48.8 min, Mr=332), formaldehyde-2,4-DNPH (tR=27.2 min, Mr=210), acetaldehyde-2,4-DNPH (tR=30.7 min, Mr=224), and acetone-2,4-DNPH (tR=33.9 min, Mr=238). Besides these oxidation products, the chromatogram shown in Fig. 2 contains three other peaks dinitroaniline (tR=21.1 min) and two unidentified products with tR=24.0 min and tR= 25.8 min. Because the same peaks were detected in a blank experiment with OH radicals but in the absence of -pinene, it is clear that these products do not result from the -pinene/OH reaction. The oxidation products could be identified from their retention times in the total ion chromatogram (TIC). The

632 Fig. 1 HPLC chromatograms (blanks; upper=DAD, 360 nm; lower =TIC) at a total pressure of 50 Torr. Initial concentrations: [C10H16]=0, [H2]=1.371013, [NO2]=0, [O2]=3.241017, each expressed in molecules cm3, microwave discharge turned off. The reaction time tr=42 ms, collection time tc=330 min. Collection method: LN2 trap coated with 2,4-DNPH solution

mass spectrum of each peak can be constructed from the TIC and compared with mass spectral data of reference compounds. The mass spectra of the reference compounds obtained from commercial sources or by synthesis are shown in Fig. 3. For all unsymmetrical carbonyl-2,4-DNPH derivatives two isomers can exist, in principal the syn and the anti isomers. Depending on the nature of the substituents, one or both isomers might be stable. For most of the carbonyl-

2,4-DNPH derivatives, however, the isomers have very similar chromatographic properties and coelute. Only for saturated carbonyls with polar substituents could two peaks be observed [18]. An analogous study was performed on the -pinene/ OH reaction. The resulting HPLCMS chromatogram for a collection experiment performed at a total pressure of 50 Torr, is shown in Fig. 4. Formaldehyde-2,4DNPH (tR=28.3 min, Mr=210) and nopinone-2,4-DNPH

633 Fig. 2 HPLC chromatograms (upper=DAD, 360 nm; lower= TIC) at a total pressure of 50 Torr. Initial concentrations: [-C10H16]=5.131012, [H2]= 1.371013, [NO2]=3.621012, [O2]=3.241017, each expressed in molecules cm3. The reaction time tr=42 ms, collection time tc=335 min. Collection method: LN2 trap coated with 2,4-DNPH solution

(tR=46.4 min, Mr=318) are the main oxidation products. Besides formaldehyde- and nopinone-2,4-DNPH it is possible to detect acetone-2,4-DNPH (tR=34.9 min, Mr=238), acetaldehyde-2,4-DNPH (tR=31.7 min, Mr=224), trans-3hydroxynopinone-2,4-DNPH (tR=38.7 min, Mr=334), perillaldehyde-2,4-DNPH (tR=49.6 min, Mr=330), and myrtanal2,4-DNPH (tR=50.1 min, Mr=332). The mass spectral data of the reference compounds nopinone-, trans-3-hydroxynopinone-, perillaldehyde- and myrtanal-2,4-DNPH are shown in Fig. 5.

Product yields For quantitative measurements the LN2 trap was coated with a 2,4-DNPH solution according to the procedure described elsewhere [26], which also gives details on how the product yields are determined. As described in the experimental section, two I.S. were added to the solution. The second I.S. was used to calculate the loss of the first I.S. and subsequently the loss of the oxidation products. It is reasonable to assume that all the oxidation products

634

Fig. 3 APCI() generated mass spectra of reference materials obtained from commercial sources (A) formaldehyde-2,4-DNPH, (B) acetaldehyde-2,4-DNPH, (C) acetone-2,4-DNPH or by synthesis (D) campholenealdehyde-2,4-DNPH, (E) pinonaldehyde-mono-2,4-DNPH, (F) pinonaldehyde-di-2,4-DNPH

635

Fig. 4 HPLC chromatograms (upper=DAD, 360 nm; lower=TIC) at a total pressure of 50 Torr. Initial concentrations: [-C10H16]= 5.081012, [H2]=1.371013, [NO2]=3.621012, [O2]=3.241017 each

expressed in molecules cm3. The reaction time tr=42 ms, collection time tc=306 min. Collection method: LN2 trap coated with 2,4DNPH solution

636

Fig. 5 APCI()-generated mass spectra of reference materials obtained by synthesis (A) nopinone-2,4-DNPH, (B) trans-3-hydroxynopinone-2,4-DNPH, (C) perillaldehyde-2,4DNPH], (D) myrtanal-2,4-DNPH]

637 Table 1 Product yields, expressed in relative units, for the -pinene/OH reaction at 50 and 100 Torr. Experimental conditions: [-C10H16]~51012, [H2]=1.371013, [NO2]=3.621012, [O2]=3.24 1017 (50 Torr), [O2]=6.481017 (100 Torr) each expressed in molecules cm3. The reaction time tr=42 ms Reactor pressure (Torr) Formaldehyde Acetaldehyde Acetone Campholenealdehyde Pinonaldehyde 50 9.70.7 1.10.1 161 112 633 100 65 0.90.5 62 5.50.7 827

Table 2 Product yields, expressed in relative units, for the -pinene/OH reaction at 50 and 100 Torr. Experimental conditions: [-C10H16]~51012, [H2]=1.371013, [NO2]=3.621012, [O2]=3.24 1017 (50 Torr), [O2]=6.481017 (100 Torr) each expressed in molecules cm3. The reaction time tr=42 ms Reactor pressure (Torr) Formaldehyde Acetaldehyde Acetone Nopinone 50 482 1.20.6 203 311 100 432 0.60.4 7.80.5 492

have the same collection efficiency on the LN2 trap. The mean value of the relative product yields (mol%) for the oxidation products of - and -pinene at 50 and 100 Torr are shown in Tables 1 and 2, respectively.

Discussion
Qualitative analysis of the -pinene/OH reaction revealed the formation of formaldehyde, acetaldehyde, acetone, campholenealdehyde, and pinonaldehyde. Formaldehyde and acetone were also identified in a large volume photochemical reaction chamber study [8] where cartridges impregnated with 2,4-DNPH were used and further analyzed by HPLCMS. Nozire et al. [9] also detected these products but with Fourier transform infra red (FTIR) spectroscopy as detection technique. Pinonaldehyde has been detected by several techniques: GCMS and GCFTIR [11], GCMS and GCFID [10], FTIR [6, 9], and protontransfer reaction mass spectrometry (PTRMS) [28]. Acetaldehyde and campholenealdehyde have not previously been identified as products of the -pinene/OH reaction under atmospheric conditions. At 50 Torr yields of the five oxidation products in relative units (mol%) were: formaldehyde (9.70.7), acetaldehyde (1.10.1), acetone (161), campholenealdehyde (112), and pinonaldehyde (633). The yields at 100 Torr were (mol%): formaldehyde (65), acetaldehyde (0.90.5), acetone (62), campholenealdehyde (5.50.7), and pinonaldehyde (827). Product yields obtained in this work can only be compared with the yields derived from experiments conducted in photochemistry reactors. Pinonaldehyde has been quantified by several research groups

[6, 9, 10, 11]. It should be pointed out that Nozire et al. [9] reported a yield for pinonaldehyde of 8720%, which is in fairly good agreement with our measurements. The yields of acetone measured in our study (161 mol% at 50 Torr and 62 mol% at 100 Torr) are of the same order of magnitude as values found by others 112.7% [29], 96% [9], and 112% [28]. The yields for formaldehyde (9.70.7 mol% at 50 Torr and 65 mol% at 100 Torr) are somewhat smaller than the value (239%) reported by Nozire et al. [9]. Qualitative analysis of the -pinene/OH reaction enabled identification of formaldehyde, nopinone, acetaldehyde, acetone, trans-3-hydroxynopinone, perillaldehyde, and myrtanal. Formaldehyde and nopinone are the most important oxidation products. Only formaldehyde and nopinone have previously been identified in other studies as oxidation products of the -pinene/OH reaction. Formaldehyde was also identified by Orlando et al. [30] and Hatakeyama et al. [6], by use of FTIR spectroscopy. The presence of nopinone has been demonstrated by use of several techniques: GCMS and GCFTIR [11], GCMS and GCFID [10], FTIR [6] and PTRMS [28]. The product yields of the -pinene/OH reaction were also determined. The relative product yields (mol%) at 50 and 100 Torr were, respectively: 482 and 432 for formaldehyde; 1.20.6 and 0.60.4 for acetaldehyde; 203 and 7.80.5 for acetone; and 311 and 492 for nopinone. Nopinone and formaldehyde have been quantified by several research groups [6, 10, 11, 28, 30]. The values reported for formaldehyde, 545 [6] and 458 [30], are in good agreement with our measurements. For nopinone, product yields of 274 [11] and 304 [10] are somewhat lower, but still in fairly good agreement with our data at 50 Torr. The only major discrepancy with other literature data is for acetone the yield of 22 [30] is at least a factor of 3 lower than our value, although in very recent PTRMS work a primary acetone yield of 132% has been reported [28]. Finally, it should be pointed out that a major advantage of the fast-flow reactor technique is that the system does not contain ozone. This is sometimes a problem in the sampling system for photochemical reactors or for ambient air, because trapped ozone can react further with other organic molecules during the analysis procedure, perturbing the measured product yields. Besides ozone, other radical intermediates might also affect our measured yields, although from kinetic simulations it could be concluded that the concentrations of the OH radical and other peroxy-radical intermediates are orders of magnitude lower than the concentrations of the stable species measured.

Conclusions
An analytical method has been developed for identification and quantification of the semi-volatile oxidation products of the reaction of -pinene and -pinene with hydroxyl radicals. The technique is based on sampling the products on a liquid-nitrogen trap with in situ derivatiza-

638

tion of the carbonyl compounds to 2,4-dinitrophenylhydrazone derivatives. Analysis of the derivatives by HPLCMS showed that pinonaldehyde is by far the major product from -pinene whereas for -pinene, nopinone and formaldehyde are the dominant products.
Acknowledgments This work is financed by the Belgian Ministry of Scientific Policy under the Federal Impulse Programme Global Change, Ecosystems and Biodiversity.

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