Chromatography 2018, 39, 119-124

Original Paper

Determination of Airborne Polycyclic Aromatic Hydrocarbons by HPLC Using SPE-Type Collection Device

Ikuo UETA*1, Naho SEKIGUCHI1, Koji FUJIMURA2, Tomotaka YOSHIMURA3, Shoji NARUKAMI3, Suguru MOCHIZUKI3, Tomohiro SASAKI3, Tetsuo KUWABARA1, Tsuneaki MAEDA4 1Department of Applied Chemistry, University of Yamanashi, 4-3-11 Takeda, Kofu 400-8511, Japan 2Shinwa Chemical Industries Ltd., 50-2 Kagekatsu-cho, Fushimi, Kyoto 612-8307, Japan 3HORIBA STEC, Co. Ltd., 11-5 Hokodate-cho, Kamitoba, Minami, Kyoto 601-8116, Japan 4Professionals' Network in Advanced Instrumentation Society, 2-6 Kanda-Awaji-cho, Chiyoda, Tokyo 101-0063, Japan

Abstract A solid-phase extraction-type collection device packed with Sunpak-H, styrene-divinylbenzene polymer particles, was used to identify 16 airborne polycyclic aromatic hydrocarbons (PAHs) using high-performance liquid chromatography (HPLC)-fluorescence detection and ultraviolet detector. The analytes were successfully collected with the collection device and quantitatively eluted using acetone as an elution solvent. The eluted PAHs were then separated by conventional HPLC and detected with a fluorescence or ultraviolet detector. The limit of detection of airborne PAHs was below 0.7 ng/m3 with an air sampling volume of 14,400 L. The relative standard deviations of the peak area were below 12%, and the method showed better repeatability than gas chromatographic analysis. The suitability of the method was confirmed by its application in identifying PAHs in tunnel and atmospheric air.

Keywords: Polycyclic aromatic hydrocarbon; Sample preparation; Semi-volatile organic compounds; HPLC-FL

1. Introduction chromatography-fluorescence (HPLC-FL) detection are Polycyclic aromatic hydrocarbons (PAHs) are organic typically used. GC offers greater resolution while MS yields compounds with more than two unsubstituted aromatic excellent qualitative information. In HPLC-FL analysis, rings. PAHs are generated by incomplete combustion of interference can be an issue [8], although this method offers organic materials such as coal, oil, and wood [1,2]. Many good sensitivity [9]. PAHs are considered hazardous to human health and are For airborne PAH collection, a sampling method using carcinogenic [1,3]. Except for naphthalene, which has two filter paper and an adsorbent is typically used. In this rings, PAHs have boiling points above 250°C and these are technique, gaseous PAHs pass through the filter paper and classed as semi-volatile organic compounds (SVOCs). are collected on an adsorbent such as polyurethane foam or Low-molecular-weight PAHs (two or three rings) exist in XAD resin (a styrene-divinylbenzene resin) [10]. The the vapor phase, while intermediate-molecular-weight collected PAHs are typically eluted by Soxhlet or ultrasonic PAHs (four rings) exist in either the vapor or the particulate extraction. However, Soxhlet extraction takes more than 16 phase, depending on the atmospheric temperature. h, and offers insufficient recovery of analyte PAHs [11,12]. High-molecular-weight PAHs (five or more rings) exist Ultrasonic extraction of PAHs is relatively rapid, although predominantly as particulates [4-6]. insufficient extraction has been reported [13,14]. To identify PAHs, gas chromatography-mass Additionally, it is difficult to reuse the collection cartridge spectrometry (GC-MS) [7] or high-performance liquid in these extraction methods.

*Corresponding author: Ikuo UETA Received: 3 August 2018 Tel: +81-55-220-8552; Fax: +81-55-220-8547 Accepted: 15 September 2018 E-mail: [email protected] J-STAGE Advance Published: 25 September 2018 DOI: 10.15583/jpchrom.2018.013

－ 119 － Chromatography 2018, 39, 119-124

Our research group developed extraction devices for The structures of the investigated PAHs are shown in Fig. 1. collecting SVOCs from air samples. To achieve simple, Acetone (>99.9%), acetonitrile (>99.9%), and rapid, and quantitative elution of collected SVOCs, we dichloromethane (>99.9%) were also purchased from Kanto introduced solid-phase extraction (SPE)-type collection Chemical Co., Inc. A standard stock solution (100 mg/L) of devices, in which distribution-based adsorbents were used each was prepared by diluting the mixed solution with to determine sesquiterpenes [15] and PAHs [16,17]. acetone. Recently, we developed a SPE-type collection device packed with Sunpak-H, styrene-divinylbenzene polymer 2.2. Collection devices particles, as an adsorbent for collecting airborne terpenes Sty-DVB polymer particles (Sunpak-H) with 50/80 mesh [18] and PAHs [19]. The device performed well in were obtained from Shinwa Chemical Industries, Ltd. collecting the investigated PAHs, which were rapidly and (Kyoto, Japan). The adsorbent had a specific surface area of quantitatively eluted from the device with 10 mL of 100-150 m2/g and an average pore diameter of 30 nm. A dichloromethane and determined by GC-MS. wire mesh (24.0 mm i.d., HORIBA STEC, Kyoto, Japan), a This manuscript reports the suitability of a collection glass fiber filter (Whatman GF/D, GE Healthcare Japan, cartridge packed with Sunpak-H for identifying airborne Tokyo, Japan), and the adsorbent (1.5 g) were packed into a PAHs in HPLC-FL analysis. The analytical performance of glass cartridge (24.0 mm i.d. and 70 mm length, HORIBA the proposed method, with respect to sensitivity and STEC). The packed length of the adsorbent was 30 mm. repeatability, was compared with GC-MS. The PAH Another glass fiber filter and a specially designed PTFE recovery during sample collection was also quantitatively stopper containing seven holes (24.0 mm in diameter, investigated. The method was used to determine PAHs in HORIBA STEC) were then packed into the cartridge. The ambient air samples. stopper had a groove around its girth and isobutylene-isoprene rubber (IIR rubber) or fluorine- 2. Experimental containing rubber was attached to the groove. A specially 2.1. Chemicals designed PTFE adapter (HORIBA STEC) was used to A mixture of 16 standard PAHs (200 mg/L each) connect the device to a gas-sampling pump. The collection containing naphthalene (Nap), acenaphthylene (Acy), devices were washed with acetone before use, and no acenaphthene (Ace), fluorene (Flu), phenanthrene (Phe), analytes were detected on the method blank. The Sunpak-H anthracene (Ant), pyrene (Pyr), fluoranthene (Flt), benz[a]- particles did not swell as a result of contact with acetone. anthracene (BaA), chrysene (Chr), benzo[b]fluoranthene The prepared collection device is illustrated in Fig. 2. (BbF), benzo[k]fluoranthene (BkF), benzo[a]pyrene (BaP), indeno[1,2,3-cd]pyrene (Ind), dibenz[a,h]anthracene (DahA), and benzo[g,h,i]perylene (BghiP) dissolved in 70 mm methanol/dichloromethane (1:1 v/v) was obtained from 30 mm Kanto Chemical Co., Inc. (Tokyo, Japan).

24.0

Sunpak-H mm (1.5 g)

1) Nap 2) Acy 3) Ace 4) Flu 5) Phe

wire mesh glass filter PTFE stopper

Fig. 2. Illustration of the collection device packed with Sunpak-H. 6) Ant 7) Pyr 8) Flt 9) BaA

2.3. Collection and elution of the analytes The collection and elution performances of the developed 10) Chr 11) BbF 12) BkF 13) BaP device were evaluated with the standard stock solution (100 mg/L). Firstly, 50 μL of the standard solution was dropped on the filter paper at the tip end of the collection device. The device was then connected to a filter holder (EMO-47, 14) Ind 15) DahA 16) BghiP GL Sciences, Tokyo, Japan) containing a 47 mm quartz fiber filter (QR-100, Advantec Toyo Kaisha, Ltd., Tokyo, Fig. 1. Structure of PAHs investigated in this study. Japan) using a PTFE tube. The device was then immersed in a water bath at constant temperature (typically 35°C) for

－ 120 － Chromatography 2018, 39, 119-124

5 min, then clean air was collected using a prototype 2018. During air collection, the collection device was gas-sampling unit (HORIBA STEC). The sampling speed covered with aluminum foil to prevent degradation of was maintained at 10 L/min. The analytes collected in the unstable PAHs. To analyze real air samples, the eluted device were eluted by passing a few milliliters of organic solvent was cleaned with a silica gel column before HPLC elution solvent. The type and volume of the organic analysis; this was performed as follows. Approximately 1 solvents were optimized in subsequent experiments. The mL of n-hexane was initially added to the concentrated solution was concentrated to 1 mL with a N2 flow. During elution solvents (100 μL). These were then transferred to a concentration, some of the Nap evaporated, while silica gel cartridge (Bond Elut SI, silica 2 g, Agilent three-to-six ring PAHs did not. The analytical method for Technologies, Santa Clara, CA, USA). The vial was rinsed the present study is shown in Fig. 3. The collection device twice with 2 mL of hexane, and this was added to the silica could be reused after drying the packed particles in a N2 gel cartridge. The PAHs were then eluted with 6 mL of flow (5 L/min) for 20 min. dichloromethane/hexane (1:9 v/v). The analytes were redissolved in acetone and concentrated to 1 mL using a N2 flow. In real sample analysis, the analytes collected on the (A) quartz fiber filter were also determined. After air collection, quartz fiber filter air the quartz fiber filter was placed in a glass vial. PAHs were then extracted by ultrasonication with 20 mL of standard elution solvent solution dichloromethane for 20 min. The filter was then transferred N2 to another glass vial and ultrasonicated again in the same manner for 20 min. The combined extraction solvent was passed through a filter paper (No. 5C, Advantec Toyo Kaisha). The solution was cleaned with a silica gel cartridge HPLC-FL measurement as described above. All the sample solutions were also analyzed by GC-MS (GC-17A/QP5050A, Shimadzu, Kyoto, Japan), and the detected analytes were identified. Sunpak-H packed collection device Table 1. Analyte PAHs and their detection wavelength. Elution λex λem Compound Abbreviation (B) order (nm) (nm) air quartz fiber filter 1 Naphthalene Nap 260 330 elution solvent 2 Acenaphthylene Acy UV 230 N2 silica gel 3 Acenaphthene Ace 260 330 column 4 Fluorene Flu 260 375

HPLC-FL 5 Phenanthrene Phe 260 375 measurement 6 Anthracene Ant 260 375

7 Fluoranthene Flt 260 440

Sunpak-H 8 Pyrene Pyr 260 380 packed collection device 9 Benz[a]anthracene BaA 265 380

10 Chrysene Chr 265 380

Fig. 3. Illustration of the analytical method. (A) standard 11 Benzo[b]fluoranthene BbF 290 440 solution and (B) real samples. 12 Benzo[k]fluoranthene BkF 290 440 13 Benzo[a]pyrene BaP 290 440 2.4. Real sample analysis and sample clean-up 14 Indeno[1,2,3-cd]pyrene Ind 290 440 Tunnel air (Atagoyama tunnel, Kofu, Japan) and atmospheric air (University of Yamanashi, Kofu, Japan) 15 Dibenz[a,h]anthracene DahA 290 440 were analyzed as real samples. The samples were collected 16 Benzo[g,h,i]perylene BghiP 293 485 at a height of 1.0 m above the ground at ambient temperature in March (17.5°C) and May (16.0°C-29.2°C)

－ 121 － Chromatography 2018, 39, 119-124

2.5. HPLC measurements acetonitrile and methanol. All the analyte PAHs collected Chromatographic separation was achieved using two on the Sunpak-H packed device were easily desorbed by JASCO PU-980 pumps (JASCO, Tokyo, Japan), a DG-980- passing acetone. Thus, 12 mL of acetone was used for 50 degasser, a CO-265 Plus column oven, a PU-2080 Plus elution, and the elution time was within 3 min. Typical UV-vis detector (JASCO), and a GL-7453A FL detector chromatogram for the determination of a standard PAHs is (GL Sciences). The excitation and emission wavelengths demonstrated in Fig. 4. The chromatogram was obtained by (λex and λem, respectively) for the PAHs are summarized in spiking standard solution (1 mg/L) onto the collection Table 1. A Chromato-PRO data integrator (Run Time device, and elution of the analytes with 12 mL of acetone. Instruments, Tokyo, Japan) was employed for data The eluted solvent was concentrated to 1 mL by N2 flow. acquisition. An Agilent ZORBAX Eclipse PAH column Therefore, the concentration of each PAH was 50 ng/mL. (particle size 5 μm, diameter 4.6 mm, length 150 mm) was employed for the separation. A Rheodyne 7725i injection 8 15 valve (Rheodyne, Cotati, CA, USA) with 20 μL of sample Chr BkF loop was used for sample injection. Separation was UV response (μV) achieved by linear gradient elution, using 50/50 6 BaP Ant 10 water/acetonitrile for 5 min then proceeding to 100% BghiP 4 acetonitrile for 20 min and maintaining for 5 min. The Flu Pyr BaA BbF DahA mobile phase flow rate was set at 1 mL/min, and the Phe Ind Nap Ace Flt 5 column temperature was maintained at 35°C. 2 Acy FL response (μV) response FL FL

UV 3. Results and discussion 0 0 3.1. Interference from the collection cartridge 0 5 10 15 20 Retention time (min) In previous studies, an IIR rubber O-ring was used to fix the PTFE stopper [19]. To evaluate interference from the Fig. 4. Typical chromatogram for the determination of PAHs eluted from the Sunpak-H packed collection device. collection cartridge, 10 mL of acetone was introduced into Spiked PAHs: 1.0 mg/L each 50 μL; elution: 12 mL of the cartridge, and the eluted solvent was concentrated to 1 acetone. mL using a N2 flow. Interference was confirmed by HPLC-FL analysis of the concentrated solution. Some peaks were observed for this solution, and two peaks 3.3. Evaluation of the method matched the retention times of Flt and Pyr. These were The collection recovery of the Sunpak-H packed identified to phthalate ester compounds via GC-MS. collection device for the 16 PAHs is described in the Therefore, a perfluoro rubber O-ring was investigated. No literature [19]. The device achieved complete collection of peaks were observed for a sample solution eluted from a Nap with a sampling volume of 10,000 L, while three-to-six cartridge attached to the perfluoro rubber O-ring; therefore, ring PAHs were collected on the device with sampling this was employed with the PTFE stopper in this study. volumes above 40,000 L at a sampling temperature of 35°C. The collection recovery was calculated by air sampling 3.2. Optimization of the desorption process from two collection devices connected in tandem after To elute the collected PAHs from the adsorbent, acetone, dropping the standard solution on a former collection acetonitrile, and methanol were evaluated as elution device. Therefore, if any PAH was detected on the latter solvents. Elution recovery was calculated by sequential collection device, the collection recovery was 100%. elution from the collection device. As shown in Table 2, However, unstable PAHs, such as Ant and BaP, were acetone exhibited improved elution recovery compared to degraded during air collection on a filter paper or an

Table 2. Elution recovery of PAHs from small device packed with Sunpak-H. Solvent Elution recovery (%) volume (mL) Nap Acy Ace Flu Phe Ant Flt Pyr BaA Chr BbF BkF BaP Ind DahA BghiP 10 100 100 100 100 100 100 100 100 100 99.7 99.6 99.5 100 100 100 100 11 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 12 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 Air sampling volume: 100 L, sampling temperature: 35°C.

－ 122 － Chromatography 2018, 39, 119-124

adsorbent [20]. The peak areas of these unstable PAHs on (A) 30 30 the former collection device also decreased when the air Nap Flu sampling volume was increased. Over 90% degradation of 25 25 UV response (μV) Ant and BaP was observed with an air sampling volume of 20 20 14,400 L (10 L/min for 24 h), even when the collection device was covered with aluminum foil during air 15 15 collection. 10 10 Phe

The limits of detection (LOD) for standard PAH solutions (μV) response FL 5 FL 5 in this study are summarized in Table 3. Compared to a UV 0 0 previous study, in which GC-MS was used as the analytical 0246810 system, the present HPLC-FL measurements exhibited two Retention time (min) 3 to fifty-fold lower LODs. LODs below 0.7 ng/m of (B) three-to-six ring PAHs in an air sample could be calculated 6 30 Nap Ace at a sampling volume of 14,400 L (10 L/min for 24 h). The 5 25 UV response (μV) relative standard deviations (RSDs) of the peak areas of the 4 20 standard analytes (n = 5) were below 12% as shown in Table 3. 3 Flu 15 2 10 Phe FL response (μV) response FL 1 FL 5 Table 3. LODs and RSDs of standard PAHs in this method. UV Compound LOD (ng/mL) LOQ (ng/mL) RSD, % 0 0 0246810

Nap 5 15 1.1 Retention time (min)

Acy 10 30 4.2 Fig. 5. Typical chromatograms for determining PAHs collected in the wide-bore collection device packed with Sunpak-H; (A) 15 8.2 Ace 5 tunnel air and (B) atmospheric air. Flu 1 3 8.2

Phe 1 3 10.1 Figure 5B shows typical chromatograms for determining Ant 1 3 3.5 PAHs collected on the collection device from an Flt 5 15 5.1 atmospheric air sample. This was collected for 24 h at 10 L/min (14,400 L). Traces of gaseous PAHs were found in Pyr 5 15 11.8 atmospheric air. However, PAHs were not found on the BaA 1 3 5.8 filter paper. The determined concentrations of the analytes Chr 0.5 1.5 4.3 are summarized in Table 4. In this study, a clean-up procedure for the eluted solution was used to remove BbF 1 3 2.4 contaminants based on the standard method. However, as BkF 0.5 1.5 2.8 shown in Fig. 5, the HPLC-FL chromatograms contained BaP 1 3 6.5 many peaks, making identification of the analyte PAHs difficult. The quantitative results for Nap in this study are Ind 5 15 1.8 inaccurate because some of the Nap evaporated during DahA 5 15 5.6 sample clean-up. BghiP 10 30 10.8

Table 4. Quantitative results for PAHs in a real air sample.

Concentration (ng/m3) 3.4. Real sample analysis The method was used to determine PAHs in real air Nap Acy Ace Flu Phe samples. Firstly, tunnel air was collected for 90 min at 10 Sunpak-H ca. 630 102 N.D. 56.8 23.4 L/min. Figure 5A shows typical chromatograms for Tunnel determining PAHs collected on the Sunpak-H packed Filter ca. 2 2.1 N.D. 0.5 4.6 collection device from the tunnel air. Relatively volatile Atmos- Sunpak-H ca. 7 N.D.

－ 123 － Chromatography 2018, 39, 119-124

4. Conclusion N. J. Chromatogr. Sci. 2007, 45, 57-62. A collection device packed with Sunpak-H was used to [10] NIOSH Manual of Analytical Methods (NMAM) determine airborne PAHs in HPLC analysis. In this method, Method 5515, "Polynuclear aromatic hydrocarbons the collected PAHs were eluted with 12 mL of acetone, by GC", U.S. National Institute for Occupational which was injected into conventional HPLC-FL. No Safety and Health, 4th ed. 1994. interference peaks were eluted from the collection device. [11] Guerin, T. F. J. Environ. Monit. 1999, 1, 63-67. Lower LOQs were obtained in the HPLC-FL method for [12] Pandey, S. K.; Kim, K. H.; Brown, R. J. C. Trends analyte PAHs than with GC-MS, with better repeatability. Anal. Chem. 2011, 30, 1716-1739. However, identifying and quantifying the analyte PAHs [13] Kuusimäki, L.; Peltones, K.; Mutanen, P.; Savela, K. would potentially be hindered by interference peaks even if Ann. Occup. Hyg. 2003, 47, 389-398. a higher sensitivity FL detector was used. Further [14] Kobayashi, A.; Kojima, Y.; Okochi, H.; Nagoya, T. application of the Sunpak-H packed collection device can Bunseki Kagaku 2010, 59, 645-652. be expected for determining airborne SVOCs by HPLC and [15] Ueta, l.; Onikata, M.; Mochizuki, S.; Fujimura, K.; GC. Sasaki, T.; Aoki, J.; Maeda, T. J. Sep. Sci. 2015, 38, 3891-3896. Acknowledgment [16] Ueta, I.; Onikata, M.; Fujimura, K.; Sasaki, T.; This work was supported by JSPS KAKENHI (Grant Yoshimura, T.; Mochizuki, S.; Maeda, T. Number 15K17875) and the Steel Foundation for Chromatography 2016, 37, 9-13. Environmental Protection Technology, Japan. One of the [17] Ueta, I.; Onikata, M.; Fujimura, K.; Yoshimura, T.; authors (I. Ueta) acknowledges support from Prof. Y. Saito, Narukami, S.; Mochizuki, S.; Sasaki, T.; Maeda, T. J. Toyohashi University of Technology and from Prof. S. Sep. Sci. 2016, 39, 4202-4208. Kawakubo and Prof. K. Tani of University of Yamanashi. [18] Ueta, I.; Fujikawa, H.; Fujimura, K.; Yoshimura, T.; Narukami, S.; Mochizuki, S.; Sasaki, T.; Maeda, T. References Chromatography 2018, 39, 27-32. [1] "Polycyclic aromatic hydrocarbons, in Air quality [19] Ueta, I.; Onikata, M.; Fujimura, K.; Yoshimura, T.; guidelines for Europe", World Health Organization Narukami, S.; Mochizuki, S.; Sasaki, T.; Maeda, T. Regional Office for Europe, Copenhagen, 2010. Anal. Sci. 2017, 33, 1175-1180. [2] Baek, S. O.; Field, R. A.; Goldstone, M. E.; Kirk, P. [20] Szulejko, J. E.; Kim, K. H.; Brown, R. J. C.; Bae, M. W.; Lester, J. N.; Perry, R. Water Air Soil. Poll. 1991, S. Trends Anal. Chem. 2014, 61, 40-48. 60, 279-300. [3] "Some non-heterocyclic polycyclic aromatic hydrocarbons and some related exposures, IARC Monogr. Eval. Carcinog. Risks Hum.", International Agency for Research on Cancer, 2010. [4] Choi, H.; Harrison, R.; Komulainen, H.; Delgado Saborit, J. M. "Polycyclic aromatic hydrocarbons", in WHO Guidelines for Indoor Air Quality: Selected Pollutants, World Health Organization, 2010. [5] Venkataraman, C.; Thomas, S.; Kulkarni, P. J. Aerosol. Sci. 1999, 30, 759-770. [6] Cochran, R. E.; Dongari, N.; Jeong, H.; Beránek, J.; Haddadi, S.; Shipp, J.; Kubátová, A. Anal. Chim. Acta, 2012, 740, 93-103. [7] Compendium Methods TO-13A, "Determination of polycyclic aromatic hydrocarbons (PAHs) in ambient air using gas chromatography/mass spectrometry (GC-MS)", US Environmental Protection Agency, Cincinnati, OH, 1999. [8] US EPA, Method 610: Polynuclear Aromatic Hydrocarbons, 1984. [9] Díaz-Moroles, N.E.; Garza-Ulloa, H. J.; Castro-Ríos, R.; Ramírez-Villarreal, E. G.; Barbarín-Castillo, J. M.; de la Luz Salazar-Cavazos, M.; Waksman-de Torres,

－ 124 －