APPLICATION NOTE 70923

Sensitive and rapid determination of

polycyclic aromatic hydrocarbons in tap water
Authors Goal
Chen Jing, Dai Zhenyu, Xu Qun, To develop an efficient high-performance liquid chromatography (HPLC)
and Liang Lina, Thermo Fisher method for sensitive and rapid determination of 20 polycyclic aromatic
Scientific, Shanghai, People’s hydrocarbons (PAHs) in environmental waters using on-line solid-phase
Republic of China extraction (SPE) for sample preparation instead of the liquid-liquid
Jeffrey Rohrer, Thermo Fisher extraction and off-line SPE specified in the U.S. Environmental Protection
Scientific, Sunnyvale, California Agency (EPA) Methods 550 and 550.1, respectively

Keywords Introduction
Hypersil Green PAH Column, Polycyclic aromatic hydrocarbons (PAHs) are a group of chemicals formed
Acclaim PA2 Column, from the incomplete combustion of organic matter. Due to their potential
HPLC, On-Line SPE, PAHs, UV carcinogenic and mutagenic properties, most countries have regulations
Detection, EPA Method 550, EPA limiting the concentrations of a variety of PAHs in drinking water, food
Method 550.1, Water Analysis additives, cosmetics, workplaces, and factory emissions.

Thermo Fisher Scientific Application Note (AN) 196 provides on-line SPE
HPLC methods to quantify low concentrations of PAHs in oil.1 The costs
for the SPE cartridge, labor, time, and reagents are significantly reduced
using these methods, and the results are more consistent. This is because
on-line SPE eliminates manual processes, such as rotary evaporation and
nitrogen-assisted evaporation in the routine liquid-liquid extraction and off-
line SPE steps described in EPA Methods 550, 550.1, and 610.2-4 However,
because the run time of the reported on-line SPE HPLC method exceeded
60 min in this study, the analytical column used in the AN was replaced to
shorten analysis time.

1
 Equipment, Software, and Consumables Conditions (Applicable to Figures 1, 2, and 6)
• Thermo Scientific™ UltiMate™ 3000 Dual Gradient On-Line SPE
Rapid Separation (RS) LC system, including: Column: Thermo Scientific™ Acclaim™
–– Thermo Scientific™ UltiMate™ 3000 DGP-3600RS PolarAdvantage II (PA2), 3 μm Analytical,
Dual Gradient Rapid Separation Pump (P/N 5040.0066) 4.6 × 50 mm (P/N 063189)
Mobile Phase: A. Water
––Thermo Scientific™ UltiMate™ 3000 SRD-3600
B. Acetonitrile
Integrated Solvent and Degasser Rack
Gradient: Table 1
(P/N 5035.9230)
Flow Rate: 0.4 and 0.6 mL/min (Table 1)
––Thermo Scientific™ UltiMate™ 3000 WPS-3000TRS Inj. Volume 1 mL on the On-Line SPE column
Rapid Separation Wellplate Sampler, Thermostatted Separation
(P/N 5840.0020) with a 1000 μL sample loop (P/N Column: Thermo Scientific™ Hypersil™ Green
6820.2429) and a 1000 μL syringe (P/N 6822.0005) PAH Column, 3 μm, 3.0 × 150 mm
(P/N 31105-153030)
––Thermo Scientific™ UltiMate™ 3000 TCC-3000SD
Mobile Phase: A. Water
Standard (P/N 5730.0010) or TCC-3000RS Rapid
B. Acetonitrile
Separation (P/N 5730.0000) Thermostatted Column
Gradient: Table 1
Compartment, equipped with one 2p–6p valve
Flow Rate: 0.8 mL/min
––Thermo Scientific™ UltiMate™ 3000 DAD-3000RS Temperature: 30 °C
Rapid Separation Diode Array Detector Detection: UV, 254 nm; Fluorescence at different
(P/N 5082.0020) excitation and emission (Ex/Em)
wavelengths for each PAH (Table 2)
––Thermo Scientific™ UltiMate™ 3000 FLD-3400RS
Rapid Separation Fluorescence Detector Note: The PAHs have good fluorescent responses, except for
acenaphthylene (Figure 1, Peak 2). Because their maximum fluorescent
(P/N 5078.0025)
responses occur at different Ex/Em wavelengths, it is necessary to
change the Ex/Em wavelengths to acquire the best detection sensitivity.
• Thermo Scientific™ Chromeleon™
These changes are dictated by the individual PAH retention times. Table
Chromatography Data System (CDS) software, 2 shows the program for wavelength changes. Although naphthalene
version 7.1 or above (Figure 1, Peak 1) has fluorescent response, EPA method 550.1 requires
it to be determined using UV detection, together with acenaphthylene
• Thermo Scientific™ Target2™ Nylon Syringe Filters,
(Figure 1, Peak 2). Figures 1 and 2 show the chromatograms of all 20
0.45 μm, 30 mm (P/N F2500-1) PAHs with UV and fluorescence detection, respectively, under the
conditions specified above.
Reagents and Standards Peaks:
• Deionized (DI) water, 18.2 MΩ-cm resistivity 1. Naphthalene 11. Benzo(a)anthracene
2. Acenaphthylene 12. Chrysene
• Methanol (CH3OH), 99.8%, HPLC Grade (Fisher 3. 1-Methylnaphthalene 13. Benzo(j)fluoranthene
4. 2-Methylnaphthalene 14. Benzo(e)pyrene
Scientific P/N AC610090040) 5. Acenaphthene 15. Benzo(b)fluoranthene
• Acetonitrile (CH3CN), HPLC Grade (Fisher Scientific 6. Fluorene 16. Benzo(a)pyrene
7. Phenanthrene 17. Benzo(k)fluoranthene
P/N AC610010040) 8. Anthracene 18. Dibenz(a,h)anthracene
9. Fluoranthene 19. Benzo(ghi)perylene
• EPA 610 PAH Solution (1000 mg/L for each, 10. Pyrene 20. Indeno(1,2,3-cd)pyrene
100
in methylene chloride) containing 18 PAH
8
standards: benzo[k]fluoranthene, acenaphthene,
acenaphthylene, anthracene, fluorene, naphthalene,
phenanthrene, benzo[a]anthracene, benzo[a] 7
pyrene, chrysene, fluoranthene, indeno[1,2,3-cd] mAU

pyrene, pyrene, benzo[b]fluoranthene, benzo[ghi]

perylene, dibenz[a,h]anthracene, benzo[e]pyrene, 12
6 11 1415
and benzo(j)fluoranthene (o2si Smart Solutions P/N 9
10 13
16 17 20

110064-01-5PAK) 1 2 345 1819

0
• 1-Methylnaphthalene (AccuStandard P/N H-001N)
• 2-Methylnaphthalene (AccuStandard P/N H-002N) –2
0 5 10 15 20 25 30 35
Minutes
Figure 1. All 20 PAHs (50 μg/L for each PAH) detected at UV 254 nm.
2
 Peaks:
1. Naphthalene 11. Benzo(a)anthracene
2. Acenaphthylene 12. Chrysene Acenaphthene Acenaphthylene Anthracene
3. 1-Methylnaphthalene 13. Benzo(j)fluoranthene Benzo(a)anthracene
4. 2-Methylnaphthalene 14. Benzo(e)pyrene
5. Acenaphthene 15. Benzo(b)fluoranthene
6. Fluorene 16. Benzo(a)pyrene
7. Phenanthrene 17. Benzo(k)fluoranthene Benzo(a)pyrene
8. Anthracene 18. Dibenz(a,h)anthracene Benzo(ghi)perylene Benzo(k)fluoranthene
9. Fluoranthene 19. Benzo(ghi)perylene
10. Pyrene 20. Indeno(1,2,3-cd)pyrene
198,8000,000
6
Dibenzo(a,h)anthracene
Chrysene

Counts 5 8
16 Benzo(ghi)perylene

Fluoranthene
15 17 Pyrene
C 10
0 12
7
4 11 14 20
B
1 Indeno(1,2,3-cd)pyrene Naphthalene Phenanthrene Fluorene
3 18
9 19
A 13
Figure 3. Structures of the 16 PAHs specified in EPA Methods
–121,200,000 550, 550.1, and 610.
0 5 10 15 20 25 30 35
Minutes

Figure 2. All 20 PAHs (50 μg/L for each PAH) detected by

CH3
fluorescence detection using programmed wavelength switching
in three parallel channels: (A) Emission_1, (B) Emission_2, (C)
Emission_3. Note: Acenaphthylene (Peak 2) is not shown here CH3
because there was no fluorescent response for acenaphthylene.
1-Methylnaphthalene
Benzo(e)pyrene Benzo(j)fluoranthene
2-Methylnaphthalene

Preparation of Standard Solutions Figure 4. Structures of four additional PAHs not specified in EPA
In addition to the 16 PAHs specified in EPA Methods Methods 550, 550.1, and 610.
550, 550.1, and 610 (Figure 3), the target analytes
in the experiments described here include four
other PAHs: benzo[e]pyrene, benzo(j)fluoranthene, of 2-Methylnaphthalene Stock Solution to a 10 mL
1-methylnaphthalene, and 2-methylnaphthalene volumetric flask, then dilute to the mark with methanol.
(Figure 4). The EPA 610 PAH Solution product The final concentration of each PAH will be 10 mg/L.
contains benzo[e]-pyrene and benzo(j)fluoranthene, This is Stock Standard Solution Mix 1.
in addition to the 16 specified PAHs.
Add 1 mL of Stock Standard Mix 1 to a 10 mL volumetric
Stock Solutions of 1-Methylnaphthalene flask and dilute to the mark with methanol. This is Stock
and 2-Methylnaphthalene Standard Solution Mix 2. The final concentration of each
In a 100 mL volumetric flask, dissolve 100 mg of PAH in Stock Standard Mix 2 will be 1.0 mg/L.
1-methyl-naphthalene in 2 mL of acetonitrile and dilute
to the mark with methanol. The final concentration of Use these mixed standard stock solutions to prepare
1-methylnaphthalene will be 1000 mg/L. Prepare a working mixed standard solutions for calibration.
1000 mg/L stock solution of 2-methylnaphthalene in the
same manner. Working Mixed Standard Solutions for Calibration
For calibration, prepare nine working standard solutions
Stock Standard Mixes 1 and 2 with different concentrations by diluting the proper
Add 100 μL of EPA 610 PAH Solution (containing amount of either Stock Standard Mix 1 or 2 with DI
18 PAH standards, 1000 mg/L each), 100 μL of water. The volumes of each solution needed to make
1-Methylnaphthalene Stock Solution, and 100 μL the calibration standards are shown in Table 3. The
3
 Table 1. Gradient programs for loading and analytical pumps.

Loading Pump Analytical Pump

Time Flow Rate %A %B Time Flow Rate %A %B

(min) (mL/min) (H2O) (CH3CN) (min) (mL/min) (H2O) (CH3CN)
0 0.6 95 5 0 0.8 60 40
4.0 0.6 95 5 5 0.8 60 40
4.5 0.4 0 100 30 0.8 0 100
25 0.4 0 100 30.5 0.8 60 40
25.5 0.6 95 5 35 0.8 60 40
35 0.6 95 5 — — — —

Table 2. Ex/Em maximums for each PAH and programmed wavelength switching times.

Time Fluorescence Ex/Em Wavelengths

PAH Peak No.
(min) Detection Channel (nm)

0.0 Emission_1 219/330 Naphthalene 1

1-Methylnaphthalene 3
Emission_1 225/333
2-Methylnaphthalene 4
13.45
Emission_2 235/332 Acenaphthene 5
Emission_3 263/310 Fluorene 6
Emission_1 247/364 Phenanthrene 7
15.50
Emission_2 247/401 Anthracene 8
Emission_1 281/453 Fluoranthene 9
17.80
Emission_2 236/389 Pyrene 10
Emission_1 281/391 Benzo(a)anthracene 11
20.50
Emission_2 264/381 Chrysene 12
Emission_1 240/510 Benzo(j)fluoranthene 13
23.50 Emission_2 283/394 Benzo(e)pyrene 14
Emission_3 249/443 Benzo(b)fluoranthene 15

Emission_1 243/412 Benzo(k)fluoranthene 16

25.40
Emission_2 260/408 Benzo(a)pyrene 17
27.50 Emission_1 290/398 Dibenz(a,h)anthracene 18
Emission_1 292/415 Benzo(ghi)perylene 19
28.70
Emission_2 246/503 Indeno(1,2,3-cd)pyrene 20

4
 Table 3. Preparation of calibration standards.

Volume of Stock Final Volume Final Concn

Volume
Stock Standard of PAHs Standard of of Calibration of Calibration
of Water
Calibration Mixture PAHs Calibration Standard Standard
(mL)
Mixture (µL) (mL) (µg/L)
100 9.9 100
Stock Standard Mix 1 (10 mg/L)
50 9.95 50
100 9.9 10
Stock Standard Mix 2 (1 mg/L)
50 9.95 5.0
100 9.9 10.0 1.0
Calibration Standard (100 µg/L)
50 9.95 0.5
100 9.9 0.1
Calibration Standard (10 µg/L) 50 9.95 0.05
10 9.99 0.01

calibration standards with concentrations of 100 the analytical column is simultaneously equilibrated with
and 10 µg/L are also used as stock standards for the the second analytical pump (labeled For Separation) of
preparation of the working mixed standard solutions the dual-pump module. After the analytes are bound to
with lower concentrations. the SPE column and impurities are washed out, the SPE
column is switched into the analytical flow path to elute
Preparation of Water Samples the bound analytes (6_1 position); then the analytes are
Tap water samples were collected from local water separated on the analytical column and detected by the
sources in the Pudong District, Shanghai, People’s UV and fluorescence detectors. This method is easily
Republic of China. accomplished using an UltiMate 3000 Dual Gradient
LC system.
Filter water samples using nylon syringe filters prior
to injection. Selection of On-Line SPE and Analytical Columns
The Acclaim PA2 column, was chosen because of its
Add 200 μL of Stock Standard Mix 2 (1.0 mg/L of each compatibility with the water matrix, while giving high
PAH) and 39.8 mL of each filtered water sample to a retentivity for the PAH compounds.
conical flask with plug. The concentration of each PAH
in the spiked water sample will be 5 μg/L.

Add 200 μL of the calibration standard with a

concentration of 10 µg/L of each PAH and 39.8 mL of
each filtered water sample to a conical flask with plug.
The concentration of each PAH in the spiked water
sample will be 0.05 μg/L.

Results and Discussion

Evaluation of On-Line SPE
Figure 5 shows a typical flow schematic of an on-line
SPE system that is directly coupled to the HPLC column
using one 6-port (2p–6p) valve. The filtered sample is
directly injected onto the system and delivered to the
SPE column for enrichment (1_2 position) using one
pump of the dual-pump module (labeled For on-line SPE); Figure 5. Flow schematic of on-line SPE.

5
 Table 4. Calibration data and MDLs for the 20 PAHs.

Concn of PAH
Linearity Standard
Regression MDL
Analyte Detection r2 Range Mixtures in
Equation (µg/L)b
(µg/L) Reagent Water
(μg/L)a
Naphthalene A = 0.0025c + 0.0136 0.9970 1.5~100 10 0.47
UV
Acenaphthylene A = 0.0034c + 0.0062 0.9989 1.5~100 10 0.72
1-Methylnaphthalene A = 7492.48c 0.9910 0.10~50 0.1 0.031
2-Methylnaphthalene A = 10516.8c 0.9982 0.10~50 0.1 0.031
Acenaphthene A = 110163c 0.9972 0.05~50 0.1 0.028
Fluorene A = 266343c 0.9966 0.05~50 0.1 0.016
Phenanthrene A = 138211c 0.9966 0.05~50 0.1 0.011
Anthracene A = 237397c 0.9973 0.05~50 0.1 0.010
Fluoranthene A = 32560.4c 0.9971 0.05~50 0.1 0.017
Pyrene A = 148615c 0.9965 0.05~50 0.1 0.012
Benzo(a)anthracene A = 100842c 0.9980 0.10~50 0.1 0.020
Chrysene Fluorescence A = 74195.7c 0.9986 0.10~50 0.1 0.024
Benzo(j)fluranthene A = 2405.81c 0.9992 0.5~100 1.0 0.156
Benzo(e)pyrene A = 13930.5c 0.9996 0.5~100 1.0 0.161
Benzo(b)fluoranthene A = 33121.4c 0.9954 0.1~50 0.1 0.034
Benzo(k)fluoranthene A = 213065c 0.9962 0.1~50 0.1 0.023
Benzo(a)pyrene A = 122278c 0.9969 0.1~50 0.1 0.035
Dibenz(a,h)anthracene A = 54922.1c 0.9997 0.1~50 1.0 0.048
Benzo(ghi)perylene A = 22558.1c 0.9998 0.1~100 1.0 0.137
Indeno(1,2,3-cd)
A = 8615.10c 0.9979 0.1~100 1.0 0.131
pyrene
a
Used for the determination of the standard deviation value for calculating MDLs
b
The single-sided Student’s t test method (at the 99% confidence limit) was used for estimating MDL, where the standard deviation of
the peak area of eight injections of tap water sample spiked with mixed PAHs standard is multiplied by 3.5 (at n = 8) to yield the MDL.

The Hypersil Green PAH column features a specially much higher sensitivity than UV detection. As previously
tailored alkyl-bonded silica with high carbon loading. discussed, each PAH has its own Ex/Em maximum, and
This column was designed specifically for the thus programmed fluorescence wavelength switching
separation of PAHs and optimized for the published (switching to the Ex/Em maximum wavelength of each
EPA methods.2–4 The column resolves benzo[e]pyrene, individual PAH when the PAH peak passes through the
benzo[j]fluoranthene, and benzo[b]fluoranthene, which fluorescence detector) is necessary to obtain the best
have similar structures (Figure 1). The structures have sensitivity for each PAH. To determine the appropriate
a different configuration, so the resolution is based on fluorescence Ex/Em wavelength for each PAH, a PAH
the steric selectivity of the Hypersil Green PAH column. standard mixture was injected and the diode array
As shown in Figures 1 and 2, using the combination detector used to obtain the maximum UV absorption
of Acclaim PA2 and Hypersil Green PAH columns (UVmax) of each PAH. This experiment showed that
under the specified conditions, on-line SPE followed all UVmax of PAHs were close to 220, 240, or 280 nm.
by HPLC can be accomplished in 35 min with baseline As a result, 220, 240, and 280 nm were used as the
separation of 20 PAHs. excitation wavelengths to perform emission scans for
each PAH in order to determine its emission maximum.
Determination of Ex/Em Maximums for PAHs Excitation scans were performed using resultant
All 20 PAHs except acenaphthylene can be detected emission maximums for all PAHs. The determined Ex/
using a fluorescence detector, which usually offers Em maximums for each PAH are shown in Table 2.
6
 Optimization of Detection Parameters 250,000
A 1
5 Samples:
Unfortunately, practical problems—as when two peaks a. Tap Water
b. Spiked Tap Water
4
elute close to one another—sometimes prevent switching c. Blank
Counts 3 7
to the appropriate Ex/Em wavelength for each PAH c
11
16 Peaks:
1. Naphthalene
b 9 13 18 19 2. Acenaphthylene
peak. When wavelength switching is programmed during a 3. 1-Methylnaphthalene
4. 2-Methylnaphthalene
the elution of a peak or even at the shoulder of a peak, 5. Acenaphthene
–40,000 6. Fluorene
the detector can be saturated and the analysis that 120,000 8
7. Phenanthrene
B 8. Anthracene
follows can be disrupted. To resolve this problem, when 5 9. Fluoranthene
10. Pyrene
two nearby peaks need to use different wavelengths, 10 11. Benzo(a)anthracene
12. Chrysene
three parallel fluorescence monitoring channels are used Counts
c
13. Benzo(j)fluoranthene
14. Benzo(e)pyrene
12 16 17
to perform the analysis (one channel for each of the b 14 20
15. Benzo(b)fluoranthene
16. Benzo(a)pyrene
nearby peaks). a 17. Benzo(k)fluoranthene
18. Dibenz(a,h)anthracene
–10,000 19. Benzo(ghi)perylene
6 20. Indeno(1,2,3-cd)pyrene
200,000
C
For example, as shown in Figure 2, benzo(j)fluoranthene
(Peak 13), benzo(e)pyrene (Peak 14), and benzo(b)
fluoranthene (Peak 15) elute in a small retention Counts c
time window; therefore, three parallel fluorescence 16
15 17
b
monitoring channels (Emmisions_1, _2, and _3) are a

used for their determination. For the same reason, –20,000

0.3 5 10 15 20 25 30 35
1-methylnaphthalene (Peak 3), 2-methylnaphthalene Minutes

(Peak 4), acenaphthene (Peak 5), and fluorene (Peak Figure 6. A tap water sample detected by fluorescence using
programmed wavelength switching in three parallel channels: (A)
6) are also monitored using three parallel fluorescence Emission_1, (B) Emission_2, (C) Emission_3.
monitoring channels. Table 2 lists the parallel
fluorescence monitoring channels used for all 20 PAHs.
was calculated using the single-sided Student’s t test
Reproducibility, Linearity, and Detection Limits method (at the 99% confidence limit). Eight consecutive
Method precision was estimated using fluorescence injections of three reagent water (DI water) samples
detection by making seven consecutive 1000 μL mixed with 0.1, 1.0, and 10 μg/L of the PAH standard
injections of a calibration standard with a concentration mixtures were used to determine the standard deviation
of 1 μg/L of each PAH. Method precision using UV values for calculating MDLs. The calculated MDLs are
detection was measured in the same manner, but with a listed in Table 4.
calibration standard having a concentration of 10 μg/L of
each PAH. Retention time reproducibilities (RSD) are all Analysis of Tap Water Samples
≤0.16 and peak area reproducibilities (RSD) are all ≤1.3, No target analytes were found in the tap water
thus demonstrating good short-term precision for this samples. Figure 6 shows chromatograms of a tap
on-line SPE HPLC method. water sample detected with fluorescence using three
parallel channels. Method recovery was investigated
Calibration linearity of 20 PAHs (using UV detection by determining the recoveries in a tap water sample
for naphthalene and acenaphthylene while using spiked at two concentrations (0.05 and 5 μg/L of
fluorescence detection for the other 18 PAHs) was each PAH). The recovery range was from 80 to 120%,
investigated by making three consecutive 1000 µL demonstrating that this on-line SPE HPLC method
injections of a mixed standard prepared at nine different combined with UV and fluorescence detections
concentrations (i.e., 27 total injections). The external provides good selectivity and suitability for the
standard method was used to establish the calibration determination of PAHs in water samples.
curve and quantify the analytes in the tap water
samples. Different linearity ranges were observed for Conclusion
the PAHs when plotting concentration versus peak area. This work describes an on-line SPE HPLC method with
Detailed calibration data calculated by Chromeleon CDS UV absorbance and fluorescence detections for rapid
software are shown in Table 4. The method detection and sensitive determination of 20 PAHs in tap water.
limit (MDL) of each PAH for UV or fluorescence detection The determination is performed on an UltiMate 3000
7
 Dual Gradient LC system controlled by Chromeleon
CDS software and combined with a Hypersil Green
PAH analytical column. The reduced MDLs for UV and
fluorescence detection enabled by on-line SPE using
the Acclaim PA2 column provide a convenient method
for determining these compounds in drinking and
environmental waters using HPLC.

References
1. Thermo Fisher Scientific Application Note 196: Determination of Polycyclic Aromatic
Hydrocarbons (PAHs) in Edible Oils by Donor-Acceptor Complex Chromatography
(DACC)-HPLC with Fluorescence Detection. Sunnyvale, CA, 2008. [Online]
https://tools.thermofisher.com/content/sfs/brochures/AN-196-Determination-
PAH-Edible-Oil-LPN1998.pdf
2. Method 550: Determination of Polycyclic Aromatic Hydrocarbons in Drinking Water by
Liquid-Liquid Extraction and HPLC with Coupled Ultraviolet and Fluorescence Detection;
U.S. Environmental Protection Agency: Cincinnati, OH, 1990.
3. Method 550.1: Determination of Polycyclic Aromatic Hydrocarbons in Drinking Water by
Liquid-Solid Extraction and HPLC with Coupled Ultraviolet and Fluorescence Detection;
U.S. Environmental Protection Agency: Cincinnati, OH, 1990.
4. Method 610: Polynuclear Aromatic Hydrocarbons; U.S. Environmental Protection Agency:
Cincinnati, OH, 1982.

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